The Study On Acid Adaptive Evolution And Fermentation Technology Of Streptomyces For ε-poly-L-lysine Production

ε-poly-L-lysine(ε-PL)is a kind of L-lysine homomer,which has been widely used in the fields of food,medicine,agriculture and electronic material design due to its superior physical and chemical properties.Due to the low synthesis ability of ε-PL producing bacteria,ε-PL has not been widely used.Therefore,improving the fermentation level of ε-PL has been a focus of research in academia and industry.In this study,Streptomyces albulus CICC 11022(a ε-PL producing bacterium)was used as the starting strain,and a high ε-PL producing strain of S.albulus F29 was screened by acid adaptation evolution.Then,the differences of intracellular physiological indexes between the high-producing and the original strains were compared to study the underlying mechanism.Finally,the fermentation process was optimized to increase the production of ε-PL,which would provide a theoretical basis for industrial production.The main research contents are as follows:(1)Taking S.albulus CICC 11022 as the starting strain,a high ε-PL producing strain of S.albulus F29 was screened by acid adaptive evolution.The production of S.albulus F29 was 1.12 g/L in shake flask fermentation,which was 80.64% higher than that of S.albulus CICC 11022.After 6 generations of cultivation,the production of ε-PL remained at 1.12±0.01 g/L.Compared to S.albulus CICC 11022,the growth rate of S.albulus F29 with initial p H4.0 was much higher and the dry cell weights(DCW)were increased by 25% at 96 h.When stressed at p H3.0 for 48 h,the survival rate of S.albulus F29 was 22.5%,which was 46.66% higher than that of S.albulus CICC 11022.After acid adaptation evolution,the ε-PL synthesis and acid tolerance abilities of S.albulus F29 have been greatly improved.(2)To disclose the high-producing mechanism,the differences of intracellular physiological levels between the high-producing and the original strains were compared in batch fermentation.The ε-PL production and DCW of S.albulus F29 were 5.62 and 26.59 g/L,respectively,which were 47.89% and 40.68%higher than those of S.albulus CICC 11022.The ε-PL yield of S.albulus F29 was0.21 g/g,which was 16.66% higher than that of S.albulus CICC 11022.After fitting the batch fermentation kinetic parameters by the Origin software,it was found that: the specific growth rate of S.albulus F29 was always higher than that of S.albulus CICC 11022,and the ε-PL specific synthesis rate of S.albulus F29 was also always higher than that of S.albulus CICC 11022 in the first 36 h.Compared to S.albulus CICC 11022,S.albulus F29 showed higher intracellular ATP,lysine,glutamate and aspartate concentrations,as well as the activities of glucose-6-phosphate dehydrogenase(G6PDH),citrate synthase(CS),phosphoenolpyruvate carboxylase(PEPC),aspartate kinase(ASK)andε-poly-lysine synthase(Pls): Among them,the intracellular ATP concentration of S.albulus F29 was 39.55 mg/g higher than that of S.albulus CICC 11022 at 30 h;at 36 h of fermentation,the intracellular lysine concentration of S.albulus F29 reached 2.71 mg/g DCW,which was 1.97 times higher than that of S.albulus CICC 11022,and the activities of G6 PDH,CS,PEPC,ASK and Pls were increased by 43.75%,31.13%,71.21%,78.02% and 237%,respectively.It was preliminarly confirmed that S.albulus F29 mainly depended on the increase of intracellular ATP,amino acids and the activity of key enzymes in cell metabolism and ε-PL synthesis pathway to achieve the high production of ε-PL.(3)In order to further improve the production of ε-PL,the fermentation parameters of S.albulus F29 were optimized in a 5 L fermenter: p H3.8 was the optimal p H for S.albulus F29 in batch fermentation,and the production of ε-PL reached 5.62 g/L after 42 h of fermentation;the optimal initial glucose concentration in the batch fermentation of S.albulus F29 was 90 g/L,and the DCW and ε-PL production of S.albulus F29 reached 21.3 and 9.7 g/L at 58 h.The optimal fermentation process was employed in the fed-batch fermentation using p H-shock strategy,the DCW and ε-PL produciton of S.albulus F29 reached 54.2and 31.6 g/L at 192 h and the ε-PL yield was 0.58 g/g,which was 42.98%,42.25%and 18.6% higher than those of the original strategy.The optimization of initial carbon source concentration and fermentation p H increased the growth andε-PL synthesis abilities of S.albulus F29,and significantly increased the biomass and the final production of ε-PL,which provided a reference for further industrial production of ε-PL.