Biosynthesis Of D-phenyllactic Acid By Redox Self-equilibrium System Of D-lactate Dehydrogenase And Glycerol Dehydrogenase

D-Phenyllactic acid（D-PLA）is a natural broad-spectrum antibacterial compound with antimicrobial activity against bacterial,yeast and other fungi.While D-phenyllactic acid exhibits higher antimicrobial activity than L-phenyllactic acid,showing good prospects in the food industry.In addition,D-phenyllactic acid could also be used as a feed additives,cosmetic active ingredients,and pharmaceutical intermediates.At present,the chemical synthesis of D-phenyllactic acid has many disadvantages,such as complicated process,serious environmental pollution,and low optical purity of the product.The fermentation and the enzyme catalysis in the biological method face the problems of low product concentration,high cost and inability to industrialize.The use of whole-cell catalysis is expected to reduce the production cost of bio-synthesis of D-phenyllactic acid,improve the catalytic efficiency,making D-phenyllactic acid more valuable.D-lactate dehydrogenase consumes coenzyme NADH,catalyzing the formation of D-phenyllactic acid from phenylpyruvate.However,the addition of coenzyme NADH increases production costs.This study firstly constructed a redox self-equilibrium system of D-lactate dehydrogenase and glycerol dehydrogenase.D-phenyllactic acid and coenzyme NAD⁺were produced by D-lactate dehydrogenase.The NAD⁺is consumed to catalyze the production of dihydroxyacetone and NADH by Glycerol dehydrogenase.So the reaction could be continuously conducted to produce D-phenyllactic acid.The strain CP001 overexpressing D-lactate dehydrogenase and glycerol dehydrogenase coud produce 0.49 g/L D-phenyllactic acid with a yield of 0.49g/g from 1 g/L phenylpyruvate by whole-cell catalysis.In order to increase the yield of D-phenyllactic acid,a dual-function fusion protein of D-lactate dehydrogenase and glycerol dehydrogenase was constructed to improve the utilization efficiency of coenzyme and realize the in-situ regeneration of coenzyme.Four D-lactate dehydrogenase and glycerol dehydrogenase fusion protein genes with flexible Linker length（GGGGS）₁,（GGGGS）₂,（GGGGS）₃,（GGGGS）₆ were constructed by Over-lap PCR.The engineering strains CP101,CP102,CP103,CP104 could produce0.54 g/L,0.59 g/L,0.64 g/L,0.52 g/L of D-phenyllactic acid.In order to further improve the production of D-phenyllactic acid by whole-cell catalysis of four fusion proteins,the conditions of whole-cell catalysis was optimized.The optimal inducer concentration was 0.2 mmol/L.The expression was induced at25°C for 10 h.The optimal transformation conditions were:reaction temperature 37°C,reaction pH 7.0,substrate phenylpyruvate 10 g/L,substrate:cell catalyst=1:1.After optimization by process,the production of D-phenyllactic acid by four engineering Escherichia coli CP101,CP102,CP103 and CP104 were increased to 4.67 g/L,4.98 g/L,5.37 g/L and 4.43 g/L,respectively.Under the optimal induction and transformation conditions,the ability of engineering strains for producing D-phenyllactic acid was discussed.The production of four flexible Linker fusion protein strains CP101,CP102,CP103 and CP104 for producing D-phenyllactic acid is 1.16-1.41 fold than the strain CP001 of single gene expressed.The strain CP103 with Linker length（GGGGS）₃ could produce the highest titer of 5.37 g/L D-phenyllactic acid with a yield of 0.54 g/g.