多基因转化构建重组工业酿酒酵母及其在木薯酒精发酵中的应用研究
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摘要
在化石能源日益枯竭和纤维素、木质素燃料酒精生产技术尚不完善的背景下,我国已将热带能源植物木薯(Manihot esulenta Crantz)列为燃料酒精生产的重要原料。木薯酒精应用受限因素主要是生产成本高。酵母是酒精工业的“灵魂”,在酵母细胞中构建淀粉代谢途径和纤维素代谢途径,部分替代木薯酒精生产过程外源酶制剂的功能,就可节省酶制剂,同时提高淀粉转化率和酒精产率,降低成本。
本研究以工业酿酒酵母Saccharomyces cerevisiae NY-K为研究对象,以提高木薯基质利用率为目标,最终构建含有三个独立表达盒的多基因酵母整合型表达载体,通过转化将葡萄糖淀粉酶基因、葡聚糖内切酶基因和β-葡萄糖苷酶基因整合到NY-K基因组,从而赋予NY-K多底物代谢活性,从而在酒精发酵阶段兼具有水解淀粉的后糖化作用和降解纤维素活性,从而达到有效利用木薯基质中的淀粉和一定程度的降解进而利用纤维素的目的,最终提高木薯发酵酒精产率。
首先,构建了一个新型酵母整合型表达载体pYES2M。根据酿酒酵母(S. cerevisiae)磷酸甘油酸激酶基因启动子Ppgk碱基序列、核糖体rDNA碱基序列、酿酒酵母抗铜基因CUP1序列和毕赤氏酵母表达载体pPIC9K为模板设计引物,分别进行PCR,扩增得到Ppgk (GenBank accession No. FJ415226)、rDNA、CUP1和信号肽α-MF片段。以酵母附加型质粒pYES2为出发质粒,将扩增得到的4个片段依次引入pYES2中,构建得到表达载体pYES2M。以铜抗性作为筛选标记,避免了新的抗药性标记的引入,在源头上提高了安全性。
建立了以铜抗性为筛选标记的工业酿酒酵母转化子筛选方法,以及菌落PCR快速鉴定工业酿酒酵母转化子的方法。
之后,通过PCR方法从泡盛曲霉(Aspergillus awamori)中扩增得到葡萄糖淀粉酶基因gal,从绿色木霉(Trichoderma viride)中扩增得到葡聚糖内切酶基因eg3和p-葡萄糖苷酶基因bgl1。
通过点突变去除gal片段上Sph Ⅰ位点,通过PCR将eg3和bgl1内含子去除,分别将修饰后的gal、eg3和bgl1插入载体pYES2M的多克隆位点,构建得到三个酵母整合型单价表达载体pYES2M-ga1、pYES2M-eg3和pYES2M-bgl1。通过电转化将上述三个线性化载体整合到宿主菌S. cerevisiae NY-K基因组。功能鉴定及SDS-PAGE电泳检测表明三个重组酵母均向胞外表达了活性重组酶蛋白
其次,通过重叠延伸PCR将特定的基因串联起来,插入pYES2M的多克隆位点,构建了2个酵母双价整合型载体pYES2M-eg3-bgl1和pYES2M-eg3-ga1。其中eg3、bgl1和gal均自带完整表达盒。通过电转化方法将线性化载体pYES2M-eg3-bgl1、pYES2M-eg3-ga1分别整合到宿主菌S.cerevisiae NY-K基因组。酶活测定及SDS-PAGE电泳检测表明两个重组酵母均向胞外表达了独立的(非融合)有活性的重组酶蛋白。
最后,将葡萄糖淀粉酶表达盒pMFgaT插入载体pYES2M-eg3-bgl1中,得到含有3个独立表达盒的酵母整合型三价表达载体pYES2M-eg3-ga1-bgl1。通过电转化将线性化多基因表达载体pYES2M-eg3-ga1-bgl1整合到宿主菌S.cerevisiaeNY-K基因组。酶活测定及SDS-PAGE电泳检测表明重组酵母向胞外表达了独立的(非融合)有活性的重组酶蛋白。
整合gal基因的重组酿酒酵母能以可溶性淀粉为底物进行酒精发酵,发酵终点时酒精浓度为1.595g/00mL～1.614g/100mL,高于整合空载体的菌株NY-K/pYES2M和对照NY-K。整合了eg3基因的重组酿酒酵母能以为羧甲基纤维素底物进行酒精发酵,发酵终点时酒精浓度为0.220～0.259g/100mL,略高于整合空载体的菌株NY-K/pYES2M和对照NY-K。整合了bgl1基因的重组酿酒酵母能以为纤维二糖底物进行酒精发酵,发酵终点时酒精浓度为0.323-0.340g/100mL,略高于整合空载体的菌株NY-K/pYES2M和对照NY-K。
三价重组酿酒酵母NY-K/pYES2M-eg3-ga1-bgl1直接发酵木薯基质,发酵终点酒精浓度为1.64g/100mL,对照宿主菌为0.42g/100mL。木薯基质中添加外源酶制剂的基础上进行酒精发酵,三价工程菌NY-K/pYES2M-eg3-ga1-bgl1发酵终点时酒精浓度为9.14g/100mL,与双价工程菌NY-K/pYES2M-eg3-ga1酒精浓度(9.17g/100mL)无明显差别,其他单价和双价重组酿酒酵母发酵木薯基质酒精浓度在8.93～9.20g/100mL之间,对照宿主菌为8.68g/100mL。
以NY-K/pYES2M-eg3-eg3-bgl1通过SSF模式和SHF模式发酵木薯基质,产生的酒精浓度分别为9.27g/100mL和9.10g/100mL,SSF出酒率高于SHF。
综合以上结果认为,SSF发酵模式下重组酿酒酵母NY-K/pYES2M-eg3-ga1-bgl1的酒精度(9.27g/100mL)、淀粉利用率(93.79%)和出酒率(53.26%)基本达到酒精工业的要求,且生产过程能节约糖化酶等酶制剂用量,具有一定的应用价值。
