improving product yield and robustness by metabolic

Yeast-based ethanol production improving product yield and robustness by metabolic engineering

Jack Pronk Delft University of Technology

The Netherlands

Chemicals from plant carbohydrates a century-old vision of a ‘bio-based’ chemical industry

inaugural address Prof. Albert Jan Kluyver Delft, January 18, 1922

www.wkimedia.org

Industrial (yeast) biotechnology towards sustainable ‘bio-based’ production of fuels and chemicals

ethanol

sucrose

Fuel ethanol production from plant carbohydrates ca. 100 Mton per year made with Saccharomyces cerevisiae

glucose

revi

stap

esq

uis

a.fa

pes

p.b

r

bee

f.un

l.ed

u

glucose, xylose, arabinose

agw

eb.c

om

wik

iped

ia.o

rg

ethanol ethanol

‘first generation - established’ ‘second generation - emerging’

Early days: serendipity hits the fan enter the elephant

PPP

pyruvate

D-xylulose-5-P

D-xylose

D-xylulose ethanol

glucose

ATP

NADH

CO2

ATP

glucose-6-P

fructose-6-P

fructose-1,6-biP

DHAP G-3-P

PEP

NADH

ATP

ATP

ATP

glycerol

NADH

CO2

2 NADPH

XI

• xylose isomerase (XI): key enzyme in (bacterial) xylose metabolism • 2003: many bacterial XI genes tested, no efficient expression in yeast

Colleagues in Nijmegen find XI gene in Piromyces fungus from elephant dung –

expression yields high activity in yeast

Harry Harhangi et al. 2003 Arch Microbiol

Marko Kuyper et al. 2003 FEMS Yeast Research

Marko Kuyper

PPP

pyruvate

D-xylulose-5-P

D-xylose

ethanol

glucose

NADH

CO2

ATP

glucose-6-P

fructose-6-P

fructose-1,6-biP

DHAP G-3-P

PEP

NADH

ATP

ATP

ATP

(xylA)

(XKS1 / XYL3)

D-xylulose

ATP

(araB)

L-arabinose

L-ribulose L-ribulose-5-P

ATP

(araD)

(araA)

Wouter Wisselink et al. 2007, 2009, 2010 Appl. Environ. Microbiol.

Marko Kuyper et al. 2004, 2005 FEMS Yeast Research

2005-2010: design, build, evolve, test isomerase-based pathways in pentose-fermenting strains

Becker and Boles 2003 Appl. Environ. Microbiol.

NADH

NADH

introduce heterologous pentose isomerase pathways

overexpress

pentose-phosphate pathway

evolve in laboratory for fast fermentation

Wouter Wisselink

Fast anaerobic fermentation of glu, xyl, ara mixtures from academia to industry

Time (h)

Sugars

, eth

anol (g

/L)

Bio

mas

(g/L

)

Glu

Ara

X

2009

• 2014: DSM-POET open full-scale plant in Emmetsburg, Iowa • Capacity ca 100 million liters of ethanol per year from corn stover • 2019: production on hold because of USA political/economic context

Mickel Jansen et al. 2017 FEMS Yeast Research

Wouter Wisselink et al. 2009 Appl. Environ. Microbiol.

2005 S. cerevisiae RWB217 XKS1 TAL1 TKL1 RPE1 RKI1 gre3 xylA (pAKX002) Multi-step strain construction (> 2 years)

Jasmine Bracher et al. 2019 FEMS Yeast Research

2019 S. cerevisiae IMU079 XKS1 TAL1 TKL1 RPE1 RKI1 gre3 xylA (pAKX002) Single-step construction < 2 weeks) (Cas9, in vivo assembly)

Marko Kuyper et al. 2005 FEMS Yeast Research

Acceleration of S. cerevisiae strain engineering in vivo assembly, CRISPR-Cas9, quality control by sequencing

