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

L-Phenyllactic acid is a natural antibacterial substance,which has a spectral inhibitory effect on a variety of pathogenic microorganisms,and is expected to become a new type of biological preservative.Phenyllactic acid is non-toxic to human and animal cells,and plays an important role in food,medicine,industry and other neighborhoods.Based on its good stability and spectral antibacterial characteristics,there is a huge potential in the production and processing process.In this study,a multi-enzyme cascade catalytic reaction system was constructed to improve the ability of Escherichia coli to synthesize L-phenyllactic acid.By co-expressing L-amino acid deaminase and phenylpyruvate reductase and coupled with glucose dehydrogenase for coenzyme regeneration,a new type of L-phenyllactic acid biosynthesis method was established.The genes were successfully expressed in E.coli,and the whole cell transformation conditions were optimized.Using RBS sequence optimization and increasing the copy amount of glucose dehydrogenase can effectively solve the inhibitory effect of the intermediate product phenylpyruvate and increase the production of L-phenyllactic acid.A simple and efficient biocatalysis method is provided,which lays a foundation for the realization of large-scale production.（1）Cloning and expressing the L-amino acid deaminase（L-AAD）gene laad from Proteus vulgaris,studying the effect of temperature and p H on its catalytic stability,the optimal reaction temperature is 35℃,and the optimal reaction p H is 8.0.The reductase from different sources was selected,and the phenylpyruvate reductase（La PPR）from Lactobacillus sp.CGMCC 9967 was selected by comparing the enzyme activity and the catalytic ability to phenylpyruvate.The study found that the optimal reaction temperature and p H were respectively 30℃,7.0.Screen the coenzyme self-circulation system and compare the selection of glucose dehydrogenase（GDH）to have a better conversion rate.（2）A multi-enzyme cascade co-expression system was preliminarily constructed.When sufficient phenylalanine and glucose are provided,the substrate phenylalanine can be consumed in a large amount.At this time,the intermediate product phenylpyruvate accumulates in a large amount,and the concentration reaches 27.89 g·L^-1.The final product L-phenyllactic acid production rate is relatively slow,gradually stabilized with the progress of the reaction,and finally only 5.67 g·L^-1 was produced.The intermediate product has a high accumulation concentration,which affects the smooth progress of the entire reaction.It may be due to the imbalance in the conversion of key enzymes in the conversion process,which affects the catalytic rate of the conversion of phenylpyruvate to L-phenyllactic acid.（3）Use the RBS analysis website to assemble RBS sequences of different strengths into the recombinant plasmid pET28a-lappr,and connect them in series with glucose dehydrogenase（GDH）to obtain different RBS strength reductase and dehydrogenase co-expression recombinant strain E.coli/pET28a-rbs_1-5-lappr-gdh.When the RBS sequence was replaced with rbs₂and rbs₃,the expression of La PPR increased significantly.The ratio of enzyme addition was adjusted,and the study found that the optimal enzyme activity ratio when the key enzyme GDH:La PPR reached the optimal catalytic reaction rate was 4:1.（4）In order to coordinate the expression levels of La PPR and GDH in the co-expression strain with one vector,the copy amount of GDH was increased.By regulating the expression of enzymes in whole-cell biocatalysts,when GDH are 2 copies,the accumulation of intermediate product phenylpyruvate is reduced,which is reduced by 84.60%,the production of L-phenyllactic acid is greatly improved,and the yield is increased by 3.77 times.The reaction conversion conditions were optimized,and the final yield reached 21.39 g·L^-1,and the molar conversion rate was 71.33%.In order to provide a low-cost and high-efficiency conversion method for biocatalytic synthesis of phenyllactic acid,it has a reference role,and it has a broad guiding significance for its production in the food and synthetic drug industries.