Oxidative Stress And Its Regulation In ?-poly-L-lysine Fermentation By Streptomyces

?-poly-L-lysine(?-PL)is a polymer of L-lysine monomers with the peptide bonds between ?-carboxyl and ?-amino groups.Because of its well water solubility and biocompatibility,non-toxicity and wide antimicrobial spectrum,?-PL has been used in food,medicine and other fields.At present,microbial fermentation is the main method of ?-PL industrial production,however,the main problem is the low product concentration and accompanying high cost.Studies applying the genome shuffling(GS)to obtain the ?-PL producing strains with an increased yield have been successful,but the corresponding mechanism remains unclear.Therefore,in this study,attempts have been made to reveal the reason for high ?-PL yield in GS strain which will benefit the improvement of ?-PL in future.The tested strains in this study included the parental strains Streptomyces sp.AS32 and Streptomyces albulus F15,and genome shuffled strain Streptomyces sp.AF3-44 with high ?-PL production.The relationship among the tolerance of pH and ?-PL,oxidative stress and ?-PL synthesis in these three strains was investigated.And exogenous substances were also supplymented to the fermentation broth to further improve the synthesis of ?-PL in AF3-44.The main contents and results are as follows:The pH and ?-PL tolerance,oxidative damage and cell viability among AF3-44,AS32 and F15 were compared.AF3-44 had the best pH and ?-PL tolerance and antioxidative ability among three tested strains,and kept good cell activity and had the highest ?-PL yield.The trehalose,catalase activity(CAT),superoxide dismutase activity(SOD)glucose-6-phosphate dehydrogenase activity(G6PDH)in AF3-44,AS32 and F15 were analyzed.Trehalose content in GS strain was higher than that in parental strains during the whole fermentation process;SOD and CAT enzyme activities were higher in GS strain than those in parental strains in the early stage of fermentation,and CAT and G6 PDH enzyme activities were higher than those in parental strains in late stage of fermentation.Higher trehalose content and enzymatic activity enabled GS strain tolerant to the acidic pH and high ?-PL concentration during the fermentation process.The cause for oxidative stress and substances counteract with the stress in ?-PL fermentation were investigated.Both pH and ?-PL can induce oxidative stress.?-PL caused the reactive oxygen species production at the earlier but pH at the later stage of fermentation.The antioxidant glutathione(GSH)significantly increased the yield of ?-PL in the shake flask fermentation.Effects of GSH on the fermentation of ?-PL and the expression of key enzymes in the ?-PL synthesis pathway were analyzed.Results showed that yield of ?-PL was 10.50 g·L-1,which was increased by 68.8% with the addition of GSH at pH 6.5 compared with the control(without GSH addition)in batch culture in the 5 L fermentor.The yield of ?-PL was 46.54 g·L-1,which was increased by 17.8% compared with the control in fed-batch fermentation.qRT-PCR analysis indicated that GSH increased the transcription level of phosphate fructokinase,citrate synthase,aspartate kinase,?-polylysine synthase enzymes in ?-PL synthetic pathway,resulting in the increase of ?-PL production.