Johan Thevelein

KU Leuven

Lab of Molecular Cell Biology

New Delhi

12 March 2019

Development of yeast

strains for production of

second-generation

bioethanol and bio-based

chemicals

2G bioethanol and bio-chemicals production

Bioenergy crops

Waste streams

Forest residues

Recycled paper

Corn cob & stover

Bagasse

Wheat straw

Empty fruit bunches + many other potential feedstocks

Rice straw

• 1G Bioethanol

• Sugar

• Biomass

1G and 2G bioethanol production

1G YEAST

2G YEAST • 2G Bioethanol

Main challenges

• Efficient xylose fermentation

• High inhibitor tolerance

Engineering of S. cerevisiae for xylose fermentation

Eubacterium species: VIB/GlobalYeast (IP)

Two pathways

• Xylose reductase (fungal pathway):

redox problem xylitol accumulation

• Xylose isomerase (bacterial pathway)

difficult expression in yeast

Xylose isomerase expression in yeast

• Proprietary xylose isomerase

• Many gene copies high xylose isomerase activity

• Few other xylose isomerases are active in yeast very tight IP space small

number of commercial proprietary 2G bioethanol strains

• proprietary 2G yeast: bottleneck 2G bioethanol + 2G bio-based chemicals

Engineering of S. cerevisiae for high inhibitor tolerance

• Acetic acid

• Furfural and hydroxymethylfurfural corresponding alcohols

high toxicity low toxicity

medium

HAc

H+ + Ac- pH ±5

pH ±7

ATP ATP

Reductases

Export pumps

Improvement by targeted and random engineering:

(site-directed) mutagenesis, evolutionary adaptation, genome shuffling,

whole-genome transformation

Cheaper pretreatment methodologies higher inhibitor

levels more robust yeast cheaper process

Pooled

Extraction of genomic DNA

Whole-genome sequence analysis (Illumina)

Polygenic analysis platform for complex traits:

pooled-segregant whole-genome sequence analysis

2 parent strains QTL mapping (Quantitative Trait Locus)

min. ± 30 segr.

Screen of S. cerevisiae strain collection

Diploid strain with superior trait

Industrial diploid strain with inferior trait

Acetic acid tolerance of fermentation

Known:

Haa1: transcription factor

involved in acetic acid

tolerance

New:

Cup2: homolog of Haa1

Dot5

Glo1

Vma7

F1 segregants

F7 segregants

HAA1* : unique

mutation in acetic

acid tolerant strain

0% Acetic acid 1% Acetic acid

2.0% Acetic acid 1.6% Acetic acid

1.4% Acetic acid 1.2% Acetic acid

Insertion of G A mutation in HAA1 (2 alleles) of T18

● T18 HAA1*

○ T18

Predictable

improvement of stress tolerance

Industrial strains with high xylose fermentation capacity

Further improvement by evolutionary adaptation, genome shuffling, targeted genetic engineering with superior alleles, whole-genome transformation steady improvement of performance

ethanol glucose

xylose

Semi-anaerobic static fermentation in bagasse

hydrolysate

Goal for commercial E2G production

> 80% of sugar in 48 h with 1 g DW yeast/L and > 5% (v/v) ethanol titer

2G bioethanol and bio-chemicals production

Other major challenges

• Pretreatment of the biomass robustness of machinery

• Enzymatic hydrolysis ±20% of the cost of the ethanol

E2G yeast with secreted enzymes

- reduce enzyme requirement

- Holy grail: ‘Consolidated bioprocessing’

yeast: enzymatic hydrolysis + fermentation

Cellulolytic enzymes:

• β-glucosidase (BGL)

• Endoglucanase (EG)

• Cellobiohydrolase I (CBH I)

• Cellobiohydrolase II (MCBH II)

Hemicellulolytic enzymes:

• β-xylosidase (β-XYL)

• Xylanase (XYN)

Types of enzymes required

0 10 20 30 400

1

2

3

4

5

6

Time (h)

Co

mp

on

en

t (%

)

HPLC analysis of components during fermentation by MD4 and AC1 at 35°C

YP + 5% Glucose + 4% Xylose + 1% Cellobiose

Cellobiose AC1

Glucose AC1

Xylose AC1

Ethanol AC1

Cellobiose MD4

Glucose MD4

Xylose MD4

Ethanol MD4

Glucose

Xylose Cellobiose

Ethanol

MD4

MD4 with beta-glucosidase

Secreted ß-glucosidase (SfBGL1) supports cellobiose

fermentation

No negative effect of

ß-glucosidase expression on

glucose or xylose fermentation

MD4

MD4 with beta-glucosidase

Expression of beta-glucosidase results in efficient cellobiose utilization

50 mL YP + Xn4%

35°C

120rpm

0 25 50 75 1000.00

0.25

0.50

0.75

1.00

1.25

1.50

Time (h)

Weig

ht

loss (

%)

Fermentation of YPXn4 by MD4, AC1 and AC235°C, 120rpm, 1g/L

MD4

AC1

AC2.1

AC2.2

MD4: parent

AC1: MD4-TrBGL1

AC2: MD4-TrBGL1-AnXlnD-TrXyn2

AC2 strain: secreted β-xylosidase (AnXlnD) and xylanase

(TrXyn2) support xylan fermentation

AC2: Xylan is consumed

Parent strains do

not degrade xylan

4% Xylan + yeast extract/peptone

0 24 48 72 96 120

144

168

192

216

0

200

400

600

800

1000

Time (h)

mg

/L

PCA

0 24 48 72 96 120

144

168

192

216

0

500

1000

1500

2000

2500

3000

Time (h)

mg

/L

Muconic acid

Production of muconic acid with glucose + xylose mixture

20 mL CSM-FWY +Glucose (2.2%) + Xylose (2%) + EtOH (1%) + citrate buffer (0.1M, pH 5.5) in shaking flasks (300 ml), 250 RPM at 30°C. Start OD660: 1

PCA (500 mg/L) and muconic acid (2300 mg/L) from glucose + xylose

Sugars PCA Catechol Muconic acid

Muconic Acid pathway

Glucose (2.2%) + Xylose (2%)

Conclusions

Efficient industrial yeast strains for second-generation bioethanol production available: xylose utilization + high inhibitor tolerance Can still be improved further for better performance in undetoxified lignocellulose hydrolysates cheaper pretreatment technologies Strong platform for secreted enzyme expression Reduction of enzyme load/cost - Consolidated BioProcessing Strong platform for cell factory strains to produce bio-based chemicals with lignocellulosic biomass

2G bioethanol and bio-chemicals production

Thank you for your

attention