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Steven Pullan, Stéphane Delmas, Matthew Kokolski, Laure Ries &
David Archer.
School of Biology, University of Nottingham
Fungal Enzymes for Saccharification of
Biomass
Bioenergy-The LACE Programme
LACE is part of the BBSRC Sustainable Bioenergy Centre
(BSBEC)
Six programmes with a total funding of £27 million
LACE funding of around £6.8 Million for 5 years
11 PDRAs, 2 technicians and with an associated cohort of 20
Ph.D students
LACE will focus on the use of Agricultural “residues” such as
wheat straw to produce 2nd generation biofuels - bioethanol
Lignocellulosic Conversion To
Ethanol (LACE)
The conversion of biomass into ethanol requires three main steps:
The scientific strands of LACE are studying aspects of each of these in particular:
o the fundamental processes behind the “hydrothermal deconstruction of the plant cell wall.
o the genetics behind the production of cell wall degrading hydrolytic enzymes by fungi.
o the selection of novel strains of yeast with improved processing capabilities such as resistance
to fermentation inhibitors .
Associated Themes
• Theme A: Energy Analysis and Environmental Life
Cycle Assessment (in association with Bath
University)
• Theme B: Agricultural Economics
• Theme C: Social and Ethical Dimensions
In addition to the “scientific” programme there
are three important associated themes:
Use A. niger as a Model Fungus
Understand how A. niger and T. reesei Respond to and Degrade Wheat
Straw
Add New Knowledge on the Enzymic Degradation of Lignocellulose
Why? Because enzymes are a major cost with second generation biofuels
Aims of the Fungal Enzymes project
Andersen et al. 2011 Genome Research 21, 885-897.
Comparative genomics of citric-acid-producing Aspergillus niger
ATCC 1015 versus enzyme-producing CBS 513.88.
Pel et al. 2007 Nature Biotechnology 25, 221-231.
Genome sequencing and analysis of the versatile cell factory
Aspergillus niger CBS 513.88.
Aspergillus niger, wheat straw and genomes
A. niger vs Trichoderma reesei
A.niger
248 Glycoside Hydrolase in 51 families
25 Carbohydrate Esterase
8 Polysaccharide Lyase
29 Hemicellulases
T. reesei
200 Glycoside Hydrolase in 48 families
16 Carbohydrate Esterase
3 Polysaccharide Lyase
16 Hemicellulases
Martinez et al. 2008 Nature Biotechnology 26, 553-560.
Andersen et al. 2011 Genome Research 21, 885-897.
Comparative genomics of citric-acid-producing Aspergillus niger
ATCC 1015 versus enzyme-producing CBS 513.88.
Pel et al. 2007 Nature Biotechnology 25, 221-231.
Genome sequencing and analysis of the versatile cell factory
Aspergillus niger CBS 513.88.
Aspergillus niger, wheat straw and genomes
8
Glucose
48h
Straw
24h
Straw + Glucose
5h
~ 19% ~1.5%
GH15 GH15
GH7
GH61
GH11 GH62
CE1
In Straw, CAZy Gene Expression Represents
a Huge Investment for A. niger
~3%
The CAZy Genes Expression of A. niger and
T. reesei Define a Core Set of Activities
GH7 endo-glucanase, cellobiohydrolase
GH11 xylanase
GH3, GH30 beta-glucanase, beta-xylosidase
GH61 Cu-dependent monooxygenase
GH62 arabinofuranosidase
CE esterase
x300
x2400
GH61s from A. niger (7) and T. reesei (3)
1% Glucose
48 h
Time after transfer to straw / h
0.5 1 2 3 6 9 12 24
TID_51773 - cbhB
TID_53159 - cbhA
TID_211595 - GH61
TID_200308 - yefC
TID_205580 - GH5
TID_200605- abfB
We have identified a subset of genes that form an initial response at 6 h,
all of which are induced by Carbon Starvation
Expression of Glycoside Hydrolyse is
sequential
1% Glucose
48 h
Time after transfer to straw / h
0.5 1 2 3 6 9 12 24
TID_51773 - cbhB
TID_53159 - cbhA
TID_211595 - GH61
TID_200308 - yefC
TID_205580 - GH5
TID_200605- abfB
The vast majority of CAZy genes are then induced at 9 h
in response to inducing molecules released from straw by the initial response
Expression of Glycoside Hydrolases is
sequential
13
Cellulase / Xylanase etc
CreA CreA CreA CreA
XlnR
Glucose Straw
48h 24h
Classical Model of XlnR / CreA Activity
CreA represses in the
presence of glucose
Cellulase / Xylanase etc
CreA CreA CreA CreA
XlnR
14
Glucose Straw
48h 24h
Classical Model of XlnR / CreA Activity
*
XlnR activates in the
presence of xylose
15
Effect of xlnR Deletion
0
2000
4000
6000
8000
Wt Glu Wt Straw XlnR Glu XlnR Straw
cbhA
cbhA
0
50
100
150
200
250
300
350
Wt Glu Wt Straw XlnR Glu XlnR Straw
gh61
gh61
Rel
ativ
e ex
pre
ssio
n
Rel
ativ
e ex
pre
ssio
n
Glucose Straw
48h 24h
0
100
200
300
400
500
600
700
Wt Glu Wt Straw XlnR Glu XlnR Straw
cbhB
cbhB
Rel
ativ
e ex
pre
ssio
n
(Early)
(Late)
(Late)
•All activated by XlnR
•Late-induced gene induction entirely
XlnR-dependent
•Still some induction of the early
expressed genes in the ΔxlnR strain
16
0
0,2
0,4
0,6
0,8
1
1,2
1,4
Wt Glu CreA Glu
cbhA
cbhB
gh61
p=0.0005
Effect of creA Deletion
Rel
ativ
e ex
pre
ssio
n
Glucose
48h
•In the ΔcreA strain the early-expressed gene cbhB is expressed at a
significantly higher level in glucose
•Early-expressed genes may be those that have a high level of basal
expression in the absence of catabolite repression, without activated XlnR
•In the presence of glucose all
are repressed in the Wt strain
17
0
5
10
15
20
Wt Glu CreA Glu
abfB
abfB
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
Wt Glu CreA Glu
gh5
gh5
p=0.04
p=0.01
Can We Predict Other Early Induced Genes?
