Metabolic Regulation Of Branched Amino Acids Promoting Natamycin Biosynthesis

Natamycin is a kind of biological preservative,and its research and use has gradually attracted people's attention,and has become a hotspot in food preservation research.Natamycin is mainly produced by Streptomyces natalensis and Streptomyces gilvosporeus through fermentation.It is an efficient and broad-spectrum polyolefin macrolide antifungal antibiotic.It can effectively inhibit the growth of yeasts and fungi.It has the characteristics of low dosage,high efficiency,odorless and odorless,wide application range of pH,good specificity and high safety.Therefore,in the food industry it is used to prevent fungal contamination.Amino acids can be used as carbon or nitrogen sources,and metabolites of amino acids,such as acetyl CoA and propionyl CoA,are precursors of macrolide antibiotics.Amino acid metabolism has an effect on biological fermentation.However,there are few studies on the regulation of biosynthesis by adding branched amino acids in natamycin-producing actinomycetes,and the specific regulation mechanism needs to be further explored.1.The effect of L-valine(L-Val)on biosynthesis pathway of natamycin by Streptomyces natalensis HW-2 was investigated.The experimental results showed that the maximal yield of natamycin reached 1.83 g/L when the adding amount of L-Val was 0.5 g/L during the fermentation of Streptomyces natalensis HW-2 for 36 h,which was 84.85% higher than that of the control.The addition of L-Val resulted in a decrease of biomass and an increase of pH,and an improvement of glucose utilization.The activities of pyruvate kinase(PK),phosphoenolpyruvate carboxylase(PEPC)and pyruvate carboxylase(PC)in intracellular enhanced,while that of citrate synthase(CS)decreased by 26.57%.Comparing with the control,the contents of pyruvic acid,oxaloacetic acid,acetyl-CoA in the broth increased by 80.50%,53.28% and 47.19%,respectively.And,the contents of acetic acid,propionic acid and ?-ketoglutaric acid increased by 16.98%,10.65% and 15.40%,respectively.But,the content of citric acid decreased by 27.01%.2.The transcriptomes of the S.natalensis HW-2 with and without L-valine added to the medium were sequenced by the RNA-Seq technique in the log phase,the late log phase and the early phase of the stationary phase.The results showed that 201 genes were up-regulated and 445 genes were down-regulated at 48 h after fermentation.After fermentation for 60 h,147 genes were up-regulated and 42 genes were downregulated.At 84 h,45 genes were up-regulated and 72 genes were down-regulated.GO analysis showed that differentially expressed genes are mainly involved in biological regulation,metabolic processes,cellular processes,membrane components,catalytic activity,transportation and other processes,mainly in the biosynthesis and catabolism of carboxylic acids,the biosynthesis and metabolism of amino acid and so on.KEGG analysis showed amino acid biosynthesis,fatty acid metabolism,carbon metabolism,C5 branched-chain dicarboxylic acid metabolism,butyrate and propionate metabolism,proline,leucine and isoleucine biosynthesis and degradation,pentose phosphate pathway and other pathways have been enriched.The transcription level of the gene was analyzed from the embden meyerh of parnas pathway,the pentose phosphate pathway,and the tricarboxylic acid cycle.After the addition of valine,the level of gene transcription changed,and the carbon flux was changed,so provided more precursors for the biosynthesis of natamycin.The transcription level of the type I polyketide synthase gene cluster encoding natamycin biosynthesis was analyzed,and it was found that the differential expression level of the gene cluster was not significant.The samples were verified by RT-PCR,and the experimental results were basically consistent with the transcriptome data,indicating that the transcriptome data was reliable.3.Verification of the function of ilvH gene in S.natalensis HW-2,according to the proline synthesis pathway and transcriptome data,select the gene acetyl lactate synthase regulatory subunit(ilvH)for overexpression of the target gene,verify gene function.The activity of acetolactate synthase is regulated by ilvH.By genetic engineering,the expression of S.natalensis HW-2 by ilvH increases the yield of natamycin.The ilvH gene sequence was found from the S.natalensis HW-2 genome,and the primer was cloned to obtain the target gene.The overexpression plasmid of ilvH was constructed based on the plasmid pIB139.The recombinant plasmid was transformed into ET12567(pUZ8002),and the target gene was integrated into S.natalensis HW-2 by E.coli-streposis transfer,and the mutant strain was overexpressed by resistance screening,and the natamycin was detected by fermentation.The yield of the fermentation broth was measured for natamycin production,and it was found that ilvH overexpression strain the yield of natamycin increased by 37.93% compared with the original strain.