improving product yield and robustness by metabolic
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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
2nd generation processes
Ro
yal N
edal
co
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
Dani González-Ramos et al. 2016 Biotechnol Biofuels
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
Dani González-Ramos et al. 2016 Biotechnol Biofuels
(‘If I can rebuild it, I do not necessarily understand it’)
After the ‘pentose rush’ challenges in yeast-based bioethanol production
• 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
2nd generation processes
flee
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
Ioannis Papapetridis et al. 2018 Biotechnology for Biofuels
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)
Ioannis Papapetridis et al. 2016 Biotechnology for Biofuels
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
Ioannis Papapetridis et al. 2017 Biotechnology for Biofuels
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
Ioannis Papapetridis et al. 2017 Biotechnology for Biofuels
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
2nd generation processes
Ro
yal N
edal
co
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
Ioannis Papapetridis, Maarten Verhoeven et al. 2018 FEMS Yeast Research
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
Ioannis Papapetridis, Maarten Verhoeven et al. 2018 FEMS Yeast Research
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
Ioannis Papapetridis, Maarten Verhoeven et al. 2018 FEMS Yeast Research
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
Maarten Verhoeven et al. 2018 FEMS Yeast Research
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…
Maarten Verhoeven et al. 2018 FEMS Yeast Research
Unexpected interactions of consortium partners additional laboratory evolution required
ara
xyl
evolution of consortium
Maarten Verhoeven et al. 2018 FEMS Yeast Research
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)
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
Ioannis Papapetridis et al. 2016 Biotechnology for Biofuels
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 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
Ioannis Papapetridis et al. 2016 Biotechnology for Biofuels
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 %
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