Under the background of fossil energy is going to be exhausted and cellulose, lignin fuel ethanol production technology is not perfect, cassava will be used as important material for fuel ethanol production in China in the present and a period of future. The main limited factor for the application of cassava ethanol is the high cost of production. Yeast is the "soul" in the ethanol industries. If constructing new metabolic pathways of starch and cellulose in yeast cells to partial substitution the function of exogenous enzymes in cassava ethanol production, then exogenous enzymes would be saved. Starch conversion and ethanol yield would be improved and production costs would be reduced.
The strain used in our study was industrial Saccharomyces cerevisiae NY-K The purpose was to improve the utilization ratio of cassava matrix. A multi-gene integrated yeast expression vector with three indivial expression cassette was constructed. Amylase gene, endoglucanase gene and β-glucosidase gene were integrated into the NY-K genome and multi-substrate metabolic activity was added to the transformants. So the transformants could perform post-saccharification to starch in alcoholic fermentation stage and have the function of degrading cellulose. The conversion of starch in cassava matrix was more efficient and cellulose could be degrade in certain degreen. At last ethanol yield was improved.
At first, a new integrated yeast expression vector pYES2M was contructed. Primers were designed based on the sequence of promoter (Ppgk) of phosphogly cerate kinase gene, ribosomal gene rDNA, copper resistance gene CUP1in S. cerevisiae and signal peptide a-MF in Pichia pastoris expression vector pPIC9K. PCR was performed and four fragments including Ppgk, rDNA,CUP1and a-MF were obtained. Ppgk sequence cloned has been submitted to GenBank, accession nummber was FJ415226. Episomal plasmid pYES2was used as the parent plasmid and the four amplified fragments were introduced into pYES2one by one. Expression vector pYES2M was contructed. Copper resistance gene was used as a selection marker in pYES2M for avoiding the introduction of a new drug-resistance selection marker. Biological security was improved in the source.
A methods of copper resistance as selection marker for screenig industrial yeast transformants was established. And a rapid identification method for industrial Saccharomyces cerevisiae transformants by colony PCR was established.
