Metabolic engineering for improved xylose utilisation of ...
Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang...
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![Page 1: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/1.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Engineering microbes for
production of biofuels and
chemicals
Gregory Stephanopoulos
MIT
Fuel Choices Summit 2016
Habima Theater Tel Aviv, Israel
3 November, 2016
![Page 2: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/2.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Forces of change
![Page 3: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/3.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
What has changed drastically during
the past 25-30 years?
• Continuous increase of the cost of fuels and
raw materials
![Page 4: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/4.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
![Page 5: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/5.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
What has changed drastically during
the past 25-30 years?
• Continuous increase of the cost of fuels and
raw materials
• Strategic challenges in securing the required
amounts of fuels and raw materials
• Grave consequences for climate change
![Page 6: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/6.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Global CO2 emissions
![Page 7: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/7.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Atmospheric Carbon Dioxide
![Page 8: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/8.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
![Page 9: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/9.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
What has changed drastically during
the past 25-30 years?
• Continuous increase of the cost of fuels and
raw materials
• Strategic challenges in securing the required
amounts of fuels and raw materials
• Serious concerns about climate change
• Development of Biotechnology and Metabolic
Engineering: Core technologies for converting
renewable resources to fuels and chemicals
![Page 10: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/10.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Technology advances:
Engineering the metabolism of
microbes to convert them to chemical
factories for the production of biofuels
and chemicals
Biotechnology beyond medicine
![Page 11: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/11.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Cells:
Little chemical
factories with
thousands of
chemical compounds
interconverted
through thousands of
chemical reactions
Main substrate:
Sugars
Products: Virtually
infinite
![Page 12: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/12.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Microorganisms
They αre found
everywhere, from the
human gut to the hot
springs of Yellowstone
Park
![Page 13: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/13.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Rxn
1
Rxn
3
Rxn
2
Rxn
5
Rxn
4
Rxn
6
Pentanol
Substrates
P 2 P 4
P 4
13
CoA activator
CoA remover
HPLC analysis
Engineering microbes to produce any
product
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Types of biofuels and biofuel feedstocks
Ethanol from corn
Biodiesel from plant seeds and vegetable oils
Ethanol from sugarcane
Other feedstocks (not competing with food):
cellulosics, algae
Other biofuels than ethanol (butanol, lipids,
hydrocarbons)
![Page 15: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/15.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Contributions from my lab
1. Improving ethanol tolerance of
yeast
Extensions:
Improving microbial tolerance
to toxicity
1. F.H. Lam, A. Ghaderi, G.R. Fink and G. Stephanopoulos, “Engineering alcohol tolerance in
yeast,” Science, 346: 71-75 (2014)
2. H. Alper, J. Moxley, E. Nevoigt, G.R. Fink and G. Stephanopoulos, “Engineering yeast
transcription machinery for improved bioethanol tolerance and production,” Science, 314:
1565-1568 (2006)
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GIM 2016
October 16-20 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Product toxicity is a major problem in
engineering microbes for production of
biofuels and biochemical products
It is important that studies aiming at
improving tolerance are conducted under
bioprocess-relevant conditions
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EtOH increased upon K-Pi supplementation
K-Pi supplementation
and pump engineering
enhances:
I. Growth despite
accumulating EtOH
II. Tolerance despite
accumulating EtOH
EtOH [g/L] 300 g/L glucose
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Despite accumulating EtOH, KCl+KOH boosts:
I. Cell growth
High KCl+KOH enhance population viability
EtOH [g/L]OD600
70% live
~80%
~11%
98% live
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High KCl+KOH enhance population viability
EtOH [g/L]OD600 VIABLE BIOMASS
~80%
Despite accumulating EtOH, KCl+KOH boosts:
I. Cell growth
II. Tolerance
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Tolerance and titer highly correlated
pH 3.6
pH 5.8
F.H. Lam, A. Ghaderi, G.R. Fink and G.
Stephanopoulos, “Engineering alcohol
tolerance in yeast,” Science, 346: 71-75 (2014)
![Page 21: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/21.jpg)
GIM 2016
October 16-20 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Product toxicity is a major problem in
engineering microbes for production of
biofuels and biochemical products
It is important that studies aiming at
improving tolerance are conducted under
bioprocess-relevant conditions
![Page 22: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/22.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Contributions from my lab
2. Engineering xenobiotic pathways
to prevent contamination
Extensions:
Eliminating the need for the
use of antibiotics
A.J. Shaw, F.H. Lam, M. Hamilton, A. Consiglio, K. KacEwen, E. E. Brevnova, E.
