High Temperature Resistance Analysis And Element Identification Of Corynebacterium Glutamicum

In the process of industrial fermentation,microorganisms are often exposed to various environmental stressors,such as heat,Acidity and alkalinity,oxidation,osmotic stimulation,etc.among them,heat stress is one of the most important factors affecting microbial growth and metabolism.It has a great impact on the solubility of fermentation medium components,the catalytic efficiency of enzymes in metabolic pathway and the reaction rate of biochemical process.Moreover,extreme temperature will also destroy the structure of cell membrane or interfere with the function of biological macromolecules such as protein and RNA,so as to destroy the integrity of cells and reduce cell viability and fermentation ability.Corynebacterium glutamicum is an important industrial member in the biotechnology production of amino acids and organic acids.Its production performance decreases with the increase of ambient temperature in the fermentation process.Therefore,in order to achieve efficient high-temperature fermentation,it is of great significance to screen and develop robust strains with ideal heat-resistant properties.This study attempts to develop a heat-resistant evolutionary strain of Corynebacterium glutamicum,and screened the functional elements with high temperature resistance by overexpressing the related stress resistant regulatory factors and molecular chaperones from Corynebacterium glutamicum,Escherichia coli,Thermococcus kodakarensis and Lactobacillus brevis.Using the adaptive laboratory evolution strategy,two strains with improved resistance to high temperature were isolated,All mutation targets were obtained by genomic analysis and tested for high temperature growth.Finally,transcriptome analysis of mutant strains carrying positive targets was carried out to explore the stress resistance mechanism of Corynebacterium glutamicum to high temperature stress.The main research contents and results are as follows:(1)Determine the temperature and pressure of the screening element.It was cultured at 30°C,32°C,35°C,37°C,40°C,42°C and 43°C for 20 hours respectively.The biomass of Corynebacterium glutamicum under different temperature and pressure was tested,and 42°C was finally determined as the best temperature for screening high temperature resistant elements.(2)Screening of high temperature resistant components.A total of 39 genes with potential high temperature tolerance from Corynebacterium glutamicum,Escherichia coli,Thermococcus kodakarensis and Lactobacillus brevis were constructed,including transcription regulators,molecular chaperones and heat shock proteins,and expressed in Corynebacterium glutamicum ATCC13032.They were cultured at 42°C for 20 hours.Through biomass test and screening,the genes hsp Q,G6 and G9 with enhanced high temperature tolerance were obtained.When induced by IPTG,compared with the control strain,its temperature tolerance to 42°C was improved by17.5%,25.1% and 20.3% respectively.(3)Adaptive laboratory evolution.Corynebacterium glutamicum ATCC13032 was selected as the parent strain for adaptive laboratory evolution under heat stress.Through continuous shake flask culture,two independent samples HTMevol-1 and HTMevol-2 were evolved.After adaptation,two individual colonies WHT4 and WHT59 with the best heat resistance were finally isolated from the heat stress screening plate.The growth curves of the two heat-resistant strains were tested at32°C and 42°C for 28 hours,and the growth tests were carried out in the temperature range of 30°C to 43°C.The results showed that the biomass of WHT4 and WHT59 strains was 2.5 and 2.4 times that of wild-type strains,and the evolved strains showed good heat-resistant ability.In order to study the genetic mutation of the adaptive strain,the whole genome of the parent strain Corynebacterium glutamicum ATCC13032 and two thermally evolved strains WHT4 and WHT59 stored in the laboratory were resequenced.The results showed that the evolved strain had 13 missense gene mutations and 3 synonymous gene mutations compared with the control.Further studies showed that the missense point mutations of some genes,especially fas R-L102 F and hrc A-L119 P,compared with wild-type strains,the high temperature tolerance was increased by 38.4% and 18.3% respectively.Through the combined expression of positive mutations of Corynebacterium glutamicum fas R-L102 F and hrc A-L119 P and the knockout high temperature test on the genome,the results showed that the high temperature tolerance of positive mutation combined expression strains increased by 55.6%,and the high temperature tolerance of ?hrc A and ?fas R increased by 22.6% and 61.7% respectively.Therefore,fas R-L102 F and hrc A-L119 P are important targets for Corynebacterium glutamate to improve heat tolerance.(4)Analysis of high temperature resistance mechanism.Through transcriptome go enrichment analysis,it was found that hrc A-L119 P and fas R-L102 F mutant strains improved heat tolerance in many biological processes,such as ATP energy metabolism,nucleotide metabolism,peptide metabolism,redox process,membrane transporter activity and so on;The enrichment of KEGG pathway showed that the expression level of genes related to RNA degradation,inositol phosphate metabolism and carbon metabolism of hrc A-L119 P mutant strain was significantly changed compared with the control strain,and the differentially expressed genes related to glycolysis,inositol phosphate metabolism,ABC transporter,propane acid metabolism and propane acid metabolism pathway of fas R-L102 F mutant strain were significantly up-regulated.The changes of these pathways may enhance the heat tolerance of Corynebacterium glutamate.In conclusion,these findings help us to understand the heat tolerance mechanism of Corynebacterium glutamicum and provide new stress-related targets for strain improvement to obtain heat tolerance.