improving formate upgrading by cupriavidus necator
TRANSCRIPT
This presentation does not contain any proprietary, confidential, or otherwise restricted information
BETO 2021 Peer ReviewNL0034713 (2.3.2.111)Improving formate upgrading by Cupriavidus necator
Christopher W. JohnsonNational Renewable Energy LaboratoryMarch 11, 2021CO2 Utilization Technology Session
NREL | 2
Quad chartTimeline• Active Project Duration: 10/1/2018 – 9/30/2021• Total Project Duration: 10/1/2018 – 9/30/2021
FY20 Active Project (FY19-21)
DOE Funding
$0Project was fully funded in FY19
$930,000
Barriers addressed • Ct-H Gas Fermentation Development• Ct-D Advanced Bioprocess Development• Ct-J Identification and Evaluation of Potential
Bioproducts
Project GoalDevelop the natural formatotroph, C. necator, as a robust microbial chassis for efficient formate conversion to value-added products in support of CO2capture.
End of Project MilestoneDemonstrate production of 2 g/L (14 mM) of 2-hydoxymuconate semialdehyde in C. necator using formate as the sole source of carbon and energy.Project Partners
Funding MechanismBioenergy Technologies Office FY19 AOP Lab Call (DE-LC-000L060) – 2018
NREL | 3
Project Overview
Background:• Waste CO2 is generated by petroleum-
based and biobased processes
• CO2 can be electrocatalytically reduced to generate formate/formic acid
• Renewable energy could enable low cost, low GHG production of formate
Anode
Cathode
Formate(HCOO-)
CO2H+
FormateStorage
ElectrocatalyticConversion
Renewableelectricity
e-H+
e-
H2OBiorefinerywaste CO2
Membrane
• Formate has been proposed as a soluble intermediate compound to store carbon and energy that overcomes challenges associated with gaseous intermediates (solubility, mass transfer, safety, storage, transport…)
NREL | 4
Project Overview
Background:
• A microbial host capable of robust conversion of formate would enable the production of fuels and chemicals and bring value to CO2
• Cupriavidus necator• Natural formatotroph• Robust formate assimilation• High cell densities (≥200 g/L)• Well studied• Amenable to genetic manipulation• Proven industrial host (≥200,000 L)
FormateWasteCO2
e-C
Microbial Conversion
Ce-
Ce-e-
Low-cost,renewableelectricity
NREL | 5
Project Overview
History:
• This project was funded by a “Rewiring the Bioeconomy: Biological Formate Upgrading Lab Call” issued by BETO in response to guidance from EERE leadership and congress.
• The focus of the call was on strain development for formate conversion:
“This Lab Call is targeting the development of platform organisms that can:1) use formate as their sole carbon source, and 2) subsequently be leveraged to produce fuels and chemicals.”
• While conversion of CO2 to formate is ultimately important to this concept, the FOA did not specify or encourage a specify a specific source of formate
• Part of BETO focus on C1 intermediates for CO2 conversion, but less developed (lower TRL) than other technologies such as CO fermentation
NREL | 6
Project Overview
Aims:
• Improve formate assimilation via the native pathway (≥ 1.2X)• Improve formate assimilation via more efficient synthetic pathway (≥ 1.1X)• Demonstrate improved conversion of formate to an exemplary product, the
polymer precursor 2-hydroxymuonate semialdehyde (2HMS) (≥ 2 g/L)• Perform techno-economic analysis (TEA) and life-cycle assessment (LCA)
Project Goal: Develop the natural formatotroph
Cupriavidus necator as a robust microbial host
for conversion of formate to value-added
products
NREL | 7
Project Overview
Heilmeier Catechism:
• What are you trying to do?o Develop a robust microbial host for
conversion of CO2-derived formate to value-added products
• How is it done today and what are the limits?o Formate bioconversion not performed at
scale today• Why is it important?
o Future carbon emissions must go negative to avoid global warming beyond 1.5°C
• What are the risks?o Scale up, toxicity of formate/formic acid
Soil leads the solutions for negative emissions in a new climate change report. Soil carbonsequestration was among the cheapest methods with the greatest potential.