Greenhagen, W.G LaTouf, C. R. South, H. van Dijken, V. Rajgarhia and G. Stephanopoulos,
“Engineering contamination resistance in industrial biosystems,” Science, 353: 583-586 (2016)
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Contributions from my lab
3. Engineering yeast to metabolize
all sugars from biomass hydrolysis
Extensions:
Use of vast amounts of lingo-
cellulosics for biofuels
Hang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase
overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering
enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae,” Metabolic
Engineering, 14: 611-622 (2012)
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24
Biofuel (ethanol) from renewables
Fuel ethanol from corn starch or sugar
Used as such or blended with gasoline
Ethanol from plant biomass
Readily fermentable starch and sucrose
Resistant lignocellulosic fractions
Biofuels
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25
Performance of the S. cerevisiae strains
Strain Description Conditions
Yields g/g Ethanol
production
g·g-1h-1
Xylose
consumption
g·g-1h-1
µmax h-1
Ethanol Xylitol
H131-A31XylA, PsXyl3, PsTal1, TKL1,
RPE1, RKI1Aerobic batch, SDX N/A N/A N/A N/A 0.031±0.022
H131E1-A31Selection of H131-A31, aerobic
sequential batchAerobic batch, SDX 0.200 <0.01 0.034 0.169 0.197±0.006
H131E3-A31Selection of H131E1-A31,
micro-aerobic sequential batch
Anaerobic batch,
2×YNB, 4% xylose0.440 <0.01 0.120 0.273 0.061±0.002
H131E5-A31Selection of H131E3-A31,
anaerobic sequential batch
Anaerobic batch,
2×YNB, 4% xylose0.410 <0.01 0.233 0.568 0.073±0.002
H131E8-A31Selection of H131E5-A31,
anaerobic chemostat
Anaerobic batch,
2×YNB, 4% xylose0.440 <0.01 0.383 0.870 0.120±0.004
H131E8-A31Anaerobic chemostat of
H131E5-A31
Anaerobic
chemostat, YNBX0.438 <0.01 0.641 1.464 0.148
RWB 217 XylA, XKS1, TAL1, TKL1, RPE1,
RKI1, gre3Δ
Anaerobic batch,
synthetic medium0.43 0.003 0.46 1.06 0.09
RWB 218 Selection of RWB 217 Anaerobic batch,
synthetic medium0.41 0.001 0.49 1.2 0.12
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Towards integrated and complete
processes for biofuel and chemical
production from renewable feedstocks
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
http://bioenergy.ornl.gov/papers/misc/bioenergy_cycle.html
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28
τ≈ Millions of years
Carbon cycle
Xτ≈ 1-100 years
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
First and foremost,
biofuels are a feedstock story
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Feedstocks must be cheap and
aggregated
Examples:1. Lignocellulosic biomass
2. Waste solids
3. Waste gases
4. Algae (cheap?)
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31
Potential of biofuels (USA)
70-100 gallons ethanol/dry ton of biomass
42-60 B gallons Ethanol/year or
28-40 B gallons of gasoline equivalent
20-30% of gasoline used
(1 ton of ethanol = 333 gallons, or
1 Gallon = 3 kgs, or 1 B Gallons = 3 M tons)
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
NEW Contributions from my lab
4. Engineering oleaginous yeast for
overproduction of lipids
Extensions:
Open up the potential for Green
Diesel or Renewable Diesel from
biomass or waste
K.J. Qiao, T.M. Wasylenko, K. Zhou, P. Hu and G. Stephanopoulos, “Rewiring metabolism to maximize
lipid production in Yarrowia lipolytica,” Nature Biotechnology (in press) (2016)
Peng Hu, S. Chakraborty, A. Kumar, B. Woolston, H. Liu, D. Emerson, and G. Stephanopoulos,
“Integrated system for biological conversion of gaseous substrates to lipids,” Proceedings of the National
Academy of Sciences, PNAS, doi/10.1073/pnas.1516867113 (2016)
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Rising global diesel demand
0.00
10.00
20.00
30.00
40.00
50.00
1960 1970 1980 1990 2000 2010 2020 2030 2040
Qu
ad
ril
lion
BT
U
Historical Projection
Air transportation
Freight services
US department of transportation, 2016.
US energy information administration, 2016.