Jasmine Bracher

After the ‘pentose rush’ challenges in yeast-based bioethanol production

Academic synthetic medium

Industrial corn stover hydrolysate

• Acetic acid tolerance in 2nd generation ethanol production

2nd generation processes

• Strategies for improving ethanol yield on feedstock 1st and 2nd generation processes

• Improving kinetics and genetic stability of mixed-sugar utilization


Ro

yal N

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Full acetic acid tolerance requires pre-adaptation in non-evolved, non-engineered S. cerevisiae

Pre-adaptation: preculture supplemented with 9 g/l acetate (pH 4.5)

Dani González-Ramos et al. 2016 Biotechnol Biofuels

Acetic acid • Integral component of

plant biomass • Strong inhibitor of

yeast performance

‘On-off’ evolution strategy for constitutive tolerance alternating batch cultures with and without acetic acid stress


1 2

Dani Ramos

Identification of causal mutations for constitutive acetic-acid tolerance of evolved strains

Whole-genome sequencing 5 - 21 single-nucleotide mutations per evolved strain 6 genes affected in multiple strains

Classical genetics Mutated alleles of 4 genes (ASG1, ADH3, SKS1, GIS4)

co-segregate with high acetic-acid tolerance

Reverse engineering Constitutive acetic-acid tolerance approaches that

of evolved strains


(‘If I can rebuild it, I do not necessarily understand it’)







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tsan

dfu

els.

com

Carbohydrate feedstock accounts for up to 70 % of production costs

Improving ethanol yield on feedstock tapping into biosynthesis-derived NADH

NADH

Generation of NADH from anaerobic

biomass formation

Glycerol as ‘inevitable’ byproduct ca. 4 % loss of sugar feedstock in industrial ethanol production

glucose

G-6P

F-6P

GAP DHAP

G-3P

glycerol

biomass

NADH

ATP

ATP

Pi

ethanol + CO2

NADH

NADH

2 ATP

Pi

Reoxidation of NADH by glycerol production

Torben Nissen et al. 2000 Metabolic Engineering

Expression of Calvin-cycle enzymes in yeast for improved bioethanol yield on sugar

Victor Guadalupe et al. 2013 Biotechnology for Biofuels

• CO2 as ‘redox sink’ • Proof-of-principle strain: 13 %

higher ethanol yield on sugar in anaerobic chemostat cultures

• Suboptimal growth rate and performance in batch cultures

Victor Guadalupe Medina

Expression of Calvin-cycle enzymes in yeast use of CO2 as electron acceptor

NADH

glucose

G-6P

F-6P

GAP DHAP

G-3P

glycerol

biomass

NADH

ATP

ATP

Pi

ethanol + CO2

ATP

Pi

ribulose-5P

ribulose-1,5-diP

ATP

NADH

3-PG

ATP

NADH CO2

PPP

NADH

PRK

Rubisco

Express spinach prk from anaerobically induced promoter

Integrate 9 Thiobacillus cbbm (Rubisco) expression cassettes

Express 2 E. coli chaperonin genes (groEL/ES)

Over-express 6 yeast genes to improve Rib5P supply

Delete GPD2 to reduce competition for NADH

Ioannis Papapetridis et al. 2018 Biotechnology for Biofuels

Victor Guadalupe et al. 2013 Biotechnology for Biofuels

Ioannis Papapetridis

Expression of Calvin-cycle enzymes in yeast performance of optimized strain in anaerobic batch cultures

IMX1443

GPD1 gpd2Δ

pDAN1-prk 9*cbbm

groEL/ES, PPP↑

IME324

‘wild type’

15 % higher ethanol yield

87 % lower glycerol yield

Near-wild-type growth rate


Acetyl-CoA as electron acceptor for NADH oxidation inspired by ‘duo teaching’ a 2nd-year BSc class with Ton van Maris

