strain engineering for microbial production of value-added ... · 2,3-butanediol (2,3-bdo)...
TRANSCRIPT
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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
Research review paper
Strain engineering for microbial production of value-added chemicals andfuels from glycerolAdam W. Westbrook⁎, Dragan Miscevic, Shane Kilpatrick, Mark R. Bruder, Murray Moo-Young,C. Perry Chou⁎
Department of Chemical Engineering, Waterloo, Ontario, Canada
A R T I C L E I N F O
Keyword:GlycerolMetabolic engineeringBiorefineryBiofuelsE. coliClostridiumKlebsiellaCitrobacterLactobacillus
A B S T R A C T
While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-basedeconomy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentallycompatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolicengineering, synthetic biology, and systems biology principles for the development of whole-cell catalyticplatforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined orcrude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbialdissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain de-velopment for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesseshave been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains anuntapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbialhosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. Webegin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and keyend metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilateglycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and system-atically review the progress made toward engineering these microorganisms for glycerol biorefinery. We alsoidentify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bot-tlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culmi-nates in the development of potential strategies to overcome the current technical limitations identified forcommonly employed strains, with an outlook on the suitability of different hosts for the production of keymetabolites and avenues for their future development into biomanufacturing platforms.
https://doi.org/10.1016/j.biotechadv.2018.10.006Received 16 December 2017; Received in revised form 3 October 2018; Accepted 10 October 2018
Abbreviations: 3-HPA, 3-hydroxypropionaldehyde; 1,2-PDO, 1,2-propanediol; 1,3-PDO, 1,3-propanediol; 1,3-PDOOR, 1,3-PDO oxidoreductase; 2,3-BDO, 2,3-bu-tanediol; 3-HH, 3-hydroxyhexanoate; 3-HP, 3-hydroxypropionic acid; 3-HV, 3-hydroxyvalerate; (R)-3-HV-CoA, (R)-3-hydroxyvaleryl-CoA; ALDH, aldehyde dehy-drogenase; ACE, Allele-Coupled Exchange; AOR, aldehyde oxidoreductase; asRNA, antisense RNA; ATP, adenosine Triphosphate; cAMP, cyclic adenosine mono-phosphate; CRE, catabolite repression element; CRISPR, Clustered Regularly Interspaced Palindromic Repeats; CRISPRi, CRISPR interference; Cas9, CRISPR-associated [protein] 9; CRP, cAMP receptor protein; dcw, dry cell weight; DHA, dihydroxyacetone; DHAK, DHA kinase; DHAP, dihydroxyacetone phosphate; DODHt,diol dehydratase; EDP, Entner-Doudoroff pathway; FAD, flavin adenine dinucleotide; FHL, formate hydrogen lyase; FDH, formate dehydrogenase; G3P, glycerol-3-phosphate; GDHt, glycerol dehydratase; GluDH, glucose dehydrogenase; GlyDH, glycerol dehydrogenase; GK, glycerol kinase; LDH, lactate dehydrogenase; MDH,malate dehydrogenase; MG, methylglyoxal; MGR, MG reductase; MGS, MG synthase; NADH, nicotinamide adenine dinucleotide; NOX, NADH oxidase; PHA, poly-hydroxyalkanoate; P(3HB-co-3HH), poly(3-hydroxyburyrate-co-3-hydroxyhexanoate); P(3HB-co-3HV), poly(3-hydroxybutyrate-co-3-hydroxyvalerate); P(3HP), poly(3-hydroxypropionate); PDU, propanediol utilization; PEP, phosphoenolpyruvate; PCK, PEP carboxykinase; PFL, pyruvate formate lyase; PDC, pyruvate decarbox-ylase; PDH, pyruvate dehydrogenase; PFOR, pyruvate:ferrodoxin oxidoreductase; PGL, phosphogluconolactonase; PPC, PEP carboxylase; PPP, pentose phosphatepathway; PTA, phosphotransacetylase; PYK, pyruvate kinase; PO, pyruvate oxidase; RBS, ribosome binding site; Sbm, sleeping beauty mutase; TCA, tricarboxylicacid; UDH, pyridine nucleotide transhydrogenase
⁎ Corresponding authors.E-mail addresses: [email protected] (A.W. Westbrook), [email protected] (C.P. Chou).
Biotechnology Advances 37 (2019) 538–568
Available online 17 October 20180734-9750/ © 2018 Elsevier Inc. All rights reserved.
T
1. Introduction
Currently, it is estimated that ~90% of the world’s energy re-quirements are met through the use of non-renewable fossil fuels suchas petroleum, natural gas, bitumens, oil shale, and coal (Parikka, 2004;Pöschl et al., 2010; Srirangan et al., 2012). These resources, particularlyoil and natural gas, are also the most important feedstock for the pro-duction of fine and commodity chemicals (Basiago, 1994; Liu et al.,2010). Nevertheless, the unrestricted use of fossil fuels is consideredunsustainable due to the finite supply and unequal distribution ofnatural reserves, coupled with climate change resulting from increasinggreenhouse gas emissions (Basiago, 1994; Börjesson, 2009; Sriranganet al., 2012). As a result, biorefinery becomes an emerging approachthrough integrating systems biology, genetic engineering, syntheticbiology, and metabolic engineering principles for the development ofwhole-cell biocatalytic platforms for manufacturing purposes (Menonand Rao, 2012; Octave and Thomas, 2009). While this appears to be apromising avenue, a major detriment to the use of biological platformsis the high cost of feedstock (Menon and Rao, 2012; Octave andThomas, 2009). Industrially, value-added products are derived fromagricultural crops (i.e. first-generation feedstock) or from lig-nocellulosic crops and agricultural wastes (i.e. second-generationfeedstock) (Srirangan et al., 2012). First-generation feedstocks (e.g.corn, starch, oilseed, and sugar) often have a high energy, oil, andcarbohydrate content and are currently used for the production ofbiodiesel (and other bio-esters), bioethanol (and other bioalcohols), andbiogas (Hein and Leemans, 2012; Srirangan et al., 2012). However,these bioprocess schemes are considered unsustainable due to theircompetition with the human food and animal feed markets, and therequirement for large arable lands (Schmidhuber, 2008). While notdirectly impacting the cost and availability of food supplies, second-generation feedstocks are not practical due to their inherent recalci-trance and the high cost of pre-treatment technologies required for theirvalorization (Luo et al., 2010; Srirangan et al., 2012).
Accordingly, several alternative biorefinery schemes have beenproposed to utilize biomass feedstock in an economically viable mannerto compete with existing petroleum-refinery technologies. One suchmodel is co-utilization of the starting feedstock and the byproductstream (Clomburg and Gonzalez, 2013; Yazdani and Gonzalez, 2007).For instance, glycerol is a major sugar alcohol byproduct associatedwith bioethanol (Raynaud, and xe, line, Sar, xe, abal, P., Meynial-Salles,I., Croux, C., Soucaille, P., 2003; Zeng and Biebl, 2002) and biodiesel(Balat and Balat, 2010) production. Transesterification of oils and fatswith an alcohol generates ~1 lb crude glycerol (with at least 80%purity) for every 10 lb biodiesel produced (López et al., 2009; Sabourin-Provost and Hallenbeck, 2009). Likewise, all first-generation-basedbioethanol programs generate significant amounts of glycerol as a fer-mentative end-product (Kim et al., 2008; Zeng and Biebl, 2002). Cur-rently, biodiesel and bioethanol represent the two largest biomass en-ergy programs in the world, and their significant growth led to a morethan 10-fold increase in global glycerol production between 2004 and2011, with ~700 million lb crude glycerol produced in the UnitedStates alone in 2011 (Clomburg and Gonzalez, 2013; López et al.,2009). Additionally, many other industries which derive products fromanimal fats and vegetable oils generate waste streams of high glycerolcontent, e.g. the oleochemical industry (Sabourin-Provost andHallenbeck, 2009; Yazdani and Gonzalez, 2007). This surplus in gly-cerol has plummeted its price to ~2.5 cents/lb in recent years, effec-tively rendering it a waste product with an associated disposal cost (Rajet al., 2008).
As a carbon source, glycerol offers several distinctive advantagesover traditional fermentable sugars. Due to the high degree of reduction(reductance κ = 4.67), glycolytic degradation of glycerol generatesapproximately twice the number of reducing equivalents (i.e. NADH)compared to xylose and glucose (κ = 4) (Murarka et al., 2008; Yazdaniand Gonzalez, 2007). Therefore, harnessing glycerol metabolism not
only results in higher yields but also expands the repertoire of chemi-cals and fuels generated from microbial systems. However, microbialdissimilation of glycerol is difficult under anaerobic conditions, as thecellular redox balance (i.e. the NAD+/NADH ratio) must be properlymaintained through terminal transfer of electrons to internally pro-duced organic compounds as opposed to molecular oxygen (Celińska,2010; Yazdani and Gonzalez, 2007). As a result, only select organisms(e.g. Clostridium pasteurianum and Klebsiella pneumoniae) are capable offermenting glycerol anaerobically. Glycerol metabolism generates in-termediates such as dihydroxyacetone (DHA) and 3-hydro-xypropionaldehyde (3-HPA), and reduced end-products such asethanol, 1,2-propanediol (1,2-PDO), 1,3-propanediol (1,3-PDO), and2,3-butanediol (2,3-BDO) (Clomburg and Gonzalez, 2013; Murarkaet al., 2008; Yazdani and Gonzalez, 2007). Accordingly, glycerol fer-mentation has been extensively explored in both native and geneticallytractable hosts for the production of various value-added chemicals andfuels, including advanced alcohols (e.g. 1-propanol), organic acids (e.g.succinic, propionic, and 3-hydroxypropionic acids), natural products(e.g. terepenes), ketones (e.g. acetone and butanone) and bio(co)poly-mers (e.g. polyhydroxyalkanoate (PHA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]). This article reviews our cur-rent understanding of glycerol metabolism and recent approaches ofstrain engineering for glycerol biorefinery.
2. Overview of glycerol metabolism in microbes
Due to the high reductance of glycerol, there is an excess of reducingequivalents that remain unbalanced such that the overall redox balancewithin a cell is a vital principle that governs the nature and quantity ofmetabolites during glycerol dissimilation (Clomburg and Gonzalez,2013). The natural fermentative metabolism of glycerol has beenthoroughly studied in the Enterobacteriaceae family (Booth, 2005;Murarka et al., 2008). Under fermentative conditions, microorganismsmust be metabolically capable of consuming glycerol in the absence ofexternal electron acceptors. Specifically, for 1.1 mM of glycerol in-corporated into cell mass, 0.6 mM reducing equivalents (i.e. NADH) aregenerated. This is not an issue for other common carbohydrates, such asglucose and xylose, since their degree of reduction is lower than that ofthe cell biomass. Furthermore, excess electrons cannot be consumedthrough the production of reduced products such as ethanol or succi-nate since their production from glycerol is already redox-balanced.Thus, to achieve redox poise, there must be a metabolic pathway gen-erating more reduced products for consuming surplus reducingequivalents. The formation of 1,3-PDO (Homann et al., 1990; Saint-Amans et al., 1994; Schütz and Radler, 1984) and 1,2-PDO (Clomburgand Gonzalez, 2011) via ATP-neutral pathways results in successfulanaerobic dissimilation of glycerol due to the highly reduced nature ofthese metabolites (κ = 5.33).
Like other small and uncharged molecules, glycerol can cross thecytoplasmic membrane via two mechanisms: (i) passive diffusion underhigh concentrations; and (ii) facilitated diffusion with the aid of atransport protein (i.e. GlpF) under low concentrations (da Silva et al.,2009; Sun et al., 2008). Intracellular glycerol is subsequently metabo-lized either in the presence of electron acceptors (i.e. respiratorybranch) or in the absence of electrons acceptors (i.e. fermentativebranch) (Gonzalez et al., 2008). Fermentative metabolism of glycerolhas been previously reported in microbes within the genera of Klebsiella(Forage and Foster, 1982; Homann et al., 1990), Citrobacter (Danielet al., 1995; Seifert et al., 2001), Clostridium (Abbad-Andaloussi et al.,1995; Biebl, 2001; Macis et al., 1998), and Lactobacillus (Talarico et al.,1990), as well as in Escherichia coli (Dharmadi et al., 2006; Murarkaet al., 2008). The natural fermentative bioconversion of glycerol to amore reduced metabolite, such as 1,3-PDO, occurs both reductively andoxidatively in Klebsiella sp., Citrobacter sp., Clostridium sp., and certainstrains of Lactobacilli sp. (da Cunha and Foster, 1992; da Silva et al.,2009). In the reductive branch, a B12-dependent glycerol dehydratase
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
539
(GDHt) dehydrates glycerol to form 3-HPA, which is subsequently re-duced to 1,3-PDO (Fig. 1) (Murarka et al., 2008). In the latter reaction,NAD+ is regenerated by NADH-dependent 1,3-PDO oxidoreductase(1,3-PDOOR), resulting in a balanced internal redox state (González-Pajuelo et al., 2006). Glycerol fermentation is the only known processfor anaerobic conversion for 1,3-PDO production (Homann et al.,1990). As 1,3-PDO is more reduced than glycerol, its formation is ac-companied by a more oxidized co-product (Clomburg and Gonzalez,2013). In Klebsiella sp. and Citrobacter sp., acetic acid is the major co-product during 1,3-PDO production under fermentative conditions(Homann et al., 1990), whereas mixed co-products are formed (i.e.butyrate, butanol, ethanol, and lactate) in Clostrodium sp. (Biebl, 2001;Clomburg and Gonzalez, 2013; Saint-Amans et al., 2001). Biosynthesisof 1,3-PDO relies on the availability of NADH, which can be naturallygenerated in Klebsiella sp. and Lactobacillus sp. via oxidation of 3-HPAinto 3-hydroxypropionic acid (3-HP) by NAD+-dependent aldehydedehydrogenase (ALDH) (Zhu et al., 2015). In the oxidative branch,NAD+-dependent glycerol dehydrogenase (GlyDH) catalyzes thetransformation of glycerol to dihydroxyacetone (DHA) (Fig. 1), which issubsequently phosphorylated by a DHA kinase (DHAK) to form dihy-droxyacetone phosphate (DHAP) (Shams Yazdani and Gonzalez, 2008).In Klebsiella sp. and Citrobacter sp., the genes encoding enzymes specific
to glycerol metabolism (i.e. DhaB/DhaBCE, DhaT, DhaD, and DhaK) arelocated in a DHA (dha) regulon (Zhu et al., 2002). In certain Clostridiumsp., such as Clostridium butyricum, the dha regulon is composed of threegenes encoding GDHt (i.e. DhaB1 encoded by dhaB1), its reactivatingprotein (i.e. DhaB2 encoded by dhaB2), and 1,3-PDOOR (i.e. DhaTencoded by dhaT) (Raynaud, and xe, line, Sar, xe, abal, P., Meynial-Salles, I., Croux, C., Soucaille, P., 2003), while the dha regulon can befound in two spatially different clusters on the chromosome of Clos-tridium perfringens (Ignatova et al., 2003) (Table 1).
E. coli can ferment glycerol anaerobically in a pH-dependent mannerwithout external electron acceptors (Booth, 2005; Dharmadi et al.,2006). The internal availability of CO2 generated by formate hydrogenlyase (FHL)-mediated oxidation of formate enables E. coli to anaerobi-cally metabolize glycerol. For E. coli to synthesize small molecules andfatty acids, it requires a steady supply of bicarbonate/CO2 substrates.Lowering the pH of the media as well as potassium and phosphateconcentrations under fermentative conditions activates the transcrip-tion of FHL and subsequently achieves the cellular requirement for CO2
upon formate accumulation (Murarka et al., 2008). Additionally, for-mate oxidation also produces H2 and, therefore, can contribute to re-ducing equivalent accumulation that other redox-balanced pathways,such as succinate and ethanol-generating pathways, cannot (Dharmadi
Fig. 1. Schematic representation of the major glycerol catabolism pathways in bacteria. Blue arrows represent reactions in the fermentative branch, displaying bothreductive and oxidative routes; green arrows represent reactions in the respiratory branch; red arrows represent reactions in the 1,2-PDO pathway; and gold arrowsrepresent reactions in the 2,3-BDO pathway. Solid line: direct bioconversion step; dashed line: multi-step bioconversion. Enzyme abbreviations: ACK, acetate kinase;ADH, bifunctional aldehyde/alcohol dehydrogenase; ae-G3PDH, aerobic glycerol-3-phosphate dehydrogenase; AKR, aldo-keto reductase; ALDH, aldehyde dehy-drogenase; ALDC, α-acetolactate decarboxylase; ALS, α-acetolactate synthase; an-G3PDH, anaerobic glycerol-3-phosphate dehydrogenase; AR, acetoin reductase;DODHt, diol dehydratase; DHAK, dihydroxyacetone kinase; FHL, formate hydrogen lyase; FRD, fumarate reductase; GDHt, glycerol dehydratase; GK, glycerol kinase;GlpF, glycerol diffusion facilitator; GlyDH, glycerol dehydrogenase; LDH, lactate dehydrogenase; MGR, methylglyoxal reductase; MGS, methylglyoxal synthase; PDH,pyruvate dehydrogenase; PduL, phosphate propanoyltransferase; PduP, propionaldehyde dehydrogenase; PduW, propionate kinase; PFL, pyruvate formate-lyase;PTA, phosphotransacetylase; PK, pyruvate kinase; 1,2-PDOOR; 1,2-propanediol oxidoreductase; 1,3-PDOOR, 1,3-propanediol oxidoreductase. Chemical inter-mediates and product abbreviations: α-AL, α-acetolactate; acetyl-P, acetyl phosphate; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; MG, methylglyoxal; PEP, phosphoenolpyruvate; 1,2-PDO, 1,2-propanediol; 1,3-PDO, 1,3-propanediol; 3-HP, 3-hydroxypropionate; 3-HP-CoA, 3-hydro-xypropionyl-CoA; 3-HP-P, 3-hydroxypropionyl phosphate.
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
540
Table1
Sum
mar
yof
enzy
mes
com
pris
ing
natu
ralp
athw
ays
for
glyc
erol
cata
bolis
min
Kleb
siella
sp.,Citrob
acter
sp.,Clostridium
sp.,La
ctob
acillus
sp.,
andE.
coli.
Locu
sta
gs,a
ndre
spec
tive
enzy
me
nam
esw
here
avai
labl
e,ar
epr
ovid
edfo
ra
repr
esen
tativ
est
rain
for
each
genu
sor
spec
ies.
Each
num
bere
dco
lum
nco
rres
pond
sto
one
ofth
enu
mbe
red
reac
tions
inFi
g.1.
