| ?-poly-L-lysine(?-PL)is a homo-polymer composed of 25 to 35 residues of L-lysine(L-Lys)with linkages between?-carboxyl group and?-amino group,which is mainly produced by aerobic microbial fermentation and extracellularly accumulated.With wide antimicrobial spectrum and high safety,?-PL has been successfully used as a food preservative in Japan,the United States,South Korea and China at present.Besides,as a safe and green biopolymer,it has also been extensively applied in the medical industry.Therefore,?-PL is a novel biotechnology product with widespread application and tremendous vendibility.In this paper,a strain of?-PL producer Streptomyces albulus M-Z18(S.albulus M-Z18)was selected as the research object.Through the establishment of two-stage fermentation strategy with low p H shock,the efficiency of?-PL fermentation with low p H shock was significantly improved.The effects of low pH shock on S.albulus M-Z18 were studied in physiological and transcriptional levels.Meanwhile,the physiological functions of two-component system Mpr AB in S.albulus M-Z18 in response to low pH shock were studied by molecular manipulation such as gene inactivation,complement and overexpression.Finally,the physiological mechanism of tolerance to low p H stress was systematically studied.It was found that urease system plays an important role in the resistance of S.albulus M-Z18to low pH stress.The major conclusions were attached as follows:(1)A two-stage fermentation strategy with low pH shock was established,which significantly improved the fermentation efficiency of?-PL.Based on the analysis of the fermentation process of the current p H shock strategy,the reasons for its low production intensity were found.Through the optimization of inoculation time,initial fermentation p H and inoculation volume,a new two-stage fermentation strategy with low pH shock was established:the first stage was seed culture stage,including seed growth,p H shock and bacterial recovery,the second stage was constant pH3.85 fermentation stage.With the new strategy,the production,yield and conversion of?-PL were reached 32.22 g/L,5.86 g/L/d and 8.2%respectively in 5 L fermentor,which were 55.12%,23.1%and 43.9%higher than the current p H shock strategy.The production of?-PL and DCW reached 31.26 g/L and 45.68 g/L in 50 L fermentor.(2)The effect of low p H shock on S.albulus M-Z18 was studied at physiological level.Through the analysis of physiological parameters of cell,it was found that low p H shock increased the activity of?-PL synthetase and the concentration of L-lysine in cell;at the same time,low pH shock increased the level of intracellular ATP by enhancing the respiratory activity of cell,which were all the key factors affecting the biosynthesis of?-PL;in addition,the low p H shock decreased the length of carbon chain and increased the unsaturation of fatty acids;the unsaturation of acid improves the fluidity of cell membrane and facilitates the transportation of substances.The low pH shock also increases the level of intracellular reactive oxygen species,but the content of malondialdehyde is relatively low,which indicates that the low p H shock enhances the anti-oxidation ability of the cell,which is beneficial for the cell to resist the long-term and low pH fermentation environment in the later fermentation.(3)The effect of low pH shock on S.albulus M-Z18 was studied at the gene transcription level.With comparative transcriptomics,we found that low pH shock caused significant changes in the transcription level of genes related to signal transduction,electronic respiratory chain,membrane transport,nitrogen metabolism, energy metabolism and redox balance,which indicated that low p H shock caused a global effect in S.albulus M-Z18.Meanwhile,the changes of gene transcription level also confirmed the analysis results of cell physiological parameters.(4)The physiological function of two-component system MprAB was studied.Based on the analysis of transcriptome data,we found that low pH shock increased the transcription of two-component system Mpr AB and serine protease PepD by 2-5 times;through further inactivation of mprA,mprB and pep D,the?-PL production of S.albulus M-Z18 decreased by 52.11%,60.56%and 55.63%respectively;and then three genes were complemented in mutant strains respec tively,and the production of ?-PL was recovered to the level of original fermentation.Finally,the overexpression of three genes in S.albulus M-Z18 significantly increased the production of?-PL.These results indicate that mprA,mpr B and pepD have positive regulatory effects on synthetase gene(pls).In addition,through q RT-PCR,it was found that mprB can positively regulate the expression of mprA,and there is a positive correlation between mprA and pepD.(5)The physiological mechanism of tolerance to low p H stress was systemly studied.The analysis of intracellular microenvironment parameters showed that S.albulus M-Z18 in low pH environment(pH4.0)could maintained the intracellular p H in neutral(p H 7.5)by increasing membrane fatty acid unsaturation and average carbon chain length,increasing H~+-ATPase activity and intracellular ATP level, accumulating more arginine,lysine and histidine;further comparative transcriptome analysis showed that in the low pH environment(p H 4.0),the glycolysis,TCA cycle and oxidative phosphorylation pathways of S.albulus M-Z18 were enhanced,while most of the amino acid synthesis pathways were weakened,but the synthesis of aspartic acid group amino acids was enhanced;the synthesis ability of long-chain fatty acids and unsaturated fatty acids was also improved;meanwhile,the ability of protein repair and degradation,a nd the ability of DNA repair were improved,which were consistent with the intracellular microenvironment parameters.In addition,we found that urease system plays an important role in the tolerance of S.albulus M-Z18 to low pH stress by constructing acid resistant system gene mutants. |
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