Adaptive Evolution Of Streptomyces Albulus To Low PH Tolerance And Mechanism Analysis

ε-poly-L-lysine（ε-PL）is a homotype amino acid polymerization.It formed by dehydration condensation of 25～35 L-lysine monomers throughα-COOH andε-NH₂.Due to its wide antibacterial spectrum and high safety,ε-PL has been approved as a natural food preservative in Japan,South Korea,the United States and China.Streptomyces albulus is the main producer ofε-PL;the optimum pH for cell growth of S.albulus is neutral,while the optimum pH forε-PL production is around 4.0.Therefore,S.albulus have to be subjected to low pH stress for a long time（about 7 days）during the fermentation process,which leads to the decline of cell viability andε-PL biosynthesis ability.In order to increaseε-PL production efficiency caused by low pH stress,the adaptive evolutionary strategy was used to enhance the low pH tolerance of S.albulus.Meanwhile,S.albulus ALE3.6,an adaptive strain with increased pH tolerance and constantε-PL synthesis ability,was obtained by combination with strain screening.Secondly,the effects of pH on the growth andε-PL synthesis of ALE3.6were investigated,and theε-PL production performances under different fermentation processes were also evaluated.Finally,the physiological mechanism of the low pH by ALE3.6 was analyzed based on comparative transcriptomics and cell physiological analysis.The main research results are as follows:（1）Through the adaptive evolution strategy of gradually lowering the pH value,the strain ALE3.6 with enhanced low pH tolerance andε-PL production was obtained.Based on the pH tolerance of the parental strain GS114 and the investigation of the growth of the bacteria at low pH environment,the initial transfer pH of the adaptive evolution was determined to be 4.0,and the transfer time was 36 h.In the process of adaptive evolution,the optimal strains ALE4.0（pH 4.0）,ALE3.8（pH 3.8）and ALE3.6（pH 3.6）.Through the evaluation of ε-PL fermentation and acid resistance performance,ALE3.6 was determined to be the optimal adaptive evolution strain.The acid tolerance of this strain was significantly enhanced,while itsε-PL production was increased by 17.9%.（2）The optimal fermentation strategy of ALE3.6 was determined at different pH levels.First,the effects of pH on the growth andε-PL biosynthesis of ALE3.6 were investigated,and the optimal pH forε-PL biosynthesis was still 4.0.Subsequently,the fermentation process of S.albulus ALE3.6 under three fermentation strategies was analyzed in detail,and the strategy of constant pH 4.0 combined with L-lysine feeding was identified to be the optimal fermentation process of ALE3.6.Under this fermentation process,theε-PL production and the synthesis capacity of unit cell reached 81.43 g·L^-1 and 1.83 g·g^-1 at 192 h,which were 20.5%and 28.9%higher than those of the parental strain GS114.（3）The physiological mechanism of S.albulus ALE3.6 tolerance to low pH stress was preliminarily analyzed at the global gene transcription level and physiological level.It was found that the cell wall of S.albulus ALE3.6 was more sensitive to acidic environment and the cell membrane integrity was improved.Genes associated with cellular antioxidant and stress resistance were also up-regulated.In addition,the synthesis and content of arginine and L-lysine increased,while the use and efflux of intracellular protons were enhanced to prevent cytoplasmic acidification.