汇报人：刘巧洁

1. Improving NADPH availability for natural product biosynthesisin Escherichia coli by metabolic engineering 汇报人：刘巧洁

2. Background • With microbial production becoming the primary choice for natural product synthesis, increasing precursor and cofactor availability has become a chief hurdle for the generation of efficient production platforms. • For industrial applications, the biosynthetic processes requiring reducing cofactors like NADH and NADPH would be prohibitively expensive if used in equimolar amounts.

3. A number of studies have focused on enzymatic systems that recycle oxidized cofactors using NAD(P)+-dependent enzymes. Berrios-Rivera et al., 2002,Metabolic Engineering

4. Central anaerobic metabolic pathway of Escherichia coli showing generation of NADH and regeneration of NAD

5. Traditionally, improvement of cofactor availability is addressed by inspection of known metabolic networks via metabolic engineering , yet these methods fail to ascertain the myriad of interaction changes occurring within the global metabolic network. • Stoichiometric-based simulations provides a means for understanding the connectivity of a genome-wide reaction network using limited parameters and assumptions

6. Introduction • we employed a stoichiometric-based model to identify combinations of gene knockouts for improving NADPH availability in Escherichia coli. • The top single, double and triple gene deletion candidates were constructed and as a case study evaluated for their ability to produce two polyphenols, leucocyanidin and (+)-catechin.

7. Genes targeted for deletion were based on the simulationresults of the CiED(Cipher of Evolutionary Design) model. CiED(Cipher of Evolutionary Design)?

8. Experiment • Bacterial strains and plasmids The top four single, double and triple knockout mutants were ranked by either biomass product coupled yield (BPCY) or NADPH production.

9. the product of biomass production and catechin production • Maximizing biomass product coupled yield(BPCY) Top single, double and triple genotypes determined by CiED with an objective function for maximizing BPCY

10. Maximizing NADPH Top single, double and triple genotypes determined by CiED with an objective function for maximizing NADPH production rate.

11. 83% • Production of leucocyanidin These results indicate the reliance on the TCA cycle to generate NADPH rather than the pentose phosphate pathway by the unmodified parent strain under aerobic conditions. 85% 93% Maximum detected levels of extracellular leucocyanidin produced from E. coli expressing DFR

12. BLD△pgi△pldA△ppc BLD△pgi△pldA△ppc

13. Production of (+)-catechin Maximum detected levels of extracellular (+)-catechin produced from E. coli expressing DFR and LAR

14. To minimize substrate inhibition of LAR ○：BL21 StarTM(DE3);□：BL△pgi; ×： BL△pgi△ppc;▲BL△pgi△pldA△ppc

15. Nicotinamide cofactor levels during exponential growth Ratios and concentrations of intracellular nicotinamide nucleotide cofactors.

16. Conclusion • Exhaustive searches through all possible gene deletion combinations using a stoichiometric model of E. coli to identify optimal strategies yielding the highest (+)-catechin production is computationally and experimentally impractical.

17. Improving NADPH availability can improve the production of leucocyanidin and (+)-catechin

19. Thank you!