Design and Evolution of Carbon Conservation and Fixation Pathways
James C. Liao Academia Sinica Taipei, Taiwan
1 • Non-Oxidative Glycolysis (NOG) Bogorad Nature 2013; Lin PNAS 2018 6”H”
C6H12O6 3 CH3COOH 3 C2H5OH • Malyl-CoA Glycerate (MCG) pathway for CO2 fixation Yu Nat Comm 2018
4”H” 8”H”
C6H12O6 + 2 CO2 4 CH3COOH 4 C2H5OH
2 o Global net CO2 emission pathway (1.5 C limit)
Reduced emission
Negative emission
2050
IPCC “Global Warming of 1.5oC” Oct 2018 3 Pathways to Life
Cytoplasm Ribulose5P ATP NADPH Ribulose1,5BP hν CO2 hν Fdox Fdred Ribose5P Xylulose5P
G3P S7P PQ 3PGA Cytochrome
b6f complex ATP PQH2 S1,7BP Pc-Cu+ ATP Photosystem II Photosystem I 2+ Xylulose5P H O Pc-Cu E4P 2 O2 Lumen e- Calvin cycle (CBB pathway)1,3BPGA Light reactions G3P F6P NADPH F1,6BP G3P
DHAP Cellulose Glucose G3P Glycolysis (EMP pathway) ATP e- TCA Cycle Acetyl-CoA Pyruvate
CO2 CO2
Fatty acid Amino acids Alcohol Alcohol Acids Lipid Acids Isoprenoids 4 CO2 loss is a common problem in cellular metabolism
C H O , C H O CO2 CO2, HCOOH, CH3OH, CH4 5 10 5 6 12 6
CBB RuMP PPP W-L EMP rTCA
Acetyl-CoA Pyruvate CO2 PDHc PFOR PFL CO2 Pyruvate Acetyl-CoA
Amino acids Isoprenoids Alcohol Fatty acid 5 Non-Oxidative Glycolysis (NOG)
C6H12O6 3 CH3COOH
6 Oxidative Glycolysis Embden-Meyerhof-Parnas (EMP) pathway
$0.4/Kg Glucose F6P FDP
DHAP G3P
2e- Partial oxidation C3 Pyruvate
2e- Carbon yield = 66% CO2 Mass yield O
= 50% SCoA Acetyl-CoA C2
$0.8/Kg Ethanol Butanol Lipids Isoprenoids 7 Theoretical Energy and Mass Yields
C6H12O6 2C2H6O + 2 CO2 energy % mass % 2540 2 x 1235 kJ/mol 97.24% 51%
C6H12O6 C4H10O + 2 CO2 2540 2455 kJ/mol 96.6% 41%
2 C6H12O6 C7H16 + 4CO2 + CO 2x 2540 4543 kJ/mol 89.4% 27%
Jet fuel 8 Non-oxidative Glycolysis (NOG)
Glucose F6P
3 F6P (C6)
Carbon 2 F6P 3 AcP 3 E4P Rearrangement111 (C2) (C4)
3 AcCoA Bifidobacterium breve.
Phosphoketolase Carbon yield = 100% Ethanol Acetate F6P AcP + E4P Butanol X5P AcP + G3P 9 Two Modes of Non-Oxidative Glycolysis (NOG)
F6P phosphoketolase X5P phosphoketolase F6P AcP + E4P X5P AcP + G3P F6P F6P
AcP E4P tal tkt tal fpk E4P AcP F6P F6P G3P S7P fpk fbp X5P fbp G3P S7P G3P F6P E4P tkt FBP FBP AcP tkt xpk tkt X5P X5P AcP fba G3P R5P fba DHAP R5P AcP tpi rpi G3P xpk rpi DHAP fpk G3P Ru5P F6P E4P Ru5P xpk tpi X5P rpe rpe G3P X5P AcP tkt
Net reaction: F6P 3 AcP
Bogorad et al. Nature 201310 Construction and Evolution of an NOG E. coli strain Glycolysis
TCA
11 Construction and Evolution of an NOG E. coli strain
Glycolysis NOG
TCA/GS
12 Lin et al. PNAS 2018 Laboratory evolution
Initial Strain
Mutations
13 Deleting Regular Use directed strain evolution to force the cell Glycolysis
“Loophole” mutations
14 Lin et al. PNAS 2018 Deleting Potential Loopholes
No loopholes But no growth
15 Lin et al. PNAS 2018 Tune-up desired pathways, Delete potential futile cycles
Growth in
Glucose + Acetate 16 Lin et al. PNAS 2018 Use pathway assays to identify limiting enzymes De-bottlenecking
Growth in Glucose 17 Lin et al. PNAS 2018 Improved 18 Lin et al. PNAS 2018 growth Pathway assay to identify limiting enzymes
19 Construction and Evolution of an NOG E. coli strain
Glycolysis NOG
TCA/GS
20 Lin et al. PNAS 2018 Important mutation occurring during evolution ∆ptsG EII-P CyaA cAMP
rpoS::transposon
21 Fine-tuning of enzyme activity Ensemble Modeling for Rubustness Analysis
22 Fine-tuning of enzyme activity
23 Installed Stronger Wt promoter Evolved away
Pck Activity
24 Lin et al. PNAS 2018 NOG21 NOG22 JCL16
