Systematic Metabolic Engineering Of Escherichia Coli To Produce L-leucine

As one of the eight essential amino acids,L-leucine has a good application prospect and market demand.Escherichia coli has a clear genetic background,fast growth,and simple nutritional requirements.E.coli W3110 as a chassis cell,combined with the basic principle of metabolic engineering,genetic engineering and molecular biology technology,finally the study constructed a L-leucine industrial strain.The results are as follows:In order to enhance the L-leucine synthesis pathway,the mutant gene ilv IH^mut was introduced to relieve the feedback inhibition of acetylhydroxyl synthase by branched amino acids.Key enzymes of L-leucine synthesis are enhanced.Leu A^CP,a mutant gene of isopropyl malate synthase with no feedback inhibition,was introduced into the plasmid p Trc99a with high copy expression.The accumulation of L-leucine reached 14.7 g/L,after 24 h of shaking-bottle fermentation.Then leu CD and leu B were expressed on plasmid p Trc99a-leu A^CP.After 24 h shaker fermentation,the accumulation of L-leucine was 16.9 g/L.the yield of L-leucine was increased by 14.9%.In order to improve the extracellular L-leucine production and promote the efflux of L-leucine,the L-leucine transporter was overexpressed in this study.Shaking flask results showed that the strains increased the L-leucine production by 33.3%.In order to modify the transamination in L-leucine synthesis pathway,the ilv E dependent on coenzyme factor NADPH was knocked out,and the leucine dehydrogenase bcd from Bacillus subtilis dependent on coenzyme factor NADH was introduced.The results showed that the introduction of bcd^Bsu gene not only benefited the synthesis of L-leucine,but also reduced the accumulation of L-valine by product.It also improved the growth.Therefore,bcd^Bsu was overexpressed.And leu11 was obtained.After 24 h shaking flask fermentation,the accumulation of L-leucine reached34.19 g/L.In order to enhance pyruvate enrichment,the ack A,pox B and ldh A were successively knocked out.Therefore,the study obtained strains leu13,leu15 and leu17.These strains were fermented in 24 h,under the condition that there was no significant effect on the biomass of the strains.Compared with leu11,the accumulation of L-leucine increased by 6.88%,12.09%and 16.03%.leu17 strain was fermented in 5 L fermenter in batches.After 30 h of fermentation,the yield of L-leucine reached 58.1 g/L and the conversion rate of sugar and acid was21%.This strain has the potential of industrial production.