fungal enzymes for saccharification of biomass

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


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


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


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


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


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


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

fungal enzymes for saccharification of biomass

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