**Recommended
Effect of Glycerin-producing Candida 25S RNA Methyltransferase BMT5 on Acetic Acid Stress Tolerance and Its Application
Author:
ZHOU Liu1,2, LU Xinyao1,2, ZONG Hong1,2, ZHU Gebin1,2*
Unit:
1 Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University.
2 School of Bioengineering, Jiangnan University, Industrial Microbiology and Polyol Research and Design Center.
**Item:
National Natural Science Program (22278187).
Abstract > Keywords
Summary:Mining functional genes and improving the tolerance of strains to environmental stress are crucial for the efficient use of cellulose hydrolysate to produce ethanol. Candida glycerinogenes is an industrial strain with multiple stress resistance, and the RNA methyltransferase gene CGBMT5 was obtained by genomic library screening. The expression of CGBMT5 in Saccharomyces cerevisiae improved acetic acid tolerance, and the ethanol yield of recombinant bacteria under stress was 605 g l, increase 177%。In cAfter overexpression of CGBMT5 in glycerinogenes, ethanol yield increased by 176%, and the yield, substrate conversion rate and production intensity of the two overexpressed cells were improved. Further, CGlycerinogenes overexpression bacteria were applied to cellulose hydrolysate fermentation, and the ethanol yield increased by 717% and a 65% increase in conversion rates0%, the production intensity increased by 1557%。Under acetic acid stress, the level of lipid peroxidation in the overexpressing bacteria decreased, and the activities of catalase (catalase) and superoxide dismutase (SOD) increased. Transcriptional analysis showed that PFK1 and ARG3 genes were up-regulated, and GPD1 and CoX3 were down-regulated, suggesting that CGBMT5 may promote acetic acid tolerance and fermentation ability of strains by reducing lipid peroxidation levels, increasing SOD and CAT activities, and affecting glucose metabolism and arginine synthesis. This study provides new biomaterials for the stress tolerance mechanism of yeast and the development of cellulosic ethanol technology.
Keywords:glycerin-producing Candida cerevisia; genetic screening; environmental stress; cellulose hydrolysate; RNA methyltransferases.
Key conclusions:
1.Effect of CGBMT5 on acetic acid tolerance and fermentation performance of Saccharomyces cerevisiae
After genome library screening and sequencing comparison, the gene CGBMT5 that promoted the tolerance of acetic acid was obtained. With the control bacteria (s.)cerevisiae p414-kan).Cerevisiae P414-kan-CGBMT5 grew better at 85 mmoll L acetic acid (Fig. 1-A), with a shortened lag period of about 12 h and ethanol yield of 605 g l, increase 177%；Ethanol yield per unit cell129 g l, 27 liters7%；Conversion rate 416%, an increase of 178%, the production intensity increased by 176%, and there was no significant change in biomass (Fig. 1-b). These results indicated that CGBMT5 could improve the growth and ethanol fermentation performance of Saccharomyces cerevisiae under acetic acid stress.
A-85 mmolL acetic acid stress results of dot plates; Ethanol fermentation performance of B-85 mmoll l acetic acid stress.
Fig.1 CGBMT5 vs. SEffect of cerevisiae acetic acid tolerance and fermentation performance.
figure 1 effects of cgbmt5 on acetic acid tolerance and fermentation performance of s. cerevisiae
2.Overexpression of CGBMT5 against CEffect of glycerinogenes stress tolerance
120 mmol l acetic acid under stress in CCGBMT5 was overexpressed in glycerinogenes, the lag time of the overexpressed strain was shortened by 24 h, and the ethanol yield was 627 g l, increase 176%, ethanol yield per unit cell is 123 g l, increase by 171%；Conversion rate 461%, an increase of 176%；Production intensity increased by 477%, and there was no significant difference in final biomass (Fig. 2-a). The results showed that in cOverexpression of CGBMT5 in glycerinogenes improved ethanol fermentation performance under 120 mmoll l acetic acid stress.
Glycerol is a by-product of ethanol fermentation with a yield of 51 g l, 1 unit bacterial glycerol yield0 g l, there was no significant difference with the control, and the production intensity increased by 205% (Figure 2-b). The results showed that overexpression of CGBMT5 could not increase glycerol accumulation in strains under acetic acid stress.
a- c.glycerinogenes ethanol fermentation; Glycerol yield, a by-product of b-ethanol fermentation.
