David B. Levin 1 , & Richard Sparling 2 1 Department of Biosystems Engineering & 2 Department of Microbiology University of Manitoba Winnipeg, MB Canada Microbial Genomics for the Development of Biocatalysts for Lignocellulosic Biorefining and Biofuels production
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Microbial Genomics for the Development of Biocatalysts for ......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
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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
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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
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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
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!
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End-Product Synthesis and Cellobiose Consumption During Growth
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•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:
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
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oa
na
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act
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sid
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phil
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SR
4
Th
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rosu
lfuri
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WC
1
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1
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bsp
. fi
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Th
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51
3
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4
Total Sequences in CAZyome 58 55 50 60 59 44 42 45 42