Metabolic Engineering of Cyanobacteria for Commodity Chemicals Production from CO2

1. 代謝工程學metabolic engineering 生質能源 (酒精等) chemicals

2. Using metabolically engineered cyanobacteria to produce commodity chemicals from CO2 ETHAN LAN 20160511 Department of Biological Science and Technology National Chiao Tung University

3. Renewable energy and chemical production can alleviate dependence on fossil fuels World Petroleum Consumption (Million barrels per Day) Alleviate dependence on fossil resources with renewable biological resources CO2 Sunlight Biofuel Biochemical Source: EIA Monthly Energy Review (Feb 2012) Petrochemical - plastics Transportation fuel

4. Bioplastics/Biopolymers & precursors Lee et al. 2011 Current Opinion in Biotechnology, 22:758–767

5. Pharmaceuticals, Nutrachemical, drug precursor, and proteins Supplements Pharmaceuticals Chiral drug precursor lycopene Artemisinin – anti-malarial Resverstrol Zhang et al., 2010 Taxol – anti-cancer Reduces cost and negative environmental impacts Pacific yew tree

6. glycolysis A metabolic pathway

7. TCA cycle

10. Fuel & Chemical Bioenergy, chemical feedstock, pharmaceuticals, fragrance, nutraceuticals, polymers, plastics, etc Biological feedstocks CO2, wasteproteins, Lignocellulose, Sugars, natural gas, etc Whole cell Catalysis Modify and regulate microbial metabolism Design and express synthetic pathways for production of desirable compounds Using native metabolic capability to utilize different biological feedstock Remove native competing pathways

11. B A C Strategies for metabolic engineering B (product) A C (byproduct) B (product) A C (byproduct)

12. Strategies for metabolic engineering product substrate product substrate co-enzyme (B form) co-enzyme (A form)

13. Outline An example for metablic engineering 1-butanol We (ETHAN LAN’s group) aim to study the engineering of cyanobacteria for the synthesis of chemicals using CO2

14. Importance and market of n-butanol plasticizers Gasoline fuel fuels Butyl phthalates 2-Ethyl hexanol Butane n-Butanol Butyraldehyde Butyrate n-Butene Butyrate esters butadiene Polyvinyl butyral solvents Butyl acylate Butyl methacrylate Butyl acetate Polybutylene Butyl glycol ethers polymers Annual production :2.9 million metric tons, $5.7 billion Market grows 4.7% a year. (not accounting for use as fuel) As a fuel: Better than ethanol because 1) Low hygroscopicityless corrosive, 2) higher energy density 3) compatible with current gasoline engine

15. History & on-going progress of Biobutanol production • 1861 – Louis Pasteur observed biological production of butanol • 1912 - 1914 – Chaim Weizmann isolated Clostridium acetobutylicum and discovered ABE fermentation • 1916 – (World War I) – ABE was commercialized in UK for making cordite (smokeless gun powder). (Acetone:Butanol:Ethanol) • 1950 - 1960s – Development of ABE became non-economical in Western countries due to competition from cheap petroleum sources. • 1970s – Renewed interest because increase in petroleum price. (Energy crisis in 1970s) • 1980s – increased research on improving ABE process • 1990s – Advances in genetic research enabled strain improvement • 2000s – Advances in genomics (and other “omics”) – C. acetobutylicum sequenced & enabled transfer of butanol pathway to other organisms – however poor expression

16. Clostridium n-Butanol pathway was poorly expressed in heterologoushosts Atsumiet al., 2008 Inui et al., 2008 Clostridium pathway Acetyl-CoA 25 10 to 20 Recombinant producer 20 Acetyl-CoA thl Native producer 15 1-Butanol titer (g/L) 10 5 1.2 Acetoacetyl-CoA 0.58 0.12 0.3 0.0025 0 NADH hbd Clostridium beijerickii BA101 Lactobacillus brevis Pseudomonas putida Bacillus subtilis Saccharomyces cerevisiae Escherichia coli 3-Hydroxybutyryl-CoA Steen et al., 2008 Berezina et al., 2010 Nielson et al., 2009 Nielson et al., 2009 Chen and Blaschek, 1999 crt Fdred Several features of the Clostridium pathway was noted: Pathway was reversible and lack significant driving force Bcd/Etf protein may require Clostridium ferredoxin for optimal function Crotonyl-CoA Fdox FADH2 bcd/ etfAB FAD 2 NADH Butyryl-CoA NAD+ NADH adhE2 n-Butyraldehyde NADH adhE2 n-Butanol

17. Synthetic driving forces increase butanol production To increase the driving force for butanol production: Replaced Bcd/EtfwithTrans-enoyl-CoA reductase (Ter), which irreversibly reduces crotonyl-CoA with NADH, effectively increaseing butanol production. Knocked out the major pathways consuming NADH and acetyl-CoA, increasing substrate pool Glycolysis Glucose NAD+ NADH NADH Lactate NADH Succinate NADH Ethanol Acetyl-CoA Acetate Acetoacetyl-CoA NADH Fdred Fdox Crotonyl-CoA NADH FADH2 FAD 2 NADH Butyryl-CoA NAD+ NADH NADH Butyraldehyde n-Butanol Shen et al. (2011). Appl Environ Microbiol

