David B. Levin1, & Richard Sparling2 1Department of Biosystems Engineering &

2Department of Microbiology

University of Manitoba

Winnipeg, MB

Canada

Microbial Genomics for the Development of Biocatalysts

for Lignocellulosic Biorefining and Biofuels production

Microbial Genomics of Biocatalysts for Biorefining

Slide 2

Outline

Biofuels from Direct Cellulose Fermentation: C. thermocellum

Improvements through medium optimization

Microbial Genomics and Metabolism

Comparative genomics: central metabolism

Bioinformatics & Proteomics reveal unexpected Pathways in C. thermocellum

Direct conversion of raw substrates

Comparative genomics: Towards high performance lignocellulose fermentation

through consolidated bioprocessing

Cellulosic Biofuels:

Current vs Alternative Approach

Slide 3

Clostridium

thermocellum Clostridium termitidis

Biofuels from Direct Cellulose Fermentation

Clostridium thermocellum: thermophilic, cellulolytic, gram +ve, anaerobic Clostridium termitidis: mesophilic, cellulolytic, gram +ve, anaerobic Degrade cellulose and synthesize: Ethanol, H2 and CO2, VFAs - acetate (formate, lactate) C. thermocellum possesses a high rate of cellulose-degradation

C. termitidis cellulose hydrolysis comparable to C. cellulolyticum

Slide 4

Clostridium thermocellum

End-Product Formation on cellulose: Medium optimization

Slide 5

Starting with a baseline medium for C. thermocellum,

one can alter the medium to enhance:

Growth rate and end-product formation rate from

cellulose

Shift production towards the generation of a specific

end-product

Clostridium thermocellum

End-Product Formation on cellulose: Batch Cultures

0.01

0.10

1.00

10.00

100.00

0 10 20 30 40 50

Time (hours)E

nd

pro

du

ct

form

atio

n (

um

ole

)

Acetate

Formate

Lactate

Ethanol

10

100

1000

0 10 20 30 40 50

Time (hours)

To

tal

Pro

tein

(u

g)

1

10

100

Ga

s p

rod

uc

ed

(u

mo

le)

Protein

H2

CO2

A) Total protein, hydrogen and carbon dioxide; B) lactate, acetate, formate and ethanol produced by

C.thermocellum within 10mL baltch tubes of 1191 media grown on 4.5g/L a-cellulose incubated at 60C

A

To

tal P

rote

in (

g)

Ga

s p

rod

uce

d (

mo

le)

En

d p

rod

uct fo

rma

tio

n (

mo

le)

B

Time (hours) Time (hours)

Slide 6

Clostridium thermocellum

Medium optimization design

Slide 7

C. thermocellum

End-Product Formation: Medium optimization

Slide 8

Cellulose fermentation:

From test tube to genome to proteome

Slide 9

Medium optimization can only go so far without

-a deeper understanding of the genome,

-an understanding of the subset of genes actually used

under specific growth conditions (e.g. proteome)

Can lead to better medium design

Can lead to genetic engineering

Understanding the genome of cellulolytic fermentative

organisms: selection of organisms

Organism Optimum temp

End Products (mol/mol hexose equivalents) Growth condition Ref

(°C) H2 CO2 Acetate Ethanol Formate Lactate

Ca. saccharolyticus DSM 8903

70 3.5 2.5 3.6 4.0

NR 1.4 1.5 1.8

2.1 1.4 1.6 NR

NR ND ND ND

NR ND ND ND

NR 0.1 ND ND

Batch, 10 g l-1 sucrose Batch, 10 g l-1 glucose Continuous, 4.1 g l-1 glucose (D=0.1 h-1) Continuous, 1.1 g l-1 glucose (D=0.09 h-1)