Then glucoarnylase gene gal was amplified by PCR based on Aspergillus awamori genomic DNA. Endolucanase gene eg3and β-glucosidase gene bgll were amplified by PCR based on Trichoderma viride totol RNA.
Restriction enzyme Sph I site in gal fragment was deleted. The intron in eg3and bgll was deleted individually. Then gal,eg3and bgll was inserted into the multiple cloning site of the vector pYES2M individually. Three yeast integrated expression vector pYES2M-gal,pYES2M-eg3and pYES2M-bgll were constructed and were introduced into the host strain S. cerevisiae NY-K by electroporation method. Functional identification and SDS-PAGE electrophoresis were performed and the three recombinant yeasts were found to produce functional extracellular recombinant enzyme.
Second, the special genes were spiced through overlap extension PCR and inserted into the multiple cloning site of the vector pYES2M. Two yeast integrated expression vector pYES2M-eg3-ga1and pYES2M-eg3-bgl1were constructed. In every vector the gene eg3,bgl1and ga1contained complete expression cassette. The linear pYES2M-eg3-ga1and pYES2M-eg3-bgl1were introduced into the host strain S.cerevisiae NY-K by electroporation method. Enzyme activity detection and SDS-PAGE electrophoresis were performed and both two recombinant yeast were found to produce independent (non-fusion) functional extracellular recombinant enzyme.
At last, expression cassette PMFgaT was inserted into the vector pYES2M-eg3-bgll and a yeast trivalent integrated expression vector pYES2M-eg3-gal-bgll was constructed. The linear pYES2M-eg3-gal-bgll was introduced into the host strain S.cerevisiae NY-K by electroporation method. Enzyme activity detection and SDS-PAGE electrophoresis were performed and recombinant yeast was found to produce independent (non-fusion) functional extracellular recombinant enzyme.
Recombinant yeast integrated gal could performed alcoholic fermentation on soluble starch, ethanol concentration was1.595～1.614g/100mL at the end of fermentation. The value was higher than the strain NY-K/pYES2M and NY-K. Recombinant yeast integrated eg3could performed alcoholic fermentation on CMC. ethanol concentration was0.220～0.259g/100mL at the end of fermentation. The value was a little higher than the strain NY-K/pYES2M and NY-K. Recombinant yeast integrated bgll could performed alcoholic fermentation on cellobiose. ethanol concentration was0.323～0.340g/100mL at the end of fermentation. The value was a little higher than the strain NY-K/pYES2M and NY-K.
Trivalent recombinant S. cerevisiae NY-K/pYES2M-eg3-gal-bgl1performed alcohol fermentation on cassava matrix directly, ethanol concentration was1.64g/100mL and0.42g/100mL at the end of fermentation for recombinant and parent strain individually. Alcoholic fermentation was performed again after enzyme were added into cassava matrix, ethanol concentration was9.14g/100mL and9.17g/100mL at the end of fermentation for recombinant strain NY-K/pYES2M-eg3-gal-bgl1and NY-K/pYES2M-eg3-ga1individually, ethanol concentration was8.93～9.20g/100mL at the end of fermentation for other recombinants and8.68g/100mL for parent strain.
NY-K/pYES2M-eg3-eg3-bgll was transfer in cassava matrix and alcoholic fermentation was performed by SSF and SHF mode, ethanol concentration of the liquor was9.27g/100mL and9.10g/100mL for SSF and SHF. ethanol concentration of SSF was higher than SHF.
Based on the results,the conclusion was that ethanol concentration (9.27g/100mL), starch utilization rate (93.79%) and ethanol production rate (53.26%) for recombinant yeast NY-K/pYES2M-eg3-eg3-bgl1arrived the requirement of ethanol industris under SSF model, and glucoamylase and other enzymes can be saved in the process of production. Transgenic yeast strain NY-K/pYES2M-eg3-eg3-bgl1might be useful for cassava ethanol industries.
