Prof. Ehud Banin

M.O.B.I May 2017

2

Of the major sustainable energy options, biofuel is most promising

Bioethanol –enzymatically produced fuel from fermentable sugars

Versatile biofuel feedstock: ALGAE

Green algae is an underexploited resource

3

Proteins Carbohydrates Lipids(10.3-17.2%) (41-61%) (1.8-3.5%)

Ulvan

(8%-29%)Cellulose Starch

(Lahaye et al. 2007)

Cell wall

polysaccharides

(38-54%)

(Robic et al. 2009)4

5

1. SACCHARIFICATION To fully utilize ULVAN, enzymatic

machinery for degradation must be identified and functional

2. FERMENTATION Once broken down, sugars can only

be fermented to ethanol by microorganisms with the appropriate tools

S S

S

S

S

S

OH

Ethanol

6

7

Four bacteria were isolated from Aplysia sp. and tested for ulvan/oligo-degrading capability

In collaboration with Dr. William Helbert from CERMAV-CNRS, France

Polysaccharide utilization locus (PUL)

Genomes of the native marine bacteria were sequenced and annotated, revealing ulvan PULs

A PUL is a set of physically linked genes that coordinates the breakdown of a specific polysaccharide

Includes genes for sensing and binding the polysaccharide, to cleavage and transport of oligosaccharides

8

Thomas et al. 2011, Front. Microbiol.

OR

F_2

OR

F_4

OR

F_12

OR

F_13

OR

F_14

OR

F_15

OR

F_16

OR

F_18

OR

F_26

OR

F_19

OR

F_20

OR

F_22

OR

F_24

25

OR

F_28

OR

F_30

OR

F_27

OR

F_29

OR

F_31

OR

F_33

OR

F_34

32

OR

F_59

OR

F_60

OR

F_69

OR

F_70

OR

F_61

62

OR

F_75

74

OR

F_78

OR

F_80

OR

F_87

OR

F_85

OR

F_86

OR

F_93

OR

F_97

OR

F_95

OR

F_96

OR

F_98

OR

F_100

OR

F_107

OR

F_108

Hypothetical protein

Polysaccharide sensing, binding and transport proteins

Proteins involved in polysaccharide degradation (GH and PL)

Xylose metabolism

Sulfatase

Rhamnose metabolism

PULs found in LOR ulvan cluster

OR

F_101

OR

F_102

OR

F_103

OR

F_104

OR

F_105

OR

F_106

OR

F_57

Moran Kopel, Yana Belnik, Vitaliy Buravenkov 9

Detection of novel ulvan lyaseThe first enzyme required for ulvan degradation

TonBDependent recp

LOR_19 LOR_20 LOR_22 LOR_24 LOR_26 LOR_27 LOR_28 LOR_29 LOR_30 LOR_31 LOR_33 LOR_34

GH 78 GH 78GH 105

hypothetically involved in oligo-ulvans degradation

incubated with ulvan

Degradation kinetics of LOR_29

analytical size-exclusion chromatography

purified LOR_29 protein Ulvan

Two main products:

DP2 – disaccharide

DP4 - tetrasaccharide

ulvan

DP4

DP2

∆GlcA-R3S

Verified by 1H-NMR spectra analysis

R3S — GlcA R3S — IduA—

— GlcA—IduA — R3S—R3S — Xyl R3S

∆GlcA-R3S-Xyl-R3S

LOR_29 homologs

StrainPair

similaritycoverage

Pseudoaltermonas sp. PLSV

76% 97%

Altermonas sp.LTR

100% 100%

Nonlabens ulvanivorans (NLR) 53% 99%

Paraglaciecola agarilytica

76% 97%

Paraglaciecola chathamensis

75% 97%

Catenovulum agarivorans

61% 96%

Pseudoalteromonas haloplanktis

57% 98%

incubated with ulvan

Second enzyme in ulvan saccharification process

β – glucuronyl hydrolase (βGH)

∆GlcA-R3S R3S GlcA

βGH

TonBDependent recp

LOR_19 LOR_20 LOR_22 LOR_24 LOR_26 LOR_27 LOR_28 LOR_29 LOR_30 LOR_31 LOR_33 LOR_34

GH 78 GH 78GH 105

incubated with ulvan-modified by LOR_29

Relates to GH105 or to GH88 families

Digests products of ulvan lyases (oligo-ulvan)

Degradation decreases absorption (235nm)

LOR_28 characterization

ulvan-modified by LOR_29 purified LOR_28

LOR_28 is able to remove unsaturated

glucuronyl residue (∆-GlcA)

LOR_28 is active on LOR_29 products

Identification of rhamnosidases

TonBDependent recp

LOR_19 LOR_20 LOR_22 LOR_24 LOR_26 LOR_27 LOR_28 LOR_29 LOR_30 LOR_31 LOR_33 LOR_34

GH 78 GH 78GH 105

GH 78 – family of proteins with rhamnosidase activity

LOR_27 – not enough soluble protein

LOR_34 – able to release 4-nitrophenol leaving group

SSS S S S S S S S

Glucose (Glu)

Xylose (Xly)

Glucuronic acid (GluA)

Rhamnose 3-S (Rha3S)Iduronic acid (IduA)

LOR_107LOR_61

Ulvan lyase

LOR_29

- Activity verified on ulvan- Biochemically characterized

SSS S

LOR_96

GH88

LOR_28

GH105

Leaving groups

Proposed ULVAN saccharification

The MAIN players: Glycoside hydrolase (GH)

16

Chapter 1:

Saccharification

OH

Ethanol

PROJECT OVERVIEW

S

Chapter 2:

Metabolic

Engineering

Chapter 3:

Consolidated Bioprocess

(CBP)

Fermentation

E. coli

S

17

18

19

Glucose

PEP

Pyruvate

Formate D(-)-Lactate

NAD+

NADH

ADP

ATP

Succinate

Fumarate

Acetyl-CoA

Acetyl~PAcetaldehyde

AcetateEthanol

NAD+

NADH

NAD+

NADH

NAD+

NADH

NAD+

NADH ADP

ATP

frdABCDldhA

pflB

pflB /

pta

ackA

pdc+

adhB+

adhB+

Metabolic engineeringOptimization of ethanol fermentation

(Mariana Klyman, unpublished)

ΔldhA

Δpta

ΔackA

20

21

Glucose

Xylose

Optimization of ethanol fermentation

Rhamnose

Glucuronic acid?

MAK1

Rhamnoseoperon

GluAoperon

kduL, kduD, kdgK, eda

Coupled Fermentation

R3S: 16.8-45.0%

GluA: 6.5-19.0%

rhaA, rhaB, rhaD, aldA, ldhL

22

Metabolic engineering of fermentation pathway

• Glucuronic acid:

kduID

kdgK

eda

kduID

2X

24

• Rhamnose:

lctO

rhaA

rhaB

rhaD

aldA2. lctO

2X

Metabolic engineeringRhamnose fermentation

Rhamnose

operon

rhaA, rhaB, rhaD, aldA, ldhL

84% 96%

24

25

Identified and expressed most of the enzymes required for saccharifcation of ulvan

Improved rhamnose fermentation

26

Bar-Ilan University (Life Sciences)Dr. Moran KopelDr. Sivan ShoshaniMariana KlymanElizabeth ForanVitally BuravenkovNaama Mizrachi

CollaboratorsWilliam Helbert (CNRS)Miroslaw Cygler (University of Saskatchewan)Aharon Gedanken (BIU)Alvaro Israel (IOLR)Avigdor Abelson (TAU)

FundingMinistry of ScienceAbeles Foundation