The Transcriptional Regulation Mechanism Of Methyl-accepting Chemotaxis Proteins In Zymomonas Mobilis Under N₂-fixing Condition

Zymomonas mobilis,a model bacteria for ethanol production,can grow and ferment with nitrogen as the only nitrogen source.Methyl-accepting chemotaxis protein?MCP?and methyltransferase?Che R?are the core proteins in the chemotaxis pathway of bacteria,which are involved in the regulation of biofilm formation,flagellum assembly,and energy metabolism.Moreover,under the condition of nitrogen fixation,MCP mediates energy metabolism through dynamic changes of FAD,and Che R is involved in the regulation of biofilm formation and aerotropism.In the early stage,the transcriptome data in our research group found that the transcription of mcp and che R genes of the ZM4 strain was significantly up-regulated.It confirmed MCP and Che R involvement in biological nitrogen fixation.However,their gene function in biological nitrogen fixation and its regulatory mechanism has not been studied.Exploring these mechanisms is helpful to understand the biological nitrogen fixation process and regulatory mechanism in Zymomonas mobilis.Therefore,this study focuses on the transcriptional regulation mechanism of methyl-accepting chemotaxis protein under the condition of nitrogen fixation.The main contents and conclusions are as follows:?1?In this study,a comparison of genomic editing methods and the construction of knockout strains encoding methyl-accepting chemotaxis protein in ZM4 were presented.The knockout efficiency of the Type I-F CRISPR/Cas system was higher than the Type?CRISPR/Cas9 efficiency.With these two gene-editing techniques,the following strains were successfully constructed:single-gene deletion strains?KOmcp1,KOmcp2,KOmcp3,KOche R?,double-gene deletion strains?DKOmcp12?and three-gene deletion strains?TKOmcp123,TKOmcp12R?.?2?Fermentation evaluation was tested in methyl-accepting chemotaxis protein defective strains.Under the condition of nitrogen fixation,the deletion of mcp and che R genes affected the growth rate of the strain in the logarithmic growth period but did not affect the ethanol production of Zymomonas mobilis.Under the condition of NH₄⁺,the deletion of mcp and che R genes did not significantly affect the growth,glucose metabolism,and ethanol production.Relative expression of related chemotaxis and flagellum assembly genes in DKOmcp12 strain were tested by real-time quantitative PCR under the nitrogen-fixing condition.The results showed that lack of mcp1 and mcp2 genes caused differential expression of genes such as chemotaxis and flagellum assembly of Z.mobilis.Therefore,it was speculated that mcp genes may regulate on transcription levels.?3?Transcriptome analysis was made in methyl-accepting chemotaxis protein defective strains.Under the condition of nitrogen fixation,mcp and che R had a wide range of effects on glucose metabolism,response to heat,oxidative stress response,redox process,and other biological processes,as well as the molecular functions such as ethanol dehydrogenase?NAD?activity,NADP binding,ion binding,redox enzyme activity,and ribosomal RNA binding.The results of pathway-significant enrichment showed that gene mcp and che R affected the expression of key genes in leucine and isoleucine biosynthesis,glycolysis and oxidative phosphorylation of Z.mobilis under nitrogen fixation conditions.Therefore,it had an effect on the growth rate during the logarithmic phase and the glucose metabolism rate of Z.mobilis under nitrogen fixation conditions.