19, 20 22 23 23

A. thermophilum 75 ✓ ✓ ✓ ✓ 21 28

C. cellulolyticum H10 37 1.6 1.8

1.0 1.1

0.8 0.8

0.3 0.4

ND ND

NR NR

Batch, 5 g l-1 cellulose Batch, 5 g l-1 cellobiose

24 24

C. phytofermentans ISDg 35-37 Major 1.0 1.6

Major 0.9 1.2

0.6 0.6 0.6

1.4 0.5 0.6

0.1 0.1 ND

0.3 NR NR

Batch, 34 g l-1 cellobiose Batch, 5 g l-1 cellulose Batch, 5 g l-1 cellobiose

18 24 24

C. thermocellum ATCC 27405

60 0.8 1.0

1.1 0.8

0.7 0.8

0.8 0.6

0.3 0.4

ND 0.4

Batch, 1.1 g l-1 cellobiose Batch, 4.5 g l-1 cellobiose

1 25

C. thermocellum JW20 60 1.8 0.6

1.7 1.8

0.9 0.3

0.8 1.4

ND ND

0.1 0.2

Batch, 2 g l-1 glucose Batch, 27 g l-1 cellobiose

26 26

T. pseudethanolicus 39E

NR 0.1

NR ✓ 2.0

0.3* 0.2 ✓ 0.1

1.3* 0.8 0.4 1.95 1.45 1.8

NR NR NR

>0.1* 1.1 ✓ 0.1

1 g l-1 xylose Batch, 20 g l-1 xylose Batch, 20 g l-1 glucose Batch, 8 g l-1 glucose

29 27 27 30 31 32

Hyd

rog

en

Eth

an

ol

ND- not detected

NR- not reported

* per xylose equivalent Slide 10

Comparative Bioinformatics analysis

Alcohol dehydrogenases G

ene

CO

G

ID

Ca

. sa

cch

aro

lyti

cus

DS

M 8

90

3

A. th

erm

op

hil

um

DS

M 6

72

5

C. ce

llu

loly

ticu

m

H1

0

C. p

hyt

ofe

rmen

tan

s

ISD

g

C. th

erm

oce

llu

m

AT

CC

27

40

5

C. th

erm

oce

llu

m

DS

M 4

15

0

T. p

seu

det

ha

no

licu

s

AT

CC

33

22

3

adhE 1454

1012

ND ND Ccel_3198 Cphy_3925 Cthe_0423 Cthe_C10_1096 Teth_390206

Fe-adh 1454 Csac_0407

Csac_0711

Csac_0622

Csac_1500

Athe_2244

Athe_0928

Ccel_1083

Ccel_0894

Ccel_3337

Cphy_2650

Cphy_1029

Cphy_1421

Cphy_2463

Cthe_0394

Cthe_2579

Cthe_0101

Cthe_C9_2833

Cthe_C3_0189

Cthe_C25_0616

Teth_391597

Teth_391979

Teth_390220

aldh 1012 ND ND ND Cphy_3041

Cphy_0958

Cphy_2418

Cphy_1178

Cphy_2642

Cphy_1428

Cphy_1416

Cthe_2238 Cthe_C58_1042 ND

adhE encodes domains necessary for complete reduction of acetyl-CoA to ethanol

Hydrogen Ethanol

Slide 11

Comparative Bioinformatics

Hydrogenases

Gen

e

CO

G

ID

Ca

. sa

cch

aro

lyti

cus

DS

M 8

90

3

A. th

erm

op

hil

um

DS

M 6

72

5

C. ce

llu

loly

ticu

m

H1

0

C. p

hyt

ofe

rmen

tan

s

ISD

g

C. th

erm

oce

llu

m

AT

CC

27

40

5

C. th

erm

oce

llu

m

DS

M 4

15

0

T. p

seu

det

ha

no

licu

s

AT

CC

33

22

3

ech NiFe H2ase

(Fd)

3260

3261

Csac_1534-1539 Athe_1082-1087 Ccel_3371-3366

Ccel_1691-1686

Cphy_1730-1735 Cthe_3024-3019 Cthe_C50_2173-2168 ND

Fe H2ase

(NADH)