本研究以工业酿酒酵母Saccharomyces cerevisiae NY-K为研究对象,以提高木薯基质利用率为目标,最终构建含有三个独立表达盒的多基因酵母整合型表达载体,通过转化将葡萄糖淀粉酶基因、葡聚糖内切酶基因和β-葡萄糖苷酶基因整合到NY-K基因组,从而赋予NY-K多底物代谢活性,从而在酒精发酵阶段兼具有水解淀粉的后糖化作用和降解纤维素活性,从而达到有效利用木薯基质中的淀粉和一定程度的降解进而利用纤维素的目的,最终提高木薯发酵酒精产率。
首先,构建了一个新型酵母整合型表达载体pYES2M。根据酿酒酵母(S. cerevisiae)磷酸甘油酸激酶基因启动子Ppgk碱基序列、核糖体rDNA碱基序列、酿酒酵母抗铜基因CUP1序列和毕赤氏酵母表达载体pPIC9K为模板设计引物,分别进行PCR,扩增得到Ppgk (GenBank accession No. FJ415226)、rDNA、CUP1和信号肽α-MF片段。以酵母附加型质粒pYES2为出发质粒,将扩增得到的4个片段依次引入pYES2中,构建得到表达载体pYES2M。以铜抗性作为筛选标记,避免了新的抗药性标记的引入,在源头上提高了安全性。
建立了以铜抗性为筛选标记的工业酿酒酵母转化子筛选方法,以及菌落PCR快速鉴定工业酿酒酵母转化子的方法。
之后,通过PCR方法从泡盛曲霉(Aspergillus awamori)中扩增得到葡萄糖淀粉酶基因gal,从绿色木霉(Trichoderma viride)中扩增得到葡聚糖内切酶基因eg3和p-葡萄糖苷酶基因bgl1。
通过点突变去除gal片段上Sph Ⅰ位点,通过PCR将eg3和bgl1内含子去除,分别将修饰后的gal、eg3和bgl1插入载体pYES2M的多克隆位点,构建得到三个酵母整合型单价表达载体pYES2M-ga1、pYES2M-eg3和pYES2M-bgl1。通过电转化将上述三个线性化载体整合到宿主菌S. cerevisiae NY-K基因组。功能鉴定及SDS-PAGE电泳检测表明三个重组酵母均向胞外表达了活性重组酶蛋白
其次,通过重叠延伸PCR将特定的基因串联起来,插入pYES2M的多克隆位点,构建了2个酵母双价整合型载体pYES2M-eg3-bgl1和pYES2M-eg3-ga1。其中eg3、bgl1和gal均自带完整表达盒。通过电转化方法将线性化载体pYES2M-eg3-bgl1、pYES2M-eg3-ga1分别整合到宿主菌S.cerevisiae NY-K基因组。酶活测定及SDS-PAGE电泳检测表明两个重组酵母均向胞外表达了独立的(非融合)有活性的重组酶蛋白。
最后,将葡萄糖淀粉酶表达盒pMFgaT插入载体pYES2M-eg3-bgl1中,得到含有3个独立表达盒的酵母整合型三价表达载体pYES2M-eg3-ga1-bgl1。通过电转化将线性化多基因表达载体pYES2M-eg3-ga1-bgl1整合到宿主菌S.cerevisiaeNY-K基因组。酶活测定及SDS-PAGE电泳检测表明重组酵母向胞外表达了独立的(非融合)有活性的重组酶蛋白。
整合gal基因的重组酿酒酵母能以可溶性淀粉为底物进行酒精发酵,发酵终点时酒精浓度为1.595g/00mL～1.614g/100mL,高于整合空载体的菌株NY-K/pYES2M和对照NY-K。整合了eg3基因的重组酿酒酵母能以为羧甲基纤维素底物进行酒精发酵,发酵终点时酒精浓度为0.220～0.259g/100mL,略高于整合空载体的菌株NY-K/pYES2M和对照NY-K。整合了bgl1基因的重组酿酒酵母能以为纤维二糖底物进行酒精发酵,发酵终点时酒精浓度为0.323-0.340g/100mL,略高于整合空载体的菌株NY-K/pYES2M和对照NY-K。
三价重组酿酒酵母NY-K/pYES2M-eg3-ga1-bgl1直接发酵木薯基质,发酵终点酒精浓度为1.64g/100mL,对照宿主菌为0.42g/100mL。木薯基质中添加外源酶制剂的基础上进行酒精发酵,三价工程菌NY-K/pYES2M-eg3-ga1-bgl1发酵终点时酒精浓度为9.14g/100mL,与双价工程菌NY-K/pYES2M-eg3-ga1酒精浓度(9.17g/100mL)无明显差别,其他单价和双价重组酿酒酵母发酵木薯基质酒精浓度在8.93～9.20g/100mL之间,对照宿主菌为8.68g/100mL。
以NY-K/pYES2M-eg3-eg3-bgl1通过SSF模式和SHF模式发酵木薯基质,产生的酒精浓度分别为9.27g/100mL和9.10g/100mL,SSF出酒率高于SHF。
综合以上结果认为,SSF发酵模式下重组酿酒酵母NY-K/pYES2M-eg3-ga1-bgl1的酒精度(9.27g/100mL)、淀粉利用率(93.79%)和出酒率(53.26%)基本达到酒精工业的要求,且生产过程能节约糖化酶等酶制剂用量,具有一定的应用价值。
Under the background of fossil energy is going to be exhausted and cellulose, lignin fuel ethanol production technology is not perfect, cassava will be used as important material for fuel ethanol production in China in the present and a period of future. The main limited factor for the application of cassava ethanol is the high cost of production. Yeast is the "soul" in the ethanol industries. If constructing new metabolic pathways of starch and cellulose in yeast cells to partial substitution the function of exogenous enzymes in cassava ethanol production, then exogenous enzymes would be saved. Starch conversion and ethanol yield would be improved and production costs would be reduced.