BY SABRINA SHANKMAN Follow @shankman
OCT 12, 2018
Capturing CO2 From Air: To Keep Global Warming Under1.5°C, Emissions Must Go Negative, IPCC Says
A Pulitzer Prize-winning, non-profit, non-partisan news organization dedicated to covering climate change, energy and the environment.
NREL | 8
Value Proposition• Enable conversion of formate generated
from low-cost renewable energy and waste CO2 to myriad fuels and chemicals
• Decarbonize or reduce the carbon intensity of processes that generate CO2
Key Differentiators• Biology can generate myriad products with
high selectivity compared to catalytic routes• C. necator is a natural formatotroph and a
proven industrial host, making it an excellent starting point for formate conversion
NREL’s Bioenergy Program Is Enabling a Sustainable Energy Future by Responding
to Key Market Needs
Market Trends
NREL | 9
1. Management
Coordination
• Strain development meetings biweekly
• Postdoc one-on-one biweekly• Meetings with other formate
conversion projects biannually• Ad hoc meetings with TEA/LCA
team
Risks
• Low transformation efficiencyo Sufficiently improved (ORNL)
• Native pathway already optimalo Improvement demonstrated
• Synthetic pathway insufficiento No-Go redirection
Management
• Christopher Johnson: PI• Michelle Reed: Project MgmtStrain Development
• Christopher Calvey, Postdoc • Carrie Eckert, Scientist
Bioprocess Development
• Violeta Sanchez, Scientist• Colin Kneucker, TechnicianTEA/LCA
• Ling Tao, Engineer
NREL | 10
2. Approach
Challenges
• Formate/formic acid is relatively toxic, complicating cultivation• Detection of 2HMS by mass spec is difficult
Go/No-Go Decision
• Demonstrate 1.1X benefit from incorporation of the synthetic formolasepathway or redirect effort toward improving assimilation via native pathway
Overall approach
• Use evolution and rational engineering to improve assimilation of formate by C. necator
• Engineer C. necator to convert formate to 2-hydroxymuconate semialdehyde (2HMS)
• Apply strategies to improved formate assimilation to improve production of 2HMS
NREL | 11
CO2 recycling in a biorefinery at scale
• A typical 40 million gallon/yr starch EtOH biorefinery generates around 15,000 kg CO2/h
• 1,775,205 L of C. necator would be needed to assimilate formate at the same rate*
• A 1.2X improvement in formate assimilation would reduce this by 355,041 L
*Conditions and assumptions: C. necator consumes formate at a rate of 12 mmol/g cells/h and 16% is assimilated (Grunwald, et al., 2014. doi:10.1111/1751-7915.12149). Assumes 100% CO2 is converted to formate. C. necator culture is 100 g/L cells. Fermenters are 2 million L.
