Effects Of Exogenous Compounds And Key Genes On Phenyllactic Acid Production By Lactobacillus Plantarum

Phenyllactic acid（PLA）is a small molecule organic acid in the metabolites of lactic acid bacteria,which has a broad-spectrum and highly effective antibacterial activity.In recent years,PLA,as a new type of natural preservative,has been applied to the preservation of food.At present,the research of PLA production is mainly focused on fermentation technology.Because the amount of PLA produced was low by the strain fermentation,and it has not reached the standard of industrial production,resulting in the limitation of the use of PLA in production and life.In this paper,the lactic acid strain Lactobacillus plantarum（Lb.plantarum）YM-4-3,which used for food production has been selected for microbial fermentation,and the production of PLA was increased by adding exogenous compounds to the fermentation medium MRS,and the production of PLA differentially expressed genes of strain Lb.plantarum YM-4-3 were subjected to heterologous expression studies to determine genes that have an important role in PLA production,which provide a reference for the future industrial production of PLA.The main research contents and results of this paper are as follows:1.Adding 4 kinds of cytokinin（6-Benzylaminopurine,6-BA）,Methyl Jasmonate,MeJA),urea and sodium humate to MRS medium,which may promote Lb.plantarum YM-4-3 for the compound with increased PLA output,the supernatant was taken by centrifugation after 48 hours of fermentation,and the PLA content was determined by high performance liquid chromatography（HPLC）.It was found that 6-BA,MeJA and urea can significantly increase PLA production.In order to further study the optimal concentration of three compounds in this process,the optimal concentration of 6-BA,MeJA,and urea were confirmed,with 3.2 μL/mL,0.4 mg/mL,4 mg/mL seperately that produced 290 mg/L,78 mg/L and 120 mg/L PLA.2.In order to explore the genes that play an important role in PLA in Lb.plantarum YM-4-3 at the molecular level,the research team conducted a transcriptome analysis of Lb.plantarum YM-4-3 in the early stage of the study.From this study,10 were selected significantly differentially expressed genes,through vector constructionand electroporation transformation,successfully obtained 4 differential genes Lactococcus lactis transformants: NZ9000/pNZ8048-ORF₂045,NZ9000/pNZ8048-ORF₀172,NZ9000/pNZ8048-ORF₀453,NZ9000/pNZ8048-ORF₀615.Bioinformatics annotations indicate that these four genes are: citrate [pro-3S]（citrate）,NADH oxidase,hypothetical protein,PTS sugar transporter subunit IIA.The four transformants were subjected to heterologous expression analysis.After the fermentation broth was induced with nisin,the content of PLA was determined by HPLC.Statistical analysis found that except for the strain NZ9000/pNZ8048-ORF₀172 PLA production 0.255 mg/L was lower than the control strains NZ9000（0.295 mg/L）and NZ9000/pNZ8048（0.425 mg/L）,the yield of PLA from the remaining 3 strains were significantly higher among the control strains,NZ9000/pNZ8048-ORF₀615 had the highest yield of 2.845 mg/L,which was 9.64 and 6.69 times that of the control strains（NZ9000 and NZ9000/pNZ8048）,respectively.Based on the above research results,in Lb.plantarum YM-4-3,the genes of OFR₂045,ORF₀453,and ORF₀615 can promote the production of PLA,and ORF₀172 may not be related to the production of PLA.