Ferm
enta
tive
met
abol
ism
Resp
irat
ory
met
abol
ism
Org
anis
m1
23
45
67
89
Kleb
siella
sp.1,
2
(K.p
neum
oniae
MG
H78
578)
GD
Ht:
Dha
B⁎,a
(RS1
8900
,RS
1889
5,RS
1889
0)
DO
DH
t:Pd
uCD
E(R
S171
90⁎ ,
RS17
195,
RS17
200)
1,3-
PDO
OR:
Dha
T⁎
(RS1
8910
),Yq
hD⁎,
b
(RS1
8380
)
ALD
H:
YdcW
⁎
(RS1
0355
),Yn
eI⁎,
c
(RS0
8785
),Fe
aB⁎
(RS0
7805
),Pu
uC⁎
(RS0
5455
),Ba
dH⁎
(RS0
3160
),G
abD
⁎
(RS0
1360
)
PduP
⁎
(RS1
7245
)
PduL
⁎
(RS1
7225
)Pd
uW⁎
(RS1
7275
)G
lyD
H:
Dha
D⁎
(RS1
8930
),G
ldA
⁎
(RS2
2860
)
DH
AK:
Dha
KI⁎
(RS1
8955
),D
haKI
I⁎
(RS1
8940
,RS
1894
5,RS
1895
0)
GK:
Glp
K⁎,d
(RS2
1635
)
Citrob
acter
sp.3,
4
(C.f
reun
dii
CFN
IH1)
GD
Ht:
Dha
BCE⁎
(RS0
2570
,RS
0256
5,RS
0256
0)
DO
DH
t:Pd
uCD
E(R
S208
65,
RS20
870,
RS20
875)
1,3-
PDO
DH
:D
haT⁎,
b
(RS0
2580
)
NA
NA
NA
NA
Gly
DH
:D
haD
⁎,c
(RS0
2600
)G
ldA
(RS0
7260
)
DH
AK:
Dha
K⁎
(RS1
9390
)
GK:
Glp
Kd
(RS0
5005
)
Clostridium
sp.5,
6
(C.p
asteurianu
mA
TCC
6013
)
GD
Ht:
PduC
DE/
Dha
BCE
(RS1
1085
,RS
1108
0,RS
1107
5)D
haB1
⁎
(Gen
bank
AY1
1298
9.1)
j
1,3-
PDO
H:
Dha
T2(R
S110
50)
Dha
T1(R
S053
50)
NA
NA
NA
NA
Gly
DH
:G
ldA
(RS0
5875
,RS
1866
5),
RS13
630,
RS16
340
DH
AK:
Dha
K(R
S200
60)
GK:
Glp
K(C
A_C
1321
)k
Lactob
acillus
sp.7,
8
(L.r
euteri
DSM
2001
6)
DO
DH
t:Pd
uCD
E⁎
(RS0
9105
,RS
0910
0,RS
0909
5)
1,3-
PDO
OR:
RS00
160⁎ ,
RS09
040⁎
ALD
H:
PduP
⁎,e
(RS0
9045
)
PduP
⁎
(RS0
9045
)Pd
uL(R
S090
70)
PduW
(RS0
9035
)G
lyD
H:
RS09
575
NA
GK:
RS05
600
E.co
liN
AA
LDR:
YqhD
⁎
(b30
11)
ALD
H:
Ald
H⁎,
c
(b13
00)
NA
NA
NA
Gly
DH
:G
ldA
⁎
(b39
45)
DH
AK:
Dha
KLM
⁎
(b12
00,
b119
9,b1
198)
GK:
Glp
K⁎
(b39
26)
Resp
irat
ory
met
abol
ism
1,2-
PDO
Succ
inat
eLa
ctat
e2,
3-BD
O
Org
anis
m10
1112
1314
1516
1718
1920
Kleb
siella
sp.1,
2
(K.p
neum
oniae
MG
H78
578)
ae-G
3PD
H:
Glp
D(R
S204
85)
an-G
3PD
H:
Glp
ABC
(RS1
4215
,
NA
NA
NA
NA
NA
FRD
:Fr
dABC
D(R
S245
50,
PK:
PykA
(RS1
2780
),
LDH
:Ld
hA⁎
(RS0
7775
),
ALS
:Bu
dB⁎
(RS1
1100
)(con
tinue
don
next
page
)
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
541
Table1
(con
tinue
d)
Resp
irat
ory
met
abol
ism
1,2-
PDO
Succ
inat
eLa
ctat
e2,
3-BD
O
RS14
220,
RS14
225)
RS24
545,
RS24
540,
RS24
535)
PykF
(RS1
1500
)Ld
h1(R
S087
70),
Ldh2
(RS2
1315
)Citrob
acter
sp.3,
4
(C.f
reun
dii
CFN
IH1)
ae-G
3PD
H:
Glp
D(R
S047
15)
an-G
3PD
H:
Glp
ABC
(RS2
2435
,RS
2244
0,RS
2244
5)
NA
NA
NA
NA
NA
FRD
:Fr
dABC
D(R
S083
35,
RS08
330,
RS08
325,
RS08
320)
PK:
RS16
640,
RS20
115
LDH
:Ld
hA⁎
(RS1
8135
)
NA
Clostridium
sp.5,
6
(C.p
asteurianu
mA
TCC
6013
)
NA
an-G
3PD
H:
Glp
A(C
A_C
1322
)k
NA
NA
NA
NA
NA
NA
PK:
RS13
430
LDH
:RS
1342
0,RS
0850
5
NA
Lactob
acillus
sp.7,
8
(L.r
euteri
DSM
2001
6)
ae-G
3PD
H:
RS01
940
NA
NA
NA
NA
NA
NA
FRD
:RS
0804
0PK
:RS
0402
5LD
Hf :
RS00
995,
RS03
840,
RS03
845,
RS08
830,
RS09
895
NA
E.co
liae
-G3P
DH
:G
lpD
⁎
(b34
26)
an-G
3PD
HG
lpA
BC⁎
(b22
41,b
2242
,b2
243)
MG
S:M
gsA
⁎
(b09
63)
AKR
:Ye
aE⁎
(b17
81),
YghZ
⁎
(b30
01),
YafB
⁎
(b02
07),
YqhE
⁎
(b30
12),
YqhD
⁎
(b30
11)
glyD
H:
Gld
A⁎
(b39
45)
MG
RYd
jG(b
1771
)
1,2-
PDO
R:Fu
cO⁎
(b27
99)
FRD
:Fr
dABC
D⁎
(b41
54,
b415
3,b4
152,
b415
1)
PK:
PykA
⁎
(b18
54),
PykF
⁎
(b16
76)
LDH
:Ld
hA⁎
(b13
80)
NA
2,3-
BDO
Etha
nol
Ace
tate
Org
anis
m21
2223
2425
2627
2829
Kleb
siella
sp.1,
2
(K.p
neum
oniae
MG
H78
578)
AR:
BudC
⁎
(RS1
1105
)
ALD
C:Bu
dA⁎
(RS1
1095
)
AR:
BudC
(RS1
1105
)
PFL:
PflA
B(R
S050
10,
RS05
015)
PDC:
RS14
705
PDH
:A
ceE
(RS0
0635
),Po
xB⁎
(RS0
4870
)D
HLA
T:A
ceF
(RS0
0640
)
ALD
H:
Ald
A⁎
(RS0
8050
)RS
0289
0,RS
0542
5,A
stD
(RS0
6540
)
AD
H:
YigB
(RS2
5235
),RS
2268
0,Yq
hDb
(RS1
8380
),A
dhE⁎
(RS1
1855
),A
dhP
(RS0
9985
)
PTA
:Pt
a⁎
(RS1
4425
),Eu
tD(R
S149
85)
ACK
:A
ckA
⁎
(RS1
4420
)Td
cD(R
S123
55)
Citrob
acter
sp.3,
4
(C.f
reun
dii
CFN
IH1)
NA
NA
NA
PFL:
PflA
(RS1
4340
)
PDH
:A
ceEF
(RS1
0115
,RS
1012
0)
ALD
H:
RS16
255,
RS20
920
Ast
D(R
S162
55)
AD
H:
RS02
285,
RS04
980,
RS15
860,
RS16
775,
RS17
775
PTA
:Pt
a(R
S226
70)
ACK
:RS
2095
0,RS
2266
5Td
cD(R
S027
95)
Clostridium
sp.5,
6N
AN
AN
AN
AA
LDH
:A
dhE
AD
H:
Adh
EA
CK:
Ack
A(con
tinue
don
next
page
)
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
542
Table1
(con
tinue
d)
2,3-
BDO
Etha
nol
Ace
tate
(C.p
asteurianu
mA
TCC
6013
)PD
H:
NifJ
(RS0
2460
)
(RS0
7340
,RS
0275
0,RS
0758
0RS
1406
5,CA
_P01
62k ,
CA_P
0162
k )
(RS0
7340
,RS
0275
0,RS
0758
0RS
1406
5,CA
_P01
62k ,
CA_P
0035
k )
PTA
:Pt
a(R
S101
15)
(CLP
A_
RS10
110)
Lactob
acillus
sp.7,
8
(L.r
euteri
DSM
2001
6)
NA
NA
NA
NA
PDH
:RS
0341
0,RS
0341
5D
HLA
T:RS
0342
0
ALD
H:
RS00
180,
RS01
680
AD
Hg :
RS07
840,
RS08
065,
RS08
320,
RS09
040,
RS09
680
PTA
:RS
0207
5A
CK:
RS02
910,
RS09
035e
E.co
liN
AN
AN
APF
L:Pfl
B⁎
(b09
03),
TdcE
⁎
(b31
14)
PDH
(E1)
:A
ceE⁎
(b01
14)
DH
LAT
(E2)
:A
ceF⁎
(b01
15)
LAD
H(E
3):
Lpd⁎
(b01
16)
ALD
Hh :
Adh
E⁎(b
1241
)A
DH
i :A
dhE⁎
(b12
41),
Adh
P⁎(b
1478
)
PTA
:Pt
a⁎
(b22
97),
EutD
⁎(b
2458
)
ACK
:A
ckA
⁎
(b22
96)
1 K.pn
eumon
iae
andK.
oxytoc
a;2
locu
stag
prefi
xis
KPN
;34
locu
stag
prefi
xis
CFN
IH;5 C
.pasteurianu
man
dC.
acetob
utylicum
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cust
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PA;7 L
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teri,
L.pa
nis,L.
collino
ides
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s,an
dL.
brevis;
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cus
tag
prefi
xis
LREU
;ain
activ
ein
K.ox
ytoc
a;b
NA
D(P
)H-d
epen
dent
;cN
AD
(P)+
-dep
ende
nt;d
Pote
ntia
llyin
activ
epa
thw
ayin
som
est
rain
s;e
hom
olog
ueno
tpre
sent
inL.
panis;
fid
entifi
edpu
tativ
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Hsw
ere
sele
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base
don
the
mos
tsig
nific
anth
omol
ogy
amon
gpu
tativ
eLD
Hsf
ound
inal
lspe
cies
incl
uded
(add
ition
alpu
tativ
eLD
Hsm
ayex
isti
nea
chsp
ecie
s);g
iden
tified
puta
tive
AD
Hsw
ere
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base
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the
mos
tsig
nific
anth
omol
ogy
amon
gpu
tativ
eA
DH
sfo
und
inal
lspe
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incl
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(add
ition
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tativ
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DH
sm
ayex
isti
nea
chsp
ecie
s);h
addi
tiona
lAD
HE
may
exis
t;iad
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nalA
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may
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2-in
depe
nden
tGD
Htf
rom
C.bu
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um;k
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C.ac
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peri
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tally
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onst
rate
d.
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
543
et al., 2006). Also, under fermentative conditions, E. coli can recycle theexcess H2 formed via FHL by functional expression of native hydro-genase isoenzymes Hyd-1 and Hyd-2 (Murarka et al., 2008; Sawerset al., 1985; Sawers and Boxer, 1986). Compared to 1,3-PDO, theconversion of glycerol to 1,2-PDO is uncommon and less effective withsignificantly lower yields (Clomburg and Gonzalez, 2013; Zeng andSabra, 2011). In the oxidative branch, 1,2-PDO production starts withthe formation of DHAP via phosphorylation of DHA. Subsequently,DHAP enters the methylglyoxal (MG) pathway and is converted to theintermediate MG by a MG synthase (MGS; Fig. 1) (Hopper and Cooper,1972). The availability of DHAP is crucial since it represents a nodewhere carbon flux between glycolysis and 1,2-PDO formation diverges(Clomburg and Gonzalez, 2011). Conversion of MG to 1,2-PDO is pos-sible through either (i) the intermediate acetol via aldehyde oxidor-eductase (AOR), or (ii) the intermediate lactaldehyde via GlyDH or MGreductase (MGR; Fig. 1) (Clomburg and Gonzalez, 2011).
Under respiratory conditions, glycerol dissimilation can advancethrough two different two-step routes to DHAP (Fig. 1): (i) the aerobicglycerol kinase (GlpK)-glycerol-3-phosphate (G3P) dehydrogenase(GlpD) pathway; or (ii) the anaerobic GlpK-anaerobic G3P dehy-drogenase (GlpABC) pathway (Blankschien et al., 2010; Durnin et al.,2009). In either case, G3P is first synthesized via phosphorylation ofglycerol by GlpK at the expense of ATP (Fig. 1) (Murarka et al., 2008),or it can be directly imported into the cell via the G3P transporter, GlpT(encoded by glpT) (Jin et al., 1982). Through the aerobic pathway,GlpD, a plasma membrane-associated homodimer, generates DHAP viaoxidation of G3P. On the other hand, DHAP is synthesized from G3P byGlpABC (encoded by glpABC) anaerobically (Fig. 1). In addition tobeing a precursor to DHAP, G3P also serves as a precursor for lipidbiosynthesis or production of other metabolites (da Silva et al., 2009).Similar to the dha regulon in the fermentative pathway, the glp regulonin E. coli contains genes encoding enzymes found in the respiratorypathway (i.e. GlpF, GlpK, GlpD, and GlpABC). Similar glp regulons arealso present in Klebsiella sp. and Citrobacter sp. (da Silva et al., 2009),while the putative partial glp regulon of Lactobacillus sp. is inactive, due,in part, to the absence of DHAK (Morita et al., 2008).
3. Biological conversion of glycerol for the production of value-added chemicals in select microbial platforms
3.1. Klebsiella sp.
3.1.1. Natural metabolismKlebsiella sp. (i.e. K. pneumoniae) own natural pathways for flexible
and efficient metabolism of glycerol (Kumar and Park, 2017; Zenget al., 1993; Zhang et al., 2008; Zhang et al., 2009). Fermentativeglycerol dissimilation in Klebsiella sp. proceeds anaerobically throughcoupled oxidative and reductive branches (Fig. 1), providing outlets forenergy generation and redox balance. In the oxidative branch, glycerolis first converted to DHA by GlyDH (i.e. DhaD encoded by dhaD), andDHA is subsequently converted to DHAP by one of two DHAKs, i.e.DhaK I (encoded by dhaK) and DhaK II (encoded by dhaK123), withDhaK II playing the dominant role (Wang et al., 2003; Wei et al., 2014).Glycerol is simultaneously converted to 3-HPA by GDHt (i.e. DhaBencoded by dhaB123), followed by reduction of 3-HPA to 1,3-PDO,which is the major redox valve for glycerol dissimilation, via the NADH-dependent (DhaT encoded by dhaT) or putative NADPH-dependent(YqhD encoded by yqhD) 1,3-PDOORs (Ashok et al., 2011; Wang et al.,2003; Zeng et al., 1993). In Klebsiella sp., which synthesize B12 natu-rally, DhaB is B12-dependent and is activated by GdrAB (encoded bygdrAB), a reactivation factor that can reactivate glycerol- or oxygen-deactivated DhaB and protect it from substrate inhibition (Sun et al.,2003). Biosynthesis of 1,3-PDO can be limited by dhaB123 expression(Ahrens et al., 1998; Wang et al., 2003) given the detrimental effects of3-HPA accumulation (Wang et al., 2003). While a glp regulon is presentin K. pneumoniae, it is dormant in certain strains (Forage and Lin, 1982;
Wang et al., 2017b). Components of the oxidative and reductive bran-ches of the fermentative pathway of glycerol dissimilation (i.e. DhaD,DhaKII, DhaB, and DhaT) were expressed under aerobic conditions in aK. pneumoniae strain lacking a functional glp regulon, although theexpression levels were markedly lower compared to those observedunder anaerobic conditions (Forage and Lin, 1982). Conversely, gly-cerol kinase (i.e. GlpK encoded by glpK) and G3P dehydrogenase (GlpDencoded by glpD) were expressed under aerobic conditions in a strainpossessing a functional glp regulon, while the expression of DhaD,DhaKII, DhaB, and DhaT were highly repressed. Note that 1,3-PDOproduction under microaerobic conditions has been studied extensively(Chen et al., 2003a, 2003b; Chen et al., 2003a, 2003b; Liu et al., 2007;Mu et al., 2006; Xiu et al., 2007; Zhang and Xiu, 2009). Interestingly,the addition of exogenous cyclic adenosine monophosphate (cAMP),the inducer of the cAMP receptor protein (CRP) that relieves cataboliterepression in the absence of preferred carbon sources (Zheng et al.,2004), significantly increased the expression of DhaD, DhaKII, DhaB,and DhaT under aerobic conditions in either strain, although the foldincrease in expression was significantly higher in the strain possessing afunctional glp regulon (Forage and Lin, 1982). As the minor oxidizedproduct derived from the reductive branch of fermentative glyceroldissimilation, 3-HP is derived from 3-HPA by the NAD+-dependent(YdcW encoded by ydcW) and NADP+-dependent (YneI encoded byyneI) ALDHs (Fig. 1) (Luo et al., 2013; Luo et al., 2011a, 2011b). 3-HPproduction can provide compensatory NADH for 1,3-PDO synthesis(Zhu et al., 2015), and alleviate the accumulation of the toxic inter-mediate 3-HPA, which is also known to inhibit DhaT in the presence ofexcess glycerol (Hao et al., 2008a; Sun et al., 2008). Moreover, theexpression of other ALDHs, including PuuC (Ashok et al., 2013a; Ashoket al., 2011; Huang et al., 2012; Luo et al., 2011a, 2011b), FeaB (Huanget al., 2012), BadH (Huang et al., 2012), and GabD (Huang et al., 2012)(encoded by puuC, feaB, badH, and gabD, respectively), can potentiallyenhance 3-HP production in K. pneumoniae. Finally, alternate pathwaysderived from the propanediol utilization (PDU) system exist for theconversion of glycerol to 1,3-PDO and 3-HP in Klebsiella sp. (Bobiket al., 1999; Cho et al., 2015; Luo et al., 2012), as is the case in Sal-monella sp. (Bobik et al., 1999) and Lactobacillus sp. (Makarova et al.,2006; Morita et al., 2008; Sauvageot et al., 2002; Sriramulu et al.,2008). In this system, glycerol is first converted to 3-HPA by the B12-dependent diol dehydratase (DODHt) PduCDE (encoded by pduCDE)(Bobik et al., 1999; Cho et al., 2015; Morita et al., 2008; Sauvageotet al., 2002) with the reactivation factor PduGH (encoded by pduGH)(Bobik et al., 1999), followed by conversion of 3-HPA to 1,3-PDO via a1,3-PDOOR (e.g. DhaT). Alternatively, 3-HPA can be converted to 3-HP-CoA via PduP, a CoA-dependent propionaldehyde dehydrogenase en-coded by pduP, followed by phosphorylation of 3-HP-CoA to yield 3-HP-P via a phosphate propanoyltransferase PduL (encoded by pduL), andfinal conversion of 3-HP-P to 3-HP via a propionate kinase PduW (en-coded by pduW; Fig. 1) (Bobik et al., 1999; Honjo et al., 2015; Luo et al.,2012).
During anaerobic growth, pyruvate metabolism is mediated by boththe pyruvate formate lyase (PFL) and pyruvate dehydrogenase (PDH)complexes, with both enzyme systems presenting similar activities,while pyruvate:ferrodoxin oxidoreductase (PFOR) does not appear to beactive (Menzel et al., 1997) except under conditions of glycerol excess(Zeng et al., 1993). The activity of PFL was observed to increase anddecrease under glycerol limitation and excess, respectively (Menzelet al., 1997). Conversely, the activity of the PDH complex increaseswith glycerol concentration (Menzel et al., 1997). The phenomena ofoscillation of biomass concentration and glycerol consumption in con-tinuous anaerobic cultures of K. pneumoniae has been, in part, attrib-uted to the feedback control between the parallel pyruvate metabolismpathways for the production of acetate, CO2, 2,3-BDO, lactate, andethanol (Menzel et al., 1996). Interestingly, the activity of PFL, which isthe presumptive major player in fermentative metabolism of pyruvate,is strongly affected by oscillations in anaerobic continuous cultures,
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
544
while the activities of pyruvate kinase (PYK) and the PDH complexwere marginally influenced (Menzel et al., 1998). Finally pyruvateoxidase (PO encoded by poxB) appears to play a significant role in bothaerobic and anaerobic pyruvate metabolism, as mutation of poxB re-sulted in excessive acetate accumulation and severely inhibited cellgrowth during aerobic cultivation, and abolished CO2 production underanaerobic conditions (Lin et al., 2016).
Other major fermentative products produced by Klebsiella sp. in-clude 2,3-BDO, acetate, lactate, succinate, and ethanol (Xu et al., 2009;Zeng et al., 1996). A substantial amount of NADH released through theactivities of DhaD and GlpD is oxidized and discharged as H2 throughpathways other than the 1,3-PDO, ethanol, lactate, and 2,3-BDO path-ways. When glycerol is in excess, the bulk of reducing equivalents areconsumed for 1,3-PDO production with a small amount being releasedas H2. Under glycerol limitation, ethanol production is more prevalentas it is energetically favorable, although ethanol is more toxic to Kleb-siella sp. than 1,3-PDO (Zeng et al., 1993).
3.1.2. Strain engineeringTable 2 summarizes various strategies for strain engineering of
Klebsiella sp. to convert glycerol to value-added products, while detailedtechnical discussion is provided in the following sections.
3.1.2.1. 1,3-PDO. To date, 1,3-PDO has been the most common targetmetabolite in engineered Klebsiella sp. The formation of 1,3-PDOsimultaneously generates 1 mol NADH per mol glycerol consumed asthe major redox valve for oxidative glycerol metabolism via theglycolytic pathway. Major strain engineering strategies to enhance1,3-PDO formation include: (1) disruption of the oxidative branch ofthe fermentative glycerol dissimilation pathway; (2) overexpression ofgenes in the reductive and/or oxidative branch of the fermentativeglycerol dissimilation pathway; and (3) inactivation of pathwaysleading to side metabolites, such as lactate, 2,3-BDO, ethanol,succinate, and acetate. Note that these strategies have been applied tobalance the NAD+/NADH ratio, increase carbon flux toward 1,3-PDO,and/or reduce toxic metabolite formation. The oxidative branch offermentative glycerol dissimilation produces DHAP, which issubsequently converted to the glycolytic intermediate glyceraldehyde3-phosphate. Glycolysis results in the synthesis of phosphoenolpyruvate(PEP), leading to the formation of succinate or pyruvate, the latter ofwhich is an intermediate in 2,3-BDO, lactate, acetate, and ethanolformation (Fig. 1). To inactivate oxidative glycerol metabolism in K.pneumoniae, dhaD and dhaK were mutated with the simultaneous use ofthe lacZ promoter from E. coli to regulate the expression of dhaB123, ordhaR, encoding the transcription factor that activates the expression ofthe dha regulon (DhaR), was mutated (Seo et al., 2009). In eitherapproach, the reductive branch of fermentative glycerol dissimilationwas also inactivated, necessitating the expression of native dhaT andgdrAB to restore 1,3-PDO production. Side metabolite formation wasmarkedly reduced, although 1,3-PDO production was not enhanced.Mutation of dhaD and dhaK with coexpression of native dhaB123 anddhaT resulted in abolished production of 2,3-BDO, lactate, and ethanol,but the 1,3-PDO titer and glycerol consumption were reduced relativeto the wild-type strain (Horng et al., 2010). In the absence of afunctional glp regulon for respiratory dissimilation of glycerol, theoxidative branch is the major pathway for fermentative glycerolmetabolism in K. pneumoniae, such that inactivation of the dharegulon potentially hinders glycerol dissimilation and reduces NADHlevels. Activating the glp regulon in K. pneumoniae via overexpression ofnative glpK significantly altered cellular metabolism with acetoinproduction being favored over reductive glycerol metabolism (Wanget al., 2017b), while reduced expression of glpK may mildly stimulateglycerol dissimilation and improve 1,3-PDO production under suitableaeration conditions. In parallel, expression of formate dehydrogenase(FDH) and/or pyridine nucleotide transhydrogenase (UDH) canpotentially regenerate NADH to restore the redox balance during 1,3-
PDO production. On the other hand, co-fermentation of glycerol andglucose is an alternate strategy to enhance 1,3-PDO production in theΔdha background, and can be achieved via mutation of crr, encodingglucose phosphotransferase protein (EIIAGlc) (Oh et al., 2013a; Wanget al., 2017c). Co-feeding limiting amounts of glucose with excessglycerol may stimulate biomass accumulation and NADH regenerationvia glucose metabolism, while glycerol can be converted to 1,3-PDO inthe absence of oxidative glycerol metabolism.
The expression of genes in the reductive branch of the fermentativeglycerol dissimilation pathway can increase carbon flux toward 1,3-PDO production with reduced 3-HPA accumulation, while expression ofgenes in the oxidative branch can improve glycerol dissimilation andNADH levels albeit at the expense of potential side metabolite forma-tion. To alleviate growth cessation associated with toxic 3-HPA, nativedhaT was overexpressed (Hao et al., 2008b). While cell growth con-tinued until glycerol was exhausted with reduced levels of all major sidemetabolites, 1,3-PDO production was not improved (Hao et al., 2008b),even in fedbatch cultures where a decrease in the growth rate wasobserved, eventually leading to growth stagnation (Zhao et al., 2009).Similarly, overexpression of native dhaD reduced ethanol and 2,3-BDOaccumulation without improving 1,3-PDO formation, suggesting thatthe redox balance in the cell is optimal for natural 1,3-PDO synthesis(Zhao et al., 2009). On the other hand, coexpression of native dhaD anddhaT significantly reduced 3-HPA accumulation, leading to a sub-stantial increase in 1,3-PDO production (Chen et al., 2009).