Fig.2 Overexpression of CGBMT5 vs. CEffect of glycerinogenes ethanol fermentation performance.
fig.2 effect of overexpression of cgbmt5 on the ethanol fermentation performance of c. glycerinogenes
To explore the mechanism of acid tolerance of the overexpressed strains, the cell membrane lipid peroxidation levels and the enzymatic activities of CAT and SOD of the overexpressed strains were further analyzed (Figure 3). Under normal conditions, the levels of lipid peroxidation were similar between the control and overexpressing strains. However, under acetic acid stress, the lipid peroxidation level of the overexpressed strain was reduced181% (Fig. 3-A), indicating that CGBMT5 may regulate cells to cope with acetic acid stress by reducing the level of membrane lipid peroxidation in overexpressed strains; The CAT and SOD activities of the overexpressed strain were 9., respectively2 u mg protein, 347 u mg protein (Figure 3-b), increase by 736% and 535%, indicating that the change of acetic acid tolerance of the strain after overexpression of CGBMT5 was related to the increase of antioxidant enzyme activity.
a-c.glycerinogenes lipid peroxidation levels; b-c.Glycerinogenes peroxidase activity.
Fig.3 Overexpression of CGBMT5 vs. CEffect of glycerinogenes lipid peroxidation levels and peroxidase enzyme activity.
fig.3 effect of overexpression of cgbmt5 on lipid peroxidation level and peroxidase activity of c. glycerinogenes
Note: The stress conditions in the figure are all 120 mmol l acetic acid.
In order to study the regulatory mechanism of CGBMT5 on intracellular metabolism of strains, the transcription levels of glycerol-3-phosphate dehydrogenase gene (GPD1), ornithine carbamoyltransferase gene (ARG3), plasma membrane P2 H+-ATPase gene (PMA1), phosphofructokinase gene 1 (PFK1) and cytochrome C oxidase subunit gene (COX3) were detected. Under acetic acid stress, these genes were partially upregulated (Figure 4), where the transcriptional level of Arg3 was increased by 48-fold, the key enzyme gene PFK1 of the glycolytic pathway was up-regulated by 15 times. These results indicated that overexpression of CGBMT5 could affect the transcription of the above acid stress response genes.
Fig.4 Transcription levels of genes related to acetic acid tolerance.
fig.4 transcription levels of genes related to acetic acid tolerance
3.The overexpressing strains fermented ethanol with cellulose hydrolysate
CGBMT5 overexpression strain CGlycerinogenesδura5 purgap-cgbmT5 was applied to cellulose hydrolysate to ferment ethanol, and the growth of the overexpressed strain was better than that of the control, and the biomass was increased by 676%；Ethanol production 218 g l, an increase of 717%；Ethanol yield per unit cell29 g l, 26 liters1%；The conversion rate is 363%, an increase of 650%, the production intensity increased by 1557%。In summary, in COverexpression of CGBMT5 in Glycerinogenes strains could significantly improve the tolerance of strains to multiple stresses and complex environments, thereby improving the ethanol fermentation performance of cellulose hydrolysate.
Fig.5 CGBMT5 overexpressing strain was fermented with ethanol using fiber hydrolysate.
fig.5 cgbmt5 overexpressed strain ferments ethanol with fiber hydrolysateh
This study confirms that CGBMT5 alleviates the damage caused by ROS to cells by affecting the activities of peroxidase SOD and CAT as well as affecting CThe transcription of glycerinogenesδura5 acid stress response genes PFK1, GPD1, COX3 and ARG3 improved the tolerance of strains to acetic acid and the ethanol fermentation performance under acetic acid stress by promoting the intracellular arginine synthesis pathway and up-regulating phosphofructokinase PFK1, and enhanced the unit cell yield and substrate conversion rate of cellulose hydrolysate ethanol, providing new biomaterials for the research of yeast stress tolerance and the development of cellulosic ethanol technology.
Meet the team. The Industrial Microbiology Laboratory of Jiangnan University was established and developed by the older generation of scientific researchers, and was one of the earliest units to carry out industrial microbial technology research in China (1963). His main research interests are the stress resistance mechanism of industrial yeast candida glycerinogenes, its chassis modification and its application in the synthesis of high value-added chemicals. The research group has won the "Second Prize of National Invention", "First Prize of Science and Technology Progress Award of China Light Industry", "First Prize of Science and Technology Award of China Cereals and Oils Society" and "First Prize of Science and Technology Progress Award of China Federation of Commerce".
Team Leader
Zhuge Bin is a professor and doctoral supervisor at the School of Bioengineering, Jiangnan University, and his main research direction is the stress resistance mechanism and chassis application of industrial yeast. Jiangsu Province "six talent peak" high-level talents, has won the national provincial and ministerial science and technology progress first prize 3, 1 third prize, municipal 1, nearly 200 domestic and foreign academic journals published **, editor-in-chief, deputy editor-in-chief or co-editor of 5 books (2 national "Eleventh Five-Year Plan" 2), 21 invention patents. He has presided over 6 national projects such as natural **, 3 provincial and ministerial projects, and a number of municipal and enterprise projects.
Citation format
Zhou Liu, Lu Xinyao, Zong Hong, etc. Effect of Glycerin-producing Candida 25S RNA Methyltransferase BMT5 on Acetic Acid Stress Tolerance and Its Application[J].Food and Fermentation Industry,2024,50(01):1-6.
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