Optimization Of Production Conditions And Preliminary Transformation Of Lactic Acid Bacteria For Phenyllactic Acid Production

Phenyllactic acid(PLA),a naturally occurring small-molecule organic acid,is widely present in various foods and exhibits multiple antibacterial activities against foodborne pathogens and spoilage molds.PLA has broad prospects for application in the food,pharmaceutical,and cosmetics industries.Many microorganisms are capable of synthesizing PLA,with high biosafety lactic acid bacteria being natural hosts for its production.Therefore,to deepen our understanding of the biosynthetic mechanism of PLA in lactic acid bacteria,it is necessary to investigate its synthesis pathway and key gene functions,providing a theoretical basis for establishing high-yield strains and improving PLA production from the perspectives of metabolic engineering and synthetic biology.This study focused on Pediococcus lactis LA52,a high-PLAproducing strain,and Lactobacillus plantarum YM-4-3,a moderate-PLA-producing strain.Single-factor optimization and Plackett-Burman design experiments were performed to screen the culture conditions and medium components of LA52,followed by response surface methodology(RSM)for the optimization of the best medium formulation.Transaminase genes patA and amino V,as well as lactic acid dehydrogenase genes ldh1 and ldh2,were heterologously overexpressed in YM-4-3,and preliminary modifications were made in LA52 through homologous overexpression.The growth status,PLA production,acid-producing ability,glucose residual content,phenylalanine content,and α-ketoglutaric acid content in the fermentation broth were dynamically monitored in YM-4-3 and LA52 wild-type strains and their overexpression strains.The main research results are as follows:1.The optimization of LA52 medium revealed that the addition of 0.5 g/L glutamic acid and 6 g/L phenylalanine to regular MRS medium resulted in the highest PLA production,reaching 718.67 mg/L,significantly higher than the maximum production of 648.12 mg/L obtained through single-factor optimization.Therefore,this formulation was determined as.2.Through heterologous overexpression of the transaminase genes patA and amino V,as well as the lactate dehydrogenase genes ldh1 and ldh2,in YM-4-3,it was found that the colony forming units(CFU)of the overexpressing strains in regular MRS medium were significantly lower than those of the wild type,while in optimized MRS medium,they were slightly higher than the wild type.This suggests that the composition and concentration of the culture medium significantly influence bacterial growth.The results of PLA production showed a significant increase in PLA yield for the strains overexpressing patA and amino V in both regular and optimized MRS,with yields being 3.3-fold,1.15-fold,1.14-fold,and 1.03-fold higher than the wild type,respectively.This confirms that transaminases are indeed the rate-limiting enzymes in PLA synthesis,and overexpression of the transaminase genes can alleviate the inhibitory effect of the rate-limiting step.Furthermore,measurements of residual glucose in the fermentation broth showed that YM-4-3 and its overexpressing strains consumed more glucose in the optimized MRS compared to regular MRS.This may be attributed to the addition of more components in the optimized MRS that affect PLA synthesis,stimulating the activity of key enzymes in the synthetic pathway and requiring more carbon sources to enhance PLA production.Additionally,the variations in phenylalanine and α-ketoglutarate levels indicated their close association with PLA synthesis.When PLA was produced in large quantities,the levels of these two substances decreased significantly,and when PLA production reached a state of saturation,their levels gradually increased with slow bacterial growth.3.Similarly,physiological and biochemical indicators were measured in LA52 strains through homologous overexpression of the transaminase genes patA and amino V,as well as the lactate dehydrogenase genes ldh1 and ldh2.The results showed that regardless of regular or optimized MRS medium,the CFU of the overexpressing strains were significantly lower than those of the wild type LA52.In terms of PLA production,the overexpression of patA,amino V,and ldh2 in both regular and optimized MRS led to a significant increase in PLA yield,with yields being 1.07-fold,1.16-fold,1.24-fold,1.16-fold,1.03-fold,and 1.08-fold higher than the wild type strains,respectively.This indicates that homologous overexpression of the transaminase and lactate dehydrogenase genes in LA52 strains,resulting in initial molecular modifications,can significantly enhance PLA production.Furthermore,the measurements of residual glucose in the fermentation broth,as well as the levels of phenylalanine and α-ketoglutarate,once again confirmed their close correlation with PLA synthesis.