3. Impact
C. necator
NREL | 12
3. Impact
Global Impact
• Decarbonizing or reducing the carbon intensity has the potential to reduce the environmental impact and improve the economics of a wide variety of CO2 emitting processes
Industrial• Bring greater value to formate (and CO2)• Improve other processing using C. necator• Anticipate at least one patent application resulting from this work
Scientific
• Anticipate at least two high-impact publications resulting from this work• In preparation: Calvey et al., Adaptive laboratory evolution of C. necator
for improved formate utilization• Contribute to our understanding of formatotrophy• Potential integration with CO2 reduction projects
NREL | 13
4. Progress and Outcomes
Aims:
• Improve formate assimilation via the native pathway (≥ 1.2X)• Improve formate assimilation via more efficient synthetic pathway (≥ 1.1X)• Demonstrate improved conversion of formate to an exemplary product, the
polymer precursor 2-hydroxymuonate semialdehyde (2HMS) (≥ 2 g/L)• Perform techno-economic analysis (TEA) and life-cycle assessment (LCA)
Project Goal: Develop the natural formatotroph
Cupriavidus necator as a robust microbial host
for conversion of formate to value-added
products
NREL | 14
Two proposed routes for formate assimilation
Figure adapted from Antonovsky, N., et al., 2017. Curr. Opin. Biotechnol. 47, 83–91. doi:10.1016/j.copbio.2017.06.006
Figure adapted from Yishai, O., et al., 2016. The formate bio-economy. Curr Opin ChemBiol 35, 1–9. doi:10.1016/j.cbpa.2016.07.005
NativeCalvin-Benson-Bassham (CBB) cycle
SyntheticFormolase pathway
NREL | 15
The formolase pathway did not improve formatotrophy
• Genes encoding the formolase pathway were integrated into the genome of C. necator and adaptive laboratory evolution was performed by serial transfer of three parallel lineages in minimal media containing 50mM sodium formate.
• Repeated 24-hour growth cycles, for 40+ generations.
No benefit was observed from the incorporation of the formolase pathway (No-Go), so resources were redirected toward improving the native pathway.
Growth
Time
n
0
0.1
0.2
0.3
0 12 24 36
OD6
00
Time (h)
Unevolved WT
Evolvedisolates
NREL | 16
Evolution improved formate assimilation
• Adaptive laboratory evolution (ALE) was performed using wild-type C. necator (WT) by serial transfer of six parallel lineages in minimal media containing 50mM sodium formate for 400+ generations.
Evolved isolates were identified that exhibit a ~1.2X rate of growth andbiomass yield on formate relative to the WT parent.
Growth
Time
n
Wholegenome
sequencing0
0.1
0.2
0.3
0.4
0 12 24 36 48
OD6
00
Time (h)
Unevolved WT
Evolvedisolates
NREL | 17
Potentially causative mutations identified
• Whole genome sequencing of 6 independently evolved isolates revealed that they shared several similar mutations, suggesting they were likely to be causative.
LineageGenome section 1
Genome section 2
Genome section 3
Transcription factor 1
Total deletion (bp)
HA6 Mutation 0
HB3 Deletion Deletion Mutation 42,177
HC8 Deletion Deletion Deletion 124,302
GD2 Deletion Deletion Deletion Mutation 120,753
GE7 Deletion Deletion Deletion Mutation 120,730
GF4 Mutation Deletion 12,282
NREL | 18
• To evaluate the potential causation of these mutations and develop a genetically defined strain that recapitulates the performance of the evolved isolates, individual and combinations of these mutations were engineered into the otherwise WT strain.
Genetically defined strains were developed that exhibit a ~1.2X rate of
growth and biomass yield on formate relative to the WT parent.
• This achieves our overall project goal of improving the growth rate or biomass yield of C. necator on formate by ≥ 1.2X
0
0.1
0.2
0.3
0.4
0 12 24 36 48
OD6
00
Time (h)
Rational engineering improved formate assimilation
Wholegenome
sequencing
Unevolved WT
Evolvedisolates
Engineeredstrains
Targetedgenome
engineering
NREL | 19
Toward a mechanistic understanding
• The deletion of several genetic elements, in particular mutations in a single transcription factor, were shown to improve formate assimilation.
• RNA-Seq transcriptomics to identify the targets of this transcription factor and elucidate the metabolic changes resulting from these deletions are ongoing.
The results of this analysis will inform our basic understanding of
formatotrophy and how it can be improved.