4624

(1894)

Csac_1860-1864 Athe_1298-1299 Ccel_2232-2233

Ccel_2303-2304

Cphy_3804-3805

Cphy_0088-0087

Cthe_0341-0342

Cthe_0429-0430

Cthe_C10_1102-1103 Teth_391457-

391456

Fe H2ase

(NADPH)

4624

(0493)

ND ND ND ND Cthe_3003-3004 ND ND

Fe H2ase

(Fd)

4624 ND ND Ccel_2467 Cphy_2056 ND ND Teth_390221

rnf 4656-

4660,2

878

ND ND ND Cphy_0211-0216 Cthe_2430-2435 Cthe_C28_0369-0375 Teth_392124-

392119

Hydrogen Ethanol

Slide 12

Comparative Bioinformatics

End-product synthesis (yes/no inventory)

NADH NAD+

NADH NAD+

ADP ATP

RNF Ech

PFO

NADH NAD+ H+ H2

NADH H2ase FdRED FdOX

Fd H2ase

NAD+ NADH

NADPH H2ase

NADP+ NADPH

ATPase

H+IN

H+OUT H+

OUT

H+IN

K+IN

K+OUT

Pyruvate Acetyl-CoA

PFL

CO2

Formate

Lactate LDH

Acetyl-P

Acetaldehyde

2 NADH 2 NAD+

ADP ATP

ALDH ADH

AdhE

PTA ACK

Pi

Acetate

Ethanol

Alcohol

A B C D E F * H

NADH NAD+

ABCDEFGH ABCDEFGH

A x x x x x * x

A – Ca. saccharolyticus DSM 8903 B – A. thermophilum DSM 6725 C – C. cellulolyticum H10 D – C. phytofermentans ISDg E – C. thermocellum ATCC 27405 F – C. thermocellum DSM 4150 G – C. thermocellum JW20 H – T. pseudethanolicus 39E

A B C D E F * H

x x C D E F * x

x x C D E F * H

A B C D E F * H x x x D E F * x

A B C D E F * x

A B C D E F * H

x x C D x x * H

x x x D E F * H

Slide 13

What genomics tell us about C. thermocellum

Slide 14

Multiple genes have same

putative annotated

function!?

Slide 15

•Glycolytic pathway utilizes both PPi and multiple ATP-dependent PFKs

•Multiple methods of interconverting PEP and pyruvate exist, but no

genomic evidence of a pyruvate kinase AND no peptides corresponding to a

clostridial PK.

•MDH and ME may be used in transhydrogenation of NADH to NADPH,

could also be used for conversion of PEP to pyruvate

•Branched product pathway uses multiple PFOs and ADHs

•The absence of ALDH suggests ADH-E is needed for EtOH synthesis

•Fd-dependent Ech hydrogenase and NADH dependent hydrogenases

are present

•What will the proteome say?

Conclusions from central metabolism genomics

in C. thermocellum:

Slide 16

CAUTION: Proteomics is a tool, you need an experimental context!

0

2

4

6

8

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12

14

16

0 2 4 6 8 10 12 14

End

-pro

du

cts

(mM

) Time (h)

H2

CO2

Acetate

Ethanol

Formate

End-Product Synthesis and Cellobiose Consumption During Growth

6.2

6.4

6.6

6.8

7.0

7.2

7.4

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12 14 16 p

H

Bio

mas

s an

d s

ub

stra

te (

mM

)

Time (h)

Biomass

CB used

pH Exp

Stat

•C. thermocellum grown under carbon-limited conditions (2g/l cellobiose) in closed batch cultures with

no pH control. End-product profiles generally follow growth with a slight increase in ethanol:acetate

ratio consistent

Slide 17

Proteomic Analysis (Shotgun and 4-Plex 2D-HPLC-MS/MS)

-Relative protein expression

Based on spectral counts (SpC) in both shotgun and 4-plex 2D-HPLC-MS/MS

runs

Given as ‘relative abundance index’ (RAI) = peptide SpC / protein Mr

-Differential protein expression Sample labeling: iTRAQ (isobaric labelling)

Tag 114 & 115 (exponential phase, biological replicates), Tag 116 & 117

(stationary phase biological replicates)

Given as total iTRAQ reporter ratios per protein (stationary/exponential)

Significance of changes in expression based ‘vector difference’ (Vdiff )

Slide 18

Shopping list, good, but targeted analysis better!

