Cre-loxP重组酶技术敲除酿酒酵母丙酮酸脱羧酶编码基因pdc1和pdc5
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摘要
目的:分别构建含有酿酒酵母丙酮酸脱羧酶基因pdc1和pdc5同源序列loxP-kanMX-loxP的质粒,敲除丙酮酸脱羧酶基因。方法:以两端含40 bp pdc1或pdc5的同源序列为引物,以pUG6质粒DNA为模板,通过PCR扩增loxP-kanMX-loxP基因敲除片段,将其分别插入pMD18-T、pEASY-T3,构建表达载体pTWCL-PDC1与pTWCL-PDC5并测序,构建后的质粒利用醋酸锂转化法转化酿酒酵母H5,经筛选鉴定后进行摇瓶发酵试验。结果:分别含有1693、1672 bp目的片段pdc1-loxP-kanMX、pdc5-loxP-kanMX的质粒pTWCL-PDC1与pTWCL-PDC5构建成功,发酵试验显示重组酿酒酵母H5-01与H5-02较原始菌株的乙醇产量分别下降了14.53%与17.54%,证明pdc1或pdc5基因被敲除。结论:利用Cre-loxP重组酶技术分别敲除了酿酒酵母pdc1和pdc5基因,为后续在酿酒酵母中连续敲除pdc1和pdc5奠定了良好的技术基础。
Objective: To construct plasmid containing homologous loxP-kanMX-loxP fragment of Saccharomyces cerevisiae pyruvate decarboxylase gene pdc1 or pdc5 to knock out pyruvate decarboxylase gene. Methods: The loxP-kanMX-loxP gene knockout fragments were amplified by PCR using homologous sequences containing 40 bp pdc1 or pdc5 at both ends as primers from pUG6 plasmid DNA template, and were inserted into pMD18-T and pEASY-T3 to construct expression vectors pTWCL-PDC1 and pTWCL-PDC5 respectively and sequenced. The constructed plasmid was transformed into S.cerevisiae H5 by a lithium acetate conversion method, and subjected to a shake flask fermentation test after screening and identification. Results: The plasmids pTWCL-PDC1 and pTWCLPDC5 containing the 1693, 1672 bp fragment pdc1-loxP-kanMX and pdc5-loxP-kanMX respectively were successfully constructed, and the fermentation experiments showed that the ethanol production of recombinant strains S.cerevisiae H5-01 and H5-02 decreased by 14.53% and 17.54% compared with the original strain respectively, demonstrating pdc1 or pdc5 gene knockout. Conclusion: pdc1 and pdc5 in S.cerevisiae were successfully knocked out by using the Cre-loxP recombinant enzyme technology, which laid a good technical foundation for the subsequent continuous knocking out of pdc1 and pdc5 in S.cerevisiae.
Objective: To construct plasmid containing homologous loxP-kanMX-loxP fragment of Saccharomyces cerevisiae pyruvate decarboxylase gene pdc1 or pdc5 to knock out pyruvate decarboxylase gene. Methods: The loxP-kanMX-loxP gene knockout fragments were amplified by PCR using homologous sequences containing 40 bp pdc1 or pdc5 at both ends as primers from pUG6 plasmid DNA template, and were inserted into pMD18-T and pEASY-T3 to construct expression vectors pTWCL-PDC1 and pTWCL-PDC5 respectively and sequenced. The constructed plasmid was transformed into S.cerevisiae H5 by a lithium acetate conversion method, and subjected to a shake flask fermentation test after screening and identification. Results: The plasmids pTWCL-PDC1 and pTWCLPDC5 containing the 1693, 1672 bp fragment pdc1-loxP-kanMX and pdc5-loxP-kanMX respectively were successfully constructed, and the fermentation experiments showed that the ethanol production of recombinant strains S.cerevisiae H5-01 and H5-02 decreased by 14.53% and 17.54% compared with the original strain respectively, demonstrating pdc1 or pdc5 gene knockout. Conclusion: pdc1 and pdc5 in S.cerevisiae were successfully knocked out by using the Cre-loxP recombinant enzyme technology, which laid a good technical foundation for the subsequent continuous knocking out of pdc1 and pdc5 in S.cerevisiae.
引文
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[14]黄琳,刘常金,王芳,等.耶氏酵母菌POX5基因的敲除[J].现代食品科技,2010,26(4):331-336.
[15]柯崇榕,吴毕莎,邵庆伟,等.酿酒酵母PDC1基因过表达菌株的构建[J].应用与环境生物学报,2013,19(4):704-708.
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[17]林晓华.酿酒酵母SNF4基因敲除缺失菌株的构建[J].生物工程学报,2011,27(4):572-578.
[18]Semkiv M V,Dmytruk K V,Sibirny A A.Development of a system for multicopy gene integration in Saccharomyces cerevisiae[J].J Microbiol Methods,2016,120:44-49.
[19]Anvari M,Motlagh M R S.Enhancement of 2,3-butanediol production by klebsiella oxytoca PTCC 1402[J].J Biomed Biotechnol,2011,2011(1):6361-6370.
[20]Celińska E,Grajek W.Biotechnological production of2,3-butanediol-current state and prospects[J].Biotechnol Adv,2009,27(6):715-725.
[21]Liu J M,Chan S H J,Theis B N,et al.Combining metabolic engineering and biocompatible chemistry for high yield production of homo-diacetyl and homo-(S,S)-2,3-butanediol[J].Metab Eng,2016,36:57-67.
[22]吴满珍,李鹏越,苏珂,等.弱化乙醇途径关键酶活性减少酿酒酵母在丙酮酸发酵过程中副产物乙醇的积累[J].食品与发酵工业,2015,41(7):7-12.
[23]Lian J,Chao R,Zhao H.Metabolic engineering of a Saccharomyces cerevisiae strain capable of simultaneously utilizing glucose and galactose to produce enantiopure(2R,3R)-butanediol[J].Metab Eng,2014,23:92-99.
[2]Kim S J,Kim J W,Lee Y G,et al.Metabolic engineering of Saccharomyces cerevisiae for 2,3-butanediol production[J].Appl Microbiol Biotechnol,2017,101(6):2241-2250.
[3]Kim S,Hahn J S.Efficient production of 2,3-butanediol in Saccharomyces cerevisiae by eliminating ethanol and glycerol production and redox rebalancing[J].Metab Eng,2015,31:94-101.
[4]Kim J W,Kim J,Seo S O,et al.Enhanced production of 2,3-butanediol by engineered Saccharomyces cerevisiae through fine-tuning of pyruvate decarboxylase and NADH oxidase activities[J].Biotechnol Biofuels,2016,9(1):265-276.
[5]张志凯,汤宏赤,樊少林,等.酿酒酵母蔗糖关键代谢途径suc2基因的敲除及其蔗糖代谢特性的变化分析[J].基因组学与应用生物学,2016,35(3):622-628.
[6]Ng C Y,Jung M Y,Lee J,et al.Production of 2,3-butanediol in Saccharomyces cerevisiae by in silico aided metabolic engineering[J].Microb Cell Fact,2012,11(1):68-81.
[7]Kim J W,Kim J Y,Seo S O,et al.Enhanced production of 2,3-butanediol by engineered Saccharomyces cerevisiae through fine-tuning of pyruvate decarboxylase and NADH oxidase activities[J].Biotechnol Biofuels,2016,265(9):1-12.
[8]Ishii J,Morita K,Ida K,et al.A pyruvate carbon flux tugging strategy for increasing 2,3-butanediol production and reducing ethanol subgeneration in the yeast Saccharomyces cerevisiae[J].Biotechnol Biofuels,2018,11(1):180-199.
[9]Kim S J,Seo S O,Park Y C,et al.Production of 2,3-butanediol from xylose by engineered Saccharomyces cerevisiae[J].J Biotechnol,2014,192:376-382.
[10]Mclellan M A,Rosenthal N A,Pinto A R.Cre-lox P-Mediated recombination:general principles and experimental considerations[J].Curr Protoc Mouse Biol,2017,7(1):1-12.
[11]Yang Y J,Singh R,Lan X,et al.Genome editing in model strain Myxococcus xanthus DK1622 by a sitespecific Cre/lox P recombination system[J].Biomolecules,2018,8(4):137-154.
[12]Gueldener U,Heinisch J,Koehler G J,et al.A second set of lox P marker cassettes for Cre-mediated multiple gene knockouts in budding yeast[J].Nucleic Acids Res,2002,30(6):23-30.
[13]Mizutani O,Masaki K,Gomi K,et al.Modified Crelox P recombination in Aspergillus oryzae by direct introduction of Cre recombinase for marker gene rescue[J].Appl Environ Microbiol,2012,78(12):4126-4133.
[14]黄琳,刘常金,王芳,等.耶氏酵母菌POX5基因的敲除[J].现代食品科技,2010,26(4):331-336.
[15]柯崇榕,吴毕莎,邵庆伟,等.酿酒酵母PDC1基因过表达菌株的构建[J].应用与环境生物学报,2013,19(4):704-708.
[16]黄守锋,裴芳艺,王长丽,等.利用酿酒酵母工程菌株生产2,3-丁二醇的研究进展[J].食品安全质量检测学报,2015,6(10):3928-3934.
[17]林晓华.酿酒酵母SNF4基因敲除缺失菌株的构建[J].生物工程学报,2011,27(4):572-578.
[18]Semkiv M V,Dmytruk K V,Sibirny A A.Development of a system for multicopy gene integration in Saccharomyces cerevisiae[J].J Microbiol Methods,2016,120:44-49.
[19]Anvari M,Motlagh M R S.Enhancement of 2,3-butanediol production by klebsiella oxytoca PTCC 1402[J].J Biomed Biotechnol,2011,2011(1):6361-6370.
[20]Celińska E,Grajek W.Biotechnological production of2,3-butanediol-current state and prospects[J].Biotechnol Adv,2009,27(6):715-725.
[21]Liu J M,Chan S H J,Theis B N,et al.Combining metabolic engineering and biocompatible chemistry for high yield production of homo-diacetyl and homo-(S,S)-2,3-butanediol[J].Metab Eng,2016,36:57-67.
[22]吴满珍,李鹏越,苏珂,等.弱化乙醇途径关键酶活性减少酿酒酵母在丙酮酸发酵过程中副产物乙醇的积累[J].食品与发酵工业,2015,41(7):7-12.
[23]Lian J,Chao R,Zhao H.Metabolic engineering of a Saccharomyces cerevisiae strain capable of simultaneously utilizing glucose and galactose to produce enantiopure(2R,3R)-butanediol[J].Metab Eng,2014,23:92-99.