RNA-Seqtranscriptomics0
0.1
0.2
0.3
0.4
0 12 24 36 48
OD6
00
Time (h)
WT
Engineeredstrains
NREL | 20
Engineering for production of 2HMS
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
β-ketoadipic acid pathway
β-ketoadipicacid
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146Ndhap
3PGformateformate assimilation
OH
OO
O
HO
Adapted from a figure by Jon Magnuson et al., PNNL
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146NFormate
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
β-ketoadipic acid pathway
β-ketoadipicacid
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146Ndhap
3PGformateformate assimilation
OH
OO
O
HO
Adapted from a figure by Jon Magnuson et al., PNNL
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
ΔpcaHG
2HMS
2HMS
praA praH2HMS pathway PCA
ΔxylG
• A restriction enzyme that enabled more efficient genome engineering was deleted (ORNL)
• Genes for production of a competing product, PHB, were deleted
• Competing pathways were deleted• ΔpcaHG, ΔxylG
• The pathway for production of 2HMS was introduced• aroGD146N, asbF, praA,
praHO
O
OH
OH
NREL | 21
• A strain engineered for production of 2HMS was cultivated in a 0.5 L bioreactor using pH-stat feeding of 25% formic acid.
• The OD reached >20 and 76 mg/L 2HMS was produced
2HMS production and robust growth were demonstrated using pH stat
control, which also mitigates toxicity of formate.
0
20
40
60
80
100
0
5
10
15
20
25
0 24 48 72 96 120 144
2HM
S (m
g/L)
OD6
00
Time (h)
pH-state Bioreactor production of 2HMS
NREL | 22
Page 3 of 11Unrean et al. Bioresour. Bioprocess. (2019) 6:49
consumed for maintenance processes was treated sepa-rately. !e synthesis pathways for isobutanol and hexade-canol were based on the recombinant pathways reported in Li et al. 2012 and Guo et al. 2016, respectively. !e synthesis pathway for isobutanol was a combination of valine biosynthesis and recombinant expression of keto-acid decarboxylases and alcohol dehydrogenases. !e hexadecanol biosynthesis was diverted from fatty acid biosynthesis via recombinant expression of fatty acyl-CoA reductase and hexadecanol dehydrogenase.
Metabolic network analysis!e recombinant network was analyzed by elementary mode analysis (EMA) and flux balance analysis (FBA) based on stoichiometric and thermodynamic constraints.
Elementary mode analysisEMA decomposed the recombinant network into all pos-sible, unique and balanced metabolic pathways, allowing for the calculation of a phenotypic space from all the pathways. EMA was simulated using publicly available program METATOOL (http://pingu in.biolo gie.uni-jena.de/bioin forma tik/netwo rks/metat ool/) and network
characteristics were examined by sorting and filtering through all identified paths based on relative flux inputs and/or product yields using Microsoft Excel spreadsheet (Microsoft). Pathway filtering was performed (1) during genetic perturbation by selecting for remaining pathways after each gene deletion, and (2) during different gas input by selecting for remaining pathways after restrict-ing relative gas input. Yield was defined as the mass flux ratio of the amount of product produced per CO2 substrate consumed. !e predicted yields were sorted to identify maximum metabolic capacity of the studied network and the efficiency of each identified metabolic pathway. Phenotypic plot was made on the basis of rela-tive gas uptake, computed as the value of the gas input rate per the value of total gas input, sum of CO2, H2 and O2 uptakes. Reaction requirements of efficient pathways, maximum yielding paths, were categorized as either (1) core reactions, ones necessary for product synthesis dur-ing growth and/or non-growth autotrophic conditions, or (2) inactive reactions, ones not requiring for product synthesis. !e core reactions were analyzed in gene over-expression simulation, while the inactive reactions were analyzed in gene deletion simulation.
Fig. 1 Recombinant network model of C. necator H16 under autotrophic growth utilizing CO2, H2 and O2 as carbon and energy sources, respectively. The recombinant pathways for synthesizing isobutanol and hexadecanol were included for investigation
TEA/LCA
• A previously published metabolic model can be used to derive the governing equations for conversion of formate to 2HMS.
• Final TEA/LCA on biological production of 2HMS from formate in FY21 will enable the DOE and other stake holders to evaluate the economic and environmental impact of this project.
