Study Of Biosynthesis Of Phenyllactic Acid By Recombinant E.coli Whole-cell Biotransformation Of L-phenylalanine

Phenyllactic acid?PLA?is a small molecule natural organic acid widely found in nature,it can inhibit a variety of foodborne pathogens.A large number of studies have shown that phenyllactic acid is a broad-spectrum antibacterial substance and the antibacterial activity of PLA is stronger than some commonly used food preservatives,so it is expected to develop into a new type of biological preservation applied to the food field.Early researches mainly used microbial metabolism to produce PLA.This production method has the disadvantages of long production cycle,non-uniform fermentation products,and low yield.Therefore,in recent years,people have shifted their research directions to the preparation of PLA by biocatalysis.Although some achievements have been made,there are still some deficiencies:?1?Phenylpyruvate?PPA?is mostly used as a substrate,but phenylpyruvic acid is very expensive and is not suitable for industrial production.?2?In the existing studies,the efficiency of the enzymes used in the synthesis of PLA was low,which resulted in a low PLA yield and conversation rate.?3?During the catalysis process of Lactate dehydrogenase in the system,NADH needs to be continuously consumed.However,the price of NADH is also very expensive,exogenous addition of NADH would cause a significant increase in cost.In order to solve the above problems,inexpensive L-phenylalanine?Phe?was used as the substrate to produce PLA.The recombinant E.coli BL21?DE3?(p RSF-aad-ldh₁₀-fdh)with high yield of PLA was constructed by co-expressing L-amino acid deaminase?L-AAD?,lactate dehydrogenase?LDH?and formate dehydrogenase?FDH?.First,the L-AAD three-point mutant D165K/F263M/L336M from Proteus mirabilis KCTC 2566 was cloned and successfully expressed in E.coli BL21?DE3?.Then,10 different sources of genes coding for LDH were screened from the GenBank database by bioinformatics techniques.In order to select the most active one from the 10 genes,we co-expressed L-AAD with the obtained lactate dehydrogenase and constructed 10recombinant E.coli BL21?DE3??p RSF-aad-ldh?.By comparing the expression of 10 kinds of LDH proteins and the ability of different recombinant E.coli to produce PLA,the LDH with the highest activity was selected,namely LDH₁₀ derived from Lactobacillus sp.CGMCC9967.In order to construct a more stable coenzyme coenzyme NADH regeneration system with fewer byproducts,the CbFDH derived from Candida boidini was selected to to regenerate NADH.LDH₁₀ and FDH were fused firstly by the method of enzyme coupling and then co-expressed with L-AAD to construct recombinant E.coli BL21?DE3?(pRSF-aad-ldh₁₀-fdh).After induction by IPTG,L-AAD,LDH₁₀ and FDH were successfully expressed in E.coli.InordertoefficientlysynthesizePLAwithrecombinantE.coli BL21?DE3?(pRSF-aad-ldh₁₀-fdh)from Phe,the RBS sequence preceding the ldh₁₀ gene was optimized.The best RBS seque nce was 5'-TTAATAAGGAGATATA-3'.In addition,the fermentation conditions and whole cell transformation conditions of recombinant E.coli BL21?DE3?(pRSF-aad-ldh₁₀-fdh)were optimized.The fermentation conditions were as follows:seeding time was 9h,inoculum amount was 4%,and the concentration of IPTG was0.4 mmol?L^-1,and induction temperature and time was at 25^o C for 10 h.The whole cell catalysis conditions were determined as follows:the pH of the transformation system was 7.0,the temperature was 37^oC,the amount of the common substrate ammonium formate was double the molar equivalent of Phe,and the ratio of the substrate Phe to the whole cell catalyst was 4:3(g?L^-1).Moreover,90 mmol?L^-1 glucoses were added when the concentration of the substrate Phe is higher than or equal to 30 g?L^-1.Under the above conditions,the recombinant E.coli BL21?DE3?(p RSF-aad-ldh₁₀-fdh)can synthesize 30 g?L^-1 PLA from Phe with a conversion rate of 100%and a space-time yield of 48.48 g?L^-1?d^-1.Finally,the stability of recombinant E.coli BL21?DE3?(p RSF-aad-ldh₁₀-fdh)was studied in this research.It was found that after three consecutive serial passages,the PLA production decreased only by 3.25%and the plasmid retention rate was 99%.After 5 passes,PLA production decreased 5.40%,in line with the requirements of engineering bacteria applied to industrial production.