Study On Metabolic Engineering Of Escherichia Coli For L-pipecolic Acid Production

L-Pipecolic acid(L-PA)is an important rigid cyclic non-protein amino acid,which can define the structure of the polypeptide and also serve as a multifunctional framework in the synthesis of different compounds,which is used for the preparation of many chiral drugs and bioactive substances.Such as Ropivacaine,Thioridazine,Rapamycin and anti-tumor Sandramycin.The bioactivity of these drugs depends on the stereochemical structure of the piperidine moiety.Therefore,the synthesis of high purity L-pipecolic acid is particularly important.At present,L-pipecolic acid is mainly synthesized by chemical methods.The production of chemical synthesis is DL-pipecolic acid.It is very difficult to obtain high purity L-pipecolic acid using chemical method.Moreover,the separation of chiral products is very difficult and highly cost.The synthesis process cause seriously environmental pollution,which is harmful to sustainable development.In this paper,Escherichia coli engineering strains were constructed by metabolic engineering technology.The production of pipecolic acid was carried out by whole-cell biocatalyst and fermentation using these engineered strains.The chiral purity of L-pipecolic acid was 100%,the cost of production was relative lower and pollution was reduced.In this paper,engineered strain was constructed and one-step biosynthesis of L-pipecolic acid was executed.The lysine cyclodeaminase gene pip A was cloned into vector pET22 b,which was introduced into BL21(DE3).The lysine cyclodeaminase was used to convert L-Lysine to L-pipecolic acid.The engineered strain ML03/pET22b-pip A with a lysine cyclodeaminase overexpression could produce 3.06 g/L L-pipecolic acid with a yield of 0.159 g/g.In order to further improve the yield of L-pipecolic acid,a multi-gene heterologous expression pathway was constructed based on E.coli strain.An E.coli strain harboring a plasmid encoding AIP from Scomber japonicus,glucose dehydrogenase from Bacillus subtilis,Pip2 C reductase from Pseudomonas putida,and Lysine permease from Escherichia coli str.K-12 substr.MG1655 was constructed.In this study,ribosome-binding sites were optimized.Promoter engineering was done and the substrate utilization was enhanced.Meanwhile,in order to block the lysine degradation pathway,cadA was knocked out.The production of L-pipecolic acid was greatly enhanced and the fermentation time was rapidly shortened by expressing lysP encoding lysine permease,which increased the rate of lysine transport into the cells.The fermentation conditions were optimized.The production and yield of L-pipecolic acid were improved.The multi-step biosynthesis of L-pipecolic acid was produced by flask fermentation and high density fermentation.The optimum fermentation medium was 40 g/L glucose,K2HPO4.3H2 O 7.5 g/L,MgSO4.7H2 O 2 g/L,(NH4)2SO4 1.6 g/L,citric acid 2 g/ L,FeSO4.7H2 O 0.0756 g/L,Na2SO4 0.02 g/L,Zn2SO4 0.0064 g/L,CoCl2.6H2 O 0.004 g/L,CuSO4.5H2 O 0.0006 g/L.After 36 h,with intermittent supplement of glucose and L-lysine HCl,the concentration of L-pipecolic acid reached 40.3 g/L,and the yield reached 0.88 g/g.In this paper,there are two innovations.For the first time,internationally,the lysine transporter was used to facilitate the multi-gene heterologous metabolic pathway,which improved the lysine transport rate and shortened fermentation time.The fermentation time was reduced to 36 h from 48 h.The L-pipecolic acid produced by fermentation for the first time was enhanced to 40.3 g/L.Compared to whole-cell biocatalyst,the former reduced production time,improved production efficiency and saved production costs.