Metabolic Engineering Study Of TIGR Regulating Synthesis Of L-Pipecolic Acid

The lack of oil resources and increasing environmental problems urgently require a renewable,metal-free,sustainable and green bio-manufacturing method.However,the potential of the biomass-derived chemical value chain is critical to fostering such an emerging bio-based industry.In this thesis,we constructed a strain of Escherichia coli which uses lysine as a substrate to synthesize a chiral drug intermediate piperidine for the expression of a foreign gene by bio-fermentation.This method is relatively simple and efficient.Chemical synthesis often does not require pure chiral pipecolic acid and requires racemic resolution.In order to further increase the yield of L-pipecolic acid,the lysine-degrading enzyme?CadA?Escherichia coli BL21?DE3?was firstly used as the host strain,and Raip,GDH,Dpka,LysP were used as the carrier.The gene was expressed in tandem to obtain a concentration of L-pipecolic acid of 30 g/L.Through the LC-MS,we found that a large amount of the intermediate product?1-piperidine-2-carboxylic acid was still present in the fermentation broth.In order to reduce the accumulation of intermediate products,we designed four different repeat sequences?A?₁₆,?C?₁₆,?AC?₈ and?AT?₈ to obtain 16 different types before the gene Dpka,GDH start codon.Combined tunable intergenic region sequence?TIGR?.The TGrc99a-containing vector was ligated to GDH and Dpka,respectively,and expressed in tandem with different combinations of LysR capable of expressing deaminase cyclase rAIP and capable of expressing lysine permease LysR to enhance metabolism of the microorganism.process.This biotechnology to produce L-pipecolic acid is a simple,economical,green technology that can replace existing chemical synthesis.In addition,the biomass-derived lysine industry can be scaled up to larger scales,and studies have shown better prospects for biomass-derived chemicals with extended value chains.By studying the effect of substrate on the production of L-pipecolic acid,L-lysine HCl was identified as a better substrate to produce L-pipecolic acid than L-lysine,which could increase the production of L-pipecolic acid by 21.28%and reduce the fermentation cost by 16.67%.The knockout of key enzyme CadA in lysine degradation pathway and overexpression of LysP improved the L-pipecolic acid titer about 52.52%and 49.88,respectively.This engineered microbial factory achieved the L-pipecolic acid production of 46.7 g/L with a yield of 0.89 g/g in 5-L fermenter for 40 h.This paper has some innovations.The introduction of TIGR into Dpka and GDH genes for the first time in the world has greatly improved the transcription level,reduced the accumulation of intermediate products,and greatly optimized the metabolic pathway of L-pipecolic acid.At the same time,by optimizing the E.coli codon knockout of the piperidine formic acid degradation pathway,etc.,it is beneficial to reduce the intermittent fermentation space and improve the effective labor efficiency.