High demands in diesel
consumption in both
industries
Diesel consumption in
the U.S. projected to grow
at 400-500 million gallons
per year
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Byproducts
Hydrotreatment
Diesel
Benefits of green diesel
0.00
0.40
0.80
1.20
1.60
2.00
2000 2010 2020 2030 2040 2050
USD
per
gal
lon
Current aviation diesel costs
Projected aviation diesel costs
Jet fuel costs
Jet fuel + carbon costs
Economical Environmental
Ideal physical and chemical properties
Air transport action group, 2011
US energy information administration, 2016
Alternative energy news, 2016
-10 10 30 50
Bioethanol
Biodiesel
Gasoline
Jet fuel
Green diesel
Energy density (MJ/kg)
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
a b c
0.1
0.12
0.14
0.16
0.18
0.2
0.22
0.24
0.26
0.28
0.3
Lip
id y
ield
(g/g
)
**
0
20
40
60
80
100
120
0 20 40 60 80
Lip
ids t
iter
/ D
ry c
ell
weig
ht
(g/L
)
Time (h)
AD DCW
AD Lipid titer
ADgy DCW
ADgy Lipid titer
ADgm DCW
ADgm Lipid titer
0
20
40
60
80
100
120
140
160
0 20 40 60 80 100
Lip
id t
iter
/ D
ry c
ell
weig
ht
(g/L
)
Time (h)
ADgm DCW
ADgm Lipid titer
ADgm-hi DCW
Adgm-hi Lipid titer
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Importance in advancing
Renewable Diesel
Base case: Sugars at $200/ton (~9c/lb)
Feedstock cost of lipids produced from sugars from
renewable biomass:
1. At a yield of 0.18g/g (state of the art):$1,100/ton
2. At a yield of 0.30g/g (our work): $660/ton
3. Oil selling price range: $700-1,100
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Alternative feed stocks
Glucose is expensive
Acetic acid is interesting alternative. Can be
supplied at large volumes from
Anaerobic digestion
Fixation of CO2 with CO or Hydrogen
using anaerobic acetogenic bacteria
VFA Yarrowia
Fermentation
Trash
Gases
TTF: Anaerobic Digestion
GTL: Anaerobic FermentationLipids, Food, Biodiesel
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4. Optimization of Nitrogen feed based on RQ/CTR feedback control
• Working volume 1.5 L• Maintain carbon at zero
![Page 40: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/40.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Importance of waste utilization
1. Waste generation: 1 ton/person (US, 2011)
2. Fermentable fraction: 25% (US)-50% (China)
(use 35%)
3. Potential for 3-5B gallons diesel/year (USA)
4. Cost of waste: can be negative at $100/ton
5. Potential depends on capacity to aggregate
waste economically
![Page 41: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/41.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Algae
Gallons GE/acre/year
Soybeans 48
Sesame 74
Jatropha 202
Cellulosic (for ethanol production) 533
Sugarcane (for ethanol) 566
Algae ~6,000
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Biofuel production by direct
photosynthesis
Algae
Sun
Biomass
Oil-alkane
production
Other biofuels
(ethanol)
Productivities are high
but cultures very dilute
Key challenge:
Cost-effective dewatering
Just growth
Metabolic Engineering;
Secretion?
Oil recovery
![Page 43: Engineering microbes for production of biofuels and chemicals Stephanopoulos - Award Winner.pdfHang Zhou, J.-S. Cheng, B. Wang, G. R. Fink and G. Stephanopoulos, “Xylose isomerase](https://reader030.fdocuments.net/reader030/viewer/2022040908/5e7ecdf9ac37ff1f9f4a9450/html5/thumbnails/43.jpg)
Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
A final word about chemicals
It is now possible to produce commodity
chemicals (as well as, of course, specialty
chemicals):
1. With cost-effective processes
2. Using renewable feedstocks
3. Small, efficient specialized units
4. Examples: Ethylene glycol, glycolic acid,
biopolymers, organic acids (diacids), others
B. Pereira, Zheng-Jun Li, M. De Mey, C.G. Lim, H. Zhang, C. Hoeltgen and Gregory Stephanopoulos,
“Efficient utilization of pentoses for bioproduction of the renewable two-carbon compounds ethylene glycol
and glycolate,” Metabolic Engineering, 34: 80-87, (2016); dx.doi.org/10.1016/j.ymben.2015.12.004 (2015)
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Fuel Choices Summit 2016
3 November 2016G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory