过表达INO1基因提高酿酒酵母乙醇耐受性
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摘要
为获得燃料乙醇生产菌株,通过基因工程改造,构建能够利用能源甘蔗汁发酵、乙醇产率高的酿酒酵母工程菌株。即过表达肌醇-3-磷酸合成酶基因ino1,敲除kanMX抗性基因,获得重组菌。对过表达菌株的乙醇耐受性进行分析。利用甘蔗汁进行发酵培养,采用气相色谱(GC)对发酵产物乙醇进行检测。结果显示过表达菌株YI2-1能够耐19%(V/V)的乙醇,利用20oBx甘蔗汁厌氧发酵乙醇积累量为13.10%(V/V),较出发菌提高了8.55%。而过表达菌株YI2-1△KP的最大乙醇积累量为13.17%(V/V),较出发菌提高了9.16%。研究表明通过过表达酿酒酵母ino1基因能够有效提高菌株细胞活力、乙醇耐受性。构建的工程菌可利用甘蔗汁发酵,具有较高的乙醇产量。
In order to obtain the strains producing fuel ethanol,the engineering strain Saccharomyces cerevisiae,which may ferment using sugarcane juice to yield high ethanol,were constructed by genetic engineering. In other words,the inositol-3-phosphate synthase gene(ino1)was over-expressed in S. cerevisiae;the antibiotic resistance gene(kanMX)were knocked out,thus recombinant S. cerevisiae was acquired.Further,the tolerances(survivability)of the over-expressed strains to ethanol were analyzed. The sugarcane juice was used as a carbon source in the fermentation,and the ethanol content in the fermentation broth of strains was detected by gas chromatography. The experimental results showed that over-expressed strains YI2-1 tolerated 19%(V/V)ethanol. The 20 oBx of sugarcane juice was employed in the anaerobic fermentation,which yielded 13.10%(V/V)of the ethanol,and this was 8.55% more than that by the initial strain. The biggest output of ethanol by the over-expressed strain YI2-1△KP was accumulated to 13.17%(V/V),and this was 9.16% more than that by the original one.This study indicates that over-expression of ino1 gene efficiently enhances the cell viability and tolerance of S. cerevisiae to ethanol and the constructed strain S. cerevisiae is proved to highly yield ethanol by the fermentation of sugarcane juice.
In order to obtain the strains producing fuel ethanol,the engineering strain Saccharomyces cerevisiae,which may ferment using sugarcane juice to yield high ethanol,were constructed by genetic engineering. In other words,the inositol-3-phosphate synthase gene(ino1)was over-expressed in S. cerevisiae;the antibiotic resistance gene(kanMX)were knocked out,thus recombinant S. cerevisiae was acquired.Further,the tolerances(survivability)of the over-expressed strains to ethanol were analyzed. The sugarcane juice was used as a carbon source in the fermentation,and the ethanol content in the fermentation broth of strains was detected by gas chromatography. The experimental results showed that over-expressed strains YI2-1 tolerated 19%(V/V)ethanol. The 20 oBx of sugarcane juice was employed in the anaerobic fermentation,which yielded 13.10%(V/V)of the ethanol,and this was 8.55% more than that by the initial strain. The biggest output of ethanol by the over-expressed strain YI2-1△KP was accumulated to 13.17%(V/V),and this was 9.16% more than that by the original one.This study indicates that over-expression of ino1 gene efficiently enhances the cell viability and tolerance of S. cerevisiae to ethanol and the constructed strain S. cerevisiae is proved to highly yield ethanol by the fermentation of sugarcane juice.
引文
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[11]张厚程,池振明. ino1基因在粟酒裂殖酵母中的表达及对蔗糖酶分泌和磷脂合成的影响[J].山东大学学报:理学版,2002, 37(6):544-547.
[12]赵华,谌文菊.基因工程在构建酿酒酵母酒精发酵菌的研究进展[J].世界最新医学信息文摘, 2017, 17(68).
[13]张苗苗,陆栋,剡倩,等.细胞膜对酿酒酵母乙醇耐受性影响的研究进展[J].中国酿造, 2016, 35(9):16-19.
[2]尤亮,丁东栋,崔志峰.乙醇耐受性酿酒酵母菌株选育的研究进展[J].工业微生物, 2016, 46(3):51-55.
[3]Voordeckers K, Kominek J, Das A, et al. Adaptation to high ethanol reveals complex evolutionary pathways[J]. PLoS Genetics, 2015,11(11):e1005635.
[4]Chi Z, Kohlwein SD, Paltauf F. Role of phosphatidylinositol(PI)in ethanol production and ethanol tolerance by a high ethanol producing yeast[J]. J Ind Microbiol Biotechnol. 1999, 22(1):58-63.
[5]季冉,袁兴中,等.外加肌醇对嗜鞣管囊酵母发酵的酒精产量及其耐酒精能力的影响[J].生物技术通报, 2008(2):163-167, 171.
[6]Furukawa Keiji, Obata Hiroshi, Kitano Hideyuki, et al. Effect of cellular inositol content on ethanol tolerance of Saccharomyces cerevisiae in sake brewing[J]. Journal of Bioscience and Bioengineering, 2004, 98(2):107-113.
[7]Hisashi S, Yusuke M. Microorganism producing inositol with high yield, and method for manufacturing inositol by using same:WO,2013125666 A1[P]. 2013-08-29.
[8]Hong ME, Lee KS, et al. Identification of gene targets eliciting improved alcohol tolerance in Saccharomyces cerevisiae through inverse metabolic engineering[J]. J Biotechnol, 2010, 149(1/2):52-59.
[9]黄贞杰,陈玲,张积森,等. Scino1基因克隆及酵母多基因多拷贝整合表达载体的构建[J].福建师范大学学报:自然科学版,2012, 28(6):100-105.
[10]黄贞杰,陈由强,陈丽霞,等.产肌醇酿酒酵母基因工程菌的构建[J].微生物学通报, 2016, 43(7):1540-1546.
[11]张厚程,池振明. ino1基因在粟酒裂殖酵母中的表达及对蔗糖酶分泌和磷脂合成的影响[J].山东大学学报:理学版,2002, 37(6):544-547.
[12]赵华,谌文菊.基因工程在构建酿酒酵母酒精发酵菌的研究进展[J].世界最新医学信息文摘, 2017, 17(68).
[13]张苗苗,陆栋,剡倩,等.细胞膜对酿酒酵母乙醇耐受性影响的研究进展[J].中国酿造, 2016, 35(9):16-19.