Screening Of ?-Polylysine-Producing Strains And Its Metabolic Regulation

?-poly-Lysine(?-PL)is mainly produced by Streptomyces albulus,it is a highly efficient and broad-spectrum homopolymer of amino acids,which can inhibit the growth of G⁺,G^-,yeasts and molds.It also has low toxicity and high bacteriostasis.As a natural microbial preservative,?-PL has gradually become a research hotspot in food storage and preservation.It has been proved that adding ethanol stress can effectively improve the secondary metabolites of microorganisms.At present,the study on the regulation of biosynthesis by adding ethanol in?-PL fermentation has not been reported,and the specific regulation mechanism needs further investigation.The main research contents in this paper are as follows:1.Three actinomycete strains were isolated based on the transparent zone formed by themethylene blue and?-PL.Itzhaki method was used to screen the strains.One of the three strains,with the highest?-PL yield(0.39 g/L),was identified by morphological and physiological characterization and sequence analyses of 16S r DNA and gyr B gene.The strain was named as Streptomyces albulus X-18.The molecular polymerization degree of?-PL from S.albulus X-18 was 25-30 L-lysine by the spectral data analysis and the comparison with the?-PL standard.It showed good antimicrobial effect on bacteria and fungi.2.The fermentation medium of?-PL by S.albulus X-18 was optimized using the single factor test and response surface method.Moreover,the stress conditions with positive regulation on?-PL yield were screened.The composition of the optimal fermentation medium was:corn starch 20.0 g/L,(NH₄)₂SO₄ 7.5 g/L,yeast extract 5.0g/L,KH₂PO₄ 0.6 g/L,CaCl₂ 0.7 g/L,MgSO₄ 7H₂O 0.5 g/L,Zn SO₄ 0.03 g/L,and Fe SO₄ 0.03 g/L.After verification,the?-PL yield was 1.23 g/L.Through the screening of the stress conditions,ethanol showed obvious positive effect.The condition with adding 1%ethanol at 6 h fermentation showed the strongest stress effect,and?-PL yield was increased by 42.6%.3.The physiological and biochemical characteristics of S.albulus X-18 were studied with ethanol stress.The results showed that the morphology of the colonies was significantly different,the colonies were with wrinkles,and the edges were neat.Biomass was increased by 6.9%and p H decreased slightly.The utilization rate was increased by 85.7%,and the production of?-PL was increased by 37.8%.The intracellular active oxygen concentration reached 27.25 U/m L at 96 h fermentation,which was 2 times that of the control.At 24 h,the intracellular soluble protein content was 1.75 times that of the control.The intracellular free amino acid analysis showed that the content of aspartic acid,asparagine,and proline were significantly increased,and the content of lysine decreased.The content of fatty acids in the cell membrane was not significantly changed;the activity of ADH was always higher than that of the control,and the ALDH enzyme activity was higher than that of the control after 48hours.At 68 hours,the enzyme activity of PFK was significantly higher than that of the control,by an increase of 24.3%.At 78 h,the enzyme activity of PEPC was significantly higher than that of the control,by an increase of 82.7%.At 96 h,HK and PK were significantly higher than that of the control,by increase of 14.1%and 66.7%;At 110 h,the enzyme activity of DHDPS was significantly higher than that of the control,by increase of 121.08%.Therefore,after ethanol stress,S.albulus X-18increased the concentration of intracellular reactive oxygen species,soluble protein and some free amino acids to adapt the bacteria to ethanol stimulation;The increased expression level of rate-limiting enzymes of EPM pathway,ethanol oxidation,PEPC,DHDPS and other enzymes is beneficial to the accumulation of oxaloacetate and L-lysine,thereby increasing the content of?-PL.4.i TRAQ technology was used to analyze the differences in extracellular proteome levels in S.albulus X-18 with or without ethanol stress.A total of 4419proteins were identified by i TRAQ,with 261 significantly different proteins,of which75 proteins were significantly up-regulated and 186 proteins were significantly down-regulated.GO analysis and KEGG metabolic pathway analysis showed that the upregulation of acetaldehyde dehydrogenase caused the flow of ethanol metabolism to acetyl Co A;Changes in the expression levels of malate 2-isopropyl ester synthase and3-oxoacyl-[acyl carrier protein]synthase III shifted the metabolic flow of the EMP and TCA pathways to the oxaloacetate aspartate pathway;the decreased expression levels of aspartate oxidase and arginine succinate synthase make the metabolism of aspartate The flow focuses on the DAP pathway,which enhances the synthesis of L-lysine.A slight increase in?-PL synthase and a small decrease in?-PL degrading enzymes are beneficial to the accumulation of?-PL.