Effect Of Different Dissolved Oxygen Concentrations On Corynebacterium Glutamicum Metabolism In The Process Of EGFP Expression

Corynebacterium glutamicum(C. glutamicum) possesses many advantages as a host for heterologous protein expression. C. glutamicum has obvious superiorities in the food safety index which is safe and no endotoxin generated. Moreover, C. glutamicum can secrete correctly folded proteins in the supernatant and enhance downstream purification efficiency.Dissolved oxygen(DO) is an essential factor in fermentation process for cell growth,maintaining, metabolism, and production. So it is significant that we study the changes of material and energy in C. glutamicum for controling process under different DO conditions.And now, the specific regulatory mechanism in C. glutamicum influenced by DO has not been elaborated. In recent years main studies had been reported for the modification of metabolic pathways in C. glutamicum to improve the production of endogenous metabolites, but studies on modifing metabolic pathways for enhancing heterologous protein expression are rarely reported.We cultivated C. glutamicum wild type(C. glutamicum BZH 001) and engineering strains expressed EGFP(C. glutamicum EGFP) under 0, 30 % and 50 % DO concentrations,to study the effect mechanism of different DO conditions on metabolism in the process of heterologous protein expression and set up a system for efficiently expressing foreign proteins.We detected the content of the intracellular and extracellular metabolites including organic acids, amino acids, as well as the activity of the key enzymes in metabolic pathways. And we also detected the expression of key genes and intracellular reduction potential and ATP to study the changes of material and energy metabolisms in the fermentation process under different DO levels. Main results were described as follows:1. DO levels had great effects on the growth of C. glutamicum BZH 001 and C.glutamicum EGFP. Biomass under DO 30 % and 50 % concentrations was similar, but with the decrease of DO concentration, bacterial growth was restricted, especially under the low DO of 0 level. Low DO level inhibited the growth of bacterial in the early fermentation stage,and high DO level was not conducive to the survival of the bacteria in the middle-late stage.2. DO levels had great effects on metabolism of C. glutamicum BZH 001 and C.glutamicum EGFP. Bacterias had high carbon flux, and could interface product quickly under the DO 30 % and 50 %. However, cellular oxidative phosphorylation was weakened, resulting in the decrease of ATP provided for life activity under low DO. Therefore, the excessive substrate-level phosphorylation was needed to generate ATP for meeting the demand of life activity. In this case, the accumulation of cellular NADH was increased; the flow of TCA cycle decreased and switched to the glycolysis pathway and glyoxylate cycle. As a result, a variety of organic acids and amino acids was generated, including lactate, valine, acetate and so on, which can further influence the production of target product under zero DO concentration.3. EGFP expression of C. glutamicum EGFP under different DO concentrations was also different. It was more suitable for the EGFP expression under DO 30 %, and protein content under low DO level was significantly lower than that under the high DO, which peak valuewas only 66.56 % of DO 50 % concentrations. However, the trend changes of protein expression were similar and there was no obvious drop in the late stages, which suggested that protein activity was stable.4. The EGFP expression also has certain influence on C. glutamicum metabolism.Growth and decline of C. glutamicum EGFP were both delayed under the condition of DO30 %, and gluconeogenesis reduced then consumpted more materials. In this case, glycolytic pathway rised, flux of carbon source into the TCA cycle increased, with more metabolites produced including glutamate, gliycine, lactic acid, and acetic acid, which the spillover of carbon source increased.5. The expression of heterologous proteins caused expression changes of many key genes on C. glutamicum metabolic pathways. Such as such as the aceE of TCA pathway rised,which made the TCA cycle strengthens; atpC encoding ATP synthase rised, which synthetized more ATP; rpmF, rpsT encoding ribosomal protein rised, which made the protein synthesis at higher levels, and also was advantageous to the foreign protein synthesis; SecG, SecE in the Sec way and NCgl0909 encoding transporters atpase rised, which made the relative block smaller of transmembrane transport, and intracellular metabolic substances secreted the extracellular more easily.Above results further illustrated the influence of different DO levels on growth and metabolism of C. glutamicum. So we can control high DO in the growth period to make its rapid growth on the optimization strategy, and supply lower DO levels in the middle-late stages to make mass production. Moreover in this study we found the effect mechanism of different DO conditions on metabolism in the process of heterologous protein expression, and found a large number of differentially expressed genes related to the metabolic flow changes,which laid the foundation for finding genomic loci to change flow of the host cell metabolism.Therefore, the research of metabolisms and transcriptome regulations on heterologous protein expression of C. glutamicum had high academic value and broad application prospects.