The strain used in our study was industrial Saccharomyces cerevisiae NY-K The purpose was to improve the utilization ratio of cassava matrix. A multi-gene integrated yeast expression vector with three indivial expression cassette was constructed. Amylase gene, endoglucanase gene and β-glucosidase gene were integrated into the NY-K genome and multi-substrate metabolic activity was added to the transformants. So the transformants could perform post-saccharification to starch in alcoholic fermentation stage and have the function of degrading cellulose. The conversion of starch in cassava matrix was more efficient and cellulose could be degrade in certain degreen. At last ethanol yield was improved.
At first, a new integrated yeast expression vector pYES2M was contructed. Primers were designed based on the sequence of promoter (Ppgk) of phosphogly cerate kinase gene, ribosomal gene rDNA, copper resistance gene CUP1in S. cerevisiae and signal peptide a-MF in Pichia pastoris expression vector pPIC9K. PCR was performed and four fragments including Ppgk, rDNA,CUP1and a-MF were obtained. Ppgk sequence cloned has been submitted to GenBank, accession nummber was FJ415226. Episomal plasmid pYES2was used as the parent plasmid and the four amplified fragments were introduced into pYES2one by one. Expression vector pYES2M was contructed. Copper resistance gene was used as a selection marker in pYES2M for avoiding the introduction of a new drug-resistance selection marker. Biological security was improved in the source.
A methods of copper resistance as selection marker for screenig industrial yeast transformants was established. And a rapid identification method for industrial Saccharomyces cerevisiae transformants by colony PCR was established.
Then glucoarnylase gene gal was amplified by PCR based on Aspergillus awamori genomic DNA. Endolucanase gene eg3and β-glucosidase gene bgll were amplified by PCR based on Trichoderma viride totol RNA.
Restriction enzyme Sph I site in gal fragment was deleted. The intron in eg3and bgll was deleted individually. Then gal,eg3and bgll was inserted into the multiple cloning site of the vector pYES2M individually. Three yeast integrated expression vector pYES2M-gal,pYES2M-eg3and pYES2M-bgll were constructed and were introduced into the host strain S. cerevisiae NY-K by electroporation method. Functional identification and SDS-PAGE electrophoresis were performed and the three recombinant yeasts were found to produce functional extracellular recombinant enzyme.
