| L-tryptophan is one of the eight essential amino acids,which is widely applied in food,medicine,feed and other industries.Compared with the traditional industrial production methods,at present,bio fermentation for L-tryptophan production has gradually occupied the mainstream of the market.However,the complex biosynthetic pathway accompanied by feedback regulation mechanisms makes it difficult to improve the conversion rate.Therefore in this study,we developed a feedback-resistant Escherichia coli as chassis strain,which is suitable for L-tryptophan production.Combined with modular engineering,metabolite profiling,and promoter engineering strategies,the final L-tryptophan titer and glucose conversion were significantly improved.The major results are described as follows:（1）Construction of the synthetic L-tryptophan chassis strain:First,knock out the repressor protein trp R and the attenuator trp L to release the repression and attenuation caused by L-tryptophan at transcription level.Secondly,introduce a feedback resistant mutant Aro GS211F,a key gene of the synthetic pathway was introduced to relieve the rate limiting bottleneck in the metabolic pathway.Finally,the resulting chassis strain E.coli TRP3 was evaluated at fermenter level,and the main by-products were then determined.After 40 h fermentation,the L-tryptophan titer reached 11.80 g·L-1and the glucose conversion reached8.25%.Meanwhile,the by-products were mainly acetic acid and lactic acid,the contents of which reached 9.63 g·L-1 and 12.41 g·L-1,respectively.（2）Reconstruction of the central metabolic pathway to increase the supply of precursor DAHP:First,according to the literature of by-product analysis results,the relevant genes of acetic acid and lactic acid synthesis including pox B,tdc D,pta,ack A,ldh A and dld,were mined.Through shake flask evaluation,the appropriate target genes（pox B and ldh A）were screened.Secondly,the key genes of precursor synthesis（pps A and tkt A）were inserted into the loci of above targets to improve the supply of PEP and E4P.Finally,over-expression of the key gene aro Gfbr enriched the carbon metabolic flow to the L-tryptophan synthesis pathway.The L-tryptophan titer of recombinant strain E.coli TRP6 reached 17.43 g·L-1 in 5-L fermenter,which was 47.71%higher than that of E.coli TRP3.The glucose conversion increased by 35.76%,reaching 11.20%.Furthermore,the titer of acetic acid and lactic acid decreased by 63.97%and 56.41%,respectively.The detection of metabolic intermediates in next module showed that 3-dehydroshikimate and shikimic acid accumulated significantly,reaching 4.52 g·L-1 and 2.50 g·L-1,respectively.However,the titer of chorismate in the metabolic node,is relatively low to 0.64 g·L-1.（3）Optimization of the shikimic acid pathway to increase chorismic acid supply:First,the key metabolic targets aro B,aro E and aro L from shikimic acid pathway to branching acid node were explored via the literature mining.Secondly,optimize the expression of each target gene through promoter engineering to regulate the metabolic level of shikimic acid pathway.Finally,the optimal results were combined and integrated into the genome to dredge the metabolic flow of shikimic acid pathway and reduce the strain burden.The production performance of recombinant strain E.coli TRP7 was evaluated in 5-L fermenter.The L-tryptophan titer and the glucose conversion reached 23.41 g·L-1 and 34.31%,respectively.The titer of 3-dehydroshikimate and shikimic acid decreased to 1.51 g·L-1 and 0.86 g·L-1,respectively.The chorismate titer increased from 0.64 g·L-1 to 3.64 g·L-1.（4）Rational modification of the L-tryptophan terminal synthesis pathway to promote L-tryptophan synthesis:First,overexpression of the L-tryptophan operon strengthened the metabolic flux of L-tryptophan terminal synthesis pathway.Secondly,promoter engineering was applied to optimize the synthetic genes serA and prs of precursors in the terminal synthesis pathway to increase the supply of L-serine and PRPP,and then the optimal expression cassettes were integrated into the genome.Finally,the production performance of strain E.coli TRP9 was evaluated in 5-L fermenter.The L-tryptophan titer reached 36.08g·L-1,and the glucose conversion reached 18.55%,which was 81.72%of the theoretical conversion. |