Reduction of acetyl-CoA an alternative approach for improving ethanol yield

NADH

glucose

G-6P

F-6P

GAP DHAP

G-3P

glycerol

NADH

ATP

ATP

Pi

ethanol + CO2

ATP

Pi

acetyl-CoA

acetaldehyde

NADH

3-PG

ATP

NADH

NADH

Victor Guadalupe et al. 2010 Appl Environ Microbiol

9 % higher ethanol yield on sugar in anaerobic cultures • Less inhibitor (acetate) • Less byproduct (glycerol) • More product (ethanol)

NADH

acetate

ethanol

Acs1,2

Adh’s

Gpd1,2

Express E. coli A-ALD (acetylating acetaldehyde dehydrogenase)

Delete GPD1, GPD2 (inactivate glycerol pathway)


Reduction of acetic acid tackling osmosensitivity of gpd1 gpd2 strains

NADH

glucose

G-6P

F-6P

GAP DHAP

G-3P

glycerol

NADH

ATP

ATP

Pi

ethanol + CO2

ATP

Pi

acetyl-CoA

acetaldehyde

NADH

3-PG

ATP

NADH

NADH

NADH

acetate

ethanol

Acs1,2

Adh’s

Replace native Gpd’s by NADPH-dependent Archaeoglobus fulgidus gpsA, controlled by GPD1 promoter. uncouple osmo-protectant and redox roles of glycerol


NADPH

glucose

acetate

glycerol

Anaerobic growth on 1 M glucose of S. cerevisiae IMX901

gpd1Δ gpd2Δ ald6Δ eutE gpsA

Osmotolerant acetate-reducing strain replacement of Gpd1,2 by NADP+-dependent gpsA

Reference strain IMX901

Ethanol yield (g/g glucose) 0.43 ± 0.01 0.49 ± 0.00 Glycerol yield (g/g glucose) 0.07 ± 0.00 <0.001 Acetate conversion (g/g glucose) 0.011 ± 0.001 0.027 ± 0.003



Academic synthetic medium

Industrial corn stover hydrolysate






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Mixed-sugar fermentation kinetics a key challenge in process intensification

Anaerobic batch culture of engineered, xylose-fermenting S. cerevisiae strain (non-evolved) on mixture of 20 g/l glucose and 10 g/l xylose)

XKS1↑ PPP↑ XI↑ gre3Δ

Slow xylose fermentation phase - Decreased volumetric productivity - Decreased inhibitor tolerance

Ioannis Papapetridis, Maarten Verhoeven et al. 2018 FEMS Yeast Research

glu

xyl

NADPH

A yeast strain design for forced glucose-xylose co-consumption


NADPH

glucose

G-6P

F-6P

GAP DHAP

ATP

ATP

Pi

ATP

Pi

3-PG

ATP

6-PG ru-5P

rib-5P

xylose

xylulose

xu-5P

se-7P

er-4P

xu-5P

NADH

NADH

pyruvate

rpe1Δ

pgi1Δ gnd1Δ gndA↑

ATP

• Deletion of PGI1 and RPE1 • Expression of XI-based xylose pathway (incl PPP↑) • NAD-dependent Gnd to prevent excess of NADPH • Model prediction (aerobic growth): qxyl : qglu = 1.4 : 1

ATP

TCA cycle

Maarten Verhoeven

Laboratory evolution of strain engineered for forced xylose-glucose co-consumption


Glucose-xylose co-consumption in aerobic batch cultures of evolved strain

glu

xyl

xyl : glu = 1.6 : 1

Co-consumption by xylose-fermenting strain upon introduction of mutations found in evolved ‘forced’ strains

XKS1↑ PPP↑ XI↑ gre3Δ

glu

xyl

• Whole genome sequencing of evolved ‘forced co-consumption’ strains • Reverse engineering of mutations into non-evolved xylose-fermenting strain • Analysis in anaerobic bioreactor cultures on glucose-xylose mixture

glu

xyl


hxk2Δ gal83::GAL83G673T

Consumption of glucose-xylose mixture in anaerobic bioreactor cultures of xylose-fermenting S. cerevisiae strains