Focus on core metabolism

LEGEND

Genomics tells us what organism can do,

not what it does do

Slide 19

Examples:

Fd (Ech) NiFe-Hydrogenase not expressed under tested growth condition

NADH-Fd bifurcating hydrogenase appears dominant

Pyruvate could be synthesized via pyruvate dikinase (PPi dependent) OR

malate shunt; both are highly expressed

PPi dependent phosphofructokinase is a major enzyme present for glycolysis

Possibility of major role for PPi in energy conservation in C. thermocellum

Need to check proteome of multiple organisms…

Bioinformatics & Proteomics Pathways of central

metabolism in C. termitidis

C. termitidis is a mesophilic, cellulolytic bacterium, isolated from the gut of the termite Nasutitermes lujae Can use cellulose, cellobiose, and other hexose sugars Major end-products: Ethanol, Acetate, H2, and CO2, but can synthesize Lactate

and Formate under certain growth conditions Reported to utilize xylose Genome sequence analysis and annotation revealed genes for pentose and glucoronate interconversion

Slide 20

Slide 21

Clostridium termitidis

Protocol for genomic and proteomic characterization of novel

organisms

Biofuels from Direct Cellulose Fermentation:

Choice of substrate

Slide 22

While there are source of “refined” cellulose wastes:

paper cups, paper plates, old news papers, pulpe waste

There are many sources of agricultural ligno-cellulosic wastes:

bagasse, wheat straw, flax shives, hemp hurds, sawmill waste

To what extent can raw substrates be fermented by consolidated

bioprocesses?

Substrates milled with 32-35 mesh to 0.5mm particle size a-cellulose

Biofuels from Direct Cellulose Fermentation:

Evaluation of Different Substrates

Slide 23

+ C. thermocellum

Biofuels from Direct Cellulose Fermentation:

Evaluation of Different Substrates

Available cellulose (mg): 19.4 11.5 23.0 46.0 15.7 15.8 18.3 15.4 17.3 7.7

Yields of fermentation end-products vary with % cellulose & substrate complexity

Normalized Yields of Ethanol and Hydrogen in C. thermocellum fermentation Reactions: 0.2% loading = 20 mg (2 g/L) each substrate in at 60 oC for 24 hrs

Slide 24

No pre-

treatment

Combination of organisms enhance breakdown of some

raw substrate

Slide 25

Magnified view of C. thermocellum

CBM

Structure of Cellulosome

Cellulosome Components

Anchoring protein Scaffoldin (cipA)

Cellulose Binding Motif (CBM) Cohesin domains

Enzymatic subunits

Dockerin domains

Dockerin

domains

Cohesion

domains

Presence or Absence of Dockerin Domains

in Conserved Glycoside Hydrolases

- 19 GHs conserved across

6 cellulolytic Clostridia:

Clocel – C. cellulovorans

Cther – C. thermocellum

Cter – C. termitidis

Cpap – C. papyrosolvens

Ccel – C. cellulolyticum

Cphy – C. phytofermentans

- C. stercorarium (Clst) genome

contains only 13 of the 19 GHs

found in other cellulolytic

clostridia

- 6 GHs not detected (ND) in Clst

genome

“+” and “++” indicate GHs with

dockerin domains

- Cphy and Clst GHs are NOT

cellulosome associated

Presence or Absence of Selected Glycoside

Hydrolases in Clst, Cter, and Cther

- GHs in Cther are mostly

cellulosomal:

- 12 GH cellulases; few

xylanses

- All GHs in Cphy and Clst are

acellulosomal;

- GHs in Cter and Cther are a

mixture of cellulosomal

and acellulosomal:

C – cellulosome-associated

A - acellulosomal

(no dockerin domains)

Remember: C. thermocellum encodes xlyanases, but does not grow on xylan hydrolysis products

CAZyome Analysis of sequenced Thermoanaerobacter spp.

