| Phenyllactic acid(PLA)is a small molecule natural organic acid that is widely found in nature.PLA has broad-spectrum inhibitory properties against bacteria and fungi and is more active than some commonly used chemical preservatives,promising a new type of safe and non-toxic biological preservative.The biosynthesis of PLA has been mainly studied in Escherichia coli,but little has been reported in Lactic acid bacteria(LAB).There are limitations in industrial production as E.coli is a conditional pathogen and is susceptible to phage contamination.In addition,PLA has a strong inhibitory effect on E.coli and further enhancement of PLA production in E.coli will be challenging.Lactobacilli are natural hosts for the production of PLA and have the potential to produce large amounts of PLA.For this reason,there is an urgent need to systematically and thoroughly study the biosynthetic pathways and regulatory mechanisms of PLA in Lactobacillus to provide theoretical support for the establishment of high yielding strains.In this study,four strains of Lactobacillus plantarum with different PLA production were used to analyse the metabolic mechanism of PLA synthesis in L.plantarum by genome,transcriptome and metabolome.Knockout of differential genes related to L.plantarum YM-4-3 PLA synthesis using homologous recombination.The importance of differential genes in PLA synthesis was determined by analysing changes in PLA yield,acid production and growth capacity in media optimised for different components.Transcriptome sequencing analysis of two PLA-related differential gene knockout strains was used to further reveal the functions of the serine acetyltransferase(Cys E)and hypothetical protein(Hpp4)genes.The main findings were as follows:1.Comparative genomic analysis revealed a complete PLA denovo synthesis pathway in S1 and the absence of the prenylate dehydrogenase gene(Phe A)in YM-4-3.A complete core pathway for PLA synthesis,including four aromatic amino acid aminotransferase and five lactate dehydrogenase genes,was present in all four strains with a high degree of homology.2.A comparative analysis of YM-4-3(YM-4-3y),which is a high producer of PLA after medium optimization,YM-4-3,which is not optimized,and S1,which is a low producer of PLA,by transcriptome and metabolome sequencing revealed that S1 has a weak denovo synthesis pathway attributed to feedback inhibition of phenylalanine,tyrosine and tryptophan substrates and that the core pathway is not enhanced resulting in low PLA yield.In contrast,YM-4-3,which does not have a complete denovo synthesis pathway,synthesizes PLA mainly in the core pathway and mediates high PLA yield by weakening the synthesis of amino acids and their derivatives and through enhanced central carbon metabolism.3.Seven knockout strains were obtained by knocking out 12 specific different expressed genes in YM-4-3y.The seven strains showed no significant differences in growth and acid production in MRS medium.?Cys E strains showed a 51.3%reduction in PLA yield in the optimised medium and a 50% reduction in cell dry weight compared to the wild type strain,but none of these phenomena were present in the MRS medium;it is assumed that a component of the optimised medium mediates the activation of the Cys E gene.The ?Hpp4 strain showed a 10% increase in fermentation supernatant and PLA yield per cell in the optimised medium,with no significant differences in growth and acid production from the wild type.4.ranscriptome analysis of the ?Cys E and ?Hpp4 strains revealed that the down-regulated differential genes of the ?Cys E strain were significantly enriched in the purine metabolism,especially in the hypoxanthine nucleotide(IMP)biosynthetic pathway and fatty acid biosynthesis pathway,and the up-regulated differential genes were enriched in the starch and sucrose metabolism pathways.It is hypothesized that deletion of the Cys E gene affects IMP biosynthetic and metabolic,leading to reduced cell replication.The deletion of the Hpp4 gene caused mainly low expression of glutamate dehydrogenase and upregulation of serine D-Ala-D-Ala carboxypeptidase.The upregulation of carboxypeptidase increased the catalytic and translocation activities of the intracellular enzyme,thereby slightly enhancing the yield of PLA. |
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