ε-poly-L-lysine Producing Strain Breeding By Genome Shuffling

ε-Poly-L-lysine（ε-PL） is a25-35L-lysine linear homopolymer, which has a uniquelinking between ε-amino and α-carboxyl. The compound is biodegradable, water soluble, andhas various function such as antimicrobial activity, antifungus, antiphage action and has lowtoxicity to animals and human. So it turns to be a safe and efficient biopreservative. ε-PL ispositive charged and has been of a broad range of other industrial application such as drugcarrier, highly water absorbale hydrogels and gene chip, etc.Streptomyces padanus LS-L5was the starting source. In order to enhance its ε-PLproductivity followed actions have been performed: firstly, Streptomyces padanus LS-L5wastreated with ultraviolet and positive mutants were screened on solid medium with succinicacid as sole carbon source; secondly, the positive mutants were undergone three rounds ofgenome shuffling and strains with higher ε-PL productivity were screened; thirdly, the basesubstitution in16S rDNA, a comparison of PEP carboxylase activity and metabolic flowvariation between LS-L5and the best fusant were performed to explain the ε-PL productivityenhancement in the fusant; at last, a optimization about nutritions for the best fusant wascarried out.Streptomyces padanus LS-L5were treated with ultraviolet. The optimal dosage was3min （8W,30cm）. Spore suspension was spread on plates with succinic acid as sole carbonsource to screen positive mutants. Six strains were selected and their average ε-PLproductivity was0.65g/L, which was35.4%higher than that of LS-L5.Three rounds of genome shuffling between the six positive mutants were carried out. Inthe first round shuffling, eight strains were obtained and their average ε-PL productivity was0.73±0.02g/L. In the second round shuffling, seven shuffled strains were obtained with anaverage ε-PL productivity0.80±0.02g/L. There was no improvement of ε-PL productivity inthe third round shuffling compared with that of the second round shuffling. It proposed thatthe PEP carboxylase activity was high enough and other enzymes activity in mutants were notchanged. Strain F2-7of the second round shuffling turned to be the best shuffled strain. ε-PLproductivity of F2-7in5liter jar batch fermentation was1.55times that of LS-L5, and whichwas2.21times in fed-batch fermentation.The differences between LS-L5and F2-7were investigated. The16S rDNA similaritybetween original strain LS-L5and shuffled strain F2-7was99.36%, and only eight baseschanged. The PEP carboxylase activity of shuffled strain F2-7was4times higher than that oforiginal strain LS-L5. The metabolic flux analysis （MFA） revealed that the flux from PEP toOAA was enhanced8.67%in F2-7than that of LS-L5.The result of Box-Behnken Design and Response Surface analysis determined theoptimal condition of nutritions for ε-PL production of F2-7. The optimal conditions weredetermined as followed: glucose48.06g/L, yeast extract5.09g/L,（NH₄）₂SO₄14.84g/L,KH₂PO₄1.00g/L, K₂HPO₄·3H₂O2.00g/L, MgSO₄·7H₂O1.00g/L. Under optimizedcondition ε-PL production of F2-7was0.93g/L in shake-flask fermentation.