Transcript Improving NADPH availability for natural product biosynthesis in

Improving NADPH availability for
natural product biosynthesis
in Escherichia coli by metabolic
engineering
汇报人：刘巧洁
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.
• 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
Central anaerobic metabolic pathway of Escherichia coli showing
generation of NADH and regeneration of NAD
• 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
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.
Genes targeted for deletion were based on the simulation
results of the CiED(Cipher of Evolutionary Design) model.
CiED(Cipher of Evolutionary Design)?
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.
the product of biomass
production
• Maximizing biomass
product
coupled
and catechin production
yield(BPCY)
Top single, double and triple genotypes determined by CiED with an objective
function for maximizing BPCY
• Maximizing NADPH
Top single, double and triple genotypes determined by CiED with an objective
function for maximizing NADPH production rate.
• Production of leucocyanidin
83%
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
BLD△pgi△pldA△ppc
BLD△pgi△pldA△ppc
• Production of (+)-catechin
Maximum detected levels of extracellular (+)-catechin produced from E. coli
expressing DFR and LAR
To minimize substrate
inhibition of LAR
○：BL21 StarTM(DE3);□：BL△pgi;
×： BL△pgi△ppc;▲BL△pgi△pldA△ppc
• Nicotinamide cofactor levels during exponential
growth
Ratios and concentrations of intracellular nicotinamide nucleotide cofactors.
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.
• Improving NADPH availability can improve
the production of leucocyanidin and (+)catechin