【作者】 郑阳阳；

【导师】 刘泽寰；

【作者基本信息】 暨南大学 ， 微生物学， 2012， 硕士

【摘要】 工业上木薯乙醇发酵普遍存在工序复杂、耗能大等问题。本研究通过基因工程技术，构建能同时高水平分泌表达α-淀粉酶与糖化酶的重组酿酒酵母菌株，并利用此重组工程菌株，进行了木薯乙醇直接发酵的工艺研究。首先提取米曲霉CICC40344和黑曲霉CICC40179的总RNA并反转录制备cDNA。参照NCBI公布的基因序列设计引物，扩增出α-淀粉酶基因amy与糖化酶基因ga。将其依次连入真核多基因表达载体pScIKP得到重组质粒pSc-ga-amy，通过电击转化重组进工业多倍体酿酒酵母AS2.489的基因组。用碘-淀粉水解圈与SDS-PAGE方法筛选出能够同时表达分泌两种酶的重组菌株，测定其α-淀粉酶与糖化酶活性分别为0.865U/ml与34.79U/ml，即酶活组成比例约为1：40。设计酶活配比试验，研究两淀粉酶协同作用，发现当α-淀粉酶与糖化酶酶活份数比例为1：5（当α-淀粉酶与糖化酶分别为1U与40U时，即份数比例为1：1）时，其水解淀粉的效果最好。利用Nhe I与Xba I同尾酶酶切位点向pScIKP载体中多次插入首尾相连的糖化酶基因表达盒，使载体上amy与ga表达盒比例为1：5，以此调节两种淀粉酶蛋白表达量比例，进而提高重组菌株的淀粉水解能力。但是构建好1个α-淀粉酶与5个糖化酶基因串联表达的重组酵母后发现，其水解淀粉的能力并未随之增强，分析原因可能与各基因启动子的相互干扰、整合随机性、rDNA功能受到破坏、位置效应、翻译极性等因素有关。从已构建的重组酵母菌株中筛选出淀粉水解能力最强菌株AS2.489/pScAG，对其生长特性、酶学性质及遗传稳定性进行研究，并通过正交试验对直接淀粉发酵条件进行优化。利用工程酵母AS2.489/pScAG于5L发酵罐中对200g/l的木薯粉进行直接发酵，4d内发酵液中乙醇体积分数达到8.68%，约为理论值的80.9%。该结果表明，获得的重组酵母发酵性能优越，在无需预处理、不添加任何商业酶的条件下对木薯的直接发酵效果良好，能够大大简化生产工艺与节能降耗、节约成本，具有重要的生产应用价值。更多还原

【Abstract】 In order to solve problems such as complex processes and large energy consumption in theindustrial alcohol fermentation from cassava, recombinant Sacharomyces cerevisiae strainsco-expressing α-amylase and glucoamylase genes were constructed. Taking advantage of thisengineered amylolytic yeast, the direct production process established showed good effect.Firstly, genes encoding α-amylase and glucoamylase were obtained by RT-PCR respectivelyfrom the total RNA of Aspergillus oryzae CICC40344and Aspergillus niger CICC40179. Theamplified genes were cloned into expression vector pScIKP in sequence to generate theco-expression plasmid pSc-ga-amy. It was then transformed into S.cerevisiae by pulse celltransfection. Amylolytic transformants were screened with clear halos by iodine staining onstarch plates, and showed α-amylase and glucoamylase activities of0.865U/ml and34.79U/ml.The mixed enzymes tests were carried out, and the cocktails with α-amylase andglucoamylase activity ratio of1:5showed the best starch hydrolysis effects. In order to increasethe starch hydrolysis capacity of the recombinant strain, using isocaudamer enzyme of NheI andXbaI ligation, the glucoamylase gene expression cassette was repeatedly inserted to pScIKPvector to make the α-amylase and glucoamylase expression cassettes reach a ratio of1:5. But theresults showed the recombinant yeasts with one α-amylase gene and five glucoamylase geneswere not of high starch hydrolyzing ability. It may be due to the mutual interference of eachpromoter, randomness of integration, restrain of rDNA function, position effect, translationpolarity and other factors.The recombinant strain AS2.489/pScAG with the highest starch hydrolyzing ability wasselected to study its growth characteristics, enzyme properties and genetic stability, and directstarch fermentation conditions were optimized by orthogonal test. Direct ethanol fermentationwas also conducted in a5L jar fermenter with200g/l of cassava using this engineered yeast.The ethanol concentration could reach8.68%(v/v), approximate to80.9%of theoretical yield.This result indicated that the strain AS2.489/pScAG could sufficiently produce ethanol directlyfrom cassava without addition of any commercial enzymes. It would significantly simplify theproduction process and reduce energy consumption.更多还原