•Other genes also display the high basal
expression in the ΔcreA strain
•Are they also expressed early after the
transfer to straw
GH61
1% Glucose
48 h
Time after transfer / h
0.5 1 2 3 6 9 12 24
abfB Early
Induced by
carbon starvation
Straw
No Carbon Source
GH61
abfB
Late
Non-responsive
Early Expressed Genes Are Induced by
Carbon Starvation
GH61
1% Glucose
48 h
Time after transfer / h
0.5 1 2 3 6 9 12 24
abfB Early
Induced by
carbon starvation
Straw
No Carbon Source
GH61
abfB
Late
Non-responsive
•Currently defining the full set of early induced genes through RNA-seq.
Interestingly, those found to date all contain a CBM
•Making targeted deletions to test the hypothesis that the early response is
required for the release of inducing molecules to induce the main response
Early Expressed Genes Are Induced by
Carbon Starvation
20
Expression of Non CAZy Genes Induced > 20x
and Expressed > 50 RPKM in Straw
21
Expression of Non CAZy Genes Induced > 20x
and Expressed > 50 RPKM in Straw
The CAZy Genes Expression of A. niger and
T. reesei Define a Core Set of Activities
2x hydrophobin and a swollenin are also induced and expressed to a high level in T. reesei
PTH11-like, membrane Proteins
Involved in Signalling?
• G-protein coupled receptors
•Involved in host surface
recognition during invasion of rice
by Magnaporthe grisea
•Sensing of hydrophobicity and/or
cutin-monomers
PTH11-like, membrane Proteins
Involved in Signalling?
• G-protein coupled receptors
•Involved in host surface
recognition during invasion of rice
by Magnoportha grisea
•Sensing of hydrophobicity and/or
cutin-monomers
1% Glucose
48 h
Time after transfer to straw / h
0.5 1 2 3 6 9 12 24
pth11
An16g01780
• 2 A. niger homologues are highly
induced upon straw (but not by
glucose or carbon starvation)
• Induced very early
•Creating deletion strains in each
TID_188224 - hsbA
1% Glucose
48 h
Time after transfer to straw / h
0.5 1 2 3 6 9 12 24
HsbA and Hydrophobin Homologues Recruit
Degradative Enzymes
Induction is concurrent with major hydrolase expression
1% Glucose
48 h
Time after transfer to straw / h
0.5 1 2 3 6 9 12 24
hsbA
cbhA
GH61
coH1
GH5
abfB
•Two of the hydrophobic binding
proteins are induced around the same
time as the majority of CAZy enzymes
•Hydrophobins are also induced in
response to wheat straw in T. reesei
hsbA and coh1 are Induced During the Second
Wave of Hydrolytic Enzyme Expression
Hyp1 and Coh1 Enhance Degradation of
Arabinoxylan and Soluble Cellulose
Glucose Straw
48h
+ AZCL Marked Complex
carbohydrate
Knife milled straw + AZCL-Arabinoxylan
Re
lati
ve D
ye r
ele
ase
aft
er
16
ho
urs
WT Δcoh1 Δhyp1 ΔxlnR
•Constructed deletion strain of 2/3 of
the induced hydrophobic binding
proteins
•Some decrease in degradation
efficiency, but phenotype is not strong
•Creating double and triple mutant
strains currently as well as targeting
potential regulator
•Collaborating with Prof. K. Abe to express HsbA in A. oryzae and characterise binding to
wheat straw and enzyme recruitment
29
Conclusions
1. Both A. niger and T. reesei respond to wheat straw at the
transcriptional level.
2. Both fungi (and others examined) up-regulate transcription of target
genes in a time-dependent manner, i.e. there is succession of
responses.
3. The regulatory responses are: detection of the straw, starvation /
de-repression of CreA (Cre1) to produce CBM-containing enzymes,
induction by XlnR (Xyr1) to produce the bulk response.
4. Not all up-regulated gene expression is CAZy-related. Genes
encoding surface-binding proteins are up-regulated.
5. Different genes encoding surface response proteins are up-
regulated in A. niger and T. reesei.
6. The model suggests ways in which the functionality of current
cellulase-rich enzyme cocktails might be improved.
Acknowledgments
• University of Nottingham
• LACE Strand 1 :
Sanyassi Gaddipati, Roger Ibbett, Greg Tucker
• Deep-seq_QMC
Sunir Malla, Martin Blythe, Aziz Aboobaker
Companies: DSM, Roal Oy
Thank you
HPLC analysis of culture media
HPLC analysis of culture media