Preliminary Study On Genes Related To Low PH Tolerance And Their Functions In Streptpmyces Albulus

Streptomyces albulus is a main strain used for ε-poly-L-lysine(ε-PL),which is always suffered pH 4.0 stress during ε-PL production.At present,there is a lack of mechanism on how S.albulus is combat to the low pH stress.Therefore,the study of genes related to acid tolerance and their functions in S.albulus is important for understanding the physiological mechanisms of S.albulus to resist to the low pH stress.In this study,we investigated the role of common acid tolerance system genes,peptidoglycan synthesis genes and ABC transporter genes in the resistance of S.albulus M-Z18 to low pH stress through gene inactivation/knockout and overexpression.The main results are as following:(1)Physiological function of acid tolerance system in S.albulus M-Z18.The response of acid tolerance system in S.albulus M-Z18 to low pH stress was investigated by inactivation/knockout and overexpression of the key enzyme genes involved in the acid tolerance system system.Acid tolerance assessment showed that the knockout of lysine decarboxylase cad A reduced the growth rate of S.albulus M-Z18 at pH 3.5,indicating that the lysine decarboxylase system(CAD)had an effect on maintaining intracellular pH homeostasis under low pH stress.Inactivating glutamic acid decarboxylase system(GAD),arginine deiminase system(ADI)and urease systems alone had no effect on the low pH tolerance of S.albulus M-Z18.Further overexpression of key enzyme genes involved in GAD,ADI,CAD systems and urease system separately showed no significant change in low pH tolerance in the overexpressed strains.(2)Effect of cell wall peptidoglycan synthesis related genes on the resistance of S.albulus M-Z18 to low pH stress.Transcriptional analysis of peptidoglycan synthesis genes in S.albulus M-Z18 at pH 4.0 showed that glycosyltransferase,transpeptidase and carboxypeptidase genes involved in peptidoglycan cross-linking and modification were significantly up-regulated,suggesting that peptidoglycan maintains cell morphology to resist low pH stress.Therefore,overexpression of peptidoglycan synthesis-related genes in S.albulus M-Z18 were performed.The results revealed that overexpression of mrc A4 and pbp2 A decreased the low pH tolerance of S.albulus M-Z18,while overexpression of mur C、fem X,mrc A2,pbp A1,pbp A3,fts I,dac A2 and pbp4b increased the low pH tolerance of S.albulus M-Z18 to some extent.(3)Effect of ABC transporter-related genes on the resistance of S.albulus M-Z18 to low pH stress.Transcriptional analysis of S.albulus M-Z18 ABC transporter genes at pH 4.0showed significant up-regulation of genes related to the molybdate transporter system,amino acid(glutamate,branched-chain amino acid)transporter system,glycine betaine transporter system,oligopeptide transporter system,phosphate transporter system,phosphopiridate transporter system and xylose transporter system.Overexpression of related genes in S.albulus M-Z18 revealed that xyl F overexpression reduced the low pH tolerance of S.albulus M-Z18,while overexpression of aap J,glu AB,liv K,liv H,opp A,das A,opu BC,pro X,pst ABCS,tbp A,tag GH1 and tag GH2 were able to promote the growth of the strain to some extent,but did not significantly improve the low pH tolerance of the recombination strain.