Coexpression of a putative 1,3-PDOOR bearing similarity to YqhDfrom E. coli and native gdrAB significantly increased the 1,3-PDO titer,relative to coexpression of native dhaT and gdrAB, in a strain with theoxidative branch of fermentative glycerol metabolism being disrupted(Seo et al., 2010). The activity of YqhD is NADPH-dependent, and, incontrast to DhaT, the overexpression of the putative 1,3-PDOOR couldpotentially mitigate the NAD+/NADH imbalance resulting from dis-ruption of the oxidative branch of fermentative glycerol metabolism.However, glycerol consumption and 1,3-PDO production rates weresignificantly lower relative to the parent strain coexpressing yqhD andgdrAB, in which the oxidative branch was intact, although the final 1,3-PDO titer was comparable (Seo et al., 2010). In addition, coexpressionof native dhaT and yqhD from E. coli reduced side metabolite formation,but provided only a marginal increase in the 1,3-PDO titer (Zhuge et al.,2010), while yqhD expression alone reduced the rate of glycerol dis-similation without affecting the 1,3-PDO titer (Oh et al., 2013b). Ex-pression of yqhD facilitated high 1,3-PDO titers in E. coli (Emptageet al., 2003a), although it seems to play a minor role in reductive gly-cerol metabolism in Klebsiella sp., and disruption of native dhaT nearlyabolishes 1,3-PDO production (Seo et al., 2009). Finally, basal expres-sion of native puuC from the leaky tac promoter increased and de-creased 1,3-PDO titers in cultures of lactate and lactate/2,3-BDO defi-cient mutants, respectively (Zhu et al., 2015). The conversion of 3-HPAto 3-HP (via PuuC) reduces 3-HPA levels, while producing NADH re-quired for 1,3-PDO production. The decrease in 1,3-PDO formation inthe lactate/2,3-BDO deficient strain was the result of pyruvate accu-mulation, which can interfere with glycerol dissimilation in 2,3-BDOdeficient mutants (Lee et al., 2014).
The major side metabolites produced during glycerol fermentationin Klebsiella sp. are lactate, 2,3-BDO, ethanol, succinate, and acetate,and inactivation of these pathways can potentially enhance 1,3-PDOproduction. Formation of acetate and 2,3-BDO during glycerol fer-mentation for 1,3-PDO production results in a surplus of 2 mol and 0.5mol NADH, respectively, per mol glycerol consumed, while formationof lactate and ethanol is redox balanced and formation of succinategenerates a deficit of 1 mol NADH per mol glycerol consumed. Whilethe formation of each metabolite, which is accompanied by ATP pro-duction for cell growth and maintenance, can relieve pyruvate accu-mulation resulting from the compromised tricarboxylic acid (TCA)cycle in Klebsiella sp. (Cabelli, 1955), reduced carbon flux toward 1,3-PDO formation may occur. Inactivating the ethanol pathway in K.
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
545
Table 2Summary of literature for Klebsiella sp. strains engineered for value-added chemical production from glycerol.
Parent strain Product Genetic strategy Titer (g/L) Cultivationmode
Reference
Kp ZG25 1,3-PDO Mutate budC to reduce 2,3-BDO formation 67 fedbatch (Guo et al., 2013)Kp TUAC01 1,3-PDO Express dhaT 20 batch (Hao et al., 2008b)Kp KG2 (lacks ldhA) 1,3-PDO Mutate poxB and pta-ackA to reduce acetate formation 77 fedbatch (Lin et al., 2016)Kp J2B ΔldhA 1,3-PDO Mutate budA, budB, budC, or all three to reduce 2,3-BDO formation 31 fedbatch (Kumar et al., 2016)Kp KG1 1,3-PDO
Express dhaD and dhaT to elucidate their roles during microaerobiccultivation
45 fedbatch (Zhao et al., 2009)
Kp AK (lacks oxidativepathway)
1,3-PDOExpress dhaB123, dhaT, or yqhD, or dhaT and yqhD, or dhaB123 anddhaT (along with gdrAB)
26 fedbatch (Luo et al., 2013)
Kp Cu 1,3-PDO -Mutate dhaD and dhaK while inserting the lacZ promoter (E. coli)upstream of dhaB123 to interrupt oxidative metabolism withoutdisturbing the reductive branch-Mutate dhaR to interrupt oxidative metabolism-Coexpress dhaT and gdrAB
5 flask (Seo et al., 2009)
Kp ΔldhA 1,3-PDO-Mutate budB and pta to reduce 2,3-BDO and acetate formation,respectively-Coexpress pdc and aldB (Z. mobilis) to stimulate conversion ofpyruvate to ethanol to restore metabolism in ΔbudB mutant
68 fedbatch (Lee et al., 2014)
Kp CICIM B0057 1,3-PDO Express yqhD (E. coli) and/or dhaT 18 batch (Zhuge et al., 2010)Ko M5aI 1,3-PDO Mutate ldhA to reduce lactate formation 63 fedbatch (Yang et al., 2007)Kp AK and Cu 1,3-PDO Express yqhD to increase NADH levels 8 flask (Seo et al., 2010)Kp WT 1,3-PDO -Mutate budC to reduce 2,3-BDO formation
-Chromosomally express fdh (P. pastoris) to regenerate NADH72 fedbatch (Wu et al., 2013)
Kp KG2 and KG3 1,3-PDOExpress puuC via leakage from Ptac promoter to enhance 3-HP andNADH formation
69 fedbatch (Zhu et al., 2015)
Kp YMU2 1,3-PDO Mutate aldA to reduce ethanol formation 71 fedbatch (Zhang et al., 2006a,2006b)
Kp WT 1,3-PDO -Mutate dhaD and dhaK to interrupt oxidative metabolism-Coexpress dhaT and dhaB123
8 flask (Horng et al., 2010)
Ko M5aI 1,3-PDO Mutate budA to reduce 2,3-BDO formation 27 fedbatch (Zhang et al., 2012)Kp HR526 1,3-PDO Mutate ldhA to reduce lactate formation 102 fedbatch (Xu et al., 2009)Kp Cu 1,3-PDO -Mutate budB and ldhA to reduce 2,3-BDO and lactate formation,
respectively103 fedbatch (Oh et al., 2012b)
Kp KG1 1,3-PDO Coexpress dhaD and dhaT 17 flask (Zhao et al., 2009)Kp ACCC 10082 1,3-PDO Coexpress dhaD and dhaT 59 fedbatch (Chen et al., 2009)Kp 2-1 1,3-PDO -Mutate ldhA and aldH to reduce lactate and ethanol formation,
respectively88 fedbatch (Chen et al., 2016)
Kp CICIM B0057 1,3-PDOReduce 2,3-BDO formation by reducing expression of budA, budB,and budC via asRNA
22 flask (Lu et al., 2016)
Kp CGMCC 1.6366 1,3-PDO-Mutate pflB or acoABCD to reduce acetate formation
16 batch (Zhou et al., 2017a,2017b)
Kp KG2 1,3-PDO -Mutate pck or ppc to reduce succinate formation 72 fedbatch (Zhang et al., 2017)Kp KCTC 2242 1,3-PDO -Introduce point mutations in DhaT via error prone PCR to increase
activity-Mutate crr to relieve catabolite repression for glycerol/glucose co-fermentation to increase biomass and NADH levels-Overexpress glpF to enhance glycerol uptake-Coexpress fdh (P. pastoris), gdh (B. subtilis), and udh to regenerateNADH
86 fedbatch (Wang et al., 2017c)
Kp ATCC 200721ΔldhA
1,3-PDO-Mutate budA, budB, or budC to reduce 2,3-BDO formation
49 fedbatch (Oh et al., 2018)
Kp WT 1,3-PDO-Mutate arcA to increase expression of enzymes in the TCA cycle-Mutate ldhA to reduce lactate formation-Mutate crr to relieve catabolite repression for glycerol/glucose co-fermentation to increase biomass and NADH levels
78 fedbatch (Lu et al., 2018)
Kp KCTC 2242ΔwabG
1,3-PDO -Mutate ldhA, pflB, and budA to reduce lactate, acetate, and 2,3-BDOformation, respectively-Mutate dhaD and glpK to prevent glycerol assimilation into biomass-Modify the 5’-UTR of mtlA to improve the efficiency of mannitolusage-Mutate dhaK123 and overexpress dhaK2 to enhance expression ofdhaT
21 batch (Lee et al., 2018)
Ko PDL-0(WT) 1,3-PDO/lactate
-Mutate budA and budB, adhE, and ackA-pta to reduce 2,3-BDO,ethanol, and acetate formation, respectively-Replace ldhD with ldhL (L. casei) to facilitate L-lactate production
70 / 100 (D-or L-lactate)
fedbatch (Xin et al., 2017)
Kp KCTC12133BP 1,3-PDO/2,3-BDO -Mutate ldhA and mdh to reduce lactate and succinate formation,
respectively
125 fedbatch (Park et al., 2017)
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Table 2 (continued)
Parent strain Product Genetic strategy Titer (g/L) Cultivationmode
Reference
Kp KCTC 2242 ΔldhA ΔptsG 1,3-PDO/2,3-BDO -Construct EDP from Z. mobilis and coexpress udh to regenerate
NADH-Coexpress dhaT
79 / 32 fedbatch (Wang et al., 2017d)
Kp DSMZ 2026 3-HP/1,3-PDO Express puuC and mutate dhaT to balance 3-HP and 1,3-PDO
production
16 / 17 fedbatch (Ashok et al., 2011)
Kp WM3 1,3-PDO/3-HP Express ALDHs from Zymomonas mobilis (adhB), Lactobacillus
collinoides (aldH and pduQ), K. pneumoniae (aldA, aldB, puuC, ydcW,etuE, feaB, gabD and badH), or E. coli (aldA, aldH and ycdW)
24 / 49 fedbatch (Huang et al., 2012)
Kp J2B 1,3-PDO/3-HP
Express KGSADH (A. brasilense) 16 / 12 fedbatch (Kumar et al., 2012)
Kp J2B 1,3-PDO/3-HP
-Express KGSADH (A. brasilense)-Mutate ldhA to reduce lactate formation
23 / 23 fedbatch (Kumar et al., 2013)
Kp DSMZ 2026 1,3-PDO/3-HP
Express dhaS (B. subtilis) 18 / 27 fedbatch (Su et al., 2015)
Kp AK 1,3-PDO/3-HP
Express aldH, dhaT amd gdrAB in mutant defective in oxidativeglycerol metabolism
23 / 7 fedbatch (Luo et al., 2011a,2011b)
Kp J2B 1,3-PDO/3-HP - Mutate ldhA, adhE, frdA, and pta-ackA to reduce lactate, ethanol,
succinate, and acetate formation, respectively-Mutate glpK or dhaD to reduce acetate without disrupting pta-ackAto improve glycerol consumption-Overexpress dhaT, or dhaB123 and gdrAB
21 / 43 fedbatch (Ko et al., 2017)
Kp J2B 3-HP-Mutate dhaT and yqhD to reduce 1,3-PDO formation-Express puuC, KGSADH (A. brasilense) or aldH (E. coli)
16 fedbatch (Ko et al., 2012)
Kp J2B ΔdhaT ΔyqhD 3-HP Mutate ahpF and adhE in a ΔdhaT ΔyqhD double mutant expressingKGSADH (A. brasilense) to reduce 1,3-PDO formation
9 fedbatch (Ko et al., 2015)
Kp DSMZ 2026 3-HP-Mutate glpK to force glycerol through the fermentative pathway inthe presence of nitrate,-Mutate dhaT to reduce 1,3-PDO formation-Overexpress puuC
23 fedbatch (Ashok et al., 2013a)
Kp DSMZ 2026 3-HP-Optimize puuC expression through promoter selection (Ptac)-Mutate ldh1 and ldh2, and pta to reduce lactate and acetateformation, respectively
84 fedbatch (Li, Y. et al., 2016)
Kp DSMZ 2026 3-HP-Coexpress dhaB123, gdrAB and puuC in a ΔdhaT ΔyqhD doublemutant
28 fedbatch (Ashok et al., 2013b)
Ko M1 2,3-BDO-Mutate pduC and ldhA to reduce 1,3-PDO and lactate formation,respectively
132 fedbatch (Cho et al., 2015)
Kp WT 1-butanol -Express ter (T. denticola), and bdhA and bdhB (C. acetobutylicum)-Express kivd (L. lactis) to facilitate the 2-keto acid pathway-Reduce 1,3-PDO formation by reducing expression of dhaB123,dhaT, and/or gdrAB via asRNA-Reduce 2,3-BDO formation by reducing expression of budB and/orbudC via asRNA
0.029 flask (Wang et al., 2014a,2014b, 2014c)
Kp WT 1-butanol-Express kivd (L. lactis), leuABCD, and adhe1 (C. acetobutylicum)-Enhance NADH regeneration via expression of fdh (P. pastoris), gdh,or udh-Reduce 1,3-PDO formation by reducing expression of dhaB123,dhaT, and/or gdrAB via asRNA
0.1 flask (Wang et al., 2015a,2015b)
Kp 13 1-butanol-Express kivd (L. lactis), leuABCD, and adhe1 (C. acetobutylicum)-Repress expression of metA, ilvI, ilvB, and alaA via CRISPRi toincrease carbon flux toward 1-butanol production
0.2 flask (Wang et al., 2017a)
Kp ATCC 200721 ΔldhA 2-butanol-Express ilvIH, ilvD, andkivd and adhA (L. lactis)-Mutate budA to reduce 2,3-BDO formation
0.3 batch (Oh et al., 2014)
GEM167 ethanol-Mutate ldhA to reduce lactate formation-Coexpress pdc and adhII (Z. mobilis)
31 fedbatch (Oh et al., 2012a)
ATCC 25955 D-lactate-Overexpress ldhA-Mutate dhaT and yqhD to reduce 1,3-PDO formation
142 fedbatch (Feng et al., 2014)
ATCC 25955 acetoin -Mutate budC, gldA and dhaD to prevent conversion of acetoin to 2,3-BDO
32 fedbatch (Wang et al., 2017b)
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pneumoniae via mutation of aldA (encoding ALDH; AldA) drasticallyreduced ethanol formation, resulting in significant increases in the yieldand titer of 1,3-PDO in a fedbatch fermentation, despite decreasedglycerol consumption and biomass accumulation. (Zhang et al., 2006a,2006b). On the other hand, mutation of aldH (encoding ALDH; AldH)and ldhA (encoding lactate dehydrogenase; LdhA) abolished ethanoland lactate formation, respectively, although the 1,3-PDO titer wasmarginally improved (Chen et al., 2016). Similarly, inactivation of ldhAsignificantly increased and decreased NADH and lactate levels, re-spectively, leading to substantial 2,3-BDO and ethanol formation, andthe 1,3-PDO titer increased slightly in a fedbatch fermentation (Xuet al., 2009). In contrast to K. pneumoniae, mutation of ldhA in Klebsiellaoxytoca slightly enhanced glycerol dissimilation, abolished lactateproduction, and significantly increased the 1,3-PDO titer, however, 2,3-BDO formation markedly increased (Yang et al., 2007).
Elevated 2,3-BDO levels can be particularly problematic since itsboiling point is similar to that of 1,3-PDO, making downstream pur-ification more difficult (Xiu and Zeng, 2008), and considerable efforthas been made to alleviate 2,3-BDO production. Mutation of ldhA andbudB, encoding α-acetolactate synthase (BudB), significantly decreasedlactate and 2,3-BDO formation, respectively, although the 1,3-PDO titerwas also reduced (Oh et al., 2012b). Inactivation of budC, encoding 2,3-BDO dehydrogenase/acetoin reductase (BudC), moderately decreasedand increased 2,3-BDO and 1,3-PDO production, respectively (Guoet al., 2013). The moderate reduction in 2,3-BDO production in culturesof the ΔbudC mutant is likely due to the promiscuity of DhaD, which cancatalyze the interconversion of acetoin and 2,3-BDO in response toelevated intracellular NADH levels during glycerol catabolism (Wanget al., 2014a, 2014b, 2014c), while an alternate/complementary ex-planation is the presence of the 2,3-BDO replenishment cycle in K.pneumoniae (Syu, 2001). Mutation of budA, encoding α-acetolactatedecarboxylase (BudA), in K. oxytoca abolished 2,3-BDO formation andmoderately increased the 1,3-PDO titer, although the acetate and lac-tate titers increased substantially (Zhang et al., 2012). Similarly, mu-tation of budA or budB in a K. pneumoniae ΔldhA mutant abolished 2,3-BDO formation, although the ΔldhA ΔbudA double mutant producedsignificantly less 1,3-PDO than the parent strain (Kumar et al., 2016).
Moreover, mutation of budC modestly increased and decreased the 1,3-PDO and 2,3-BDO titers, although mutation of the entire bud operon(i.e. budABC) resulted in the lowest 1,3-PDO titer. While the cultureperformance of the ΔbudABC mutant was significantly improved, i.e.production of the target metabolite (1,3-PDO) increased, by increasingthe buffering capacity of the medium in a shake flask, the 1,3-PDO titerdeclined significantly due to excessive pyruvate accumulation during afedbatch fermentation (Kumar et al., 2016), indicating inefficient op-eration of the TCA cycle. Similarly, reduced expression of budA andbudB via antisense RNA (asRNA)-mediated transcriptional interferencesignificantly decreased 2,3-BDO production without substantially af-fecting the 1,3-PDO titer, and repression of budC expression did notappreciably affect culture performance (Lu et al., 2016). In contrast,mutation of budA, budB, or budC in a ∆ldhA mutant reduced 2,3-BDOand 1,3-PDO production, with the ΔldhA ΔbudA double mutant pro-ducing significantly less 1,3-PDO than all other strains (Oh et al., 2018).To reduce pyruvate accumulation through increased ethanol synthesis,pdc and aldB, encoding pyruvate decarboxylase (PDC) and ALDH (AldB)of Zymomonas mobilis, respectively, were coexpressed in a ΔldhA ΔbudBdouble mutant (Oh et al., 2012b), resulting in restored glycerol dis-similation and 1,3-PDO production. These studies underscore the deli-cate balance between 1,3-PDO and 2,3-BDO production in Klebsiella sp.,and further suggest the unstable nature of the TCA cycle, which is acritical issue to be addressed.
Inactivating acetate pathways to enhance 1,3-PDO production hasalso been explored. Mutation of pta (encoding phosphotransacetylase;PTA) and ackA (encoding acetate kinase; AckA) restored cell growth,reduced acetate production, and modestly increased the 1,3-PDO titerunder the ΔldhA ΔpoxB background (Lin et al., 2016). Similarly, mu-tation of pflB, encoding PFL (PflB), decreased acetate production butdid not significantly improve the 1,3-PDO titer, while mutation ofacoABCD, encoding the PDH complex (AcoABCD), increased the pro-ductivity but decreased the yield of 1,3-PDO due to significantly in-creased formate and ethanol formation (Zhou et al., 2017a, 2017b).Finally, in an attempt to alleviate succinate formation, pck (encodingPEP carboxykinase; PCK) was inactivated, resulting in improved growthand reduced acetate and 2,3-BDO formation, albeit without significant
Table 2 (continued)
Parent strain Product Genetic strategy Titer (g/L) Cultivationmode
Reference
-Express glpK to activate silent glp operon-Express nox (L. brevis) to increase NAD+/NADH ratio
ATCC 25955 P(3HP) -Express dhaB123, gdrAB, and aldH (E. coli) to enhance 3-HPformation-Express prpE and phaC to convert 3-HP to P(3HP)-Mutate dhaT and yqhD to reduce 1,3-PDO formation
0.2 flask (Feng et al., 2015)
ackA, acetate kinase; acoABCD, pyruvate dehydrogenase complex; adhII, aldehyde/alcohol dehydrogenase; adhA, alcohol dehydrogenase; aldB, aldehyde dehy-drogenase; adhE, bifunctional acetaldehyde-CoA/alcohol dehydrogenase; alaA, alanine transaminase; ahpF, alkyl hydroperoxide oxidoreductase; aldH, aldehydedehydrogenase; bdhA, butanol dehydrogenase A; bdhB, butanol dehydrogenase B; budA, α-acetolactate decarboxylase; budB, α-acetolactate synthase; budC, 2,3-BDOdehydrogenase/acetoin reductase; dhaB123, glycerol dehydratase; crr, glucose phosphotransferase protein; dhaD, glycerol dehydrogenase; dhaK, dihydroxyacetonekinase; dhaR, transcriptional activator of the dha regulon; dhaS, aldehyde dehydrogenase; dhaT, NADH-dependent 1,3-PDO oxidoreductase; fdh, formate dehy-drogenase; frdA, succinate dehydrogenase; gdh, glucose dehydrogenase (Bacillus subtilis); gdrAB, glycerol dehydratase reactivation factor; gldA, glycerol dehy-drogenase; glpF, glycerol transporter; glpK, glycerol kinase; ilvB, acetolactate synthase isozyme 1 large subunit; ilvD, dihydroxyacid dehydratase; ilvI, acetolactatesynthase isozyme 3 large subunit; ilvIH, keto-acid reducto-isomerase; KGSADH, α-ketoglutaric semialdehyde dehydrogenase; kivD, α-ketoisovalerate decarboxylase;lacZ, β-galactosidase; ldh1, lactate dehydrogenase; ldh2, lactate dehydrogenase; ldhA, D-lactate dehydrogenase; ldhD, D-lactate dehydrogenase; ldhL, L-lactate de-hydrogenase; leuA, 2-isopropylmalate synthase; leuB, 3-isopropylmalate dehydrogenase; leuCD, 3-isopropylmalate dehydratase subunits C and D; mdh, malate de-hydrogenase; metA, pck, carboxykinase; nox, NADH oxidase; pdc, pyruvate decarboxylase; homoserine O-succinyltransferase; pduC, diol dehydratase large subunit;pflB, pyruvate formate lyase; phaC, polyhydroxyalkanoate synthase; poxB, pyruvate oxidase; ppc, phosphoenolpyruvate carboxylase; prpE, propionyl-CoA synthetase;pta, phosphotransacetylase; puuC, aldehyde dehydrogenase; ter, trans-2-enoyl-CoA reductase; udh, pyridine nucleotide transhydrogenase; yqhD, NADPH-dependent1,3-PDO oxidoreductase; other terms: ALDH, aldehyde dehydrogenase; 1,3-PDO, 1,3-propanediol; 2,3-BDO, 2,3-butanediol; 3-HP, 3-hydroxypropionic acid; asRNA,antisense RNA; CRISPRi, Clustered Regularly Interspaced Palindromic Repeats interference; EDP, Entner-Doudoroff pathway; Ko, Klebsiella oxytoca; Kp, Klebsiellapneumoniae; NADH, nicotinamide adenine dinucleotide; P(3HP), poly(3-hydroxypropionate); PCR, polymerase chain reaction.