Unrean, P., Tee, K.L., Wong, T.S., 2019. Metabolic pathway analysis for in silico design of efficient autotrophic production of advanced biofuels. Bioresources and Bioprocessing 1–11. doi:10.1186/s40643-019-0282-4
Electrification to Formate
Formate fermentation
In situ product recovery and
distillation
Cogeneration of heat and
power (CHP) Wastewater treatment (WWT)
Sludge
Biogas
Residual solids
CO2
Electricity
Air N, P
2HMS
Wastewater
Process heat/work
Electricity (surplus)
NREL | 23
Future Work
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
β-ketoadipic acid pathway
β-ketoadipicacid
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146Ndhap
3PGformateformate assimilation
OH
OO
O
HO
Adapted from a figure by Jon Magnuson et al., PNNL
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
β-ketoadipic acid pathway
β-ketoadipicacid
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146Ndhap
3PGformateformate assimilation
OH
OO
O
HO
Adapted from a figure by Jon Magnuson et al., PNNL
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
ΔpcaHG
2HMS
2HMS
praA praH2HMS pathway PCA
ΔxylG
O
O
OH
OH
Δpyk Δppc
Formate
• Additional strain engineering and bioprocess development to improve production of 2HMS (Δpyk, Δppc)
NREL | 24
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
β-ketoadipic acid pathway
β-ketoadipicacid
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146Ndhap
3PGformateformate assimilation
OH
OO
O
HO
Adapted from a figure by Jon Magnuson et al., PNNL
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146NFormate
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
β-ketoadipic acid pathway
β-ketoadipicacid
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146Ndhap
3PGformateformate assimilation
OH
OO
O
HO
Adapted from a figure by Jon Magnuson et al., PNNL
co2co2
co2
co2
co2
co2
co2
succ
fum
mal-Loaa
cit
icit
akg
glx
accoa
6pg
g6p
f6p
dhap
pyr
pep
r5p
ru5p-D
g3p
e4pxu5p-D
s7p
3pg
fdp
glcnglc-D
Glucose
2dhglcn
6p2dhglcn
2ddg6p
2dda7p
3dhq
quin-kt 3dhsk 34dhbz
Quinic acid
CCbuttc 2c25dho
co25odhf2a
3oxoadpAdipicacid
Extracellular
Periplasm
Intracellular
Peripheral pathway
EMP pathway
PP pathway
ED pathway
TCA cycle
Gluconeogenesis
Adipic acid pathway
Quinic acid pathway
Shikimatepathway
)pgi-1, )pgi-2
)pykA, )pykF
)ppc)pcaI, )pcaJ
asbF
aroGD146N
ΔpcaHG
2HMS
2HMS
praA praH2HMS pathway PCA
ΔxylG
Δpyk Δppc
O
O
OH
OH
• Additional strain engineering and bioprocess development to improve production of 2HMS (Δpyk, Δppc)
• Improvements to formate assimilation will be leveraged to improve conversion of formate in strains engineered to produce 2HMS
• TEA and LCA will define the potential economic and environmental impacts of this and related technologies
Future Work
0
0.1
0.2
0.3
0.4
0 12 24 36 48
OD6
00
Time (h)
NREL | 25
Value Proposition• Enable conversion of formate generated
from low-cost renewable energy and waste CO2 to myriad fuels and chemicals
• Decarbonize or reduce the carbon intensity of processes that generate CO2
Key Accomplishments• Demonstrated production of 2HMS and
robust growth on formate • Improved growth rate and biomass yield of
C. necator on formate by ≥1.2X• Contributing to our understanding of how
formate conversion can be improved
NREL’s Bioenergy Program Is Enabling a Sustainable Energy Future by Responding
to Key Market Needs
Summary
www.nrel.gov
Q&A
This work was authored by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Funding provided by the U.S. Department of Energy Office of Energy Efficiency and Renewable. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.
