Breeding Of Excellent Strain Of Natamycin Production And Analysis Of Gene Differential Expression

Natamycin is a natural macrocyclic antifungal polyene produced by microorganisms.It has been adopted as a food preservative in more than 50 countries and regions around the world.It is widely employed in the food industry,agriculture,animal husbandry and other industries.At present,because the production of natamycin is low,the industrial application is affected.Therefore,to increase the production of natamycin,Streptomyces natalensis HW-2 was bred by mutagenesis,and the fermentation process of the mutant strain was optimized in the study.Also,the high-yield mechanism of mutant strain was studied by transcriptome.The study will provide a theoretical basis for the further development of the strain.The main contents of this paper are as follows:1.S.natalensis HW-2 was breeding by UV-ARTP-DES compound mutagenesis and streptomycin resistance screening method.One mutant strain,S.natalensis DES-26 with good genetic stability was obtained.Results showed that the production of natamycin of DES-26 in the flask was 1.64 g/L,which was increased by 86.36%compared with the initial strain.2.S.natalensis DES-26 was used in this study.The coupling technology of fermentation and macroporous resin extraction was established,and the fermentation medium composition of natamycin was optimized.The results showed that the natamycin yield was 2.37 g/L,which was increased by 65.73%,with the conditions of adding 4 g/30 m L HPD450 resin at 48 h of fermentation.Through single-factor and Box-Behnken test,the most optimal fermentation medium composition was as follows:glucose 35 g/L,beef extract 10 g/L,yeast extract 1.5 g/L,Ca CO₃2.5 g/L,Na Cl 0.3g/L,and blend oil 1.4 g/L.At the conditions,the yield of natamycin was increased by63.36%.3.RNA-Seq method was used to analyze the gene differential expression of S.natalensis DES-26 and S.natalensis HW-2,and the mechanism of high yield of natamycin in the mutant strain was studied from the perspective of transcriptome.The results showed that there were 295 and 860 differential expressed genes(DEGs)at 48h and 72 h,respectively.GO and KEGG analysis showed that glucose metabolism was regulated by up-regulating the gene expression levels of pyruvate kinase,gluconate kinase and isocitrate dehydrogenase;fatty acid metabolism was regulated by up-regulating the gene expression level of type I polyketone synthase and down-regulating the gene expression levels of 3-hydroxy Co A dehydrogenase and acetyl Co A acetyl transferase;the synthesis,modification and transport of natamycin were regulated by up-regulate of the gene expression levels of PKSI,AT,pim B,pim C,pim D,pim E,pim I,pim K,and pim R.Therefore,transcriptional changes of genes involving in glucose,fatty acid metabolism and natamycin biosynthesis pathway promoted the biosynthesis of natamycin in S.natalensis DES-26.