18. Butanol production in cyanobacteria is difficult under photosynthetic condition Lan and Liao. (2011). Metabolic Engineering Glycogen Cytoplasm NADPH hν hν Fdred Fdox Acetyl-CoA Challenge: Increase Driving force Oxygen sensitivity Unfavorable reaction Keq = 10-5 Cytochrome b6f complex Acetoacetyl-CoA Pc-Cu+ Photosystem I Photosystem II Pc-Cu2+ Lumen H2O O2 NADH • Less than 1 mg/L of n-butanol observed in culture medium under photosynthetic condition • Butanol was observed only under anoxic incubation PQ Crotonyl-CoA NADH PQH2 Butyryl-CoA NADH NADH Butyraldehyde n-Butanol Oxygen sensitivity prohibits enzyme functions under oxygenic photosynthetic conditions

19. Learning from nature: similarity to fatty acid synthesis and degradation Claisen condensation of two acetyl-CoA is thermodynamically unfavorable… cyanobacteria are more difficult to manipulate acetyl-CoA pool (as opposed to E. coli) CoA n-butanol pathway Ketoacyl-ACP synthase III (KASIII) Acc CO2 CO2 ATP A metabolic pathway very similar to fatty acid degradation in reverse Fatty Acids ΔGo’ ≈ 6.8 kcal/mol Keq≈ 1.1 x 10-5 -7.3 kcal/mol ΔGo’ ≈ ATP hydrolysis is the energy input! for chain elongation! Keq≈ 5.6 x 105 ΔGo’ < 0 Keq > 1

20. Redesigning butanol pathway with ATP driving force We also expressed NADPH dependent dehydrogenase instead of NADH dependent ones Cytoplasm NADPH hν hν Fdred Fdox Acetyl-CoA Malonyl-CoA CO2 Cytochrome b6f complex Acetoacetyl-CoA Pc-Cu+ Photosystem I Photosystem II Pc-Cu2+ Lumen H2O O2 NADPH Fdred Fdox PQ Crotonyl-CoA NADH FADH2 PQH2 FAD 2 NADH Butyryl-CoA NAD+ NADPH O2 NADPH Butyraldehyde n-Butanol Lan and Liao (2012) PNAS

21. In vitro validation of PduP oxygen tolerance and butyryl-CoA specificity Leal et al. Arch Microbiol (2003) 180 : 353–361 Assay condition: His-tag purified 30 °C, aerobic assay 100 mMTris-HCl pH 7.0 500 µM NADH 600 µM acyl-CoA NADH NAD+, CoA PduP under aerobic condition • Lan and Liao. (2013) Energy Environ Sci. 6, 2672-2681

22. Expression of PduP & YqhD in cyanobacteria achieved first demonstration • of acetyl-CoA based ethanol production NADH NADPH Plasmid DNA Ptrc pduP yqhD specR 3’-NSI TrrnB 5’-NSI lacIq Acetaldehyde Acetyl-CoA Ethanol yqhD pduP Recombination S. elongatus 7942 genomic DNA Neutral Site I All strains expressing PduP homologues with YqhD achieved ethanol production under photosynthetic conditions. This result indicated that PduP is functionally expressed in cyanobacteria for conversion of acyl-CoA to aldehyde. Strain (PduP expressed) ETOH-LB (PduP_L.brevis) ETOH-KP (PduP_K.pneumoniae) ETOH-SE (PduP_S.enterica) ETOH-LM (PduP_L.monocytogenes) • Lan and Liao. (2013) Energy Environ Sci. 6, 2672-2681

23. Redesigning butanol pathway with ATP driving force 300 mg/L 10-fold increase Acetyl-CoA Malonyl-CoA CO2 Acetoacetyl-CoA Summary: ATP driving force and oxygen tolerance are important factors for achieving direct photosynthetic n-butanol production. NADPH Fdred Fdox Crotonyl-CoA NADH FADH2 FAD 2 NADH Butyryl-CoA NAD+ NADH O2 NADPH Butyraldehyde n-Butanol • Lan and Liao. (2013) Energy Environ Sci. 6, 2672-2681

24. Functional expression E. coli ATP driving force – direct photosynthetic production Elimination of oxygen sensitivity Clostridium pathway ATP driving force Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA ATP ATP accABCD accABCD CO2 CO2 Acetyl-CoA Acetyl-CoA atoB thl Malonyl-CoA Malonyl-CoA Acetyl-CoA Acetyl-CoA nphT7 nphT7 CO2 CO2 Acetoacetyl-CoA Acetoacetyl-CoA Acetoacetyl-CoA Acetoacetyl-CoA NADPH NADPH NADH NADH hbd hbd phaB phaB 3-Hydroxybutyryl-CoA 3-Hydroxybutyryl-CoA 3-Hydroxybutyryl-CoA 3-Hydroxybutyryl-CoA Universal e- donor & Irreversible trap crt crt phaJ phaJ Fdred Crotonyl-CoA Crotonyl-CoA Crotonyl-CoA Crotonyl-CoA Fdox FADH2 NADH NADH NADH bcd*/ etfAB ter ter ter FAD 2 NADH Butyryl-CoA Butyryl-CoA Butyryl-CoA Butyryl-CoA NAD+ NADH NADH NADH NADH adhE2* adhE2* Bldh* pduP n-Butyraldehyde n-Butyraldehyde n-Butyraldehyde n-Butyraldehyde NADH NADPH NADH NADPH adhE2* adhE2* yqhD yqhD n-Butanol n-Butanol n-Butanol n-Butanol Oxygen tolerance * Indicates oxygen sensitivity