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changes in 1,3-PDO or succinate production, and mutation of ppc (en-coding PEP carboxylase; PPC) did not affect culture performance(Zhang et al., 2017). A considerable body of literature suggests thatcompletely abolishing side metabolite formation is not feasible inKlebsiella sp., potentially due to their incomplete TCA cycle that canlead to accumulation of inhibitory levels of pyruvate (Kumar et al.,2016). Prior work indicates that the flavin adenine dinucleotide (FAD)binding subunit of the succinate:quinone oxidoreductase complex, en-coded by sdhA (SdhA), and fumarase A, encoded by fumA (FumA),which successively convert succinate to fumarate and fumarate tomalate in the TCA cycle, are weakly expressed in K. pneumoniae(Cabelli, 1955; Kumar et al., 2016). Fumarase A and fumarase B (en-coded by fumB; FumB) contain [4Fe-4S] clusters that are sensitive tooxidative damage, while fumarase C (encoded by fumC; FumC) is in-sensitive to oxygen (Liochev and Fridovich, 1993). Accordingly, ex-pression of the appropriate fumarase isozyme from E. coli, selectedbased on the aeration conditions employed for glycerol fermentation,may activate the TCA cycle in Klebsiella sp., in turn, reducing sidemetabolite formation and promoting glycerol dissimilation. Similarly,expression of the succinate:quinone oxidoreductase complex, encodedby sdhABCDE (SdhABCDE), may also improve culture performance. Itmay be necessary to perform more extensive transcriptional analysis ofgenes encoding enzymes comprising the TCA cycle during glycerolfermentation to identify other gene targets in Klebsiella sp. for metabolicmanipulation.
Coproduction of 1,3-PDO with a second major metabolite is an al-ternate approach to completely eliminating side metabolite production.1,3-PDO/lactate co-production is a redox balanced process that canresult in high yields of both metabolites in K. oxytoca (Xin et al., 2017).2,3-BDO, ethanol, and acetate formation were either abolished or sig-nificantly reduced via mutation of budA and budB, adhE (encoding al-dehyde/alcohol dehydrogenase; AdhE), and ackA-pta, respectively,leading to a high 1,3-PDO/D-lactate yield of 0.95 mol/molglycerol.Moreover, optically pure L-lactate was co-produced with 1,3-PDO at asimilar yield by replacing the native D-LDH (encoded by ldhD) with L-LDH from Lactobacillus casei (encoded by ldhL) (Xin et al., 2017). High-level 1,3-PDO/2,3-BDO co-production from glycerol was recently de-monstrated in K. pneumoniae. 1,3-PDO/2,3-BDO co-production wassignificantly improved in K. pneumoniae via mutation of ldhA, whilemutation of mdh, encoding malate dehydrogenase (MDH), significantlyreduced succinate formation in the ΔldhA mutant with only a modestimprovement in the 1,3-PDO/2,3-BDO yield (Park et al., 2017). Con-struction of the Entner-Doudoroff pathway (EDP) from Z. mobilis in K.pneumoniae with coexpression of native dhaT and udh (encoding UDH)significantly improved 1,3-PDO/2,3-BDO co-production during gly-cerol/glucose co-fermentation (Wang et al., 2017d). This strategyaimed to increase NADPH levels via glucose metabolism through theEDP from Z. mobilis with subsequent regeneration of NADH fromNADPH via native UDH (Wang et al., 2017d). However, 1,3-PDO/2,3-BDO co-production may be practically infeasible due to the difficultyassociated with separating these compounds, and carbon loss is sig-nificant during 2,3-BDO production due to the successive decarbox-ylations from pyruvate to acetoin (Fig. 1).
Other strain engineering strategies to improve 1,3-PDO productionfrom glycerol include protein engineering of key enzymes, globallyupregulating the expression of enzymes in the TCA cycle, relievingcatabolite repression to enable co-fermentation of glycerol and glucose,the expression of glycerol transporters to enhance glycerol uptake, andthe expression of enzymes that can regenerate NADH. Transformationof integration cassettes generated with error prone PCR resulted inpoint mutations to DhaT (D41G and H57Y) that moderately increasedits activity and improved 1,3-PDO production (Wang et al., 2017c).Creating large mutant libraries via error prone PCR, or rational proteinengineering may be effective strategies to further enhance the activityof DhaT, or to relieve the inhibition of DhaT by 3-HPA. The ArcA-ArcBtwo component system negatively regulates the expression of genes
encoding enzymes of the TCA cycle in response to decreasing levels ofreduced quinones (Bekker et al., 2010; Shalel-Levanon et al., 2005).Mutation of arcA in K. pneumoniae resulted in a ≥9-fold increase in thetranscription of gltA (encoding citrate synthase; GltA), icd (encodingisocitrate dehydrogenase; ICD), sdhC, and sucC (encoding succinyl-CoAsynthetase β subunit; SucC), resulting in a significant decrease inacetate accumulation and the intracellular NAD+/NADH ratio, and anincrease in the specific 1,3-PDO titer (Lu et al., 2018). However, lactateproduction increased substantially, which was resolved by mutatingldhA in the ΔarcA mutant, resulting in complete cessation of lactateaccumulation and a slight increase in the 1,3-PDO titer. In the presenceof glucose or other preferred carbon sources, EIIAGlc is in the depho-sphorylated state and will bind to GlpK to inhibit glycerol uptake(Deutscher et al., 2006). Accordingly, mutation of crr in the ΔarcAΔldhA double mutant enabled co-fermentation of glucose and glycerol,resulting in a moderate increase in the 1,3-PDO titer, albeit with re-duced rates of cell growth and glycerol consumption, and increasedacetate and lactate production (Lu et al., 2018). Similarly, mutation ofcrr in K. pneumoniae moderately improved 1,3-PDO production, al-though, in this case, side metabolite formation was reduced (Wanget al., 2017c). Also, co-fermentation of glycerol and untreated molassessignificantly improved the 1,3-PDO titer in a ΔldhA Δcrr double mutant(Oh et al., 2013a). Moreover, overexpression of native glpF significantlyenhanced the glycerol consumption rate and modestly increased the1,3-PDO titer in a fedbatch fermentation (Wang et al., 2017c). Finally,expression of fdh, encoding FDH from Candida boidinii, in K. oxytoca,slightly increased the 1,3-PDO titer while ethanol production increasedsubstantially (Zhang et al., 2009). However, the intracellular NAD+/NADH ratio was not affected by the activity of the NADH regenerationsystem, as was the case in E. coli (Berrı,́ S.J., San, K.-Y., Bennett, G.,2002), suggesting that either K. oxytoca can efficiently recycle NADHthrough native pathways, expression levels of fdh were not sufficient toaffect NADH regeneration, or substantial conversion of formate to H2
and CO2 by the formate hydrogen lyase (FHL) complex occurred. Si-milarly, chromosomal insertion of fdh from Pichia pastoris in the budClocus of K. pneumoniae significantly increased biomass accumulationand decreased 2,3-BDO production, although the 1,3-PDO titer wasmodestly improved (Wu et al., 2013), while coexpression of fdh from P.pastoris, gdh (encoding glucose dehydrogenase; GluDH) from Bacillussubtilis, and native udh increased both 1,3-PDO and lactate production(Wang et al., 2017c).
Recently, multiple parallel strain engineering strategies led to a veryhigh 1,3-PDO yield of 0.76 mol/molglycerol in a K. pneumoniae strain inwhich wabG, encoding a protein involved in the transfer of galacturonicacid (WabG), was mutated to prevent lipopolysaccharide synthesis (Leeet al., 2018). Mutation of ldhA and pflB in the ∆wabG mutant abolishedlactate and acetate production, respectively, with modest increases inthe 1,3-PDO titer, while mutation of budA nearly eliminated 2,3-BDOproduction at the expense of increased acetate production and severelyreduced glycerol consumption and 1,3-PDO production. To preventglycerol assimilation into biomass, dhaD and glpK were mutated in the∆wabG ∆ldhA ∆pflB ∆budA quadruple mutant, and the resulting strainKMK-23 was cultured with glycerol and glucose as co-substrates toenable growth on glucose with glycerol being converted to 1,3-PDO at asignificantly higher yield than the parent strain (Lee et al., 2018).Subsequently, mannitol was selected as the most suitable co-substratewith glycerol, and the 5’- untranslated region (UTR) of mtlA, encodingthe mannitol-specific transporter (MtlA), was modified in the genome ofKMK-23 to improve the efficiency of mannitol utilization, which, inturn, improved cell growth and 1,3-PDO production in the resultingstrain KMK-23M. Finally, based on the observation that DhaL, i.e. ahomologue of the subunit of DhaKII encoded by dhaK2, positivelyregulates DhaR, in turn, increasing the expression of DhaT in E. coli(Bächler et al., 2005), dhaK123 was mutated in KMK-23M, resulting instrain KMK-46, followed by overexpression of dhaK2 in KMK-46, re-sulting in strain KMY, which produced significantly more 1,3-PDO than
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KMK-23M. Interestingly, 1,3-PDO production was abolished in KMK-46,and could not be rescued by simply overexpressing dhaR. Moreover, theoverexpression of dhaT in KMK-23M or KMK-46 resulted in significantlylower 1,3-PDO production, compared to KMY (Lee et al., 2018), sug-gesting the complex and poorly understood regulation of glycerol me-tabolism in K. pneumoniae.
3.1.2.2. 3-HP. Due to its use in the synthesis of many industriallyimportant chemicals, 3-HP has received increasing attention as a targetmetabolite in engineered Klebsiella sp. 3-HP production from glycerolgenerates 1 mol NADH per mol glycerol consumed, such that co-production with 1,3-PDO is favorable due to redox balance, or anexogenous electron acceptor can improve the 3-HP/1,3-PDO selectivity(Ashok et al., 2013a). Strain engineering strategies employed toenhance the marginal levels of naturally produced 3-HP primarilyfocused on enhancing 3-HPA conversion to 3-HP via expression ofALDHs, and inactivating 1,3-PDOORs to increase available 3-HPA.Balanced 3-HP/1,3-PDO co-production was achieved viaoverexpression of native puuC and mutation of dhaT and, relative tothe parent strain, the 3-HP titer was improved by more than 10-foldwith a similar 1,3-PDO titer (Ashok et al., 2011). The existence ofputative oxidoreductases which convert 3-HPA to 1,3-PDO is welldocumented in Klebsiella sp., making elimination of 1,3-PDOformation difficult (Ko et al., 2015; Ko et al., 2012). Similarly,overexpression of native puuC increased the 3-HP titer by 5.5-fold ina strain with an inactivated oxidative branch of fermentative glycerolmetabolism, however, no effect was observed in the wild-type strain,suggesting that puuC overexpression was merely generatingcompensatory NADH in light of disruption of oxidative glycerolmetabolism (Luo et al., 2011a, 2011b). The ydcW and aldH genesfrom E. coli and native ydcW were selected out of 14 ALDHs evaluatedto enhance 3-HP production (Huang et al., 2012). The 3-HP titer wasincreased by 10-fold in a fedbatch fermentation of the aldH-expressingstrain, while the 1,3-PDO titer declined somewhat (Huang et al., 2012).Similarly, expression of heterologous α-ketoglutaric semialdehydedehydrogenase from Azospirillum brasilense, encoded by KGSADH,significantly increased both the 3-HP and 1,3-PDO titers, relative tothe overexpression of native puuC, in a fedbatch fermentation of theΔdhaT mutant (Ko et al., 2012). The dhaT mutation respectivelyincreased and decreased 3-HP and 1,3-PDO production in strainsexpressing puuC or KGSADH, while mutation of yqhD had nosignificant effect on culture performance. 3-HP/1,3-PDO co-production was subsequently investigated in a wild-type strainexpressing KGSADH (Ko et al., 2017). Mutation of ldhA, adhE, andfrdA (encoding succinate dehydrogenase; FrdA) significantly reducedlactate and ethanol formation, while succinate and 3-HP/1,3-PDO co-production were only slightly affected. Due to a marked increase inacetate formation observed in the ΔldhA ΔadhE ΔfrdA triple mutant, pta-ackA were mutated resulting in significantly decreased acetateformation, albeit at the expense of glycerol consumption and 3-HPproduction (Ko et al., 2017). Accordingly, acetate formation wasreduced without disrupting pta-ackA by mutating glpK in the ΔldhAΔadhE ΔfrdA triple mutant, resulting in significantly increased glycerolconsumption and 3-HP/1,3-PDO co-production, while mutation of dhaDimpeded culture performance. Moreover, overexpression of dhaT, ordhaB123 and gdrAB, increased and decreased 3-HP/1,3-PDO co-production in the ΔldhA ΔadhE ΔfrdA triple mutant, and all geneticstrategies employed in this study either reduced or did not affect 3-HPproduction (Ko et al., 2017). High-level 3-HP/1,3-PDO co-productionwas also achieved via expression of KGSADH from A. brasilense in aresting cell system (Kumar et al., 2012), and mutation of ldhA couldsignificantly improve both 3-HP and 1,3-PDO titers (Ashok et al.,2013b). In contrast to the aforementioned ΔldhA ΔadhE ΔfrdA ΔglpKquadruple mutant expressing KGSADH, a ΔglpK single mutantoverexpressing native puuC produced less 3-HP relative to the wild-type strain expressing puuC. On the other hand, mutation of dhaT
increased the 3-HP titer significantly in the wild-type strainoverexpressing puuC, while 3-HP production was highest in culturesof a ΔglpK ΔdhaT double mutant supplemented with nitrate as anelectron acceptor (Ashok et al., 2013a). Subsequently, inactivation ofdhaT and yqhD neither eliminated 1,3-PDO production nor substantiallyincreased 3-HP formation in the wild-type strain, while coexpression ofnative puuC, dhaB123, and gdrAB significantly increased 3-HPproduction in the ΔdhaT ΔyqhD double mutant, relative to puuCoverexpression alone, during fedbatch operation (Ashok et al.,2013b). Further efforts to reduce 1,3-PDO formation for enhanced 3-HP production in a ΔdhaT ΔyqhD double mutant expressing KGSADHfrom A. brasilense via mutation of adhE and ahpF, encoding alkylhydroperoxide oxidoreductase (AhpF), were unsuccessful (Ko et al.,2015), suggesting that additional 1,3-PDOORs have yet to be identified.Finally, the 3-HP titer increased by 3.9-fold and the 1,3-PDO titerdecreased substantially upon expression of dhaS, encoding ALDH fromB. subtilis (DhaS), but with significantly reduced glycerol consumptionand biomass accumulation (Su et al., 2015).
For more effective 3-HP production, the strain engineering strate-gies described above can be integrated with bioprocessing strategies.For example, while overexpressing native puuC from the strong tacpromoter, the 3-HP titer increased by 6-fold relative to that obtainedunder the lac promoter, and an extremely high 3-HP titer of 84 g/L wasobtained with a high 3-HP/1,3-PDO selectivity by manipulating aera-tion conditions (Li et al., 2016a, 2016b). It has been demonstrated thatthe selectivity of 3-HP to 1,3-PDO is highly dependent on aerationconditions, with fully aerobic operation abolishing both metabolitesdue to insufficient B12 synthesis, fully anaerobic conditions favoring1,3-PDO formation, and microaerobic conditions being conducive to co-production (Ashok et al., 2011; Ashok et al., 2013b; Li et al., 2016a,2016b; Su et al., 2015). While an improved 3-HP/1,3-PDO selectivity isachievable without disrupting 1,3-PDOOR expression, 1,3-PDO pro-duction remains significant such that identification and mutation ofadditional 1,3-PDOORs may improve culture performance for 3-HPproduction. Advanced genome engineering technologies such as theCRISPR-Cas9 (Clustered Regularly Interspaced Palindromic Repeats-CRISPR-associated [protein] 9) system facilitate multiplexed gene mu-tations in various microbial hosts (Jiang et al., 2015; Westbrook et al.,2016), and CRISPR interference (CRISPRi) has been applied to meta-bolic engineering in K. pneumoniae (Wang et al., 2017a). Accordingly,multiplexed mutation of genes encoding putative 1,3-PDOORs via theCRISPR-Cas9 system may be a convenient and effective approach toabolish 1,3-PDO production. Moreover, expression of NADH oxidase(NOX) can regenerate NAD+ to maintain the redox balance in a 1,3-PDO-deficient mutant without sacrificing carbon flux to side metaboliteformation.
3.1.2.3. Other products. High level 2,3-BDO production from glycerolwas achieved in K. oxytoca through inactivation of pduC, encoding theDODHt large subunit, and ldhA (Cho et al., 2015). The 2,3-BDO titerreached 132 g/L in the ΔpduC ΔldhA double mutant, owing tosignificantly increased glycerol dissimilation and decreased sidemetabolite formation, relative to the parent strain (Cho et al., 2015).Interestingly, the ratio of (2S,3S)-2,3-BDO to meso-2,3-BDO was higherfor the ΔpduC ΔldhA double mutant, suggesting that it consumed moreNADH per mole of 2,3-BDO produced than the parent strain. Duringaerobic growth, a significantly lower intracellular NAD+/NADH ratiowas observed in a K. pneumoniae strain in which the NADH:quinoneoxidoreductase I complex (NDH-1) was inactivated, resulting inmarkedly improved 2,3-BDO production without any significantchange in 1,3-PDO levels (Zhang et al., 2018). Strain engineering for2,3-BDO production has recently been reviewed (Yang et al., 2017;Yang and Zhang, 2018).
Production of 1-butanol from glycerol was achieved in K. pneumo-niae although titers were in the mg/L range (Wang et al., 2014a, 2014b,2014c). The ter and bdhAB genes, encoding trans-2-enoyl-CoA reductase
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from Treponema denticola (Ter) and butanol dehydrogenases A (BdhA)and B (BdhB) from Clostridium acetobutylicum, respectively, were co-expressed to construct a modified CoA-dependent 1-butanol pathway,while coexpression of kivD, encoding α-ketoisovalerate decarboxylasefrom Lactococcus lactis (KivD), to facilitate the 2-keto acid pathwaysignificantly increased 1-butanol production. In addition, asRNAs weredesigned to target dhaB123, dhaT, gdrAB, budB, and budC to reduce theformation of 1,3-PDO and 2,3-BDO, although silencing gdrB expressionalone was most effective in enhancing 1-butanol production (Wanget al., 2014a, 2014b, 2014c). 1-Butanol production from glycerol in K.pneumoniae via the 2-keto acid pathway was significantly enhancedthrough overexpression of native leuABCD (encoding 2-isopropylmalatesynthase, 3-isopropylmalate dehydrogenase, and 3-isopropylmalatedehydratase subunits C and D), kivD from L. lactis, and adhE1 (encodingaldehyde/alcohol dehydrogenase; AdhE1) from C. acetobutylicum(Wang et al., 2015a, 2015b). Moreover, coexpression of fdh from P.Pastoris, and native udh and gdh to regenerate NADH, or simultaneoustranscription of asRNAs targeting the 1,3-PDO pathway markedly in-creased the 1-butanol titer in K. pneumoniae, and side metabolite for-mation was greatly reduced (Wang et al., 2015a, 2015b). In addition,repression of expression of metA (encoding homoserine O-succinyl-transferase; MetA), ilvI (encoding acetolactate synthase isozyme 3 largesubunit; IlvI), ilvB (encoding acetolactate synthase isozyme 1 largesubunit; IlvB), and alaA (encoding alanine transaminase; AlaA) viaCRISPRi significantly increased 1-butanol production in K. pneumoniae,with repression of ilvB expression alone increasing the titer by 2.5-fold(Wang et al., 2017a). A number of strain engineering strategies thathave been applied to enhance 1-butanol production in E. coli, for ex-ample (Atsumi et al., 2008; Dellomonaco et al., 2011; Dong et al., 2017;Ohtake et al., 2017), may also prove successful in Klebsiella sp. Mutationof pta can increase acetyl-CoA availability to drive acetoacetyl-CoAproduction and simultaneously reduce acetate accumulation (Ohtakeet al., 2017), and optimization of the ribosome binding site (RBS)translation initiation rate of AdhE2 (Dong et al., 2017; Ohtake et al.,2017), which is the most common bifunctional aldehyde/alcohol de-hydrogenase employed for 1-butanol production, may enhance 1-bu-tanol production in Klebsiella sp. Moreover, disruption of the FHLcomplex, in combination with overexpression of fdh, can mitigate theredox imbalance imposed by 1-butanol production in Klebsiella sp.(Dong et al., 2017).
Metabolic engineering of a ΔldhA K. pneumoniae mutant for 2-bu-tanol production from glycerol was explored through overexpression ofnative ilvIH (encoding keto-acid reducto-isomerase; IlvIH) and ilvD(encoding dihydroxyacid dehydratase; IlvD), and kivD and adhA (en-coding alcohol dehydrogenase; AdhA) from L. lactis, (Oh et al., 2014).The 2-butanol titer increased significantly upon mutation of budA, andthe ldhA mutation was essential for 2-butanol production which beganupon cessation of cell growth (Oh et al., 2014).
Enhanced ethanol production from glycerol was investigated in a K.pneumoniae mutant generated by γ-irradiation (Oh et al., 2011; Ohet al., 2012a). Coexpression of pdc and adhII (encoding aldehyde/al-cohol dehydrogenase; AdhII) from Z. mobilis moderately increased theethanol titer during fedbatch fermentation of crude glycerol (Oh et al.,2011), while mutation of ldhA significantly improved ethanol produc-tion in the strain coexpressing pdc and adhII (Oh et al., 2012a).
High-level production of optically pure D-lactate from glycerol wasachieved by expressing native ldhA in a ΔdhaT ΔyqhD double mutant ofK. pneumoniae (Feng et al., 2014). Overexpression of ldhA with andwithout mutation of dhaT and yqhD increased the lactate titer by 11.5-fold and 5.3-fold, respectively, and the lactate titer reached 142 g/L in afedbatch fermentation under suitable aeration conditions (Feng et al.,2014).
Acetoin production from glycerol has also been explored in en-gineered K. pneumoniae (Wang et al., 2017b). Mutation of budC, gldA(encoding GlyDH; GldA), and dhaD prevented conversion of acetoin to2,3-BDO, albeit with reduced glycerol consumption and no acetoin
production. On the other hand, activation of the silent glp regulon viaexpression of native glpK further hindered glycerol dissimilation, butenabled acetoin production. Moreover, expression of nox, encodingNOX from Lactobacillus brevis, significantly improved the acetoin titer,which reached 32 g/L in a fedbatch fermentation (Wang et al., 2017b).
Finally, biopolymer production from glycerol was achieved in en-gineered K. pneumoniae (Feng et al., 2015). Poly(3-hydroxypropionate)[P(3HP)], a polyhydroxyalkanoate (PHA) possessing high rigidity,ductility, and tensile strength in drawn films, is considered an attractivealternative to petroleum-based plastics (Andreeßen et al., 2010). P(3HP) production was achieved by first enhancing 3-HP biosynthesisthrough coexpression of native dhaB123 and gdrAB, and aldH from E.coli. 3-HP was converted to 3-hydroxypropionyl-CoA (3-HP-CoA) whichwas subsequently polymerized into P(3HP) upon expression of prpE(encoding propionyl-CoA synthetase; PrpE) from E. coli, and phaC(encoding PHA synthase; PhaC) from Ralstonia eutropha. The 3-HP titerincreased by 6.4-fold in a ΔdhaT ΔyqhD double mutant, though thespecific yield of P(3HP) was low, suggesting that 3-HP is not an idealsubstrate for PrpE, which natively produces propionyl-CoA from pro-pionate (Feng et al., 2015).
