Selection Of Strains Producing ?-polylysine And Study On The Synthesis Pathway

In recent years,food safety issues have received high attention from the government and society.Needless to say,food preservatives are also one of the key factors in food safety.?-Poly-L-lysine(s-PL)is a homopolymers of L-lysine.Due to its wide range of inhibitory,good solubility,thermal stability,biodegradable,highly safety,s-PL is excellent and effective choice as food preservatives.?-PL,produced by fermentation,but low yield is the main factor limiting its industrial production.Therefore,it is very important to improve the production of ?-PL by microbial fermentation.In this study,through screening of high-yielding strains,optimizing the fermentation process and adjusting the intermediate metabolites to improve the yield of ?-PL.According to transcriptome sequencing to analyse different gene expression profiling and synthetic pathway of ?-PL.The strain SAB was used as the original strain,through morphology observation,physiological and biochemical experimental analysis and molecule identification of 16S rRNA,was identified to Streptomyces albulus.Through the ARTP method,combining with ribosome engineering and streptomycin resistant plates,glycine and ?-PL resistant plates,we obtained high-yielding strains SAY6 with ?-PL production of 2.096g/L.And increased by 118.6%,compared with the original strain SAB(0.959g/L).Through the orthogonal experiment,obtained the optimization medium:glucose 5.0%,yeast extract 0.50%,(NH4)2SO4 1.0%.The optimum fermentation conditions were as follows:the time of seed liquid culture 20h,the volume of inoculation 10%,the volume of medium 80mL/500mL,the shaker speed 200r/min,the cultivate time 72h,and while reach to 24h adjusting pH to 4.5;and e-PL yield will reach 2.536g/L,increased by 20.1%.The effects of citric acid,L-aspartic acid(L-Asp)and L-lysine(L-Lys)on the synthesis of ?-PL were studied,and we got the optimum proposal of adding intermediate metabolites:addition of 1.0g/L citric acid at the start of fermentation,0.35g/L L-Aspartic acid and 1.0g/L L-Lysine in 12h of fermentation could increase production of ?-PL to 31.5%,Compared with the original strain SAB increased by 247.7%.According to transcriptome sequencing,differentially expressed genes were found in strain SAY6,773 were up-regulated,714 were down-regulated,and most of the differentially expressed genes were enriched in amino acid synthesis and transport pathways.Analysis of synthetic pathway of ?-PL,it was found that the main pathway of s-PL synthesis may be glutamic acid to lysine,and the excess product inhibits the aspartic acid to lysine pathway,the expression of differentially expressed genes in lysine degradation pathway was down-regulated,inhibiting the degradation of lysine,and accumulating more substrates for s-PL synthesis.The results showed the reason of ?-PL production increased at molecular level,which provided the basis to improve the yield of s-PL by molecular biology method.