Synthesis Of Shikimic Acid Catalyzed By Recombinant Escherichia Coli Whole Cell Biotransformation

Shikimic acid has very important research value in the field of medicine.With the increasing demand in recent years,traditional commercial production methods are difficult to meet market demand due to low extraction efficiency and serious pollution.To solve this problem,this study used whole-cell catalysis to prepare whole-cell catalysts that can be applied to industrialization.Explore and optimize the expression ability and stability of recombinant strains at the plasmid and genome level.The goal is to construct a cost-effective and environmentally friendly industrial method for the biosynthesis of shikimic acid.To solve the problem of insufficient reducing power,the activity of shikimate dehydrogenase(Aro E)is low,which affects the smooth conversion of 3-dehydroshikimate(DHS).In this paper,exogenous glucose dehydrogenase(GDH)is introduced to construct an NADPH regeneration system,and the expression vector containing gdh and aro E genes is transferred into Escherichia coli BL21(DE3).It is overexpressed under the induction of isopropyl-β-D-thiogalactoside(IPTG),and the GDH activity from Bacillus megaterium is screened out.Secondly,the catalytic conditions are preliminarily optimized,and 34°C is determined as the optimum catalytic temperature.When the p H is 7,it has the best catalytic effect,and at the same time,the stability of the plasmid is strengthened,and the addition of antibiotics in the fermentation is reduced or even eliminated.The experiment also further analyzed the effect of gene expression elements on protein expression and plasmid stability:in the dual-promoter expression vector,exchanging key gene positions has the most significant effect on the activity of the aro E gene.Compared with the first multi-cloning site,aro E at the second site,its enzymatic activity is 569.5 U/mg prot,which is about 8.6 times that of the former.When the gene is expressed by the co-promoter,its expression intensity decreased to varying degrees.Finally,the strain is fermented in a 5 L fermenter,which further expanded the whole-cell catalytic system.It is finally determined that in an open environment,20 OD/L bacteria,using 1.1 M industrial glucose,could convert 3 L of 3-dehydroshikimate fermentation stock solution within 2.5 h,and the conversion rate is as high as 97%.Compared with catalytic synthesis,fermentation synthesis has the advantages of simpler process and lower cost,so attempts are made to modify the metabolic pathway of E.coli on the genome.After knocking out shikimate kinase(Aro L)and weakening shikimate kinase(Aro K),under the premise of ensuring normal metabolism,SA accumulation in shake flask fermentation is 1.02 g/L,and 5 L fermentation tank fermentation yield is 7.74 g/L.To enhance the reducing power required for Aro E,promoters of different strengths are used to integrate gdh into the genome and shake flask fermentation yielded 1.98 g/L of SA.This study not only provides a simple and efficient biosynthesis method for solving the problems of high energy consumption and high pollution in traditional industrial production,but also lays a foundation for realizing the large-scale production mode of shikimic acid.