3.2. Citrobacter sp.
3.2.1. Natural metabolismCitrobacter sp. are capable of fermentative glycerol dissimilation
through coupled oxidative and reductive pathways (Fig. 1). Glycerol isoxidized to DHA by a soluble NAD+-dependent GlyDH (i.e. DhaD en-coded by dhaD) and is subsequently phosphorylated into DHAP byDhaK (encoded by dhaK) (Daniel and Gottschalk, 1992; Daniel et al.,1995). While ATP-dependent DhaK of Citrobacter freundii showsminimal homology to enzymes in other microorganisms (Daniel et al.,1995), it contains K- and L-domains displaying similar functions toDhaK and DhaL of E. coli, respectively (Siebold et al., 2003). Note thatenzymes encoded by the dha regulon (i.e. DhaB/DhaBCE, DhaD, DhaK,and DhaT) from Klebsiella sp. and Citrobacter sp. share 80-90% sequencesimilarity, compared to 30-80% between Citrobacter sp. and Clostridiumsp. (Sun et al., 2003). In parallel to fermentative glycerol oxidation,glycerol is converted to 3-HPA by GDHt (i.e. DhaBCE encoded bydhaBCE) (Seifert et al., 2001) and further reduced to 1,3-PDO by DhaT,encoded by dhaT (Ainala et al., 2013). Analogous to DhaB in Klebsiellasp., the activity of DhaBCE is dependent on B12, which is naturallysynthesized in Citrobacter sp. (Keuth and Bisping, 1994). Upon theconversion to 3-HPA, glycerol inactivates DhaBCE by disrupting thecobalt-carbon (Co-C) σ-bond of the attached B12 (Seifert et al., 2001),resulting in a closer binding of the modified coenzyme to the active site(Toraya, 2000). This altered dehydratase complex is reactivated byDhaF and DhaG, encoded by dhaF and dhaG, respectively (Seifert et al.,2001). In C. freundii, dhaF is located downstream of dhaBCE, whereasdhaG is located downstream of dhaT with an opposite transcriptionaldirection to dhaBCE (Seifert et al., 2001). These findings indicate thatdhaF is co-transcribed with dhaBCE, while dhaG is transcribed sepa-rately with dhaT to prevent accumulation of 3-HPA (Jiang et al., 2016;Seifert et al., 2001).
3.2.2. Strain engineeringDue to poor genetic tractability, Citrobacter sp. has been unpopular
as a host for bio-based production. However, some progress has beenmade toward strain engineering of Citrobacter sp. for 1,3-PDO produc-tion. Preliminary attempts to produce 1,3-PDO in C. freundii focused onfermentation strategies using purified (Boenigk et al., 1993) or crude(Metsoviti et al., 2013) glycerol. As the first approach for strain en-gineering, expression of dhaT from Shimwellia blattae in C. freundiisignificantly increased 1,3-PDO production compared to the wild-typestrain during a fedbatch fermentation, with significant lactate forma-tion observed as the cells entered the stationary phase (Celińska et al.,2015). Accordingly, focus should be placed on minimizing lactate
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synthesis in C. freundii to further enhance 1,3-PDO production, as wassuccessfully achieved in Citrobacter werkmanii (Maervoet et al., 2016).C. werkmanii is also a native host for high-level 1,3-PDO production(Maervoet et al., 2012a, 2012b) and preferentially utilizes glycerol forgrowth, even in the presence of multiple carbon sources (Maervoetet al., 2014). Accordingly, dhaD was mutated to force dissimilatedglycerol through the reductive pathway during co-fermentation with asecond carbon source that could also support cell growth. Of several co-substrates tested, glycerol/glucose co-fermentation was considered fa-vorable due to a moderate increase in the 1,3-PDO yield and the rela-tively low cost of glucose, although insufficient NADH levels in theabsence of glycerol oxidation resulted in 3-HPA accumulation(Maervoet et al., 2014). Moreover, ethanol, lactate, and acetate for-mation was significant during glycerol/glucose co-fermentation(Maervoet et al., 2014), leading to subsequent inactivation of ldhA andadhE to relieve the formation of these side metabolites (Maervoet et al.,2016). During glycerol/glucose co-fermentation, the ∆dhaD ∆adhEdouble mutant and ∆dhaD ∆ldhA ∆adhE triple mutant produced 2.2-foldand 2.7-fold more 1,3-PDO, respectively, compared to the ∆dhaD singlemutant. In addition, 3-HPA accumulation was significantly reduced inthe ∆dhaD ∆adhE double mutant, compared to the ∆dhaD single mutant,and was undetectable in the ∆dhaD ∆ldhA ∆adhE triple mutant(Maervoet et al., 2016). Lastly, mutation of arcA resulted in a sig-nificant increase in acetate, lactate, and succinate formation with 1,3-PDO production being only slightly improved, whereas the ∆dhaD∆ldhA ∆arcA triple mutant did not produce any 1,3-PDO (Maervoetet al., 2016). As previously noted for Klebsiella sp., another potentialstrategy to increase NADH levels for enhanced 1,3-PDO production inCitrobacter sp. is to simultaneously inactivate the FHL complex andoverexpress FDH. In addition, 2,3-BDO formation has not been reportedin Citrobacter sp. engineered for enhanced 1,3-PDO production and thisrepresents a major advantage over other natural 1,3-PDO producerssuch as Klebsiella sp. due to potentially reduced purification costs forseparating 1,3-PDO and 2,3-BDO.
Violacein, a blue-purple pigment possessing anti-bacterial, anti-viral, and anti-tumoral properties, is produced by diverse genera ofbacteria, including Chromobacterium, Collimonas, Duganella,Janthinobacterium, Microbulbifer, and Pseudomonas (Choi et al., 2015;Durán and Menck, 2001; Rettori and Durán, 1998). Biological pro-duction of violacein has been relatively ineffective, with the most stu-died strain Chromobacterium violaceum generating small amounts ofcrude violacein (Mendes et al., 2001; Sánchez et al., 2006). Expressionof the violacein biosynthesis operon, i.e. vioABCDE, encoding L-tryp-tophan oxidase (VioA), 2-imino-3-(indol-3-yl) propanoate dimerase(VioB), deoxyviolaceinate synthase (VioC), protodeoxyviolaceinatemonooxygenase (VioD), and a protein of unknown function (VioE),from C. violaceum, resulted in negligible violacein production in en-gineered E. coli and Streptomyces albus (Pemberton et al., 1991; Sánchezet al., 2006). On the other hand, expression of the violacein biosynth-esis operon from Duganella sp. in C. freundii resulted in a crude violaceintiter of 1.7 g/L from glycerol in shake flask cultures supplemented withthe precursor L-tryptophan (Jiang, P.-x., Wang, H.-s., Zhang, C., Lou, K.,Xing, X.-H., 2010), and the titer reached 4.1 g/L in a fedbatch culti-vation (Yang et al., 2011).
3.3. Clostridium sp.
3.3.1. Natural metabolismDespite the ability to utilize a wide variety of carbon sources for
Clostridium sp. (Tracy et al., 2012), natural catabolism of glycerol as thesole carbon source appears to be confined to a small subset, of which C.butyricum and C. pasteurianum are biotechnologically important. Ad-ditionally, strains of Clostridium beijerinckii, Clostridium diolis, Clos-tridium saccharobutylicum, and Clostridium propionicum were also de-rived for glycerol dissimilation (Dobson et al., 2012; Tracy et al., 2012;Wang et al., 2014a, 2014b, 2014c). Clostridium sp. normally consume
glycerol under anaerobic conditions via coupled oxidative and re-ductive branches of fermentative glycerol dissimilation, channelingglycerol into glycolysis to generate energy and reducing equivalentswhich are consumed through the production of reduced metabolitessuch as 1,3-PDO (Tracy et al., 2012; Wang et al., 2015a, 2015b), withother acids and alcohols, including acetate, propionate, butyrate, lac-tate, ethanol, and butanol being co-produced (Oh et al., 2014; Tracyet al., 2012). Similar to glycerol dissimilation in other anaerobic mi-croorganisms, certain Clostridium sp. predominantly channel glycerol toglycolysis through the intermediates DHA and DHAP. As such, Clos-tridium sp. encode GlyDH (i.e. GldA or DhaD) and DHAK (i.e. DhaK).Concurrently, glycerol is converted to 3-HPA and 1,3-PDO by GDHt(encoded by dhaB, pduD, or pduE) and 1,3-PDOOR (encoded by dhaT),respectively, to maintain redox poise (Kubiak et al., 2012; Sandovalet al., 2015; Sun et al., 2003). While GDHt is typically B12-dependent(Pyne et al., 2016a, 2016b; Saint-Amans et al., 2001; Siebold et al.,2003), GDHt from C. butyricum (i.e. DhaB1 encoded by dhaB1) is B12-independent, a phenomenon that appears to be uncommon in nature (Liet al., 2016a, 2016b). The completed genome sequence of C. diolis DSM15410 also identified a putatively B12-independent GDHt (Wang et al.,2013a, 2013b). Interestingly, putative genes encoding enzymes in-volved in glycerol dissimilation, i.e. glpK and glpD, have been identifiedin several members of Clostridium, including Clostridium kluyveri, C.propionicum, and C. acetobutylicum (Nölling et al., 2001; Poehlein et al.,2016; Seedorf et al., 2008). This is notable as these glycerol dissimila-tion pathways are predominantly found in aerobic microorganisms.Moreover, while various enzymes involved in glycerol dissimilationhave been annotated in other Clostridium sp., these pathways appear tobe incomplete or dormant (Biebl, 2001; Sun et al., 2003; Vasconceloset al., 1994). Despite having genes associated with glycerol dissimila-tion, most Clostridium sp. are unable to metabolize glycerol as the solecarbon source, ostensibly due to the absence or deficiency of a 1,3-PDOpathway (González-Pajuelo et al., 2005; Seedorf et al., 2008). C. acet-obutylicum will utilize glycerol in the presence of a more oxidizedcarbon source such as glucose or pyruvate, or an exogenous electronacceptor such as methyl viologen or neutral red, suggesting that itsatypical glycerol dissimilation pathway is still active (Girbal et al.,1995; Girbal and Soucaille, 1994; Peguin et al., 1994; Vasconceloset al., 1994).
3.3.2. Strain engineeringDue to limited genetic tools for Clostridium sp. and the fact that
glycerol is a recalcitrant carbon source under anaerobic conditions(Jang et al., 2012; Pyne et al., 2014), strategies directed toward im-proving glycerol utilization for the production of value-added meta-bolites in Clostridium sp. remain uncommon. Random mutagenesis hasbeen widely applied to improve glycerol dissimilation and, as such,mutants with improved phenotypes remain uncharacterized geneti-cally. A C. pasteurianum mutant was capable of consuming glycerol at arate almost double that of the parent strain, resulting in enhancedproduction of 1,3-PDO and 1-butanol (Jensen et al., 2012). Similarly, aC. pasteurianum mutant with improved glycerol tolerance and growthrate produced significantly more 1-butanol than the parent strain(Malaviya et al., 2012), while 1-butanol tolerance and production in-creased in another mutant, even though glycerol consumption was notaffected (Gallardo et al., 2017). Genome shuffling was applied to C.diolis to improve 1,3-PDO tolerance, resulting in a marked increase inthe 1,3-PDO yield with enhanced glycerol utilization compared to theparent strain (Otte et al., 2009). A C. pasteurianum mutant with im-proved tolerance to crude glycerol was derived through random mu-tagenesis followed by directed evolution under increasing concentra-tions of crude glycerol, resulting in substantially increased 1-butanolproduction and reduced 1,3-PDO formation, and the improved cultureperformance was found to be associated with a large deletion in spo0A,encoding a transcriptional regulator for sporulation (SpoOA) (Sandovalet al., 2015). In agreement with this work, mutation of spoOA
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significantly improved glycerol dissimilation with a concomitant in-crease in 1-butanol production (Schwarz et al., 2017). Mutation of dhaTin C. pasteurianum led to a significant decrease in 1,3-PDO production,however, glycerol dissimilation persisted with 1,2-PDO formation as analternative redox valve (Pyne, Michael E. et al., 2016). Although theidentification of genes encoding putative enzymes related to the 1,2-PDO pathway suggests that Clostridium sp. such as C. acetobutylicum,Clostridium difficile, and C. beijerinckii may synthesize 1,2-PDO or itsprecursors, this was the first experimental demonstration of 1,2-PDOproduction in Clostridium sp. (Huang, K.-x., Rudolph, F.B., Bennett,G.N., 1999; Liyanage et al., 2001; Pyne et al., 2016a, 2016b). 1,3-PDOproduction was recently abolished in C. pasteurianum by deleting theentire dhaBCE operon using Allele-Coupled Exchange (ACE), although1-butanol and side metabolite formation were not significantly affected(Schwarz et al., 2017). In contrast, mutation of rex, encoding the redox-responsive regulator (Rex) that represses expression of genes in the 1-butanol pathway in response to low NADH levels, significantly de-creased 1,3-PDO and butyrate formation with markedly improved 1-butanol production, in spite of reduced glycerol utilization. Finally,mutation of hydA, encoding ferredoxin dehydrogenase (HydA), sig-nificantly increased acetate, lactate, and ethanol formation, with a re-duction in butyrate and 1,3-PDO production, and a modest increase inthe 1-butanol titer (Schwarz et al., 2017). The results are in agreementwith an earlier study of asRNA-mediated repression of hydA expressionin C. pasteurianum grown in a complex medium with glucose (Pyneet al., 2015).
As DhaB1 of C. butyricum is B12-independent, 1,3-PDO productionusing this organism is of significant interest (Raynaud, and xe, line, Sar,xe, abal, P., Meynial-Salles, I., Croux, C., Soucaille, P., 2003; Saint-Amans et al., 2001). Due to the lack of genetic tools, improving 1,3-PDO production in C. butyricum has been limited to derivation of 1,3-PDO-tolerant mutants via random mutagenesis (Abbad-Andaloussiet al., 1995; Reimann et al., 1998; Reimann and Biebl, 1996). The 1,3-PDO pathway of C. butyricum was introduced into C. acetobutylicum(González-Pajuelo et al., 2005) which has more advanced genetic tools(Pyne et al., 2014). The resulting engineered strains metabolized gly-cerol anaerobically, resulting in a moderate increase in the 1,3-PDOtiter relative to the natural producer C. butyricum in fedbatch fermen-tations (González-Pajuelo et al., 2005). Improved 1,3-PDO productionfrom glycerol was achieved in C. beijerinckii via coexpression of gldAand dhaKLM from E. coli and, compared to the wild-type strain, theengineered strain also had a greatly increased specific growth rate(Wischral et al., 2016). While the tools available for genetic manip-ulation in Clostridium sp. are lagging behind other industrially im-portant bacteria, recent advances such as ACE and CRISPR can accel-erate the pace of strain development. Notably, 74% of Clostridium sp.harbor CRISPR-Cas machinery, and native CRISPR-Cas systems can beexploited for genome editing with significantly greater editing effi-ciency than heterologous platforms (Pyne et al., 2016a, 2016b). Abol-ishing 1,3-PDO production in C. pasteurianum did not significantly altermetabolite profiles, such that systems level in silico analysis of genemutations combined with an efficient toolkit for genome editing may benecessary to dramatically reduce the labor of strain optimization forhigh-level 1-butanol production. In the case of C. pasteurianum, high-efficiency transformation protocols have been developed (Pyne et al.,2013; Schwarz et al., 2017), however, low transformability still remainsan issue for many promising Clostridium sp. (González-Pajuelo et al.,2006; Li et al., 2016a, 2016b) and must be addressed prior to the ap-plication of advanced genome engineering technologies for metabolicengineering purposes.
3.4. Lactobacillus sp.
3.4.1. Natural metabolismCertain Lactobacillus sp. strains are industrially important for lactate
production through anaerobic dissimilation of glycerol with sugar co-
substrates (Schütz and Radler, 1984; Talarico et al., 1990). Glycerol isconverted to 1,3-PDO via the reductive glycerol dissimilation pathwayto regenerate NAD+ consumed through the pentose phosphate pathway(PPP) (da Cunha and Foster, 1992; Garai-Ibabe et al., 2008). However,oxidative glycerol dissimilation does not occur naturally under fer-mentative and respiratory conditions due to incomplete and inactivepathways, respectively, in Lactobacillus sp. such that they cannot con-sume glycerol in the absence of a more oxidized carbon source (Kanget al., 2014a, 2014b; Morita et al., 2008; Talarico et al., 1990). Putativeglycerol kinase (GK) and NADP+-dependent G3P dehydrogenase arecommon to many Lactobacillus sp., although they are presumably dor-mant or weakly expressed as is the case in certain Clostridium sp.(Nölling et al., 2001; Vasconcelos et al., 1994) and K. pneumoniaestrains (Forage and Lin, 1982; Wang et al., 2017b). Putative GlyDH wasidentified in the genomes of several Lactobacillus sp., although DHAK isnot present, preventing oxidative glycerol dissimilation under fermen-tative conditions (Morita et al., 2008). The reductive pathway of fer-mentative glycerol dissimilation begins with the conversion of glycerolto 3-HPA, a process initially thought to occur via either of two distinctB12-dependent enzymes, i.e. GDHt and DODHt (Talarico andDobrogosz, 1990). However, the three subunits of a putative dehy-dratase from Lactobacillus reuteri JCM1112 show sequence similarity toDODHt of Salmonella typhimurium (i.e. PduCDE encoded by pduCDE)(Morita et al., 2008). Moreover, a metabolosome-associated DODHt(i.e. PduCDE encoded by pduCDE) was identified in L. reuteri DSM20016 that required pre-incubation with 1,2-PDO to achieve high 3-HPA production from glycerol (Sriramulu et al., 2008). Similar pduoperons have also been found in Lactobacillus collinoides (Sauvageotet al., 2002) and L. brevis (Makarova et al., 2006), indicating thecommon absence of a dedicated GDHt for Lactobacilli. The naturalability to synthesize B12 is an attractive feature of many L. reuteri andcertain Lactobacillus panis strains (Kang et al., 2014a; Morita et al.,2008; Ricci et al., 2015; Santos et al., 2011; Taranto et al., 2003), al-though it is not common to all Lactobacillus sp. (Pflugl et al., 2014). Thereduction of 3-HPA to 1,3-PDO is catalyzed by one or more 1,3-PDOORs, of which the activity and expression are sensitive to growthconditions. L. reuteri DSM 20016 expresses two putative 1,3-PDOORs,encoded by lr_0030 and lr_1734, which are active during exponentialand stationary growth phases, respectively (Stevens et al., 2011). Inaddition, lr_0030 is down-regulated upon depletion of glucose, resultingin 3-HPA accumulation, a phenomenon also observed in L. panis (Kanget al., 2013) and L. collinoides (Sauvageot et al., 2000). Among lactate-producing bacteria, L. reuteri has the unique capability to produce andexcrete 3-HPA in large quantities. The 3-HPA/1,3-PDO ratio decreasesas the glucose/glycerol ratio increases in L. reuteri cultures (Luthi-Penget al., 2002), although 1,3-PDO formation is hindered when glycerol ispresent in excess (~30 g/L) (Bauer et al., 2010). The latter observationis not surprising given that expression of dhaT is repressed under highglycerol loading in L. panis (Kang et al., 2013), a process aggravated bythe 3-HPA-mediated inhibition of DhaT (Kang et al., 2014b). Con-versely, the 1,3-PDO titer increased as the glycerol concentration wasincreased up to 70 g/L during batch fermentations of Lactobacillusdiolivorans (Pflugl et al., 2012).
In general, cell growth can be maintained or enhanced upon theaddition of glycerol to anaerobic glucose cultures of Lactobacillus sp. (daCunha and Foster, 1992; El-Ziney et al., 1998; Pflugl et al., 2012) with ametabolic shift from ethanol and lactate to 1,3-PDO and acetate (daCunha and Foster, 1992; El-Ziney et al., 1998; Luthi-Peng et al., 2002;Pflugl et al., 2012). The formation of 1,3-PDO replaces ethanol and, to alesser extent, lactate for NAD+ replenishment, enabling the productionof additional ATP via acetate synthesis (da Cunha and Foster, 1992).Lactate can also be oxidized to generate acetate under low sugar con-centrations, with 1,3-PDO serving as the major redox outlet (da Cunhaand Foster, 1992). Production of 3-HP has also been observed in L.reuteri (Ramakrishnan et al., 2015), L. collinoides (Garai-Ibabe et al.,2008), and L. diolivorans (Garai-Ibabe et al., 2008), serving to restore
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
553
the cellular redox balance upon depletion of fermentable sugars (Luoet al., 2011a, 2011b; Sriramulu et al., 2008). Expression of pduP from L.reuteri in K. pneumoniae markedly enhanced 3-HP production relative tothe wild-type strain (Luo et al., 2011a, 2011b), indicating that PduPmay be associated with the 3-HP pathway in Lactobacillus sp. as is thecase in Klebsiella sp. (Luo et al., 2012). Strains of L. diolivorans and L.collinoides isolated from bitter tasting ciders produced 3-HP as a majormetabolite during fermentation on fructose and glycerol (Garai-Ibabeet al., 2008). High sugar concentrations favored 1,3-PDO production,while equimolar quantities of 3-HP and 1,3-PDO were obtained underfructose limitation (Garai-Ibabe et al., 2008).
3.4.2. Strain engineeringEngineering Lactobacillus sp. to enhance glycerol dissimilation has
emerged recently for synthesis of 1,3-PDO and 3-HP. An attempt toimprove 1,3-PDO production in engineered L. reuteri was made throughexpression of yqhD from E. coli, resulting in a significant increase in thespecific 1,3-PDO productivity relative to the wild-type strain(Vaidyanathan et al., 2011). However, a shift from acetate and biomassto ethanol and lactate formation was observed, presumably due to thepreference of YqhD for NADPH, diminishing the activity of the nativeNADH-dependent 1,3-PDOOR (i.e. PduQ encoded by pduQ) and, inturn, disturbing the natural redox balance (Vaidyanathan et al., 2011).As part of an assessment of heterologous recombinases in L. reuteri,mutation of the catabolite repression element (CRE) located upstreamof the pdu operon resulted in a 3-fold increase in 3-HPA productionwhen cells grown overnight in a medium containing glucose weretransferred to a medium containing glycerol (van Pijkeren et al., 2012).The CRE mutant was subsequently evaluated for 3-HP/1,3-PDO co-production in a fedbatch resting cell fermentation and the specificproductivities of 3-HP and 1,3-PDO increased significantly, relative tothe wild-type strain, with essentially all glycerol being converted to thetarget metabolites (Dishisha et al., 2014).