Second, the special genes were spiced through overlap extension PCR and inserted into the multiple cloning site of the vector pYES2M. Two yeast integrated expression vector pYES2M-eg3-ga1and pYES2M-eg3-bgl1were constructed. In every vector the gene eg3,bgl1and ga1contained complete expression cassette. The linear pYES2M-eg3-ga1and pYES2M-eg3-bgl1were introduced into the host strain S.cerevisiae NY-K by electroporation method. Enzyme activity detection and SDS-PAGE electrophoresis were performed and both two recombinant yeast were found to produce independent (non-fusion) functional extracellular recombinant enzyme.
At last, expression cassette PMFgaT was inserted into the vector pYES2M-eg3-bgll and a yeast trivalent integrated expression vector pYES2M-eg3-gal-bgll was constructed. The linear pYES2M-eg3-gal-bgll was introduced into the host strain S.cerevisiae NY-K by electroporation method. Enzyme activity detection and SDS-PAGE electrophoresis were performed and recombinant yeast was found to produce independent (non-fusion) functional extracellular recombinant enzyme.
Recombinant yeast integrated gal could performed alcoholic fermentation on soluble starch, ethanol concentration was1.595～1.614g/100mL at the end of fermentation. The value was higher than the strain NY-K/pYES2M and NY-K. Recombinant yeast integrated eg3could performed alcoholic fermentation on CMC. ethanol concentration was0.220～0.259g/100mL at the end of fermentation. The value was a little higher than the strain NY-K/pYES2M and NY-K. Recombinant yeast integrated bgll could performed alcoholic fermentation on cellobiose. ethanol concentration was0.323～0.340g/100mL at the end of fermentation. The value was a little higher than the strain NY-K/pYES2M and NY-K.
Trivalent recombinant S. cerevisiae NY-K/pYES2M-eg3-gal-bgl1performed alcohol fermentation on cassava matrix directly, ethanol concentration was1.64g/100mL and0.42g/100mL at the end of fermentation for recombinant and parent strain individually. Alcoholic fermentation was performed again after enzyme were added into cassava matrix, ethanol concentration was9.14g/100mL and9.17g/100mL at the end of fermentation for recombinant strain NY-K/pYES2M-eg3-gal-bgl1and NY-K/pYES2M-eg3-ga1individually, ethanol concentration was8.93～9.20g/100mL at the end of fermentation for other recombinants and8.68g/100mL for parent strain.
NY-K/pYES2M-eg3-eg3-bgll was transfer in cassava matrix and alcoholic fermentation was performed by SSF and SHF mode, ethanol concentration of the liquor was9.27g/100mL and9.10g/100mL for SSF and SHF. ethanol concentration of SSF was higher than SHF.
Based on the results,the conclusion was that ethanol concentration (9.27g/100mL), starch utilization rate (93.79%) and ethanol production rate (53.26%) for recombinant yeast NY-K/pYES2M-eg3-eg3-bgl1arrived the requirement of ethanol industris under SSF model, and glucoamylase and other enzymes can be saved in the process of production. Transgenic yeast strain NY-K/pYES2M-eg3-eg3-bgl1might be useful for cassava ethanol industries.
引文
Ahmed A S, Hamdan S, Annaluru N, et al. Conversion of waste agriculture biomass to bioethanol by recombinant Saccharomyces cerevisiae. Journal of Scientific Research,2010,2(2):351-361
Kosugi A, Kondo A, Ueda M,et al.Production of ethanol from cassava pulp via fermentation with a surface-engineered yeast strain displaying glucoamylase. Renewable Energy, 2009,34(5):1354-1358
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Hadfied C, Jordan BE, Mount RC, et al. G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces carevisiae. Genet,1990,18:303-313
Lee CC. Screening assays for biomass-degrading enzymes. Biofuels,2010,1(4):575-588
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Dobson MJ, Tuite MF, Roberts NA, et al. Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae. Nucl. Acids Res.1982,10,2625-2637
Wong D, Batt S, Robertson G,et al. Chromosomal integration of both an a-amylase and a glucoamylase gene in Saccharomyces cerevisiae for starch conversion. Industrial Biotechnology.2010,6(2):112-118
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Ghang DM, Yu L, Lim MH,et al.Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and a-amylase genes from Debaryomyces occidentalis Biotechnology Letters,2007,29(8):1203-1208,
Fernandez-Gonzalez M, Ubeda JF, Cordero-Otero RR, et al. Engineering of an oenological Saccharomyces cerevisiae strain with pectinolytic activity and its effect on wine. Int. J. Food. Microbiol.,2005,102(2):173-83.
