酿酒酵母甘油-3-磷酸脱氢酶基因沉默表达
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摘要
通过构建酿酒酵母沉默表达载体PURH-SGPD1,使GPD1基因在PGK强启动子、CYC1终止子在特定区域内进行干扰和表达.酿酒酵母SY01突变菌株与出发菌株Y01发酵实验结果相比,甘油脱氢酶酶比活比Y01下降了10.52%,甘油含量下降了13.2%;乙醇量提高了12.06%.结果表明通过反义干扰GPD1基因5'UTR序列,能有效干扰酵母工程菌株GPD1转录与表达,削弱甘油积累途径,促进乙醇代谢途径.
The aim of this study was to interfere and repress expression the glycerol 3-phosphate dehydrogenase( GPD1) gene in a specific region by constructing a silencing expression vector PURH-SGPD1. The silencing expression vector containing GPD1 gene,PGK strong promoter and CYC1 terminator. Saccharomyces cerevisiae( S. cerevisiae) SY01 mutant gave 10. 52% less glycerol-3-phosphate dehydrogenase activity,13. 2% lower glycerol production,and 12. 06% higher ethanol production compared with the original strain. Results show that the interference by antisense GPD1 gene 5' UTR sequence,can interfere with the yeast engineering strains GPD1 transcription and expression,weaken the glycerol accumulation and promote ethanol metabolic pathways.
The aim of this study was to interfere and repress expression the glycerol 3-phosphate dehydrogenase( GPD1) gene in a specific region by constructing a silencing expression vector PURH-SGPD1. The silencing expression vector containing GPD1 gene,PGK strong promoter and CYC1 terminator. Saccharomyces cerevisiae( S. cerevisiae) SY01 mutant gave 10. 52% less glycerol-3-phosphate dehydrogenase activity,13. 2% lower glycerol production,and 12. 06% higher ethanol production compared with the original strain. Results show that the interference by antisense GPD1 gene 5' UTR sequence,can interfere with the yeast engineering strains GPD1 transcription and expression,weaken the glycerol accumulation and promote ethanol metabolic pathways.
引文
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[6]HE W J,YE S C,XUE T,et al.Silencing the glycerol-3-phosphate dehydrogenase gene in Saccharomyces cerevisiae results in more ethanol being produced and less glycerol[J].Biotechnology Letters,2013,35(12):1375-1377.
[7]KAJIWARA Y,OGAWA K,TAKASHITA H,et al.Enhanced glycerol production in Shochu yeast by heat-shock treatment is due to prolonged transcription of GPD1[J].Journal of Bioscience and Bioengineering,2000,90(1):121-123.
[8]MICHNICK S,ROUSTAN J L,REMIZE F,et al.Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase[J].Yeast,1997,13(9):783-793.
[2]ISHII J,YOSHIMURA K,HASUNUMA T,et al.Reduction of furan derivatives by overexpressing NADH-dependent Adh1 improves ethanol fermentation using xylose as sole carbon source with Saccharomyces cerevisiae harboring XR-XDH pathway[J].Applied Microbiology and Biotechnology,2013,97(6):2597-2607.
[3]ALBERTYNl J,VAN T A,PRIOR B A.Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae[J].FEBS Lett,1992,308(2):130-132.
[4]BONOLI M,GRAZIOLA M,POGGI V,et al.RNA complementary to the 5'-UTR of mRNA triggers effective silencing in Saccharomyces cerevisiae[J].Biochem Biophys Res Commun,2006,339(4):1224-1231.
[5]曹罗元.酿酒酵母ALD4基因敲除与GPD1基因沉默研究[D].福州:福建师范大学,2011.
[6]HE W J,YE S C,XUE T,et al.Silencing the glycerol-3-phosphate dehydrogenase gene in Saccharomyces cerevisiae results in more ethanol being produced and less glycerol[J].Biotechnology Letters,2013,35(12):1375-1377.
[7]KAJIWARA Y,OGAWA K,TAKASHITA H,et al.Enhanced glycerol production in Shochu yeast by heat-shock treatment is due to prolonged transcription of GPD1[J].Journal of Bioscience and Bioengineering,2000,90(1):121-123.
[8]MICHNICK S,ROUSTAN J L,REMIZE F,et al.Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase[J].Yeast,1997,13(9):783-793.