L. panis has been engineered by expressing yqhD from E. coli toenhance 1,3-PDO production from thin stillage with a significant gly-cerol content (Kang et al., 2014b). The L. panis strain was further en-gineered to dissimilate glycerol as the sole carbon source through me-tabolic introduction of a respiratory pathway (Kang et al., 2014a,2014b). However, coexpression of glpK, glpF, glpD, and tpi, encodingtriosephosphate isomerase (Tpi), from E. coli resulted in poor cellgrowth and minimal glycerol consumption with lactate as the majorend metabolite, indicating that NADH generated through the syntheticrespiratory pathway was not effectively recycled. Accordingly, coex-pression of yqhD from E. coli was employed to restore redox poise (Kanget al., 2014a, 2014b). It was anticipated that YqhD could reduce acetateto acetaldehyde, which could be further reduced to ethanol via an en-dogenous alcohol dehydrogenase, while simultaneously enhanced 3-HPA conversion to 1,3-PDO would occur (Kang et al., 2014a). The celldensity and glycerol utilization increased dramatically upon coexpres-sion of yqhD, relative to the control strain expressing the synthetic re-spiratory pathway, and ethanol and 1,3-PDO were the major reducedmetabolites. Enhanced 1,3-PDO production was also demonstrated in L.diolivorans by overexpressing a native putative NADPH-dependent 1,3-PDOOR (i.e. PDO-DH (NADPH)) (Pflugl et al., 2013). The approachresulted in slightly improved cell growth and a moderate increase in1,3-PDO formation, indicating that the putative 1,3-PDOOR readilyuses NADH or NADPH as a cofactor to convert 3-HPA to 1,3-PDO.
While engineering of glycerol metabolism in Lactobacillus sp. has notbeen studied extensively, many of these microorganisms have beengranted the Generally Recognized as Safe (GRAS) status from the FDA,USA, making them ideal hosts for the conversion of glycerol to value-added products on an industrial scale. In particular, further explorationof 1,3-PDO production in Lactobacillus sp. is warranted due to theirnatural production potential and apparent minimal 2,3-BDO formationas a side metabolite. For example, to overcome the NADH accumulationissue upon the introduction of a respiratory pathway for dissimilation of
glycerol as the sole carbon source in L. panis (Kang et al., 2014a,2014b), cofactor engineering via expression of NOX could regenerateNAD+ without forming competing side products (i.e. ethanol), andexpression of DhaT from Klebsiella sp. or Clostridium sp. may improveconversion of 3-HPA to 1,3-PDO while consuming NADH. Moreover,expression of NOX may also alleviate NAD+ depletion in Lactobacillussp. engineered to overproduce 3-HP. To further enhance glycerol dis-similation, the native respiratory pathway could be activated and co-expression of glpF from a Gram-positive organism, e.g. a facultativeanaerobe such as B. subtilis, may enhance the functional expression ofthe membrane-bound permease, relative to GlpF from a Gram-negativebacterium. Similarly, an engineered fermentative pathway containingthe native putative GlyDH may facilitate glycerol dissimilation in theabsence of a more reduced carbon source. Finally, in light of the sub-stantial number of putative LDHs and aldehyde/alcohol dehy-drogenases in Lactobacillus sp., knocking out relevant genes by theCRISPR-Cas9 system could simplify the elimination of lactate andethanol production to enhance target metabolite formation.
3.5. Escherichia coli
3.5.1. Natural metabolismE. coli can naturally dissimilate glycerol aerobically and anaerobi-
cally via respiratory and fermentative pathways (Dharmadi et al.,2006). Glycerol is transported into E. coli through facilitated diffusionby GlpF (Richey and Lin, 1972), which can also transport polyols (e.g.erythritol), urea, glycine, and glyceraldehyde (Heller et al., 1980). Inthe cytosol, glycerol enters the respiratory or fermentative pathways,either of which produce DHAP as an intermediate of glycolysis. In therespiratory pathway, glycerol is phosphorylated to G3P by GK (i.e. GlpKencoded by glpK) (Hayashi and Lin, 1967), the presumed limiting stepfor aerobic dissimilation of glycerol (Lin, 1976a, 1976b; Zwaig et al.,1970). Subsequently, if oxygen acts as an electron acceptor, G3P isconverted to DHAP by aerobic-G3PDH (i.e. GlpD encoded by glpD), oranaerobic-G3PDH (i.e. GlpABC encoded by glpABC) in the presence ofelectron acceptors other than oxygen (i.e. nitrate and fumarate). In thefermentative pathway, glycerol is oxidized to DHA via NAD+-depen-dent GlyDH (i.e. GldA encoded by gldA) (Tang et al., 1979), followed byphosphorylation of DHA to DHAP via PEP-dependent DHAK (i.e.DhaKLM encoded by dhaKLM) (Gonzalez et al., 2008). The expressionof enzymes in the respiratory and fermentative pathways is under acollective control of the global regulators cAMP-CRP, Cra, ArcA, Fnr,and RpoS encoded by crp, cra, arcA, fnr, and rpoS, respectively (Iuchiand Lin, 1993; Martínez-Gómez et al., 2012).
In the absence an exogenous electron acceptor, E. coli cannotnaturally maintain redox poise through the production of reducedcompounds other than 1,2-PDO (Dharmadi et al., 2006). The net NADHproduced through the lactate, ethanol, succinate, and 1,2-PDO path-ways are 1, 0, 0, and -1 mol NADH per mol glycerol consumed, re-spectively, such that only 1,2-PDO formation can regenerate NAD+. Inaddition to redox balance, glycerol fermentation is dependent on pHand the availability of CO2 (Durnin et al., 2009). E. coli requires CO2 inthe form of bicarbonate (HCO3
-) for growth and biosynthesis of varioussmall molecules and cellular components (Dharmadi et al., 2006).Under fermentative conditions, CO2 production mainly relies on theactivity of the FHL complex (encoded by fdhF and hycBCDEFG) for theconversion of formate to CO2 and H2 (Iuchi and Lin, 1993). Tran-scription of the genes encoding the FHL complex requires formate andan acidic pH (Sawers et al., 2004). An alkaline environment causes anequilibrium shift toward HCO3
-, which cannot be transported across thecellular membrane of E. coli, thus reducing CO2 availability inside thecell (Dharmadi et al., 2006). H2 is co-produced with CO2 and can act asan electron donor, further contributing to the redox imbalance duringglycerol fermentation and potentially impairing growth (Dharmadiet al., 2006). Other glycerol fermentation products of E. coli includeethanol, succinate, and acetate (Gonzalez et al., 2008), and ethanol
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
554
Table3
Sum
mar
yof
liter
atur
efo
rE.
coli
stra
ins
engi
neer
edfo
ren
hanc
edna
tura
lmet
abol
itepr
oduc
tion
from
glyc
erol
.
Parent
strain
Product
Geneticstrategy
Titer(g/L)
Cultivationmode
Reference
ATC
C873
9et
hano
l-M
utat
efrdA
BCD,
ackA
-pta
andpo
xB,a
ndldhA
tore
duce
succ
inat
e,ac
etat
e,an
dla
ctat
efo
rmat
ion,
resp
ectiv
ely,
and
mut
ategldA
andglpK
for
cont
rolle
dex
pres
sion
-Ove
rexp
ress
dhaK
LM,g
ldA
,glpK,
andad
hEfo
rin
crea
sed
carb
onflu
xan
det
hano
lfor
mat
ion
47Fe
dbat
ch(W
ong
etal
.,20
14)
LY18
0et
hano
lFo
smid
cont
aini
ngm
etag
enom
icfr
agm
ent
isol
ated
from
anae
robi
cre
acto
r75
Batc
h(L
oace
set
al.,
2016
)M
G16
55et
hano
l-M
utat
efrdA
,pta
,and
fdhF
tore
duce
succ
inat
e,ac
etat
e,an
dCO
2,an
dH
2fo
rmat
ion,
resp
ectiv
ely
-Ove
rexp
ress
gldA
anddh
aKLM
for
incr
ease
dca
rbon
flux
thro
ugh
the
ferm
enta
tive
bran
ch10
Batc
h(Y
azda
nian
dG
onza
lez,
2008
)
BW25
113
H2
-Mut
atehy
cA,h
yaB,
hybC
,hyc
E,hy
fG,p
flB,f
hlA
,fdh
F,gldA
,orglpA
toaff
ect
H2
form
atio
nN
/ACr
imp-
top
vial
s(S
anch
ez-T
orre
set
al.,
2013
)BW
2511
3H
2M
utat
earoM
,gatZ,
ycgR
,oryfgI
toen
hanc
eH
2fo
rmat
ion
See
refe
renc
eCr
imp-
top
vial
s(T
ran
etal
.,20
15)
BW25
113
H2
Mut
atefrdC
,ldh
A,f
dnG
,ppc
,narG
,mgsA,a
ndhy
cAto
enha
nce
H2
form
atio
n15
8μm
ol/m
gpr
otei
nCr
imp-
top
vial
s(T
ran
etal
.,20
14)
ATC
C873
9su
ccin
ate
-Mut
atepfl
Bto
redu
cefo
rmat
e,et
hano
l,an
dac
etat
efo
rmat
ion
-Mut
ateptsI
tosh
iftPE
Pflu
xaw
ayfr
omD
HA
K-M
utat
ion
inpr
omot
orre
gion
ofpc
kge
neto
incr
ease
expr
essi
onle
vel
12Fl
ask
(Zha
nget
al.,
2010
)
MG
1655
succ
inat
e-M
utat
ead
hE,p
taan
dpo
xB,a
ndldhA
tore
duce
etha
nol,
acet
ate,
and
lact
ate
form
atio
n,re
spec
tivel
y-M
utat
epp
cto
rem
ove
sele
ctio
nre
quir
emen
t-E
xpre
sspy
c(L.lac
tis)
for
succ
inat
epr
oduc
tion
from
pyru
vate
node
14Fl
ask
(Bla
nksc
hien
etal
.,20
10)
BL21
(DE3
)su
ccin
ate
-Mut
atepp
can
dch
rom
osom
alex
pres
sion
ofgl
yoxy
late
shun
tgen
esac
eBAK
follo
wed
bydi
rect
edev
olut
ion
fori
ncre
ased
glyo
xyla
tesh
unt
activ
ityan
dre
activ
atio
nof
ppc
-Mut
atesdhC
DAB
tore
duce
prod
uctd
egra
datio
n-C
hrom
osom
alex
pres
sion
ofsucA
Bto
redu
ceα-
keto
glut
arat
efo
rmat
ion
43Fe
dbat
ch(L
iet
al.,
2013
)
MG
1655
D-la
ctat
e-M
utat
epta,
adhE
,and
frdA
tore
duce
acet
ate,
etha
nol,
and
succ
inat
efo
rmat
ion,
resp
ectiv
ely
-Mut
atedld
tore
duce
D-la
ctat
ede
grad
atio
n-O
vere
xpre
ssglpD
andglpK
toin
crea
seca
rbon
flux
thro
ugh
the
resp
irat
ory
path
way
45Fl
ask
(Maz
umda
ret
al.,
2010
)
BL21
(DE3
)D
-lact
ate
-Mut
atepta,
adhE
,frdA
,pflB
,and
pflCD
tore
duce
side
met
abol
itefo
rmat
ion
–Mut
atemgsA
anddld
tore
duce
MG
form
atio
nan
dD
-la
ctat
ede
grad
atio
n,re
spec
tivel
y-D
irec
ted
evol
utio
nfo
rin
crea
sed
crud
egl
ycer
olut
iliza
tion
-Chr
omos
omal
expr
essi
onof
glpD
andglpK
toin
crea
seca
rbon
flux
thro
ugh
the
resp
irat
ory
path
way
,and
chro
mos
omal
expr
essi
onof
focA
andldh
(L.h
elvetic
us)
toin
crea
sela
ctat
eex
port
and
form
atio
n,re
spec
tivel
y
105
Fedb
atch
(Wan
get
al.,
2015
a,20
15b)
MG
1655
L-la
ctat
e-M
utat
epfl
B,pta,
adhE
,and
frdA
tore
duce
side
met
abol
itefo
rmat
ion
-Mut
atemgsA
andldhA
topr
even
tD
-lact
ate
form
atio
n,an
dm
utat
elld
Dto
redu
ceL-
lact
ate
degr
adat
ion
-Ove
rexp
ress
glpK
andglpD
toin
crea
seca
rbon
flux
thro
ugh
the
resp
irat
ory
path
way
-Chr
omos
omal
expr
essi
onof
ldh
(S.b
ovis)
for
L-la
ctat
efo
rmat
ion
50Fl
ask
(Maz
umda
ret
al.,
2013
)
BW25
113
L-la
ctat
e-M
utat
eac
kA,p
ta,p
ps,p
flB,p
oxB,
adhE
,and
frdA
tore
duce
side
met
abol
itefo
rmat
ion
-Mut
ateldhA
andlld
topr
even
tD
-lact
ate
form
atio
nan
dpr
oduc
tdeg
rada
tion,
resp
ectiv
ely
-Chr
omos
omal
expr
essi
onof
ldh
(B.c
oagu
lans
)fo
rL-
lact
ate
form
atio
n
142
Fedb
atch
(Tia
net
al.,
2013
)
W31
10L-
phen
ylal
anin
e-M
utat
etyrA
tore
duce
L-tr
ypto
phan
form
atio
n-M
utat
epy
kAan
dpy
kFto
incr
ease
avai
labl
ePE
P,an
dm
utat
ephe
Aan
daroF
for
cont
rolle
dex
pres
sion
-Ove
rexp
ress
aroF
BLan
dfe
edba
ckre
sist
antp
heAfbr fo
rin
crea
sed
shik
imat
ean
dL-
phen
ylal
anin
efo
rmat
ion,
resp
ectiv
ely
-Fee
dla
ctat
ean
dam
mon
ia
13Fe
dbat
ch(W
eine
ret
al.,
2014
)
BL21
(DE3
)fu
mar
ate
-Pre
viou
sly
evol
ved
succ
inat
epr
oduc
ing
stra
inco
ntai
ning
ppc
mut
atio
nan
dex
pres
sing
chro
mos
omal
aceB
AK
-Mut
atefumABC
andaspA
tore
duce
fum
arat
ede
grad
atio
n-O
vere
xpre
sspp
cto
incr
ease
fum
arat
esy
nthe
sis
42Fe
dbat
ch(L
iet
al.,
2014
)
MG
1655
form
ate
-Mut
atefrdA
,pta
,and
fdhF
tore
duce
side
met
abol
itefo
rmat
ion
-Ove
rexp
ress
gldA
anddh
aKLM
for
incr
ease
dgl
ycer
olut
iliza
tion
7Ba
tch
(Yaz
dani
and
Gon
zale
z,20
08)
MG
1655
FFA
s-M
utat
efadD
andptsG
toen
hanc
eFF
Afo
rmat
ion,
and
mut
atefabR
tore
mov
ere
pres
sion
offa
tty
acid
synt
hesi
sge
nes
-Ove
rexp
ress
nadK
andpn
tAB
toin
crea
sein
trac
ellu
lar
redo
xeq
uiva
lent
s,an
dex
pres
san
acyl
-ACP
thio
este
rase
(R.c
ommun
is)fo
rin
crea
sed
FFA
form
atio
n
5Fl
ask
(Wu
etal
.,20
14)
BL21
(DE3
)D
HA
-Coe
xpre
ssno
x(E
.fae
calis
)an
dgldA
for
incr
ease
dN
AD
+re
gene
ratio
nan
dD
HA
form
atio
n,re
spec
tivel
y-E
xoge
nous
supp
lyof
NA
D+
2Te
sttu
be(Y
ang
etal
.,20
13)
Lin4
3ac
etol
-Mut
ategloA
tore
duce
MG
degr
adat
ion
-Exp
ress
yqhD
for
incr
ease
dac
etol
synt
hesi
s5
Flas
k(Z
huet
al.,
2013
)
(con
tinue
don
next
page
)
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
555
formation is essential as it generates ATP and is redox-balanced(Murarka et al., 2008). Although succinate production is also redox-balanced, it contributes minimally to ATP generation (Unden andKleefeld, 2004), making it non-essential during glycerol fermentation(Murarka et al., 2008), whereas acetate production is often marginalduring glycerol fermentation.
The highly reduced nature of glycerol makes it an ideal feedstock forthe production of reduced compounds and derived value-added pro-ducts. Anaerobic conditions favor the production of reduced com-pounds as oxygen is not available as an electron acceptor. However,anaerobic glycerol fermentations require supplementation of richmedia components to counteract the production of reducing equiva-lents generated via biomass formation (κglycerol=4.7; κbiomass=4.3) andthe low activity of the 1,2-PDO pathway (Durnin et al., 2009). Incontrast to anaerobic fermentation, microaerobic conditions introducelimiting amounts of oxygen, effectively acting as an electron acceptorfor redox equivalents generated from biomass, such that rich mediacomponents are no longer required while the production of reducedcompounds is maintained (Durnin et al., 2009).
3.5.2. Strain engineeringTable 3 and Table 4 summarize strategies for strain engineering of E.
coli for enhanced production of natural and non-native metabolitesfrom glycerol, respectively. Selective target products are used fortechnical illustration in this section.
3.5.2.1. 1,3–PDO. Preliminary attempts to produce 1,3-PDO in E. coliby expressing the dha regulon from either K. pneumoniae (Sprengeret al., 1989; Tong et al., 1991) or C. freundii (Daniel and Gottschalk,1992) resulted in substantially lower 1,3-PDO titers than those obtainedwith natural production hosts (Cameron et al., 1998; Chotani et al.,2000). As a result, more advanced strain engineering strategies weredeveloped to enhance 1,3-PDO production in E. coli, primarilyincluding: (1) the improved expression of genes involved in thereductive branch of glycerol dissimilation from natural 1,3-PDOproducers; (2) the expression of native putative NADPH-dependent1,3-PDOOR, YqhD; and (3) reducing the formation of the toxicmetabolites MG, G3P, and 3-HPA. MG is an intermediate of 1,2-PDOproduction, and concentrations exceeding 0.5 mM can inhibit cellgrowth (Ackerman et al., 1974; Booth et al., 2003; MacLean et al.,1998a, 1998b). Anaerobic fermentation of glycerol by E. coli strainsexpressing the dha regulon from K. pneumoniae resulted in theaccumulation of MG to toxic levels with lowered 1,3-PDO production(Zhu et al., 2001). As MG can be converted to S- lactoylglutathinone byglyoxalase I, and subsequently to lactate by glyoxalase II (MacLeanet al., 1998a, 1998b), expression of glyoxalase I, i.e. Glo1 (encoded byglo1 from Pseudomonas putida), reduced intracellular MG levels andmoderately enhanced 1,3-PDO production (Zhu et al., 2001). Similarly,both cell growth and 1,3-PDO production were inhibited when theintracellular G3P concentration exceeded 10 mM in E. coli (Lin, 1976a,1976b; Zhu et al., 2002), and the addition of an exogenous electronacceptor such as fumarate could increase the activity of GlpABC and, inturn, curb G3P accumulation and dramatically improve 1,3-PDOproduction (Zhu et al., 2002).
The reductive branch of glycerol dissimilation from K. pneumoniae,C. freundii, and C. butyricum have been introduced in E. coli to facilitate1,3-PDO production. While dhaB123 and dhaT are divergently ex-pressed from individual promoters in K. pneumoniae, this arrangementresulted in a lower 1,3-PDO titer in E. coli, compared to the coexpres-sion of dhaB123 and dhaT from a single promoter (Skraly et al., 1998).Furthermore, native YqhD could convert 3-HPA to 1,3-PDO more effi-ciently than DhaT from K. pneumoniae (Emptage et al., 2003b), and thepreference of YqhD for NADPH may have contributed to higher 1,3-PDO titers by altering the reduced/oxidized cofactor ratios (Nakamuraand Whited, 2003). Coexpression of gdrAB and dhaB123 from K.pneumoniae with native yqhD significantly increased 1,3-PDOTa
ble3
(con
tinue
d)
Parent
strain
Product
Geneticstrategy
Titer(g/L)
Cultivationmode
Reference
MG
1655
1,2-
PDO
-Mut
ateac
kAan
dpta,
andldhA
tore
duce
acet
ate
and
lact
ate
form
atio
n,re
spec
tivel
y-M
utat
edh
aKan
dex
pres
sdh
aKL
(C.f
reun
dii)
for
ATP
depe
nden
tfu
nctio
nalit
y(c
ompa
red
tona
tive
PEP
depe
nden
ce)
-Exp
ress
msgA
,gldA
,and
yqhD
for
incr
ease
d1,
2-PD
Ofo
rmat
ion
6Ba
tch
(Clo
mbu
rgan
dG
onza
lez,
2011
)
Gen
epr
oduc
ts:a
ckA
,ace
tate
kina
se;a
ceBA
K,is
ocitr
ate
lyas
e,m
alat
esy
ntha
se,a
ndis
ocitr
ate
dehy
drog
enas
eki
nase
/pho
spho
ryla
se;a
dhE,
alde
hyde
-alc
ohol
dehy
drog
enas
e;aroF
BL,3
-deo
xy-D
-ara
bino
-hep
tulo
sona
te-7
-ph
osph
ate
synt
hase
isoe
nzym
e,3-
dehy
droq
uina
tesy
ntha
se,
and
shik
imat
eki
nase
;aroM
,pr
otei
nA
roM
;aspA
;as
part
ate
amm
onia
-lyas
e;dh
aKLM
,D
HA
kina
se,
PEP-
depe
nden
tdi
hydr
oxya
ceto
neki
nase
AD
P-bi
ndin
gsu
buni
t,an
dpr
otei
n-ly
sine
deac
etyl
ase;
dld,
quin
one-
depe
nden
tD-la
ctat
ede
hydr
ogen
ase;
fabR
,HTH
-type
tran
scri
ptio
nalr
epre
ssor
;fad
D,lo
ng-c
hain
fatt
yac
idCo
Alig
ase;
fdhf
,for
mat
ede
hydr
ogen
ase
H;fdn
G,f
orm
ate
dehy
drog
enas
eN
subu
nit
alph
a;fhlA
,fo
rmat
ehy
drog
enly
ase
tran
scri
ptio
nal
activ
ator
;focA
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rmat
etr
ansp
orte
r1;
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mar
ate
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ctas
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nsu
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naer
obic
);frdA
BCD
,fum
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ere
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ase
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me
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plex
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nit
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ndC;
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gato
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isph
osph
ate
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nit;
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cero
ldeh
ydro
gena
se;g
loA
,lac
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athi
one
lyas
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erob
icgl
ycer
ol-3
-pho
spha
tede
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ogen
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nitA
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erol
kina
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yaB,
hydr
ogen
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rge
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ydro
gena
se-2
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ech
ain;
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,for
mat
ehy
drog
enly
ase
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lato
rypr
otei
n;hy
cE,f
orm
ate
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ogen
lyas
esu
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t5;
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com
pone
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;ldh,
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ate
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drog
enas
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,la
ctat
ede
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ogen
ase
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D,L-
lact
ate
dehy
drog
enas
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ethy
lgly
oxal
synt
hase
;na
dK,
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Dki
nase
;na
rG,
resp
irat
ory
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ate
redu
ctas
e1
alph
ach
ain;
nox,
NA
DH
oxid
ase;
pck,
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carb
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inas
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rmat
eac
etyl
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sfer
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CD,p
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ate
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ate
lyas
esu
buni
tsC
and
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heA
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rote
in;p
ntAB,
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ein
PntA
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xB,p
yruv
ate
dehy
-dr
ogen
ase;
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carb
oxyl
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phos
phot
rans
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ylas
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syst
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ucos
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ecifi
cEI
ICB
com
pone
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EPph
osph
otra
nsfe
rase
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,pyr
uvat
eca
rbox
ylas
e;py
kA,p
yruv
ate
kina
seII;
pykF
,pyr
uvat
eki
nase
I;sdhC
DAB,
succ
inat
ede
hydr
ogen
ase
com
plex
;suc
AB,
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a-ke
togl
utar
ate
dehy
drog
enas
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rote
in;y
cgR,
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llar
brak
epr
otei
n;yfgI
,unc
hara
cter
ized
prot
ein;
yqhD
,NA
D(P
)H-d
epen
dent
1,3-
prop
aned
iold
ehy-
drog
enas
e.A
bbre
viat
ions
:1,2
-PD
O,1
,2-p
ropa
nedi
ol;a
cyl-A
CP,a
cylc
arri
erpr
otei
n;A
TP,a
deno
sine
trip
hosp
hate
;CO
2,ca
rbon
diox
ide;
DH
A,d
ihyd
roxy
acet
one;
DH
AK,
dihy
drox
yace
tone
kina
se,F
FA,f
ree
fatt
yac
ids;
H2,
hydr
ogen
gas;
MG
,met
hylg
lyox
al;N
AD
+,n
icot
inam
ide
aden
ine
dinu
cleo
tide;
PEP,
phos
phoe
nolp
yruv
ate.
