| ?-poly-L-lysine??-PL?is a cationic homopolymer with a monomer L-lysine polymerization degree in the range of 25-35.?-PL has broad-spectrum antibacterial activity and high safety,and has been approved for use as a new type of biological food preservative in Japan,the United States,South Korea and China.?-PL is mainly obtained by aerobic fermentation of Streptomyces albulus.Traditional breeding methods to enhance the production capacity of?-PL synthesis is an important way to improve its fermentation level.However,the intracellular metabolism differences between high and low production yield strains are rarely discussed,there may be key information for rapidly increasing?-PL production.This article takes Streptomyces albulus M-Z18?The first generation of high-yield bacteria?and Streptomyces albulus GS114?The second-generation high-yield bacteria,shake flask output is about twice that of Streptomyces albulus M-Z18.?as the research objects,and takes the fermentation samples of different fermentation stages?48 h,96 h,144 h?as the research basis,with the help of metabolomics and molecular biology and other research methods,initially proved the high-yield metabolic mechanism of S.albulus GS114 relative to S.albulus M-Z18 in the cell metabolic level,and identified the promote effect of the exogenous addition of differential metabolites to the?-PL synthesis of S.albulus GS114 and S.albulus M-Z18,while studying the effect of?-polylysine synthase gene?pls?overexpression combined with the addition of precursor substances.The main research results obtained are as follows:?1?The metabolic differences between S.albulus GS114 and S.albulus M-Z18 were studied at the metabolic level.Based on relative quantitative metabolomics analysis.The intermediate metabolite flux of S.albulus GS114 on glycolysis?Fructose 6-phosphate,3-Phospho-glycerate?was significantly higher than that of S.albulus M-Z18,indicating the sugar utilization efficiency of S.albulus GS114 is higher.The intermediate metabolite flux of S.albulus GS114 on the TCA cycle?L-Malate,L-Succinate,Acetyl-CoA?was significantly higher than that of S.albulus M-Z18,indicating S.albulus GS114 is more active in energy metabolism.Amino acids??-aminobutyric acid,L-leucine,L-phenylalanine,L-methionine,L-Proline,L-Glutamine?anabolic pathway flux in S.albulus GS114 is significantly higher than S.albulus M-Z18,indicating the cell metabolism of nutrients in S.albulus GS114 is more sufficient.The stearic acid flux of S.albulus GS114 in the fatty acid synthesis pathway is significantly lower than that of S.albulus M-Z18,indicating that S.albulus GS114 may have higher cell membrane fluidity and less precursor and energy expenditure.At the same time,AMP in S.albulus GS114 is significantly higher than that in S.albulus M-Z18,indicating that S.albulus GS114 has a higher enzyme content or vitality in?-polylysine synthetase?Pls?.Above research results confirmed that the overall cell activity of S.albulus GS114 is stronger than that of S.albulus M-Z18 at the intracellular metabolism level,and it is more concentrated and efficient than S.albulus M-Z18 in terms of precursors and energy utilization.it proves the high-yield mechanism of S.albulus GS114 from the metabolic level to a certain extent.?2?Exogenous addition based on metabolome data and supplementary analysis.The added differential metabolites include:difference between groups:L-Malate,L-Succinate,?-Aminobutyric acid,L-Leucine,L-Phenylalanine,L-Methionine,L-Proline,L-Glutamine;difference in groups:L-Glutamate,L-Aspartate;direct precursor:L-Lysine.The verification results shows that the added effective substances are?based on a constant pH system of citric acid-sodium citrate?:L-Proline,L-Glutamine,L-Glutamate,L-Aspartate,L-Lysine.The best combination is L-Aspartate 50 mM+L-Lysine 10 mM.Zoomed in the 5-L bioreactor,the results showed that the production of S.albulus M-Z18?144 h?increased from 35.75 g·L-11 to46.74 g·L-1,and the production of S.albulus GS114?144 h?increased from 41.39 g·L-1 to49.31 g·L-1.when scale-up in a 50-L bioreactor,the results showed that the production?144 h?of S.albulus GS114 increased from 33.66 g·L-11 to 45.09 g·L-1.?3?Overexpression of pls gene on S.albulus M-Z18.Pls is a key enzyme for?-PL synthesis.The pls gene was overexpressed by genetic means and the results showed that pls transcription factor was up-regulated by 16.8 times.The production of S.albulus M-Z18/pIB139-pls in the shake flask was significantly higher than that of S.albulus M-Z18-pIB139,and reached to 2.74±0.23 g·L-1.?-PL production of S.albulus M-Z18/pIB139-pls achieved 40.62 g·L-1?144 h?in the 5-L bioreactor.In order to further improve the production capacity of overexpressing strains,we optimized the L-Lysine and ATP exogenous addition concentration,the final establishment of S.albulus M-Z18/pIB139-pls in 5-L bioreactor was simultaneously added with 5.0 g·L-11 L-lysine and1.0 mM ATP.?-PL production was achieving 45.09 g·L-1?144 h?,which was 20.8%higher than the control group.It can be seen that the overexpression of pls gene combined with supplementary precursor L-lysine and energy cofactor ATP is an effective strategy to increase the fermentation level of?-PL. |
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