酿酒酵母ALD4基因敲除与GPD1基因沉默研究
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摘要
乙酸与甘油是酿酒酵母乙醇发酵过程主要的副产物。本研究通过敲除酿酒酵母Y01乙醛脱氢酶ALD4基因,获得了ALD4基因缺失的突变体DY01；通过构建反义表达载体抑制酿酒酵母甘油脱氢酶关键基因GPD1的转录,以促进乙醇发酵。结果如下：
1.采用以PCR为基础的loxP-KanMX-loxP序列组件和Cre/loxP删除系统删除了酿酒酵母工程菌Y01乙醛脱氢酶ALD4基因,获得突变株DY01；酿酒酵母ALD4缺失菌株DY01发酵液乙醇比出发菌株酿酒酵母Y01提高了6.88%。
2.通过构建酿酒酵母实验菌株INVScl甘油脱氢酶GPD1基因5'UTR反义表达载体pYES2.0-GPD1,实验组比对照组的3-磷酸甘油脱氢酶(GPDH)的比活力最大下降了24.03%；甘油产量含量下降幅度最大达到了25.43%,而且甘油含量的下降滞后于3.磷酸甘油脱氢酶活性的下降。
3.通过构建酿酒酵母突变株DY01甘油脱氢酶GPD1基因5'UTR反义表达载体pYES2.0-GPD1/KanMX,获得酿酒酵母DRY01菌株；酿酒酵母DRY01菌株的甘油脱氢酶(GPDH)的比活力比出发菌株DY01下降了20.04%；甘油含量下降幅度最大达到了19.14%；同时,酿酒酵母DRY01发酵液乙醇含量比酿酒酵母DY01提高了9.74%,比原始菌株Y01乙醇含量提高了14.01%。
建立在酵母高效同源重组机制的loxP-KanMX.loxP删除组件与Cre/loxP系统是进行酿酒酵母基因改良育种与酿酒酵母基因功能相关研究的有效方法,突破了工业酿酒酵母缺乏有效选择标记与遗传多样性的限制。酿酒酵母ALD4基因的缺失,有利于乙醇代谢途径。酿酒酵母工程菌株DY01 GPD1基因mRNA 5'UTR的反义RNA能够有效的抑制酿酒酵母工程菌株DY01GPD1基因的转录,触发沉默该基因的表达；为提高甘蔗等生物质发酵燃料乙醇效率迈出重要一步,为酿酒酵母工程菌的基因改良奠定了基础。
Acetic acid and glycerol are the major by-product in Saccharomyces cerevisiae ethanol fermentation.In this paper,S.cerevisiae Y01 aldehyde dehydrogenase ALD4 gene was disrupted.ALD4 gene deletion mutants DY01 was obtained.The production of S.cerevisiae ethanol was increased by GPDl silenced via antisense expression vector. The results were described as follows:
1, PCR-based loxP-KanMX-loxP and Cre/loxP system apply to delete the industrial strain S.cerevisiae Y01 aldehyde dehydrogenase ALD4 gene.The production of ethanol was improved with 6.88% compared to the original strain Y01.
2, Glycerol dehydrogenase GPDl gene 5'UTR Antisense expression vector pYES2.0-GPD1 was constructed for S.cerevisiae INVScl. When compared to the original strain, glycerol phosphate dehydrogenase (GPDH) activity 24.03% reduction and glycerol formation 25.43% reduction respectively.
3, Antisense expression vector pYES2.0-GPDl/KanMX was constructed for glycerol dehydrogenase GPD1 gene 5'UTR of S.cerevisiae mutant strain DY01.When compared to the original strain DY01, S.cerevisiae DRY01glycerol dehydrogenase (GPDH) activity 20.04% reduction, glycerol formation 19.14% reduction respectively. At same time, the production of S.cerevisiae DRY01 ethanol fermentation increased by 9.74% compared with DY01.
LoxP-KanMX-loxP gene disruption cassette and Cre/loxP system is an effective way to genetically modified via homologous integration,which breaks through difficulties of genetic engineers in industrial S.cerevisiae. We are confornted with lacking effective selectable marker and genetic diversity in industrial S.cerevisiae strain. ALD4 gene deleting is good for ethanol metabolism pathway in industrial S.cerevisiae.Antisence RNA complementary the 5'UTR of GPD1 mRNA triggers effective silencing in S.cerevisiae industrial strain DY01.
