Breeding And Metabolic Flux Analysis Of ε-poly-L-lysine Tolerant High Yield Strains

ε-poly-L-lysine(ε-PL) is a polymer that consists of 25~30 L-lysine monomers with the peptide bonds between α-carboxyl and ε-amino groups. Because of its wide antimicrobial spectrum, safety and well thermal stability, ε-PL has been widely used in many fields, such as food, health care, environment protection and so on. However, the current modification of ε-PL producing bacteria focuses mainly on the non-rational breeding method such as UV irradiation, composite mutagenesis and genome shuffling(GS), while this study attempted to apply the Genome shuffling method to obtain the ε-PL strain with an increased yield, then analyze the mechanism of the high yield through the metabolic flux analysis method, and lay a theoretical basis for further directional transformation of the ε-PL producing strain.In this study, a high ε-PL producing strain Streptomyces sp. AF3-44 was obtained by genome shuffling method. The ε-PL synthetic metabolic network of Streptomyces sp. was constructed using flux analysis method and the metabolic flux was analyzed in batch fermentation. Taking ε-PL yield as the indicator, the effect of six exogenous supplements on ε-PL production by AF3-44 was investigated based on the analysis of metabolic network. The main contents and results are as follows:Streptomyces sp. AS32 and Streptomyces albulus F15 were selected as the parental strains. After three rounds of genome shuffling, a resistant and high ε-PL yield strain AF3-44 with 3.1 g·L-1 ε-PL productivity in shake-flask, which was increased by 34% and 29%compared with those of parental strains AS32 and F15 respectively was obtained. Moreover, the hereditary character of AF3-44 was stable by five successive subculture experiments.Metabolic and enzyme activity analysis of the mutant strain AF3-44 and its parental strains was conducted in 3 L bioreactor in the same condition. The mechanism of ε-PL biosynthesis by Streptomyces sp. was investigated. The fluxes to the pentose phosphate pathway(PPP) and the tricarboxylic acid cycle(TCA) were in the middle level among parental strains. While flux to biosynthesis of ε-PL increased by 75.6% and 49.9% compared with their parental strains AS32 and F15 respectively. The metabolic flux analysis showed that higher flux to the precursor lysine and ε-PL, and the redistribution of metabolic fluxes in PPP and TCA contributed to the improvement of ε-PL in the shuffled strain.The fermentation of ε-PL with AF3-44 was regulated by adding calcium gluconate, Ca Cl2, L-lysine, L-asparagine, fumaric acid and succinic acid based on the analysis of metabolic network. Results showed that addition of 4 g·L-1 calcium gluconate improved the production of ε-PL, which was increased by 15%. Its effect on key enzyme activities of glucose metabolism and ε-PL synthesis was studied. The result showed that calcium gluconate could lead to the improvement of the flux to the synthesis of ε-PL, enhance the activities of both hexokinase and pyruvate kinase in the latter stage of fermentation which enhanced ε-PL biosynthesis.