| Engineering microorganisms with excellent stress-resistant phenotypes are an important guarantee for improving the yield and quality of biological fermentation products in industrial fermentation production.Corynebacterium glutamicum is an important traditional industrial microorganism that has been used to produce a variety of amino acids,such as glutamic acid,lysine,arginine,and other compounds.In the fermentation process,high salt,acid and alkali,osmotic pressure and other environmental stresses seriously affect the growth and metabolism of bacteria,thus reducing the biological manufacturing efficiency of the strain.At present,the potential mechanism of C.glutamicum against high osmotic pressure is still unclear.Therefore,analyzing the physiological adaptation mechanism of C.glutamicum against high osmotic pressure,mining and identifying the functional elements and metabolic pathways involved in the high osmotic pressure tolerance process of the strain will provide new ideas for the genetic transformation and physiological performance optimization of industrial strains.In this study,wild-type C.glutamicum ATCC13032 was used as the starting strain to investigate the effects of long-term and short-term high osmotic stress on gene expression and metabolic pathway changes of C.glutamicum.In addition,through the analysis and verification of significantly up/down-regulated genes or metabolic pathways,potential targets of C.glutamicum against high osmotic pressure have been explored.The main research contents and results of this paper are as follows:(1)In order to determine the screening pressure of anti-hypertonic elements,the cell mass of Corynebacterium glutamicum was tested under the pressures of 0 g/L,80g/L,120 g/L,160 g/L,180 g/L and 200 g/L lysine hydrochloride,and finally the screening pressure of anti-hypertonic elements was determined as 160 g/L lysine hydrochloride.Under the selective pressure of 160 g/L lysine hydrochloride,a variety of anti-hypertonic elements were tested,including c AMP phosphodiesterase,response regulator,heat shock protein,chaperone protein,periplasmic acid stress chaperone protein and multi-stress resistant outer membrane protein of Escherichia coli,Heat shock protein,DNA protective protein and molecular chaperone of Corynebacterium glutamicum,membrane proteins,pyridoxal biosynthesis lyase,oxidoreductase,acetic acid-coa ligase,NADP oxidoreductase,zinc-dependent protease,t RNA acetyltransferase,glyceraldehyde-3-phosphatedehydrogenase,fructose-1,6-bisphosphat ase,transcriptional regulators and osmotic pressure-inducing proteins of Thermococcus kodakarensis.The results showed that overexpression of heat shock protein Hspq of Escherichia coli can increase the cell mass of Corynebacterium glutamicum by 20-30% under high osmotic pressure,and over-expression of anti-hyperosmotic elements of Thermococcus kodakarensis had no significant improvement on osmotic pressure tolerance of Corynebacterium glutamicum.(2)Through transcriptome analysis,we analyzed the metabolic pathways of significant changes in Corynebacterium glutamicum under long-term high osmotic pressure and short-term high osmotic pressure conditions,including glycolysis,TCA cycle,oxidative phosphorylation,pentose phosphate pathway,amino acid biosynthesis,sulfur metabolism,arginine biosynthesis,signal transduction pathway and folic acid synthesis pathwayFor DNA repair pathway,the biomass of most knockout strains decreased in different degrees compared with the control strains under high osmotic pressure,among which the biomass of (?)Uvr A,(?)Uvr D,(?)Nei,(?)Nth,(?)Mut T,(?)Uncs and (?)Rec J strains decreased significantly,which were 14.5%,20.7%,19.6%,10%,30%,23%and 12.8%,respectively.In addition,as for the (?)mut T strain,after expressing the mut T gene of Corynebacterium glutamicum on the vector p EC-XK99 E,the biomass of the (?)mut T strain was supplemented by 34.2%.Compared with the control strain,the biomass of oxidative phosphorylation pathway gene knockout strain (?)Ndh decreased in different degrees under various osmotic pressures,especially under the pressure of 160 g/L lysine hydrochloride,which decreased by 90% compared with the control strain.Then,after the strain (?)Ndh expressed Corynebacterium glutamicum ndh gene on the vector p EC-XK99 E,the biomass of (?)Ndh strain was supplemented by 231.8%.Compared with the control strain,the biomass of (?)Oxy R1,(?)Oxy R3 and(?)Oxy R5 gene knock-out strains with active oxygen scavenging transcription regulators decreased by 15.6%,10.4% and 25.5%,respectively.In summary,DNA repair pathway,reactive oxygen species removal of transcription regulator Oxy R,oxidative phosphorylation pathway and heat shock protein are important targets of Corynebacterium glutamicum against high osmotic pressure stress by verification analysis. |
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