Discoveries and improvements of microbes and enzymes for lignocellulosic ethanol production

Tuan-hua David Ho Sumay YuDepartment of Biology Institute of Molecular Biology Washington University Academia SinicaWashington University Academia SinicaSt. Louis Taipei

How much energy is stored in cellulose?

Cellulose is made of glucose and it is th t b d t bi l ththe most abundant biopolymer on earth

The Taiwan case:

• 1.5 M tons of rice straws are routinely burned by farmers every year• One ton of rice straws can be converted to 250• One ton of rice straws can be converted to 250 liters of ethanol• Sufficient to produce E3 for all cars in Taiwan

Basic procedure of lignocellulosic ethanol production

CO2Seeds EnergyPhotosynthesis

“Carbon Neutral”

EthanolFermentationSaccharification

(Hydrolysis)PretreatmentRaw material

CelluloseHemi-celluloseLignin

6C Sugars5C Sugars

EthanolButanol

6C6C5C

Key enzymes involved in lignocellulosic conversion

E d l….G-G-G-G-G-G-G-G-G-G-G-G…..

Endo-glucanaseExo-glucanase

G-Gβ-glucosidase

G

Hemi-cellulases

LaccasePeroxidase

Where to discover newWhere to discover new enzymes/microbes?

• Wood or straw composts• Wood or grass eating insectsWood or grass eating insects • Gut tissues of grass eating animals

W d tti f i• Wood rotting fungi

2005.09.19 (AVRDC)

A highly active and thermostable endo-glucanase from the thermophilic bacterium Geobacillus

Isolation of Geobacillus from rice straw compost and cloning of endo-glucanase gene, CelA •1st Geobacillus endo-glucanase gene cloned

• 10 X more active than commercial Trichoderma i d lreesei endo-glucanase

• Stable and active over a broad temperature range

• Activity doubled in 2 mM MnSO4

Expression of CelA in E. coli

• Paper submitted and patent filedOriginal bacterial colony Cloning and exp of Cel A

1 2 3 4 5 6

170130

95

Expression of CelA in E. coliCelA active and stable up to 70ºC

(%) 100

120

95

72

55

43R

elat

ive

activ

ity (

20

40

60

80

(●) Optimal temp.(□) Thermostability (6 hr)

34

26

Temp (oC)

40 50 60 70 800

20 (□) Thermostability (6 hr)

Metagenomics ApproachIsolation of RSC-EG1

Fresh compost samples

by screening λphage libraryconstructed from rice straw compost Optimum Temperature

ty(%

)

80

100

120

DNA extraction Subcloning of RSC-EG1

Rel

ativ

e ac

tivit

20

40

60

Metagenomics4 kb

Sequencing revealed a novel endo-glucanase

Temperature20 30 40 50 60 70 80 90

0

20CMCase

Optimum pH

Cloning & expression

q g g

Expression and purification from E.coli

ctiv

ity(%

)

80

100

120

expression49kDa

Rel

ativ

e ac

20

40

60

100mM sodium acetate buffer (pH3.5-6.0)100mM sodium phosphate buffer (pH6.0-8.0)100mM Tris-HCl buffer (pH8.0-9.0)

Enzyme characterization

Optimal temp is 70Optimal pH is 5.5

pH3 4 5 6 7 8 9 10

0

Novel cellulase genes from white spotted longicorn beetle Anoplophora malasiaca

Gene GHF Type of cellulase Identidy(%)

G

Cloning and sequence homology of newly isolated AmEGases

• 6 longicorn cellulase genes cloned so far

• All AmEGs appear to be eukaryotic type

• An efficient silkworm expression systemAmEG 1 45 Endo-β-1,4-glucanase 75.7

AmEG 2 5 Endo-β-1,4-glucanase 80.4

AmEG 3 5 Endo-β-1,4-glucanase 58.3

AmEG 4 5 Endo-β-1,4-glucanase 56.3

An efficient silkworm expression system developed for production of AmEGs

• 10X higher yield of AmEGs in whole silkworm body than in body fluid

AmEG 5 5 Endo-β-1,4-glucanase 55.8

AmEG 6 48(9) Cellobiohydrolase (exo-glucanase)

65• AmEG1 enhances efficiency of

commercial Trichoderma reesei enzymes

• AmEG1 has an optimal pH at 4

10X higher yield of AmEG1 in wholeSilkworm body than in body fluid

Body Body fl id

Activity assay of AmEG 1 from silkworm body fluid with T. reesei(CMC by DNS)

70

80

y

55-

40-

fluidKD

20

30

40

50

60

70

Red

ucin

g su

gar

(mg/

ml)

35-

25-AmEG 1+T.reesei

T.reesei T.reesei+Novo 188AmEG 1

0

10

20R

Cellulases of Xylariaceae fungiFilter paper digestion p p g

(3 hr at 50ºC)

Xylariaceae secretes 2 endoglucanases, 1 exo-glucanase and 1 β-glucosidase

1 2

glucanase and 1 β-glucosidase

Four cellulase-related genes have been isolated from Xylariaceae

H2OFungal culturemedium

110 kDa

72 kDa

55 kDa

MM

1-1 Cellulase [Melanocarpus albomyces]Identity: 64%, Positive: 77%1 2

5

-glucosidase

4 55 kDa

43 kDa

34 kDa

26 kDa

12

3

1-2 Cellulose 1,4-beta-cellobiosidase [Acremonium thermophilum]Identity: 57%, Positive: 71%

2 Cip2 [Hypocrea jecorina]

(Exo-glucanase)

SDS-PAGECMCZymogram

26 kDa Cip2 [Hypocrea jecorina]Identity: 48%, Positive: 64%

3 Endoglucanase [Talaromyces emersonii]Identity: 62%, Positive: 78%

Highly active laccase of Rigidoporus microporusA Taiwanese fungus R. microporussecreting high level laccasesecreting high level laccase

Lcc35 laccase is a novel and abundant secretory

enzyme in R microporusenzyme in R. microporus.

