Genome Shuffling And Metabolic Regulation Analysis Of ε-poly-l-lysine Producing Strains

ε-poly-L-lysine(ε-PL), a microbial metabolite of homo-poly-amino acid where lysine is polymerized between ε-amino and α-carboxyl groups by ε-PL synthetase, can suppress many kinds of microbial growth and with potential applications in the fields of food, medicine and others. However,?for the wild-type microorganisms reason of self-protection, ε-PL synthesis is subject to strict regulation, it is necessary to be genetically transformed to increase production of ε-PL.In this paper, we enhanced ε-PL production in the ε-PL tolerant strain by genome shuffling and investigated the mechanism of improvement. The main contents and results are as follows:(1) The initial mutant library was constructed by three rounds diethyl sulfate(DES) mutagenesis and four mutants(D3-81, D3-90, D3-239, D-347) were isolated. The ε-PL productivity of these four mutants in shake flask from triplicate experiments was 2.05±0.04, 2.02±0.02, 2.06±0.02, 1.98±0.04 g·L-1, respectively. After four rounds of protoplast recursive fusion, a resistance and high yield of ε-PL strain F4-22 with 3.11 g·L-1 ε-PL productivity in shake-flask, 1.81-fold in comparison with that of parent strain M-Z18, was obtained. Moreover, the hereditary character of F4-22 was stable by five successive subculture experiments.(2) Through optimizing the fermentation conditions in a 5-L fermentater, F4-22 was able to produce 32.7% more ε-PL(39.96 g·L-1) under a constant p H 4.0 than that of M-Z18(30.11 g·L-1) which was obtained under optimized two-stage culture at the final fermentation time of 173 h.(3) The meliorative counteractive to ε-PL of F4-22 is better than that of M-Z18, which was verified by means of cell stainning, the membrane fatty acid composition analysis and the structure of mycelia observation via transmission electron microscope. Furthermore, comparisons of the activities of enzymes, which are supposed to be the key enzymes in ε-PL synthetic pathway in F4-22 and M-Z18, activities at different time in shake-flask fermentation revealed that increased activity of aspartate kinase(ASK) and vitality of pellets in high concentration of ε-PL accounted for the enhanced productivity of F4-22.