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
556
production relative to the strain expressing only dhaB123 and yqhD,(Hong et al., 2015), indicating that glycerol- and/or oxygen-mediatedinactivation of DhaB was a critical issue in these E. coli strains. Ad-ditionally, supplementation of the TCA cycle intermediates succinate,fumarate, and malate significantly reduced the NAD+/NADH ratio andfurther improved 1,3-PDO production (Hong et al., 2015). Subse-quently, gapN, encoding NADP+-dependent glyceraldehyde-3-phos-phate dehydrogenase (GAPDN) from C. acetobutylicum, was coexpressedwith native yqhD, and dhaB123 and gdrAB from K. pneumoniae, to re-generate NADPH, resulting in significantly improved 1,3-PDO produc-tion relative to the strain coexpressing yqhD, dhaB123, and gdrAB (Yanget al., 2018). Fine-tuning the expression of gapN via modification of its5’-UTR led to significant improvements to culture performance, whichwas further enhanced by mutating ptsG, encoding the phospho-transferase system (PTS) glucose-specific EIICB protein, and over-expressing galP (encoding galactose permease; GalP) and glk (encodingglucokinase; GLK) to relieve carbon catabolite repression when glyceroland glucose were provided as co-substrates (Yang et al., 2018). Relativeto the expression of genes from the reductive branch of glycerol dis-similation from K. pneumoniae, coexpression of native yqhD and dhaBCEfrom C. freundii resulted in significantly improved 1,3-PDO production
(Zhang et al., 2006a, 2006b). On the other hand, a considerable amountof acetate was generated by engineered E. coli expressing dhaBCE anddhaFG from C. freundii and dhaT from K. pneumoniae, leading to cellgrowth inhibition and decreased 1,3-PDO production (Przystałowskaet al., 2015). Finally, coexpression of native yqhD with dhaB1 anddhaB2 from C. butyricum resulted in high-level 1,3-PDO production withthe titer reaching 104 g/L in an anaerobic fedbatch fermentation, al-though acetate accumulation eventually limited culture performance(Tang et al., 2009). This strain engineering approach is particularlyattractive, as the expression of DhaB1 from C. butyricum does not re-quire B12.
Although large-scale 1,3-PDO production using engineered E. colihas been demonstrated (Celińska, 2010), strategies to eliminate bio-processing limitations and further enhance culture performance requireto be developed. For example, complete growth inhibition of E. colioccurred for 1,3-PDO concentrations exceeding 120 g/L (Cameronet al., 1998), such that improving the 1,3-PDO tolerance of E. colithrough directed evolution or rational strain engineering may be aneffective strategy to improve 1,3-PDO production. Cell membrane en-gineering was applied to improve rates of biocatalysis in E. coli (Chen,2007), protein secretion (Cao et al., 2017) and biopolymer production
Table 4Summary of literature for E. coli strains engineered to produce 1,3-PDO and 3-HP from glycerol.
Parent strain Product Genetic strategy Titer (g/L)
Cultivation mode Reference
AG1 1,3-PDO Express dha regulon (K. pneumoniae) N/A Flask (Tong et al., 1991)ELC707 1,3-PDO Express dha regulon (C. freundii) 5 Flask (Daniel and Gottschalk, 1992)AG1 1,3-PDO Coexpress dhaB and dhaT (K. pneumoniae) from trc promoter 6 Fedbatch (Skraly et al., 1998)AG1 1,3-PDO -Express dha regulon (K. pneumoniae)
-Express glo1 (P. putida) to reduce MG formation2 Flask (Zhu et al., 2001)
AG1 1,3-PDO -Express dha regulon (K. pneumoniae)-Mutate glpK and feed fumarate to reduce G3P formation
4 Flask (Zhu et al., 2002)
JM109 1,3-PDO -Coexpress dhaB and gdrAB (K. pneumoniae), and yqhD-Feed succinate to decrease NAD+/NADH ratio
13 Fedbatch (Hong et al., 2015)
JM109 1,3-PDO Coexpress dhaBCE (C. freundii) and yqhD 41 Fedbatch (Zhang et al., 2006a, 2006b)BL21 1,3-PDO Coexpress dhaBCEFG (C. freundii) and dhaT (K. pneumoniae) 11 Fedbatch (Przystałowska et al., 2015)K-12 ER2925 1,3-PDO Coexpress dhaB12 (C. butyricum) and yqhD 104 Fedbatch (Tang et al., 2009)JM109 1,3-PDO -Coexpress yqhD, and dhaB and gdrAB (K. pneumoniae)
-Coexpress gapN (C. acetobutylicum) to generate NADPH, and fine-tuneexpression via 5’-UTR engineering-mutate ptsG and overexpress galP and glk to relieve catabolite repressionduring glycerol/glucose co-feeding
14 Flask (Yang et al., 2018)
BL21 3-HP Coexpress dhaB123 (K. pneumoniae) and aldH 0.6 Fedbatch (Raj et al., 2008)BL21 3-HP Coexpress dhaB123 (K. pneumoniae) and aldH from low-copy and high-copy
plasmids, respectively31 (Raj et al., 2009)
BL21 3-HP Coexpress dhaB and dhaR (L. brevis) and aldH 14 Flask (Kwak et al., 2013)W3110 3-HP -Coexpress dhaB123 and gdrAB from K. pneumoniae, and aldH
-Overexpress glpF to increase glycerol uptake-Mutate ackA-pta, yqhD, and glpR to reduce acetate, 3-HP, and G3P formation,respectively
42 Fedbatch (Jung et al., 2014)
BL21 3-HP -Coexpress dhaB and dhaR (L. brevis), and PSALDH (P. aeruginosa)-Mutate glpK and yqhD to reduce G3P and 3-HP formation, respectively
57 Fedbatch (Kim et al., 2014)
W 3-HP Coexpress dhaB and gdrAB (K. pneumoniae), and KGSADH (A. brasilense) 42 Fedbatch (Sankaranarayanan et al., 2014)W3110 3-HP -Coexpress dhaB and gdrAB (K. pneumoniae), and engineered gabD4 (C. necator)
with point mutations E209Q and E269Q-Mutate pta-ackA and yqhD to reduce acetate and 3-HP formation, respectively
72 Fedbatch (Chu et al., 2015)
BW25113 3-HP -Implement metabolic toggle switch to conditionally repress the expression ofgapA in ΔgapA-Coexpress dhaB123 and gdrAB (K. pneumoniae), and araE (A. brasilense)-Mutate yqhD and to reduce 1,3-PDO formation
6 Fedbatch (Tsuruno et al., 2015)
B (ATCC11303) 3-HP Coexpress dhaB123 and gdrAB (K. pneumoniae), KGSADH (A. brasilense), andpduPLW (K. pneumoniae)
5 Fedbatch (Honjo et al., 2015)
Gene products: ackA, acetate kinase; aldH, aldehyde dehydrogenase; araE, α-ketoglutaric semialdehyde dehydrogenase; dhaB123, glycerol dehydratase; dhaBCDEFG,glycerol dehydratase and reactivation factors; dhaBT, glycerol dehydratase and 1,3-propanediol oxidoreductase; dhaR, dihydroxyacetone reductase; dhaT, 1,3-propanediol oxidoreductase; gabD4, aldehyde dehydrogenase; gapA, glyceraldehyde 3-phosphate dehydrogenase; gdrAB, glycerol dehydratase reactivase; glo1,glyoxylase 1; glpF, glycerol uptake facilitator protein; glpK, glycerol kinase; glpR, glycerol-3-phosphate regulon repressor; KGSADH, alpha-ketoglutarate semialdehydedehydrogenase; pduPLW, aldehyde dehydrogenase, phosphate propanoyltransferase, and propionate kinase; PSALDH, semialdehyde dehydrogenase complex; pta,phosphotransacetylase; yqhD, NAD(P)H-dependent 1,3-propanediol dehydrogenase. Abbreviations: 3-HP, 3-hydroxypropionate; G3P, glycerol-3-phosphate; MG,methylglyoxal; NAD+, nicotinamide adenine dinucleotide.
A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568
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(Westbrook et al., 2018) in B. subtilis, and solvent and acid tolerance inC. acetobutylicum (Zhao et al., 2003) and E. coli (Tan et al., 2017), and,hence, may be an effective strategy to enhance the 1,3-PDO tolerance ofE. coli. To restore the potential cofactor imbalance arising from theconversion of 3-HPA to 1,3-PDO by YqhD, native zwf, encodingNADP+-dependent glucose 6-phosphate-1-dehydrogenase (Zwf), can beoverexpressed to regenerate NADPH with low-level glucose supple-mentation. Moreover, the accumulation of acetate to inhibitory levelsobserved during high-level 1,3-PDO production (Tang et al., 2009) maybe relieved by mutation of ptsH, encoding the histidine protein of thesugar PTS that participates in the phosphorylation of DHA by DhaKLM,which significantly reduced acetate formation during anaerobic succi-nate production from glycerol (Zhang et al., 2010).
3.5.2.2. 3-HP. Strain engineering strategies to enhance 3-HPproduction in E. coli have primarily focused on balancing theactivities of GDHt and ALDH in the synthetic 3-HP pathway (Fig. 1),and limiting side metabolite formation. Low-level 3-HP production wasfirst demonstrated in E. coli via coexpression of DhaB from K.pneumoniae with one of four ALDHs, of which AldH4 from S.cerevisiae, encoded by aldH4, was most effective for converting 3-HPAto 3-HP (Suthers and Cameron, 2005). Excessive 3-HPA accumulationwas observed to hinder 3-HP production in a strain coexpressingdhaB123 from K. pneumoniae and native aldH (encoding AldH) fromindividual low-copy plasmids, due to an imbalance between theactivities of DhaB and AldH (Raj et al., 2008). Subsequent workrevealed that the activity of GdrAB was critical to prevent theinactivation of DhaB, and the coexpression of native aldH, anddhaB123 and gdrAB from K. pneumoniae improved the 3-HP titer by6-fold (Rathnasingh et al., 2009) relative to the corresponding strainwithout gdrAB coexpression (Raj et al., 2008). Moreover, the expressionof KGSADH from A. brasilense, in place of native aldH, further increasedthe 3-HP titer by 2.5-fold (Rathnasingh et al., 2009). Fine-tuning theexpression of dhaB123 from K. pneumonia by engineering its 5’- UTRalso served to manage 3-HPA accumulation (Lim et al., 2016). KGSADHfrom A. brasilense was expressed from a relatively strong promoter in ahighly 3-HPA-tolerant E. coli strain that also expressed dhaB123 withengineered 5’-UTRs from a separate promoter, resulting in a substantialincrease in the 3-HP yield (Lim et al., 2016). Lastly, parallel expressionof the partial PDU pathway (i.e. pduPLW) from K. pneumoniae and araE,encoding α-ketoglutaric semialdehyde dehydrogenase from A.brasilense (AraE), in a strain also expressing dhaB123 and gdrAB fromK. pneumoniae resulted in a significantly increased 3-HP titer, comparedto a strain that did not express pduPLW, suggesting that conversion of 3-HPA to 1,3-PDO may have decreased due to increased 3-HPA fluxthrough the partial PDU pathway (Honjo et al., 2015).
Several strategies were explored to minimize the formation of majorside metabolites, including acetate, lactate, succinate, and 1,3-PDO, forenhanced 3-HP production in engineered E. coli. Inactivating ackA-ptaand yqhD in a strain coexpressing dhaB123 and gdrAB from K. pneu-moniae and native aldH, significantly decreased acetate and 1,3-PDOproduction (Jung et al., 2014). 3-HP production increased by 2-fold inthe ΔackA Δpta ΔyqhD triple mutant compared to the parent straincoexpressing dhaB123, gdrAB, and aldH, while mutation of glpR, en-coding the repressor of the glp regulon (GlpR), and overexpression ofglpF modestly enhanced 3-HP production (Jung et al., 2014). On theother hand, inactivating respiratory glycerol dissimilation via mutationof glpK significantly improved 3-HP production in a strain coexpressingdhaB and dhaR, encoding the reactivation factor of DhaB (DhaR), fromL. brevis, and native aldH (Kim et al., 2014; Kwak et al., 2013). More-over, mutation of yqhD significantly improved 3-HP production in theΔglpK mutant, and the expression of PSALDH, encoding semialdehydedehydrogenase from Pseudomonas aeruginosa, in place of aldH, furtherenhanced culture performance (Kim et al., 2014). In silico analysis ofgene mutations based on a genome-scale metabolic model identifiedtpiA, encoding triosephosphate isomerase (TpiA), and zwf as targets for
inactivation to enhance 3-HP production from glycerol (Tokuyamaet al., 2014). Mutation of tpiA in a strain coexpressing dhaB123 andgdrAB from K. pneumoniae and native aldH increased the 3-HP titer by2-fold, while mutation of zwf had only a minor effect on culture per-formance. Moreover, mutation of yqhD significantly reduced 1,3-PDOformation and, in turn, 3-HP production was markedly increased,compared to the ΔtpiA Δzwf double mutant coexpressing dhaB123,gdrAB, and aldH (Tokuyama et al., 2014). Similarly, mutation of ackA-pta and yqhD significantly reduced acetate and 1,3-PDO formation, witha concomitant increase in 3-HP production in a strain coexpressingdhaB123 and gdrAB from K. pneumoniae and native aldH (Chu et al.,2015). Moreover, 3-HP production was further enhanced by expressinggabD4, encoding ALDH from C. necator (GabD4), in place of native aldH,while expression of engineered GabD4, with point mutations E209Qand E269Q, significantly improved 3-HP production during a fedbatchco-cultivation using glycerol and glucose (Chu et al., 2015). Finally,repressing the expression of genes in central metabolic pathways canredirect carbon flux toward target metabolite production withoutcompromising host cell viability. A previously engineered metabolictoggle switch (Gardner et al., 2000) was used to conditionally repressthe expression of gapA, encoding glyceraldehyde 3-phosphate dehy-drogenase (GapA), in a ΔgapA mutant coexpressing dhaB123 and gdrABfrom K. pneumoniae and araE from A. brasilense to redirect carbon fluxfrom glycolysis to the synthetic 3-HP pathway, while minimizinggrowth inhibition observed in ΔgapA mutants (Tsuruno et al., 2015).The conditional repression of gapA expression resulted in a significantincrease in 3-HP production compared to the strain coexpressingdhaB123, gdrAB, and araE, whereas mutation of yqhD dramatically re-duced 1,3-PDO formation and, in turn, improved the 3-HP titer andyield (Tsuruno et al., 2015).
Other technical issues to be addressed for enhanced 3-HP produc-tion from glycerol in E. coli include recycling of NADH and cir-cumventing the requirement of B12-dependent GDHt. Excess NADH mayaccumulate during 3-HP production, potentially resulting in the for-mation of reduced side metabolites. While NADH can be oxidized byNOX using molecular oxygen without diverting carbon from 3-HPproduction, B12-independent DhaB1 from C. butyricum may not functionin the presence of oxygen (Andreeßen et al., 2010; Tang et al., 2009),such that a NAD+ regeneration system functional under anaerobicconditions would be required in this context. NADH:quinone oxidor-eductase II, encoded by ndh in E. coli, oxidizes NADH during nitraterespiration (Tran et al., 1997), such that coexpression of native ndh, anddhaB1 and dhaB2 from C. butyricum (in addition to ALDH) may facil-itate anaerobic production of 3-HP without B12 if exogenous nitrate isprovided. Alternatively, protein engineering of DhaB1 to reduce itssensitivity to oxygen may enable B12-independent 3-HP productionunder microaerobic conditions. A similar strategy was employed foraerobic 1-propanol production through the L-threonine degradationpathway, whereby engineered aero-tolerant AdhE successively con-verted propionyl-CoA to propionaldehyde and 1-propanol (Choi et al.,2012). In the absence of B12 dependency, E. coli ΔyqhD mutants aresuperior hosts for 3-HP production relative to other microbial hostsdiscussed herein due to the minimal formation of 1,3-PDO.
3.5.2.3. Bio(co)polymers. PHAs are a family of biologically producedpolyesters composed of various (R)-hydroxycarboxylates as themonomers (Mozzi et al., 2006; Teeka et al., 2012) and have emergedas substitutes for conventional plastics due to their thermoplastic andelastomer properties, biodegradability, non-toxicity, and structuraldiversity (Leong et al., 2014; Reddy et al., 2003; Teeka et al., 2012).Unlike most PHAs, P(3HP) is not naturally produced by bacteria(Linares-Pastén et al., 2015), leading to the exploration of syntheticpathways in engineered hosts such as E. coli. Marginal P(3HP)production was achieved in E. coli coexpressing dhaB1 from C.butyricum, pduP from Salmonella enterica, and phaC from C. necator,from either pure or crude glycerol (Andreeßen et al., 2010). On the
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other hand, coexpressing dhaB123 and gdrAB from K. pneumoniae withpduP from S. typhimurium and phaC from C. necator resulted in a highspecific P(3HP) yield using glucose and glycerol as co-substrates in afedbatch cultivation (Wang et al., 2013a, 2013b). In spite of the highspecific P(3HP) yield, plasmid instability was an issue that wasaddressed using a plasmid addiction system for the coexpression ofpduP from S. typhimurium and phaC from C. necator, and chromosomalexpression of dhaB123 and gdrAB from K. pneumoniae, resulting in ahigh specific P(3HP) yield of 68% of dry cell weight (dcw) in a fedbatchcultivation (Gao et al., 2014). To circumvent the requirement of B12-dependent GDHt, P(3HP) was produced via the β-alanine pathway in E.coli (Wang et al., 2014a, 2014b, 2014c). In this engineered redox-neutral pathway, native L-aspartate decarboxylase (i.e. PanD encodedby panD) generated β-alanine that was converted to P(3HP) throughsubsequent actions of a β-alanine-pyruvate transaminase from P. putida(i.e. GabT encoded by gabT), native 3-hydroxyacid dehydrogenase (i.e.YdfG encoded by ydfG) and PrpE, and PhaC from C. necator. Althoughexogenous B12 was not required, P(3HP) production was low (Wanget al., 2014a, 2014b, 2014c), with only a modest improvement obtainedby expressing panD from Corynebacterium glutamicum in place of nativepanD (Lacmata et al., 2017). Lastly, engineered E. coli was used in atwo-stage cultivation with resting cells of L. reuteri for P(3HP)production from glycerol (Linares-Pastén et al., 2015). Due to a high3-HPA tolerance, L. reuteri was used in the first stage to convert glycerolto 3-HPA, which was, in turn, used as a substrate by E. coli coexpressingpduP from L. reuteri (Sabet-Azad et al., 2013) and phaC fromChromobacterium sp. (Bhubalan et al., 2011), leading to a highspecific P(3HP) yield (Linares-Pastén et al., 2015). Although notdirectly related to engineering of glycerol metabolism, other studieshave shown the potential of using pure or crude glycerol for theproduction of poly(3-hydroxybutyrate) (de Almeida et al., 2007;Ganesh et al., 2015; Shah et al., 2014; Shalel Levanon et al., 2005).
Due to high crystallinity, homopolymer PHAs are too brittle and stifffor many industrial applications (Leong et al., 2014), and such technicaldrawbacks can be overcome by the use of copolymers (Singh andMohanty, 2007). P(3HB-co-3HV) was produced in engineered E. coliwith a high specific yield of 80% of dcw via coexpression of bktB, en-coding β-ketothiolase (BktB), phaB, encoding acetoacetyl-CoA re-ductase (PhaB), and phaC, from R. eutropha, in addition to native sucCD,using glycerol as the primary carbon source with supplemental succi-nate and propionate (Bhatia et al., 2015). Previous studies have re-ported high C3 metabolite production from glycerol due to moreNADPH and ATP generation relative to glucose, favoring threoninebiosynthesis and directing carbon flux toward propionyl-CoA, a pre-cursor to (R)-3-hydroxyvaleryl-CoA [(R)-3-HV-CoA] (Tseng et al.,2010). High-level propionyl-CoA and, in turn, 1-propanol productionwas achieved from glycerol without supplemental succinate and pro-pionate by activating the native genomic sleeping beauty mutase (Sbm)operon (Srirangan et al., 2013; Srirangan et al., 2014). This approachwas extended to P(3HB-co-3HV) production by mutating dhaK andcoexpressing bktB and phaCAB from C. necator in the propanogenic E.coli strain, resulting in a high specific P(3HB-co-3HV) yield of 66% ofdcw (Srirangan et al., 2016b). Moreover, inactivating respiratory gly-cerol dissimilation via mutation of glpD in the strain coexpressing bktBand phaCAB increased the 3-hydroxyvalerate (3-HV) content by 2.6-fold to 18.5 mol%, which is the highest reported level for E. coli-based P(3HB-co-3HV) biosynthesis using an unrelated carbon source (Sriranganet al., 2016b). Lastly, poly(3-hydroxyburyrate-co-3-hydroxyhexanoate)[P(3HB-co-3HH)] was synthesized in E. coli coexpressing phaAB from C.necator, and phaJ (encoding (R)-specific enoyl-CoA hydratase; PhaJ)and phaC from Aeromonas hydrophila, although P(3HB-co-3HH) pro-duction was low on pure glycerol (Phithakrotchanakoon et al., 2013)and was negligible when crude glycerol was used(Phithakrotchanakoon et al., 2015).
