| Phenyllactic acid is a new kind of broad-spectrum antimicrobial small molecule discovered in recent years.It has a good application prospect because of its safety and stability are better than the chemical preservatives widely used at present,and it can not only be used in the food industry to inhibit a variety of pathogens,but also can be used in the medicine and even the chemical industry.PLA can be metabolized by most lactic acid bacteria(LAB).Most of LAB are recognized as Generally Recognized as Safe(GRAS)microorganisms.It has a better application prospect in the food industry to take it as the starting strain to metabolize PLA,which avoids the formation of toxic intermediates by chemical synthesis method and avoids the food safety problems caused by the fermentation of non-food-borne engineering bacteria to produce PLA.As one of the natural metabolites of LAB,it is of great significance to study its metabolic mechanism.However,at present,the yield of PLA produced by LAB metabolism is relatively low,usually less than 100 mg/L,which has become an important factor limiting its wide application.1.In this study,the characteristics,applications and biosynthesis of PLA were reviewed to form a comprehensive understanding.2.A strain of Lactobacillus casei with PLA production ability was screened by calcium carbonate solubility circle screening and HPLC detection method.The strain was used as the starting strain for the experiment,through NCBI database comparison,it was found that the sequence homology was up to 99% which from aromatic aminotransferase gene arat in PLA metabolic pathway of several Lactobacillus casei strains,indicating that this gene was highly conserved.The primers were designed based on the sequence information provided by the gene.The knockout plasmid arat-plcnick was constructed by overlapping PCR.Finally,arat gene of this stain was knocked out by CRISPR/Cas9 gene editing system,and an arat-deficient strain L.casei1.8727?arat was obtained.3.The yield of PLA was analyzed by HPLC.It was showed that after 72 h fermentation,the yield of PLA and Phe increased by 66.7% and 57.8% compared with the original strain,respectively.The results showed that the deletion of arat gene had a significant effect on the yield of PLA and Phe in L.casei1.8727,indicating that the enzyme could control the synthesis of PLA and Phe.This study is the first to knock out the arat gene in L.casei 1.8727 by CRISPR/Cas9 system.The results showed that the strain had the ability to metabolize Phe.As a key enzyme in the PLA metabolic pathway,the deletion of this gene did not reduce the PLA yield,but significantly promoted the anabolism of PLA and Phe.The results indicate that there may be other compensatory pathways in Lactobacillus casei.The changes in metabolic flow caused by the deletion of arat gene eventually lead to the increase of the production of PLA and Phe.Meanwhile,the anabolism of PLA may involve multiple genes and is a complex metabolic network.The findings of this study are of great significance for the in-depth study of the mechanism of PLA anabolism,and further promote the study of PLA anabolism theory.At the same time,the gene editing system and method used in this study can be well applied to the gene knockout operation of Lactobacillus casei,which lays a foundation for further studies on PLA metabolism and synthesis in the future.At the same time,the future research direction is prospected... |
PDF Full Text Request