Gundllapalli SB, Pretorius IS and Cordero Otero RR. Effect of the cellulose-binding domain on the catalytic activity of a beta-glucosidase from Saccharomycopsis fibuligera. J. Ind. Microbiol. Biotechnol.,2007;34(6):413-421.
Henry VB. GCR of Saccharomyces cerevisiae encodes a DNA binding protein whose binding is abolished by mutations in the CTTCC sequence motif. Proc. Nail. Acad. Sci., 1991,88,9443-9447
Nakazawa H, Okada K, Kobayashi R,et al. Characterization of the catalytic domains of Trichoderma reesei endoglucanase Ⅰ, Ⅱ, and Ⅲ, expressed in Escherichia coli. Applied Microbiology and Biotechnology,2010,81(4):681-689
Nakazawa H, Okada K, Onodera T,et al. Directed evolution of endoglucanase III (Cell 2A) from Trichoderma reesei. Applied Microbiology and Biotechnology,2011,83(4):649-657
Toda H, Takada S, Oda M, et al. Gene cloning of an endoglucanase from the basidiomycete Irpex lacteus and its cDNA expression in Saccharomyces cerevisiae. Biosci. Biotechnol. Biiochem.,2005,69(7); 1262-1269
Hitzeman RA, Hagie FE, Hayflick JS, et al. The primary structure of the Saccharomyces cerevisiae gene for 3-phosphoglycerate kinase. Nucleic Acids Res.,1982,10 (23),7791-7808
Klabunde J, Diesel A, Waschk D, et al. Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha. Appl. Microbiol. Biotechnol., (2002) 58:797-805
Rathjen J and Mellor J. Characterisation of sequences required for RNA initiation from the PGK promoter of Saccharomyces cerevisiae. Nucleic Acids Research,1990,18 (11) 3219-3223
Klabunde J, Kunze G, Gellissen G, et al. Intergation of heterologous genes in several yeast species using vectors containing a hansellula polymorpha-derived rDNA-targeting element. FEMS Yeast Research,2003(4):185-193
Jia XN, Li W, Shen JL,et al. Construction of multi-gene expression vector and tobacco transformation based on Cre-LoxP recombination system. Journal of Beijing Forestry University,2010,32(5):121-125
Kim JH, Kim HR, Lim MH,et al. Construction of a direct starch-fermenting industrial strain of Saccharomyces cerevisiae producing glucoamylase, α-amylase and debranching enzyme. Biotechnology Letters,2010,32(5):713-719
Song JZ, Liu BD, Liu ZH,et al. Cloning of two cellobiohydrolase genes from Trichodermaviride and heterogenous expression in yeast Saccharomyces cerevisiae. Molecular Biology Reports,2011,37(4):2135-2140
Skryabin KG, Eldarov MA, Larionov VL, et al. Structure and function of the nontranscribed spacer regions of yeast rDNA. Nucleic Acids Research,1984,12(6):2954-2968
Karube I, Timiya E, Matsuoka H. Transformation of Saccharomyces serevisiae spheroplasts by high electric pulse. FEBS Letters,1985,182(1):90-94
Reddy LVA, Reddy OVS and Basappa SC. Potentiality of yeasts in the direct conversion of starchy materials to ethanol and its relevance in the New Millennium. Yeast Biotechnology: Diversity and Applications,2009, Ⅲ,515-549
Ziskaa LH, Runionb GB, Tomeceka M,et al. An evaluation of cassava, sweet potato and field corn as potential carbohydrate sources for bioethanol production in Alabama and Maryland. Biomass and Bioenergy,2009,33(11):1503-1508
Lopes TS, Hakkaart GJAJ, Koerts BL,et al. Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces recevisiae.Gene,1991,105:83-90
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