Mechanism Of PpGpp Synthetases To Control The Synthesis Of Toyomycin In Streptomyces Diastatochromogenes

Stringent response means that when bacteria face amino acid and lipid starvation,they actively adjust the expression levels of different genes through the key intracellular signaling molecule ppGpp（guanosine 5’-disphosphate3’-disphosphate）,so as to improve the bacterial metabolism.Stress response to survivability in adverse environments.When a stringent response occurs,microbial growth-related genes in primary metabolism are inhibited,while secondary metabolic pathway-related genes are activated.Thus,in Streptomyces this reaction can be used to activate silent gene cluster expression and accumulate secondary metabolites.However,there are no studies on the regulation of toyomycin synthesis by stringent reactions.Therefore,in this study,starting from the correlation between stringent response and toyomycin yield with Streptomyces diastatochromogenes 1628,and through nutrient stress conditions,it was found that under the condition of nitrogen source deprivation,the synthesis gene of toyomycin in the early logarithmic growth phase,the late logarithmic growth phase and the stationary phase was found.The transcriptional level of toyomycin was significantly increased,proving that the stringent response can activate the toyomycin synthesis pathway.Then,the rsh gene sequence was determined by gene sequence alignment,and the evolutionary relationship of the rsh gene in related species was analyzed.The rsh-overexpressing 1628＿rsh+and knockout strains 1628＿Δrsh were constructed,and it was found that the yield of 1628＿Δrsh decreased to 89.27mg/L on the 4th day,a decrease of 42.14%.The TM production of 1628＿rsh+increased by 4.22%to 160.79 mg/L.Further analysis of the intracellular ppGpp content found that although the intracellular ppGpp content of 1628＿rsh+was slightly higher than that of the wild type,there was no significant difference.It is speculated that due to the existence of domains of synthesizing and hydrolyzing ppGpp in RSH,single overexpression of RSH increases the activity of synthesis and hydrolysis,and cannot accumulate ppGpp in cells.Based on sequence alignment and structure-function analysis,the synthesis of toyomycin was regulated by overexpressing the synthetic domain RelA.The RelA strain 1628＿relA_Sc+derived from Streptomyces coelicolor A3（2）and the RelA strain 1628＿relA_Sd+derived from S.diastatochromogenes 1628 amylase were constructed.However,overexpression of RelA single-function ppGpp synthetic protein in S.diastatochromogenes 1628 bacteria can greatly increase the TM yield,and the highest yields of 1628＿relA_Sc+and 1628＿relA_Sd+reached654.78 mg/L and 3459.74 mg/L,respectively.Intracellular ppGpp content analysis showed that the intracellular ppGpp content of 1628＿relA_Sc+and1628＿relA_Sd+were 106.67%and 160.00%of the wild type.Finally,the in vitro catalytic properties of the three pure proteins were initially explored,and it was found that RSH played a bifunctional role in the synthesis/hydrolysis of ppGpp,and under natural conditions,RSH mainly played a role in hydrolysis,while RelA only played a role in ppGpp synthesis.Since there is a regulatory domain that engages with ribosomes in RSH,the synthesis and hydrolysis activities were measured by breaking cells and extracting ribosome fractions.It was found that ribosomal fractions could increase the synthase activity of RSH and reduce the hydrolysis activity of RSH.It shows that ribosomal components can activate the synthetic activity of RSH.However,the regulatory mechanism between RSH-related proteins in cellular components is still unclear,and further exploration and interpretation are needed.