As is the case for 3-HP production, the regeneration of reducingequivalents and elimination of the B12-reliance for functional
expression of GDHt are technical challenges to overcome for enhancedbiopolymer production in E. coli. Excess NADH can accumulate during P(3HP) production due to the conversion of 3-HPA to 3-HP-CoA viaPduP, potentially hindering culture performance (Andreeßen et al.,2010), while NADPH is consumed through the conversion of β-keto-valeryl-CoA to 3-HV-CoA by PhaB during P(3HB-co-3HV) production.Accordingly, the same strategies proposed to recycle reducing equiva-lents during B12-independent 3-HP and 1,3-PDO production may beeffective for P(3HP) and P(3HB-co-3HV) production, respectively.Protein engineering is also a potential strategy to alter bio(co)polymercomposition. Expression of engineered PhaJ from A. hydrophila con-taining a V130A point mutation substantially increased the 3-hydro-xyhexanoate (3-HH) fraction in P(3HB-co-3HH) produced in E. coli,without compromising the specific P(3HB-co-3HH) yield (Hu et al.,2007). Moreover, bio(co)polymer content can be modified by manip-ulating glycerol dissimilation through either the fermentative or re-spiratory pathways as previously noted (Srirangan et al., 2016b).
3.5.2.4. Other products. Production of 1-propanol from glycerol in E.coli was demonstrated using two distinct pathways. In the firstapproach, the natural 1,2-PDO pathway of E. coli was modified usingan ATP-dependent DHAK, encoded by dhaK from S. blattae, andextended using components of the pdu operon from K. pneumoniae(Matsubara et al., 2016). 1,2-PDO was converted to propionaldehydevia PduCDE with the reactivation factor PduGH, and then to 1-propanolvia PduQ. In contrast to native PEP-dependent DhaKLM, ATP-dependent DHAK decreased reliance on pyruvate production and, inturn, increased flux into the 1-propanol pathway (Matsubara et al.,2016). The second strategy to achieve 1-propanol production fromglycerol involved the activation of the dormant Sbm operon of E. coli(Srirangan et al., 2013; Srirangan et al., 2014). This pathway beginswith the conversion of succinyl-CoA to L-methylmalonyl-CoA via amethylmalonyl-CoA mutase (Sbm encoded by sbm), followed by theformation of propionyl-CoA from succinyl-CoA by a methylmalonyl-CoA decarboxylase (YqfG encoded by yqfG), with a propionyl-CoA::succinate transferase (YgfH encoded by ygfH) facilitating theinterconversion between succinyl-CoA and propionyl-CoA. Theactivation of the Sbm operon introduced an intracellular carbon fluxcompetition between the C2-fermentative pathway (with ethanol andacetate as the major metabolites) and C3-fermentative pathway (with 1-propanol and propionate as the major metabolites), and glycerol wasidentified as a suitable carbon source favoring direction of thedissimilated carbon flux toward the C3-fermentative pathway(Srirangan et al., 2014). The propionyl-CoA derived from the Sbmoperon and acetyl-CoA were fused by either PhaA or BktB from C.necator to form 3-ketovaleryl-CoA, which was further converted tobutanone via a ketone-formation pathway containing acetoacetyl-CoA:acetate/butyrate:CoA transferase (i.e. CtfAB encoded by ctfAB)and acetoacetate decarboxylase (i.e. Adc encoded by adc) from C.acetobutylicum (Akawi et al., 2015; Srirangan et al., 2016a).Manipulating various genes involved in the glycerol dissimilationpathway potentially enhanced the production of propionate (Akawiet al., 2015) and butanone (Srirangan et al., 2016a) from glycerol undervarious fermentation conditions.
Small amounts (up to 154 mg/L) of 1-butanol were produced fromglycerol by reconstructing the 1-butanol pathway from C. acet-obutylicum in E. coli (Zhou et al., 2014). To use glycerol as a carbonsource, expression of native fdh1, encoding FDH, and inactivation ofadhE, ldhA, and frdBC associated with side metabolite production aimedto overcome the NADH deficit resulting from 1-butanol synthesis (Zhouet al., 2014). Significantly higher 1-butanol titers of up to 6.9 g/L wereobtained from crude glycerol in a ΔadhE ΔldhA ΔfrdA Δpta quadruple E.coli mutant expressing the 1-butanol pathway from C. acetobutylicum,with phaA from C. necator and ter from T. denticola expressed in place ofthil and bcd-etfAB, respectively, by engineering parallel NADH re-generation systems (Saini et al., 2017). Coexpression of the engineered
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native NADH-insensitive PDH complex (encoded by aceEF-lpdA⁎), andnative pgl (encoding 6-phosphogluconolactonase; PGL) and zwf re-spectively stimulated the decarboxylation of pyruvate to acetyl-CoA,and increased flux through the PPP to regenerate NADPH, which wasconverted to NADH via overexpressed native UDH (encoded by udhA),resulting in a significant increase in the 1-butanol titer. Similarly, co-expression of native gldA and dhaKLM presumably increased NADHlevels and, in turn, 1-butanol production, which could be further en-hanced by restricting carbon flux into the TCA cycle via promoter en-gineering to reduce the expression of gltA to conserve acetyl-CoA (Sainiet al., 2017).
E. coli was also engineered to produce 2,3-BDO isomers from gly-cerol. For the production of (R,R)-2,3-BDO, alsS, encoding α-acet-olactate synthase (AlsS) that converts pyruvate to (S)-2-acetolactate,and alsD, encoding α-acetolactate decarboxylase (AlsD) that converts(S)-2-acetolactate to (R)-acetoin, from B. subtilis were coexpressed withadh, encoding secondary alcohol dehydrogenase that converts (R)-acetoin to (R,R)-2,3-BDO from C. beijerinckii, resulting in a (R,R)-2,3-BDO titer of 9.5 g/L (Shen et al., 2012). Similarly, coexpression of budA,encoding α-acetolactate decarboxylase (BudA) that converts (S)-2-acetolactate to (R)-acetoin, and budC, encoding 2,3-BDO dehy-drogenase/acetoin reductase that converts (R)-acetoin to meso-2,3-BDO, from K. pneumoniae resulted in a meso-2,3-BDO titer of 6.9 g/Lfrom crude glycerol (Lee et al., 2012).
Production of up to 6.2 g/L 2-hydroxyisovalerate (an industriallyimportant surfactant, emulsifier, and intermediate for the synthesis ofpolymers, solvents, and fertilizers) from glycerol was achieved in E. coliby exploiting the native L-valine biosynthetic pathway (Cheong et al.,2018). First, two pyruvate molecules were condensed to generate 2-acetolactate by AlsS from B. subtilis, followed by its sequential con-version to 2,3-dihydroxyisovalerate, 2-ketoisovalerate, and finally 2-hydroxyisovalerate by native acetohydroxy-acid isomeroreductase (i.e.,IlvC encoded by ilvC), native IlvD, and 2-hydroxyacid dehydrogenase(i.e., PanE endoded by panE) from L. lactis, respectively. Inactivation ofcompeting mixed-acid pathways resulted in a significant decrease in 2-hydroxyisovalerate production, highlighting the importance of thesepathways for maintaining the overall intracellular redox balance(Cheong et al., 2018).
β-carotene production from glycerol was achieved in E. coli byconstructing an alternative glycerol utilization pathway in a previouslydeveloped strain (Ye et al., 2016) (Guo et al., 2018). Glycerol was se-quentially converted to D-glyceraldehyde and D-glycerate via aldehydereductase (i.e., Alrd encoded by alrd) and ALDH (i.e., AldH encoded byaldH) from C. necator, respectively, and 2-phospho-D-glycerate wasderived from D-glycerate by a native glycerol kinase (i.e., GarK encodedby garK). The synthetic glycerol utilization pathway simultaneouslyincreased the rate of glycerol consumption and the supply of earlyisoprenoid precursors, i.e., G3P and pyruvate, enabling the productionof 75 mg/L of β-carotene (Guo et al., 2018).
Production of 5-hydroxytryptophan (5-HTP), a precursor of ser-otonin used to treat depression, insomnia, and headaches, from glycerolwas recently demonstrated in E. coli (Wang et al., 2018). As a precursorof 5-HTP biosynthesis, tetrahydrobiopterin (BH4) was produced in athree-step pathway via GTP cyclohydrolase (GCHI), 6-pyruvate-tetra-hydropterin synthase (PTPS), and sepiapterin reductase (SPR), encodedby mtrA from B. subtilis, human PTPS, and human SPR, respectively. 5-HTP was synthesized from BH4 and L-tryptophan by tryptophan hy-droxylase I or II, i.e., TPH1/2 encoded by human TPH1/2, and BH4 wasregenerated via a two-step pathway comprised of pterin-4α-carbinola-mine dehydratase (PCD) and dihydropteridine reductase (DHPR), en-coded by human PCD and DHPR, respectively (Wang et al., 2018). Toavoid the requirement for exogenous L-tryptophan supplementation,the native L-tryptophan biosynthetic pathway, including engineeredfeedback resistant trpE⁎ (encoding tryptophan-insensitive anthranilatesynthase subunit TrpE⁎) and aroH⁎ (encoding tryptophan-insensitive 3-deoxy-7-phosphoheptulonate synthase AroH⁎), were coexpressed in the
5-HTP-producing strain. 5-HTP production was further enhanced byexpressing engineered TPH2, i.e. TPH145 containing deletions of 145N-terminal and 24 C-terminal amino acids, and by reducing the copynumber of the plasmid containing L-tryptophan biosynthesis genes andselecting different promoters to drive the synthesis and regeneration ofBH4. These strain engineering strategies resulted in the highest 5-HTPtiter ever reported for bio-based production (5.1 g/L) (Wang et al.,2018).
L-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of dopamineused in the treatment of Parkinson’s disease and dopamine-responsivedystonia, was synthesized from glycerol with an E. coli strain expressinga modified Shikimate pathway (Das et al., 2018). L-DOPA is derivedfrom L-tyrosine, which is the major effector of TyrR (L-tyrosine re-pressor encoded by tyrR)-based repression of aromatic amino acidbiosynthesis (Keseler et al., 2016), such that tyrR was mutated to in-crease L-tyrosine levels. To further boost L-tyrosine overproduction,feedback- resistant native 3-deoxy-D-arabino-heptulosonate synthase(i.e., DAHP⁎ encoded by aroG⁎), shikimate kinase (i.e., AroL encoded byaroL), chorismate synthase (i.e., AroC encoded by aroC), 3-phos-phoshikimate 1-carboxyvinyltransferase (i.e., AroA encoded by aroA),and L-tyrosine aminotransferase (i.e., TyrB encoded by tyrB) were co-expressed in a tyrR mutant also containing mutations to pykF (encodingpyruvate kinase I; PykF) and serA (encoding phosphoglycerate dehy-drogenase; SerA) to block competing amino acid biosynthetic path-ways. The direct conversion of L-tyrosine to L-DOPA was achieved bycoexpressing native 4-hydroxyphenylacetate-3-hydrolase (i.e., HpaBCencoded by hpaBC) in the L-tyrosine overproducing mutant, resulting inthe production of up to 12.5 g/L L-DOPA, with a marked reduction inacetate accumulation compared to previous studies in which L-DOPAwas derived from glucose (Das et al., 2018).
Itaconate is a versatile biological used in the production of resins,plastics, rubbers, paints, surfactants, and lubricants (Dwiarti et al.,2007). Itaconate production from glycerol was demonstrated in E. coliexpressing engineered cis-aconitate decarboxylase from Aspergillus ter-reus (CadA encoded by cadA), which converts cis-aconitate (a TCA cycleintermediate) to itaconate (Jeon et al., 2016). A library of synonymouscodon variants was constructed by introducing random point mutationsin the first 10 codons of CadA, and inclusion body formation was nearlyabolished when certain engineered CadA variants were expressed, re-sulting in up to 4-fold increases in the itaconate titer, which reached 7.2g/L in a fedbatch cultivation (Jeon et al., 2016). Moreover, inactivationof icd and coexpression of codon optimized cadA from A. terreus, pyc(encoding pyruvate carboxylase; Pyc) from C. glutamicum, and nativeacnB (encoding aconitase; AcnB) and gltA resulted in a high itaconatetiter of 43 g/L in a fedbatch cultivation (Chang et al., 2017).
Deoxyviolacein, a drug with antagonistic activity against tumors,Gram-positive bacteria, and fungal plant pathogens (Rodrigues et al.,2014), was first produced from glucose in engineered E. coli expressinga truncated deoxyviolacein gene cluster, i.e. vioABCE with vioD omitted,from C. violaceum (Rodrigues et al., 2013). The deoxyviolacein-produ-cing strain was further modified via inactivation of araBAD, eliminatingL-arabinose metabolism, and, among various carbon sources, glycerolwas determined to be most effective for deoxyviolacein production upto 1.6 g/L (Rodrigues et al., 2014).
Valerenadiene is an anxiolytic compound derived from the medic-inal herb Valeriana officinalis, and has been produced in small quantitiesin engineered E. coli (Nybo et al., 2017). Coexpression of codon opti-mized vds, encoding valerenadiene synthase from V. officinalis (VDS),and genes comprising the mevalonic acid pathway from S. cerevisiaeresulted in a valerenadiene titer of 62 mg/L from glycerol (Nybo et al.,2017).
Small amounts (up to 38 mg/L) of toxic acrylic acid was synthesizedfrom glycerol using a previously constructed P(3HP)-producing mutant,in which 3-HP-CoA was derived via coexpression of dhaB123 and gdrABfrom K. pneumoniae and pduP from S. typhimurium (Tong et al., 2016).Acrylyl-CoA was derived from 3-HP-CoA via an enoyl-CoA hydratase
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from the propionyl-CoA synthase complex of Chloroflexus aurantiacusand the CoA moiety was subsequently removed by a putative en-dogenous CoA transferase (Tong et al., 2016).
Serinol production (up to 3.3 g/L) from glycerol was achieved viaexpression of a bifunctional DHAP aminotransferase/dihy-drorhizobitoxine synthase from Bradyrhizobium elkanii, which mediatesDHAP conversion to serinol phosphate and subsequent depho-sphorylation (Andreeßen and Steinbüchel, 2012).
4. 4.0 Future perspectives
4.1. Selecting the appropriate production host for glycerol biorefinery
Factors associated with the selection of a proper microbial platformfor target metabolite production from glycerol include growth re-quirements, product tolerance, pathogenicity, and genetic tractability.While K. pneumoniae is genetically amenable and has a high capacity forthe conversion of glycerol into diols and organic acids with minimalnutritional requirements, the pathogenicity limits its application tobiomanufacturing, particularly for human consumption products. Onthe other hand, E. coli possesses all of the same positive attributes, witheven greater ease of genetic manipulation and a proven track record inbiomanufacturing, and has been engineered to dissimilate glycerol intomany non-native metabolites with similar efficiency to natural produ-cers. For example, using E. coli, 1,3-PDO is produced commercially fromglucose (Celińska, 2010), and B12-independent production of 1,3-PDOfrom glycerol has been achieved on a similar scale (Tang et al., 2009).Moreover, side metabolite formation appears less problematic in E. coli,potentially due to its relatively stable TCA cycle, and the insignificant2,3-BDO production during glycerol fermentation can simplify pur-ification of 1,3-PDO. E. coli is also an ideal candidate for 3-HP pro-duction due to its high production capacity and lack of redundant 1,3-PDOORs, although the engineered strains often express B12-dependentGDHt which is a technical challenge difficult to overcome.
Certain Clostridium sp. are particularly well suited to the productionof specific metabolites from glycerol, e.g. C. pasteurianum and C. bu-tyricum as 1-butanol and 1,3-PDO producers, respectively. However,they are obligate anaerobes that are recalcitrant to genetic manipula-tion and, thus, may be restricted to the production of native productswith limited strain improvement. While Citrobacter sp. have not beenwell characterized and, therefore, lack advanced genetic tools, certainstrains are attractive for glycerol biorefinery. In particular, C. werkmaniireadily utilizes co-substrates with a preference for glycerol, such thatblocking oxidative pathways for glycerol dissimilation to channel thecarbon flux into the reductive branch as well as engineering of cofactorregeneration systems that exploit glucose metabolism may be effectivestrategies in this host for target metabolite formation. Moreover, ex-ceptionally high 1,3-PDO yields from co-substrates have been reportedin cultures of C. werkmanii relative to common 1,3-PDO producer suchas Klebsiella sp. (Maervoet et al., 2012a, 2012b). Finally, Lactobacillussp. are highly alcohol and acid tolerant and many have been grantedGRAS status, making them suitable hosts for the production of biofuelsand organic acids (Bosma et al., 2017). Moreover, the genomes ofLactobacillus sp. are substantially smaller than other bacteria discussedherein (Bosma et al., 2017). For example, K. pneumoniae MGH 78578has a 5.32 Mb genome encoding 4,996 proteins, whereas L. reuteri DSM20016 has a 2 Mb genome encoding 1,917 proteins. Accordingly, it isanticipated that minimizing side metabolite formation or overcomingcomplex regulatory mechanisms that control flux distribution would besignificantly easier in L. reuteri. Note that certain species of this genusare fastidious in their nutritional requirements, although other speciessuch as L. reuteri can proliferate in less complex media (Bosma et al.,2017). Overall, while E. coli is the most preferred host for bio-basedproduction from glycerol, certain organisms are inherently specializedfor the production of specific metabolites. Hence, the development ofmore sophisticated genetic tools and comprehensive understanding of
global metabolism may facilitate the derivation of superior productionhosts.
4.2. Strategies to enhance target metabolite production from glycerol
General strategies to enhance target metabolite production fromglycerol are universal among the hosts discussed herein, although thespecific approach and relative importance depend on the strain andproduct. For example, further improvements to 1,3-PDO production inE. coli or K. pneumoniae will likely hinge on improving the producttolerance of these strains and reducing inhibitory metabolite formation.In general, improving product tolerance may be achieved via cellmembrane engineering (Tan et al., 2017; Zhao et al., 2003), directedevolution (Sandoval et al., 2015), or modulating the host stress re-sponse via overexpression of heat shock proteins (Zingaro andPapoutsakis, 2012). Reducing side metabolite formation in E. coliduring 1,3-PDO production can be effectively achieved by inactivatingkey genes (e.g. ptsH), whereas engineering of the TCA cycle in K.pneumoniae may be required to alleviate pyruvate accumulation in 2,3-BDO-deficient mutants. On the other hand, engineering cofactor re-generation systems that rely on co-substrate utilization should be a keyapproach to improve 1,3-PDO production in Citrobacter sp. and Lacto-bacillus sp. In the case of Lactobacillus sp., partially activating dormantfermentative or respiratory pathways for oxidative glycerol dissimila-tion may also serve to restore redox poise.
A major disadvantage in using E. coli for the production of 3-HP andP(3HP), or other oxidized metabolites derived from 3-HPA, is the re-quirement for exogenous B12. Biosynthesis of B12 in E. coli required thecoexpression of 22 genes contained in six operons and three plasmids(Ko et al., 2014), which, if implemented in a strain engineered toproduce a second major metabolite (e.g. 3-HP), could pose a largeburden on the host cell and compromise culture performance. As pre-viously outlined, NAD+ can be regenerated anaerobically via NADH:-quinone oxidoreductase II with supplemental nitrate, which may fa-cilitate 3-HP and P(3HP) production in strains expressing B12-independent DhaB1 from C. butyricum. Alternatively, protein en-gineering of DhaB1 to reduce or abolish its sensitivity to oxygen maycircumvent the requirement of anaerobic operation, and this was suc-cessfully achieved with AdhE from E. coli for aerobic 1-propanol pro-duction (Choi et al., 2012). Yet, another possibility is the selection of aproduction host that can naturally synthesize B12 such as Citrobacter sp.,certain Lactobacillus sp., and K. pneumoniae. While K. pneumoniae is apathogen, attenuated strains lacking endogenous endotoxins have beendeveloped through rational strain design (Huynh et al., 2015; Junget al., 2013), which is an approach that led to the development of non-hemolytic Streptococcus equisimilis for commercial hyaluronic acidproduction (Kim et al., 1996; Liu et al., 2011), and endotoxin-free E. colifor recombinant protein production (Mamat et al., 2015). Finally, theapplication of mixed microbial cultures is another potential strategy toenhance the bioconversion of glycerol to value-added products, and hasbeen studied extensively for the production of 1,3-PDO (Jiang et al.,2018; Jiang et al., 2017; Sun et al., 2018; Zhou et al., 2017a, 2017b;Zhou et al., 2018).
While the strategies proposed herein may resolve certain limitationsidentified through various strain engineering approaches, a systemsbiology approach that incorporates metabolomics, proteomics, andtranscriptomics may serve to elucidate the multiple layers of regulatoryarchitecture that orchestrate glycerol metabolism. Evidence suggeststhat regulatory mechanisms such as post-transcriptional regulation (i.e.via non-coding RNAs), post-translational regulation (e.g. phosphoryla-tion), and allosteric regulation significantly affect metabolic flux dis-tribution, which is another important topic that has recently been re-viewed (Liu et al., 2017). A systems level approach combined with next-generation genome editing tools, such as the CRISPR-Cas9 systemwhich has been implemented in various microorganisms (Choi and Lee,2016), is a logical step toward engineering microbial platforms for
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industrial-scale glycerol biorefinery. However, the functionality of theCRISPR-Cas9 system in a given host might not necessarily result indramatic improvements to the frequency with which desired genomeediting events occur. Moreover, it has been our experience that higherorder multiplexing of gene mutations may be limited by the efficiencyof the host recombination system, such that down-regulating the ex-pression of gene targets via CRISPRi (Qi et al., 2013), which reliesprimarily on the binding affinity of dCas9 to its assigned target, may bea more effective approach to large-scale pathway modulation.
Acknowledgements
Natural Sciences and Engineering Research Council, Canada.
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