1.采用以PCR为基础的loxP-KanMX-loxP序列组件和Cre/loxP删除系统删除了酿酒酵母工程菌Y01乙醛脱氢酶ALD4基因,获得突变株DY01；酿酒酵母ALD4缺失菌株DY01发酵液乙醇比出发菌株酿酒酵母Y01提高了6.88%。
2.通过构建酿酒酵母实验菌株INVScl甘油脱氢酶GPD1基因5'UTR反义表达载体pYES2.0-GPD1,实验组比对照组的3-磷酸甘油脱氢酶(GPDH)的比活力最大下降了24.03%；甘油产量含量下降幅度最大达到了25.43%,而且甘油含量的下降滞后于3.磷酸甘油脱氢酶活性的下降。
3.通过构建酿酒酵母突变株DY01甘油脱氢酶GPD1基因5'UTR反义表达载体pYES2.0-GPD1/KanMX,获得酿酒酵母DRY01菌株；酿酒酵母DRY01菌株的甘油脱氢酶(GPDH)的比活力比出发菌株DY01下降了20.04%；甘油含量下降幅度最大达到了19.14%；同时,酿酒酵母DRY01发酵液乙醇含量比酿酒酵母DY01提高了9.74%,比原始菌株Y01乙醇含量提高了14.01%。
建立在酵母高效同源重组机制的loxP-KanMX.loxP删除组件与Cre/loxP系统是进行酿酒酵母基因改良育种与酿酒酵母基因功能相关研究的有效方法,突破了工业酿酒酵母缺乏有效选择标记与遗传多样性的限制。酿酒酵母ALD4基因的缺失,有利于乙醇代谢途径。酿酒酵母工程菌株DY01 GPD1基因mRNA 5'UTR的反义RNA能够有效的抑制酿酒酵母工程菌株DY01GPD1基因的转录,触发沉默该基因的表达；为提高甘蔗等生物质发酵燃料乙醇效率迈出重要一步,为酿酒酵母工程菌的基因改良奠定了基础。
Acetic acid and glycerol are the major by-product in Saccharomyces cerevisiae ethanol fermentation.In this paper,S.cerevisiae Y01 aldehyde dehydrogenase ALD4 gene was disrupted.ALD4 gene deletion mutants DY01 was obtained.The production of S.cerevisiae ethanol was increased by GPDl silenced via antisense expression vector. The results were described as follows:
1, PCR-based loxP-KanMX-loxP and Cre/loxP system apply to delete the industrial strain S.cerevisiae Y01 aldehyde dehydrogenase ALD4 gene.The production of ethanol was improved with 6.88% compared to the original strain Y01.
2, Glycerol dehydrogenase GPDl gene 5'UTR Antisense expression vector pYES2.0-GPD1 was constructed for S.cerevisiae INVScl. When compared to the original strain, glycerol phosphate dehydrogenase (GPDH) activity 24.03% reduction and glycerol formation 25.43% reduction respectively.
3, Antisense expression vector pYES2.0-GPDl/KanMX was constructed for glycerol dehydrogenase GPD1 gene 5'UTR of S.cerevisiae mutant strain DY01.When compared to the original strain DY01, S.cerevisiae DRY01glycerol dehydrogenase (GPDH) activity 20.04% reduction, glycerol formation 19.14% reduction respectively. At same time, the production of S.cerevisiae DRY01 ethanol fermentation increased by 9.74% compared with DY01.
LoxP-KanMX-loxP gene disruption cassette and Cre/loxP system is an effective way to genetically modified via homologous integration,which breaks through difficulties of genetic engineers in industrial S.cerevisiae. We are confornted with lacking effective selectable marker and genetic diversity in industrial S.cerevisiae strain. ALD4 gene deleting is good for ethanol metabolism pathway in industrial S.cerevisiae.Antisence RNA complementary the 5'UTR of GPD1 mRNA triggers effective silencing in S.cerevisiae industrial strain DY01.
引文
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[3]柳羽丰,王滨生,王佳祥.非粮燃料乙醇发展综述[J].化学工程师.2009,166(07)：53-55.
[4]Steen EJ, Chan R, Prasad N, et al. Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol[J]. Microb Cell Fact.2008,7:36.
[5]Cordier H, Mendes F, Vasconcelos I, et al. A metabolic and genomic study of engineered Saccharomyces cerevisiae strains for high glycerol production[J]. Metab Eng.2007, 9(4):364-378.
[6]Kong QX, Gu JG, Cao LM, et al. Improved production of ethanol by deleting FPS1 and over-expressing GLT1 in Saccharomyces cerevisiae[J]. Biotechnol Lett.2006, 28(24):2033-2038.
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[8]Norbeck J, Blomberg A. Metabolic and Regulatory Changes Associated with Growth of Saccharomyces cerevisiae in 1.4 M NaCl. EVIDENCE FOR OSMOTIC INDUCTION OF GLYCEROL DISSIMILATION VIA THE DIHYDROXYACETONE PATHWAY[J]. Journal of Biological Chemistry.1997,272(9):5544.
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