A cautionary tale Table

Clade 3 Clade 2 Clade 1

Th

erm

oa

na

erob

act

er

sid

ero

phil

us

SR

4

Th

erm

oa

na

erob

act

er

ther

mo

hyd

rosu

lfuri

cus

WC

1

Th

erm

oa

na

erob

act

er

wie

gel

ii R

t8.B

1

Th

erm

oa

na

erob

act

er

ita

licu

s A

b9

Th

erm

oa

na

erob

act

er

ma

thra

nii

A3

Th

erm

oa

na

erob

act

er

bro

ckii

su

bsp

. fi

nn

ii A

ko

-1

Th

erm

oa

na

erob

act

er

pse

ud

eth

ano

licu

s 3

9E

Th

erm

oa

na

erob

act

er

sp. X

51

3

Th

erm

oa

na

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act

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sp. X

51

4

Total Sequences in CAZyome 58 55 50 60 59 44 42 45 42

Glycoside Hydrolases Unique Gene Sequences 29 26 22 32 30 21 24 23 24

Unique Classes 17 15 15 19 19 15 16 16 16

Glycosyltransferases Unique Gene Sequences 15 11 15 15 13 11 11 11 12

Unique Classes 6 6 7 6 7 5 5 5 6

Carbohydrate Esterases Unique Gene Sequences 4 5 4 4 6 3 3 3 3

Unique Classes 2 2 2 3 5 2 2 2 2

Polysaccharide Lyases Unique Gene Sequences 0 0 0 1 0 0 0 0 0

Unique Classes 0 0 0 1 0 0 0 0 0

Carbohydrate Binding Modules Unique Gene Sequences 6 8 6 5 5 5 0 5 0

Unique Modules 4 4 4 4 4 3 0 3 0

Multi-Component Proteins Unique Gene Sequences 4 5 3 3 5 4 4 3 3

Unique Combinations 4 5 3 3 5 4 4 3 3

Extracellular Proteins* 5 7 3 5 6 2 3 2 2

*Excluding proteins annotated to be involved with cell wall hydrolysis

Extracellular glycoside hydrolases in the genus

Thermoanaerobacter

Slide 30

Microbial Genomics and Cellulosic Biofuels

Direct fermentation of cellulose by cellulolytic bacteria

Requires intimate knowledge of the metabolic pathways and their

regulation, in order to control the fermentation

Possible enhancements using molecular techniques

Ethanol is not the only or best possible biofuel… nor the only value

added product possible

Development of other products using molecular techniques

Combinations of organisms enhance total glycoside hydrolase

cocktail available for deconstruction, and enhances fermentation

Slide 31

Acknowledgements

Faculty

David Levin

Richard Sparling

Nazim Cicek

MSc Graduate Students

Chris Dartiaihl

Hon Wai Wan

PhD Students

Val Agbor

Carlo Carere

Jilagamazhi Fu

Eftekhar Hossain

Rumana Islam

Matt McCandless

Riffat Munir

Umesh Ramachandran

Tom Rydzak

Ryan Sestric

Marcel Taillefer

Tobin Verbeke

Scott Wuske

Biofuels Research Group

Research Associates

Parveen Sharma

Jaime Park

Serpil Ozmihci

PDFs

John Schellenberg

Sadhana Lal

Slide 32

Manitoba Proteomics Centre

Vic Spicer

Oleg Krokhin

John Wilkins

Bioinformatics

Justin Xiang

Brian Firstensky

page 11

Thank-you

Questions?