Physiological Mechanism Of Streptomyces Albulus Response To Low PH

?-Poly-L-lysine??-PL?is a novel biological food preservative.It is mainly produced by Streptomyces albulus through aerobic fermentation and is strictly regulated by low pH.Thus,the study of physiological response mechanism of Streptomyces albulus in low pH environment has important theoretical and practical values for revealing the mechanism of?-PL biosynthesis and enhancing its biosynthesis capbility.In this thesis,Streptomyces albulus M-Z18 was used as the research object,and the chemostat cultivation(D=0.04 h^-1)at different pH?pH 4.0,5.0 and 5.5?was used as the research platform.Through microbial physiology,transcriptome and molecular biology,the physiological response of S.albulus M-Z18 to low pH stress was preliminarily explored.Moreover,the role of some common acid tolerance systems in S.albulus M-Z18 towards the low pH were identified.The main results are as follows:?1?The effects of low pH stress on S.albulus M-Z18 were studied at the microenvironment level.Based on the chemostat cultivation using glucose as a limiting carbon source,the analysis of intracellular pH?pH_i?value showed that the pH_i was about 7.5when the environmental pH values at 4.0-5.5,indicating that S.albulus M-Z18 had the mechanism of stabilizing pH_i in low pH stress environment.The analysis of H⁺-ATPase activity and intracellular ATP concentration showed that the activity of H⁺-ATPase and intracellular ATP concentration at pH 4.0 were 3.02 and 2.67 folds higher than these at pH 5.5,respectively.Further analysis showed that low pH stress also led to the increase of fatty acid unsaturation and average carbon chain length of cell membrane.Moreover,the accumulation of basic amino acids,such as L-lysine and L-arginine,in cells were also improved.The above results showed that S.albulus M-Z18 could stabilize pH_i under low pH stress by increasing H⁺-ATPase activity and enhancing the accumulation of intracellular basic amino acids.At the same time,S.albulus M-Z18 could maintain its primary metabolic under low pH stress by changing cell membrane fluidity and accelerating the exchange of substances and energy inside and outside cells.?2?The effects of low pH stress on S.albulus M-Z18 were analyzed at the level of gene transcription.Based on comparative transcriptome analysis,it was found that the second stage of glycolysis,TCA cycle and oxidative phosphorylation were strengthened under low pH stress,while the non-oxidative stage of pentose phosphate pathway was weakened,which resulted in the preference of glucose for energy producing.Meanwhile,the overall synthesis of amino acids was weakened,but the synthesis of L-lysine and L-arginine was strengthened as a whole.However,the transcription level of genes involved in the degradation of long-chain fatty acids was significantly decreased,and the transcription levels of genes encoding fatty acid desaturase was significantly increased,which resulted in the increase of fatty acid unsaturation and average carbon chain length of cell membranes.In addition,low pH stress led to a significant increase in the transcription levels of genes encoding molecular chaperones?such as DnaK,DnaJ and GroES?and ATP-dependent proteases?Clp proteases?,which enhanced the ability of protein repair and degradation.A significant increase in the transcription levels of genes related to nucleotide excision repair and non-homologous terminal junction repair,which enhanced the ability to repair severely damaged DNA.The transcriptional levels of genes related to iminase system?ADI?,glutamic acid decarboxylase system?GAD?and urease system were significantly increased,which enhanced the ability to maintain pH_i homeostasis.The above results not only confirm the physiological response of S.albulus M-Z18 at the microenvironment level,but also reveal the potential acid-resistant system.?3?The effects of ADI,GAD,lysine decarboxylase system?CAD?,?-PL synthesis and urease system on the resistance of S.albulus M-Z18 to low pH stress were studied based on the construction of gene inactivation and overexpression mutants and external addition experiment.The insertion inactivation of arcA?coding arginine deaminase?and gadB?coding glutamic acid decarboxylase?resulted in a decrease in biomass of S.albulus M-Z18 under low pH stress,which was 8.3%and 6.4%lower than that of the control,respectively.Overexpression of the two genes resulted in a slight increase in biomass,suggesting that ADI and GAD systems might participate in pH_i stabilization,but their contribution was limited.Knockout of cadA?coded lysine decarboxylase?and insertion inactivation of pls?coded epsilon-polylysine synthase?hardly affected the growth of S.albulus M-Z18 under low pH stress.S.albulus M-Z18 has urease system and can decompose urea to make environmental pH rise rapidly.The above results indicate that the common amino acid-dependent acid-tolerant system is not the main acid-tolerant pathway of S.albulus M-Z18,but the urease system may be an important acid-tolerant pathway.