Team Members
• Christopher Calvey• Carrie Eckert• Violeta Sanchez• Colin Kneucker• Ling Tao• Michelle Reed
Agile BioFoundry CapabilitiesThe ABF platform unites the unique abilities of DOE national laboratories with targeted outcomes such as an improvement in Design-Build-Test-Learn biological engineering cycle e! ciency, new microbial host organisms, and market transformation through the transfer of intellectual property and manufacturing technologies to U.S. industry.
ABF’s core capabilities include:
• Engineering biology
• Computer-aided bioprocessdesign
• Microbial host development
• Multi-scale cultivation
• Deep multi-omics analysis
Industry Partnerships to Advance the U.S. BioeconomyThe success of the ABF platform requires critical feedback from industry concerning:
• R&D challenges facing the bioengineering andbiomanufacturing industry
• Contracting and intellectual property mechanismsthrough which industry and the ABF consortium interact
• Key performance indicators for the ABF
Current public and private partnerships that have helped ABF advance the bioeconomy include:
Agilent | Kiverdi | LanzaTech | Lygos | TeselaGen | University of California San Diego | University of Georgia | Visolis | ZymoChem
Learn more about the Agile BioFoundry consortium, its partners, and impact on the bioeconomy at agilebiofoundry.org.
Front page photo: Panoramic view of the Fermentation Laboratory at the Advanced Biofuels and Bioproducts Process Development Unit at Lawrence Berkeley National Laboratory. Photo by Roy Kaltschmidt, LBNL
agilebiofoundry.org
DOE/EE-1970 • April 2019
Agile BioFoundry consortium core capabilities integrate e! cient biological engineering cycles with TEA and LCA, microbial host onboarding, and process integration and scale-up.
Desig
n Build
Learn Test
Host Onboarding
Scale-up
TEA/LCA
Integration
BIOENERGY TECHNOLOGIES OFFICE
agile-biofoundry-draft2.indd 2 4/17/19 10:25 AM
NREL | 27
Additional Slides
NREL | 28
Responses to Previous Reviewers’ Comments
• “The performers are advised to deprioritize work on formolase (and focus on CBB) unless this enzyme's activity can be significantly improved.” and “The performers should validate if they can demonstrate any flux using the extremely slow formolase enzyme and consider just focusing on improving the CBB pathway, though this will likely be extremely challenging and should be low priority. ”o Introduction of the fomolase pathway into C. necator followed by
adaptive laboratory evolution did not result in improved growth rate or biomass yield during growth growth on formate (No-go) so resources were redirected toward improving growth via the native CBB cycle.
o Adaptive laboratory evolution resulted in the identification of mutations that improved the growth rate and biomass yield on formate via the native CBB cycle by 1.2X
NREL | 29
Responses to Previous Reviewers’ Comments• “The safety and viability of the process can be better considered. Formate
toxicity as an issue could be discussed.”o We have demonstrated growth of C. necator on 100 mM and 50 mM
formate, but the biomass yield on 100 mM was less than on 50 mM, suggesting toxicity negatively impacted growth. Accordingly, 50 mM has been used in most of our experiments.
5
6
7
8
9
0 8 16 24 32 40 48
Biom
ass
Time (h)
o In pH-stat bioreactors, formate is fed at the rate it is consumed, keeping the formate concentration low and, consequently, avoiding toxic effects.
o Formic acid is caustic and solutions above 85% are flammable, but normal chemical safety measures can mitigate the risks this introduces.
50 mM
100 mM
NREL | 30
Publications, Patents, Presentations, Awards, and Commercialization
• We anticipate at least two high-impact publications will result from this work.• Adaptive laboratory evolution of C. necator for improved formate
utilization • Metabolic engineering of C. necator for upgrading of formate to 2-
hydroxymuconate semialdehyde
• We also anticipate at least one patent application related to novel metabolic engineering strategies to improve formate assimilation will result from this work.