Bioenergy and Resource Management Centre Cranfield University, UK

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1 Bio-based Production of Platform Chemical 3-Hydroxypropanoic Acid Dr Vinod Kumar Lecturer in Bioenergy/Biomass Systems 25 th October 2017 Bioenergy and Resource Management Centre Cranfield University, UK

2 Education Date of Graduation July 2010 Degree (Major) PhD (Biochemical Engineering & Biotechnology) Institute Indian Institute of Technology, Delhi May 2002 M.Sc (Chemistry) Indian Institute of Technology, Delhi May 2000 B.Sc (Honors) Chemistry Hindu College, University of Delhi Doctoral Research: Completed Doctoral thesis entitled Studies on Production and Application of Plant Growth Promoting Root Endophyte Piriformospora indica under the supervision of Prof. V.S. Bisaria and Dr. Vikram Sahai (retired) at the Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, India

3 Post Doctoral Research January 2015 December 2016: Marie Curie Fellow in Nottingham BBSRC/EPSRC Synthetic Biology Research Centre at The University of Nottingham under Prof Nigel P Minton on Project entitled Development of a Sustainable Route to the Important Platform Chemical 3- Hydroxypropanoic Acid Using Synthetic Biology and a Geobacillus Chassis". December 2013-December 2014: Research Fellow at School of Biosciences, Bioenergy and Brewing Science Building, The University of Nottingham, Sutton Bonington Campus, Loughborough, United Kingdom under Prof Gregory Tucker on the Bioethanol Production from Lignocellulosic Biomass. June 2010-June 2011: Post-Doctoral Fellow at Laboratory of Biochemical and Chemical Engineering, Polytech Clermont Ferrand, University of Blaise Pascal, France under Prof Christian Larroche on the Project entitled Valorization of volatile fatty acids through production of single cell oil using oleaginous yeast strains. August September 2013: Post-Doctoral Researcher at Laboratory of Metabolic Engineering of Microorganisms, School of Chemical and Biomolecular Engineering, Pusan National University, South Korea under Prof Sunghoon Park on the Enhanced Production of Biochemicals through Metabolic Engineering and from October- November 2013 continued as Research Professor.

4 3-Hydroxypropionic acid (3-HP) 3-HP is a platform chemical and according to US Department of Energy (DOE), it is among the top twelve chemicals which can be obtained from the biomass. It is a C3 bi-functional molecule and structural isomer of lactic acid. O OH OH Being a platform chemical, 3-HP can be converted into variety of useful chemicals which have enormous industrial applications. The potential of biological 3-HP production can be evaluated based on the market of acrylic acid. At present, around 4.5 million ton of acrylic acid per annum (worth US$11 billion) is produced and its growth rate is 4 % every year. Kumar et al. 2013

5 Kumar et al HP A Platform Chemical

6 Glycerol-based Production of 3-Hydroxypropanoic Acid

7 Glycerol and Klebsiella pneumoniae Glycerol A cheap and abundant carbon source OH HO OH Glycerol is more reduced than traditional carbohydrates. NADH + H O 2 NAD + + H 2 O NADH + H + + NO 3- NAD + + NO - 2 NADH + H + + CH 3 CHO NAD + + CH 3 CH 2 OH Klebsiella pneumoniae possesses the most established glycerol metabolism. The bacterium assimilates glycerol via two distinctive pathways, oxidative and reductive pathway.

8 Kumar et al Oxidative pathway

9 Kumar et al Reductive pathway

10 Biochemical route for 3-HP Production When a proper aldehyde dehydrogenase is overexpressed, the reductive pathway results in the production of 3-hydroxypropionic acid (3-HP) from glycerol. Kumar et al. 2016

11 Metabolic engineering approaches for enhancing 3-HP production Inactivation of respiratory route (glpk). Diversion of more glycerol carbon towards 3-HP by blocking PDO formation (dhat & yqhd). Overexpression of enzymes responsible for 3-HP production (DhaB & AldH). Continuous availability of redox cofactor. Elimination of byproducts (lactate, acetate, ethanol, succinate & 2,3-butanediol).