>3 X more active than commercial laccases

Paper to be submitted and patent filed

Laccase is the single most abundantStrain ABTS assay

(U/mg)SGZ assay

(U/mg)Laccase is the single most abundant protein secreted by R. microporus Lcc35 Rigidoporus sp. 3800 (U/mg) 1700 (U/mg)

Laccase (Fluka co.) Trametes versicolor

1300 (U/mg) 500 (U/mg)

L ( Thi l i 1020 490 k /Laccase (patent application US2006/0063246A1)

Thielavia arenaria

1020 nkat/mgor61 (U/mg)*

490 nkat/mgor29 (U/mg)*

Laccase 51003 140 11.4 (Novozymes)** (U/mg)** (U/mg)**

*16.67 nkat = 1U** Laccase 51003 provided by 永豐餘

Fast growing and highly productive R. microporus mutants

20

25Basal mediumFast growing mutants of R. microporus

H5

activ

ity (U

/ml)

15H6mutant

H7H8 WT Lacc

ase

a

5

10

WT

H6

Time (day)0 1 2 3 4 5 6 7 8 9 10

0

•R. microporus mutant H6 grows twice as fast as wild type

•H6 mutant produces more than twice as much laccase as wild type

Time (day)

p yp

•H6 mutant culture does not have viscous metabolites in medium

Dramatic structural changes on rice straws after pretreatment with specific fungi

Non-treatedTreated withstrain 40-3for 3 weeks

Treated withTreated withstrain 40 5 strain 001

for 6 weeksstrain 40-5for 3 weeks

Challenges ahead

• Low cost collection of raw materials• Lowering energy inputLowering energy input• Lowering cost of enzymes and microbes

M ffi i t f t ti• More efficient fermentation• Nonezymatic conversion (pyrolysis)

Output

• Papers-- 2 submitted (Ho, Yu and Tong)

2 under preparation (each for Ho/Yu/Tong and Chao)-- 2 under preparation (each for Ho/Yu/Tong and Chao) • Patents

-- 3 filed (Ho, Yu and Tong; 2 US and 1 ROC)1 t b li d (Ch )-- 1 to be applied (Chao)

• Licensing agreements-- 2 under preparation for Ho,Yu and Tong (bio-pretreatment; f l ll l d ti )fungal cellulase production process)

• Collaborations-- INER (bioconversion)-- Scandinavian Biofuel (bio-pretreatment; bioconversion) -- Yuen-Foong Yu 永豐餘 (paper pulping; brightening)

Development of fungal enzyme formulations tested in INER pilot plant

Fusarium proliferatum (F.P) cellulasesGlucose yield (per g cellulosic materials) 24h 48h 72hy (p g )Cellulast 1.5L (commercial enzymes) 0.410 0.550 0.670Cellulase-F.P (this project) 0.489 0.511 0.528

200

250

Relat *Local strain isolated at AVRDC Taiwan

FPase activity核研所 assessment of our enzyme formulations

100

150

tive activit

Local strain isolated at AVRDC, Taiwan* Has a full set of cellulases to hydrolyze rice straws*Crude enzyme preparation converts 50%

of pretreated rice straws to glucose

0

50

ty (%)

of pretreated rice straws to glucose*Crude preparations can be produced using

low-cost feedstocks such as rice straws*Some en me acti ities displa s nergistic effectSome enzyme activities display synergistic effect

with commercial enzymes

Development of bacterial enzyme formulations with synergistic effect

Gl f Th bifid f (T F)

Avicelase

%) 120

140

100%106% 111%

115%125%

%) 100

CMCase FPase

Glucanases from Thermobifida fusca (T.F)

ve a

ctiv

ity (%

60

80

100

e ac

tivity

(%

60

80

Rel

ativ

0

20

40

0% Rel

ativ

e

0

20

40

01:0 1:1 βgl1:2 1:101:4

Weight ratio (crude T.F enzyme/ βgl)

Molar ratio (E4/ βgl)

1 : 0

1 : 0.

251 :

0.5

1 : 1

1 : 2

1 : 4

1 : 10

1 : 20

1 : 60

0

Up to 25% enhancement of T.F avicelase activity with supplement

Up to 70~100% enhancement of CMCase and FPase activities by combining purified E4 cellulaseof β-glucosidase (βgl) combining purified E4 cellulase(with both endo- and exo- act) with T.F-β-glucosidase (βgl)

Discovery of highly active fungal β-glucosidases

Fungi from Dr YM Ju (IPMB)Screening for β-glucosidase producing fungi

se (U

/ml)

120

160

200Fungi from Dr. YM Ju (IPMB)D2 D3

β -gl

ucos

ida s

40

80

PC E9

A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6 C1

C2

C3

C4

D1

D2

D3

D4

D5

D6

D7

D8

D9

D10

D11

D12 E1 E2 E3 E4 E5 E6 E7 E9 E10

E11

E13

E15

E16

PC

0

• 6 fungi with excellent β-glucosidase activity

β-glucosidase zymogramD2 D3 PC E9 N188

6 fungi with excellent β glucosidase activity• D2 is most promising in hydrolyzing cellobiose to glucose

(Min-1) (Min-1)