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ARPAe Carbon workshop
September 26, 27, 2019G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Carbon Optimized Bioconversion Platforms for the
Bioeconomy
An ARPA-E WorkshopChicago, IL
September 26, 27, 2019
Fixing and converting CO2 and
renewable electrons to liquid fuels
Gregory Stephanopoulos
MIT
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ARPAe Carbon workshop
September 26, 27, 2019G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Point 1:
What is the cost of electrons?
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ARPAe Carbon workshop
September 26, 27, 2019G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Conclusion 1:
Hydrogen at $1/kg is equivalent to
glucose at $130/ton (5c/lb)
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ARPAe Carbon workshop
September 26, 27, 2019G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Point 2:
A two=stage process for production of
lipid (or alkanes)
Presented and demonstrated in
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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5
Two-stage system for CO2 fixation into lipids
CO2
H2
acetate
Moorellathermoacetica
Yarrowialipolytica
lipids
Goal: Produce an infrastructure compatible fuel (biodiesel) from CO2 and H2
Asset: Oleaginous microbe with extremely high yields, productivities, and titers
Strategy: Fix CO2 and H2 by acetogenic bacteria and feed acetate so produced
to Oleaginous microbe
Challenges: Achieve high rates of growth of acetogenic bacteria, and acetate
production
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ARPAe Carbon workshop
September 26, 27, 2019G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Integrated production of acetate and
lipid from waste gas
CO2
H2 Acetate lipid
Moorellathermoacetica Yarrowia lipolytica
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ARPAe Carbon workshop
September 26, 27, 2019G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Alternative feed stocks
Glucose is expensive, may compete with food
Acetic acid is interesting alternative. Can be sourced
at large volumes from: Anaerobic digestion
Fixation of CO2 with CO or Hydrogen using anaerobic
acetogenic bacteria
Oxidation of methane using novel Rhodium catalyst (Tufts
Univ. Nature, 2018)
VFA Yarrowia
Fermentation
Trash
Gases (CO2 + H2, CH4)
TTF: Anaerobic Digestion
GTL: Anaerobic Fermentation, or,
catalytic conversion of methane Lipids, Green Diesel,
Biodiesel
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ARPAe Carbon workshop
September 26, 27, 2019G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Point 3:
Convert dilute acetic acid to
lipids at high yields and
productivities
Jingyang Xu, Nian Liu, K.J. Qiao, S. Vogg and G. Stephanopoulos, “Application of metabolic controls for
maximization of lipid production in oleaginous yeast,” Proceedings of the US National Academy of Sciences,
DOI: :10.1073/pnas.1703321114/-/DC Supplemental (2017)
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ARPAe Carbon workshop
September 26, 27, 2019G. 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
K.J. Qiao, T.M. Wasylenko, K. Zhou, P. Hu and G. Stephanopoulos, “Rewiring metabolism to
maximize lipid production in Yarrowia lipolytica,” Nature Biotechnology, DOI: 10.1038/nbt.3763 (2017)
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4. Feed based on pH control, metabolic model, RQ
Jingyang Xu, Nian Liu, K.J. Qiao, S. Vogg and G. Stephanopoulos, “Application of
metabolic controls for maximization of lipid production in oleaginous yeast,” Proceedings
of the US National Academy of Sciences, DOI: :10.1073/pnas.1703321114/-
/DCSupplemental (2017).
Purdue-Biochemical Horizons
November 17, 2017G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
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4. Optimization of Nitrogen feed based on RQ/CTR feedback control
• Working volume 1.5 L• Maintain carbon at zero
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ARPAe Carbon workshop
September 26, 27, 2019G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Conclusion 3:
It is possible to convert dilute VFA to
very dense intracellular oil that is
easily separated by filtration and
extraction
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ARPAe Carbon workshop
September 26, 27, 2019G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Point 4:
Exploring synergies by substrate co-feeding
See
J.O. Park, Nian Liu, K.M. Holinski, D.F. Emerson, K.J. Qiao, B.M Woolston, J. Wu, Z.
Lazar, M.A. Islam, C. Vidoudez, P.R. Girguis and G. Stephanopoulos, “Synergistic substrate
cofeeding stimulates reductive metabolism,” Nature Metabolism (2019)
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14
Some issues must be resolved when using acetic acid
3-HB
2 AcCoA + 1 NADPH per elongation
“carbon intensive”
Glucose Acetate
Ymax,PHB 0.48 g/g 0.67 g/g
Ymax,C18:0 0.28 g/g 0.28 g/g
Ymax,C18:0+biomass 0.21 g/g 0.16 g/gNADPH
NADH
ATP
ATP
NADPH
ATP ATP
AcCoA
AcCoA
CO2
CO2
Fatty acid
1 AcCoA + 2 NADPH + 1 ATP per elongation
“Primarily cofactors”
CO2
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Can we help the cells?
oxPPP
15
Acetate
GNG
Glycerol
Fructose
Glucose Gluconate
Z. Lazar et al. Trends in Biotechnology 2018N. Liu et al. Metabolic Engineering 2016
NaAceglucose
23 μmol/hr
(+30% oxPPP)
1.2 mmol/hrTCA
FA
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Glcn provides NADPH through recursive use of oxPPP
PGI
G6PDH
GND
PPP
16
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Sourcing the acetate from CO2
(2CO2+4H2 CH3COOH+H2O)
17
CO2
fixation
ATPe-
Acetate
Glucose
CO2
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18
Glucose
Pyruvate2X
2e-
ATP
Biomass precursors
2e-CO2
Glycolysis
-CH2-O=CH- =CH-
H2O
PO43-
ADP
O=C=O -CH3
THF
ATP
Co-CH3
C≡O
PO43-
ADP
CoA
ATP
2e- 2e- 2e-
Formyl
-THFMethenyl
-THF
Methylene
-THF
Methyl
-THF
Acetic acid
Methyl branch
Ca
rbo
nyl
bra
nch
Wood-Ljungdahl
pathway
Decouple e- supply from glucose decarboxylation
GlucoseH2
ATP
CO2
fixation
e-
CO2
Media(no organic carbon)
Glucose
CO2/H2
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Synergistic cofeeding enhances CO2 fixation
19
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20
H2 (100%)
Acetic acid (95%)
Biomass
(14%)
Lipids (38%)
Synergistic cofeeding enables efficient lipid synthesis from CO2
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ARPAe Carbon workshop
September 26, 27, 2019G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Conclusion 4:
Many fold increases in CO2 fixation
and acetate production are possible
with judicious substrate co-feeding.
Acetate production can surpass that
of ethanol by yeast
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ARPAe Carbon workshop
September 26, 27, 2019G. Stephanopoulos, MITMEL- Metabolic Engineering
Laboratory
Acknowledgements
1. Cofeeding: Nian Liu, Jun park
2. Yarrowia Engineering: K.J Qiao, Mitchell Tai
3. CO2 fixation: Jingyang Xu, Peng Hu, David
Emerson
4. Funding: US Department of Energy, ARPAe