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13 Development of recombinant K. pneumoniae glpk dhat overexpressing Puuc Ashok et al. 2013a

14 Designing of recombinant K. pneumoniae dhat yqhd overexpressing DhaB and PuuC Ashok et al. 2013b

15 Simultaneous production of 3-HP and PDO 3-HP yqhd 1,3-PDO Kumar et al. 2012

16 Elimination of Byproducts

17 3-HP production by different K. pneumoniae strains Strain K. pneumoniae DSM 2026 dhat_ puuc K. pneumoniae J2B _ KGSADH K. pneumoniae glpk dhat_puuc K. pneumoniae dhat yqhd_dhab_puuc K. pneumoniae WM3 puc18kan_aldhec K. pneumoniae J2B Carbon source(s) Aeration condition Titer* (g/l) 3-HP yield on glycerol Productivity Reference (g/l. h) Glycerol Microaerobic 16.0 (16.8) (mol/mol) 0.23 Glycerol Anaerobic 11.3 (15.9) Glycerol Anaerobic 22.0 (5.9) Glycerol Aerobic 28.1 (3.3) Ashok et al. 2013b Glycerol Microaerobic 48.9 (25.3) Glycerol Microaerobic 22.7 (23.4) Huang et al Kumar et al ldha _ KGSADH K. pneumoniae DSM 2026 Glycerol MicroAerobic 83.8 (22.1) 0.54 Δ ldh1δldh2δpta_puuc (ptac) *The values shown in the bracket are the PDO concentration obtained along with 3-HP Ashok et al Kumar et al Ashok et al. 2013a 1.16 Li et al. 2016

18 Metabolic Engineering of E. coli for 3-HP Production Strain Carbon source(s) Aeration condition Titer* (g/l) 3-HP yield on glycerol Productivit y (mol/mol) (g/l. h) Reference E. coli BL21_dhaB_aldH Glycerol Aerobic E. coli SH254_dhaB_KGSADH Glycerol Aerobic E. coli (BX3_0240) Glucose Aerobic Glucose & glycerol Aerobic 14.3 (3.9) Kwak et al Glucose & glycerol Aerobic Kim et al Glucose & glycerol Aerobic Chu et al E. coli BL21_dhaB_dhaR_aldH E. coli BL21star(DE3) glpk yqhd_dhab_dhar_psal DH E. coli W3110 ΔackA-pta ΔyqhD_dhaB_gabD4 *The values shown in the bracket are the PDO concentration obtained along with 3-HP. Raj et al Rathnasingh et al Lynch et al. 2011

19 Fed-batch cultivation of recombinant K. pneumoniae strains (A) K. pneumoniae (ptac-puuc) Li et al (B) K. pneumoniae Δldh1 Δldh2 Δpta (ptac-puuc)

20 Problems associated with glycerol based microbial 3-HP production o Vitamin B12 supply o Low yield o Redox balance o Toxicity of 3-HPA o Toxicity of 3-HP

21 Advantages of Sugar-based Production of 3-Hydroxypropanoic Acid 3-HP is product of oxidative pathway Free from Vitamin B12 supply requirement All pathways are redox balanced No involvement of cytotoxic 3-HPA

22 3-hydroxypropionate pathway (shown in black) and 3-hydroxypropionate/4hydroxybutyrate cycle (shown in green) 22

23 Malonyl-CoA pathway Fukui et al. 2009; Lynch et al. 2011; Rathnasingh et al. 2012; Keller et al. 2013

24 3-HP Production via Malonyl-CoA pathway Strain Carbon source(s) Aeration condition Titer (g/l) 3-HP yield on glucose Productivity Reference (g/l. h) E. coli Glucose Aerobic 49 (mol/mol) 0.46 E. coli Glucose Aerobic CO2 Anaerobic Glucose Aerobic Chen et al CO2 Anaerobic Wang et al Pyrococcus furiosus Saccharomyces cerevisiae Synechocystis sp. PCC Lynch et al Rathnasingh et al Keller et al. 2013

25 Beta-Alanine pathway Wang et al. 2014; Borodina et al. 2015

26 3-HP Production by Saccharomyces cerevisiae using Beta-Alanine pathway Titer-13.7 g/l Yield (mol/mol) Productivity-0.17 g/l. h Borodina et al. 2015

27 Simultaneous Conversion of Glucose and Xylose to 3-HP Jung et al. 2016

28 Fed-Batch Fermentation for Simultaneous Conversion of Glucose and Xylose to 3-HP Titer-29.7 g/l Yield (g/g) Productivity-0.54 g/l. h Dry cell mass: Closed square; Glucose: Closed circle; Xylose: Open circle Glycerol: Closed triangle; 3-HP: Open triangle

29 Future Plans Development of Integrated Biorefineries Agro-industrial wastes as feedstock Application of Metabolic Engineering and Synthetic Biology tools Sustainable production of biofuels and biochemicals through second generation biorefinery

30 THANK YOU