Alternative approach to mixed-sugar utilization: division of labour consortia of specialist strains for fermentation of sugar mixtures

Maarten Verhoeven et al. 2018 FEMS Yeast Research

G X A

D-glucose L-arabinose D-xylose

ethanol

+ +

Potential advantages of consortia over ‘generalist’ strains: • Resource allocation to single pathway should enable faster fermentation • Stability of strain performance during repeated batch cultivation (no selection for ‘glucose specialists’)

Degeneration of ‘generalist’ strain performance repeated batch cultivation in anaerobic bioreactors

Glucose-xylose-arabinose generalist strain IMS0010 (previously constructed and evolved): a. Initial growth cycle in anaerobic bioreactor – 20 g/L glucose, 10 g/L xylose, 5 g/L arabinose b. Increased cycle duration during 36 cycles of repeated batch cultivation on same mixture c. Deteriorated fermentation pentose fermentation kinetics after 36th cycle


Selecting consortium members Laboratory evolution of individual pentose specialists on sugar mixtures

Glucose specialist: laboratory strain

Xylose specialist: hexose-phosphorylation-negative xylose-fermenting strain evolved for anaerobic growth on glucose-xylose-arabinose mixture (20 g/L each)

Arabinose specialist: hexose-phosphorylation-negative arabinose-fermenting strain evolved for anaerobic growth on glucose-xylose-arabinose mixture (20 g/L each)

Evolving a xylose specialist…


Unexpected interactions of consortium partners additional laboratory evolution required

ara

xyl

evolution of consortium


Stable/improved performance of consortium during prolonged repeated batch cultivation on sugar mixtures

Repeated anaerobic batch cultivation of consortium

20 g/L glu, 10 g/L xyl, 5 g/L ara

Repeated anaerobic batch cultivation of generalist strain 20 g/L glu, 10 g/L xyl, 5 g/L ara

Conclusions

• Large-scale industrial application of yeasts for 2nd generation bioethanol production is technically feasible (but meets some economical/political headwind)

• Integrated engineering of carbon and redox metabolism enables improvements

in ethanol yield (acetyl-CoA reduction, Rubisco)

• Combination of laboratory evolution and targeted strain engineering is a

powerful approach (mixed-substrate utilization, tolerance, consortia)

• Division of labour is an extremely interesting approach for improving stability and

performance of microbial processes

Research in the Industrial Microbiology group @TUDelft is financially supported by:

graciasthanksdziękujębedanktmercidankeobrigado Colleagues, students and collaborators in Delft and elsewhere

Fellow PI’s Pascale Daran-Lapujade

Jean-Marc Daran Robert Mans

Ton van Maris (now at KTH Stockholm)

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Reduction of acetyl-CoA also in the absence of external acetic acid

NADH

glucose

G-6P

F-6P

GAP DHAP

G-3P

glycerol

NADH

ATP

ATP

Pi

ethanol + CO2

ATP

Pi

acetyl-CoA

acetaldehyde

NADH

3-PG

ATP

NADH

NADH

NADH

ethanol

Adh’s

Gpd1,2

Express E. coli A-ALD (acetylating acetaldehyde dehydrogenase)

Engineer redox-neutral pathway from glucose to acetyl-CoA (e.g. via pyruvate-formate lyase)

Decrease in vivo flux towards glycerol

Improving ethanol yield and acetate conversion in acetate-reducing strains

NADH

glucose

G-6P

F-6P

GAP DHAP

G-3P

glycerol

NADH

ATP

ATP

Pi

ethanol + CO2

ATP

Pi

acetyl-CoA

acetaldehyde

NADH

3-PG

ATP

NADH

NADH

NADH

acetate

ethanol

Acs1,2

Adh’s


NADPH

6-PG PPP

NADPH NADH

Delete ALD6 – block alternative source of NADPH

NADPH

Ald6

Make 6-PG dehydrogenase reaction NAD+-dependent

Ethanol yield +9.4 % +12.3 %

improving product yield and robustness by metabolic

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