8 JCL16(Wt) 6 NOG21 600 4 OD 2
0 0 20 40 60 Time (h) 1.0 ) 1 - JCL16 NOG21
0.5 Growth (h Growth rate
0.0
Glucose minimal Glucose minimal LB medium with medium aerobic medium anaerobic glucose aerobic 25 acetate lactate formate succinate
100%
80% 66% EMP Limit 60%
40%
20% Carbon distributionCarbon (mol/molfed carbon ) 0% JCL16 NOG21
26 An E. coli strain that uses solely NOG for sugar catabolism
27 Economy of oxidative and non-oxidative glycolysis for making ethanol
Traditional fermentation 1.5 C6H12O6 3C2H6O + 3 CO2 sugar price = $0.4/Kg ethanol price = $0.8/Kg http://www.nasdaq.com/markets/sugar.aspx
NOG C6H12O6 + 6 H2 3 C2H6O + 3 H2O
sugar price 12 When (= 0.133) Then it makes sense to use NOG + H2. H2 price > 90 H2 price < $3/Kg
28 Reductive Fermentation
(H2-assisted CO2 fixation) Traditional fermentation
C6H12O6 2C2H5OH + 2 CO2
+ 6 H2 + 2CO2 C2H5OH + 3 H2O
NOG: Reductive fermentation C6H12O6 + 6 H2 3 C2H5OH + 3 H2O
29 CO2 Saving
Non-oxidative (NOG) C6H12O6 + 6 H2 3 C2H5OH + 3 H2O Methane steam reforming 1.5 CH4 + 3 H2O 1.5CO2 + 6 H2
C6H12O6 + 1.5 CH4 3 C2H5OH + 1.5 CO2
Traditional Fermentation 1.5 C6H12O6 3 C2H5OH + 3 CO2
30 Methane upgrading
Traditional C H O 2 C H OH + 2 CO Fermentation 6 12 6 2 5 2
+ Methane 0.5 CO + 1.5 CH C H OH upgrading 2 4 2 5
NOG + Steam C6H12O6 + 1.5 CH4 3 C2H5OH + 1.5 CO2 Reforming
31 Methane upgrading
∆Go Energy* Carbon# Reactions Comment (kJ/mole) efficiency yield
1) C6H12O6 --> 2 C2H6O + 2 CO2 -228.4 0.97 66.70% Current fermentation Proposed Methane 2 100.05 1.03 133% ) 0.5 CO2 + 1.5 CH4 --> C2H6O upgrading (∆Go >0) Proposed reductive 3) = 1)+2) C H O + 1.5 CH --> 3 C H O + 1.5 CO -128.35 0.99 80% 6 12 6 4 2 6 2 fermentation
4) 2CH4 + O2--> C2H6O+ H2O -309.1 0.77 100% REMOTE
*Energy efficiency is calculated from the lower heating value (LHV) of combustion of products divided by the LHV of the reactants. o o ! ∆Gf and ∆H LHV values were taken from Table 2 # Carbon yield calculation does not include CO2.
32 Malyl-CoA Glycerate (MCG) pathway
for CO2 fixation
C6H12O6 + 2 CO2 + 4”H” 4 CH3COOH
Pyruvate + CO2 2 Acetyl-CoA
Glyoxylate Acetyl-CoA
33 Comparison of different carboxylases
Carboxylase Carbon FunctioninnaturalCO fixation Oxygensensitivity SpecificActivity Cofactor 2 abbrev. species pathway (μmol/min/mg) 3.5[b] RuBisco CO2 No Yes No, but has (CBB) oxygenaseactivity HCO - ATP, biotion Yes No 18[b] ACC 3 (HP bi, HP/HB ) - [b] PCC HCO3 ATP, biotion Yes No 29.6 (HP bi, HP/HB ) - [b] PYC HCO3 ATP, biotion No No 32.4 (mainly in anaplerosis ) - [b] PPC HCO3 No Yes No 35.2 (DC/HB)
PFOR CO2 ferredoxin, TPP Yes Yes <1 (WL, rTCA, DC/HB)
KOR CO2 ferredoxin, TPP Yes Yes <1 (rTCA) [b] CCR CO2 NADPH No No 130 (found in ethylmalonyl-CoA pathway) Using Ppc for CO2 fixation
Ppc： Phosphoenolpyruvate carboxylase Problem： Efficient • OAA can not be converted to general Favor carboxylation direction building blocks without carbon loss No oxygen sensitivity
Simple CO2 CO2 Pyruvate (C3) Acetyl-CoA (C2)
- HCO3 In most cells Asp Lys Met Thr Ppc PEP (C3) OAA (C4)
35 Reversal of glycoxylate shunt allows fixation of CO2 using Ppc
From CBB
C6H12O6 + 2 CO2 + 4”H” 4 CH3COOH
36 Constructing MCG in E. coli
37 MCG in E. coli
38 39 MCG augments CBB pathway
40 MCG pathway recycles photorespiration product
Photorespiration MCG in S. elongates increased CO2 fixation
42 Summary and Acknowledgement • Hong Yu – MCG, Serine cycle • Paul Po-hen Lin – NOG • Igor Bogorad – NOG • Alec Yeager - NOG
43