【作者】 郭忠鹏；

【导师】 石贵阳；

【作者基本信息】 江南大学 ， 发酵工程， 2011， 博士

【摘要】 随着石化燃料的日益减少,酒精作为一种清洁可再生的新型能源成为当前各国研究的热点。利用基因工程及代谢工程技术对工业酒精酵母进行改造,可提高菌种的发酵性能,降低酒精的生产成本。本论文以此为出发点,将Cre/loxp重组酶系统与rDNA位点同源重组相结合应用于工业酒精酵母,从以下三个方面对工业酒精酵母进行改造,并在此基础上建立适用于工业酒精酵母的多基因改造策略。首先,在酿酒酵母（Saccharomyces cerevisiae）发酵生产乙醇的过程中,甘油是一种主要的副产物。减少甘油产量可提高乙醇产率和原料利用率。本研究中通过实验设计出最佳的甘油途径改造策略,在敲除工业酒精酵母甘油合成途径关键基因GPD1的同时表达来源于蜡状芽孢杆菌的以NADP+为辅酶的非磷酸化3-磷酸甘油醛脱氢酶（GAPN）,随后应用Cre/loxp重组酶系统剔除了构建重组菌时引入的抗性标记基因,接着通过rDNA位点同源重组在该重组菌中过量表达了海藻糖合成酶（TPS）及海藻糖磷酸化酶（TPS）,获得了重组工业酒精酵母AG1A1 (gpd1△: :P_PGK1-gapN, P_PGK1-TPS1-TPS2),实现了工业酒精酵母的多基因改造。在葡萄糖浓度为25%的底物发酵中,重组菌AG1A1的甘油得率下降了76.0±0.2%,酒精产量从113.3 g/L提高到123.4 g/L,糖醇转化率提高8.9±0.1%。更为重要的是重组菌AG1A1的最大比生长速率和葡萄糖消耗速率与出发株工业酒精酵母相比基本不变,且该重组菌表现出了更好的耐高糖,耐酒精能力。其次,作为目前生产酒精的主要原料,淀粉质原料如玉米,饲用大麦等含有较高浓度的蛋白质（玉米: 10-13%;饲用大麦: 15-18%）。而酿酒酵母无外分泌蛋白酶,不能利用原料中的可溶性蛋白。为提高酒精产率和原料利用率,在本实验中,通过酿酒酵母表面工程技术在工业酒精酵母细胞表面分别呈现表达了其自身以及来源于粗糙脉孢霉（Neuospora crassa）的酸性蛋白酶基因,获得重组菌APB2 (P_PGK1-PEP4-AG1)和SA3 (P_PGK1-Asp-AG1)。在以玉米淀粉为基质的酒精发酵实验中,重组菌APB2和SA3生长速率和酒精发酵速率均高于原始酒精酵母,酒精得率分别提高了6.5±0.2%和5.7±0.1%。发酵结束时,重组菌APB2及AS3的酒精产量分别达到126.0 g/L和125.0 g/L,而出发株工业酒精酵母仅为118.2 g/L。由于酵母来源的酸性蛋白酶更适合于目前的同步糖化发酵工艺,因此选择该蛋白酶并继续应用Cre/loxp系统与rDNA位点同源重组在重菌株AG1A1 (gpd1△: :P_PGK1-gapN, P_PGK1-TPS1-TPS2)细胞表面进行表达,获得重组菌AGS1 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2-PEP4-AG1)。另一方面,酒精发酵的糟液中含有一定量的纤维二糖未能被酿酒酵母有效利用。在工业酿酒酵母中引入纤维二糖代谢途径,提高原料利用率的同时也为以后的纤维素酒精发酵打下基础。本研究中,分别在工业酒精酵母的细胞内,胞外和细胞表面表达了来源于扣囊复膜孢酵母（Saccharomycopsis fibuligera）的?-葡萄糖苷酶BGL1基因,并对所得的重组菌SBA1 (P_PGK1-BGL1),SBB1 (P_PGK1-αF-BGL1),SBC2 (P_PGK1-αF-BGL1-AG1)进行了有氧条件下的纤维二糖发酵实验,初步说明工业酒精酵母不具有转运纤维二糖的能力。而同时表达纤维二糖透过性酶和β-葡萄糖苷酶的重组菌BPS3 (P_PGK1-BGL1-bglP)不仅具有较好的转运纤维二糖的能力,且该菌株对纤维二糖的利用效率有了明显的提高,在96 h内几乎用尽了10 g/L的纤维二糖并产生4.4 g/L的酒精。因此应用Cre/loxp系统和rDNA位点同源重组,在AGS1 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2-PEP4-AG1)中同时表达纤维二糖透过性酶和β-葡萄糖苷酶,赋予该重组酵母利用纤维二糖的能力,最终获得重组菌AGPB3 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2-PEP4-AG1-bglP-BGL1),构建了具有多方面优良性能的新型工业酒精酵母。在以木薯粉为基质的酒精发酵实验中,对重组菌AGPB3以及工业酒精酵母进行发酵性能的比较,结果表明重组菌AGPB3不仅能够利用纤维二糖,且表现出较快的生长速率,耗糖速率和产酒精速率,甘油得率降低了76.8%,酒精产量从118.5 g/L提高到129.3 g/L,酒精得率提高7.5%,达到理论产率的97%。更多还原

【Abstract】 As the reserves of petroleum decrease, ethanol, which is renewable, has become the focus of many countries as an alternative liquid fuels to gasoline. Improvement of the fermentation competence of yeast strain by gene engineering and metabolic engineering will decrease the costs of ethanol production. In the present study, combined Cre/loxp system with rDNA site homologous recombination, we have conducted research on industrial ethanol-producing yeast from three respects to establish the multi-gene modification strategy suitable used in industrial ethanol-producing yeast.First, since glycerol is a main by-product consuming up to 4%～10% of the carbon source in industrial ethanol fermentation, to reduce the production of glycerol and lead carbon source flux towards the synthesis of ethanol is an important way to improve the ethanol yield. Thus, the GPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of Saccharomyces cerevisiae, was deleted. Simultaneously, a non-phosphorylating NADP+-dependent glyceraldehydes-3-phosphate dehydrogenase （GAPN） from Bacillus cereus was expressed in the obtained GPD1 deleted mutant. And then, trehalose was over-synthesized in above recombinant strain by expression of trehalose synthesis genes TPS1 and TPS2 by using the Cre/loxp system. The resultant recombinant strain AG1A1 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2) exhibited a 76.0±0.2% （relative to the amount of substrate consumed） decrease in glycerol production and a 8.9±0.1% （relative to the amount of substrate consumed） increase in ethanol yield compared with the parent strain. Besides, the maximum specific growth rate （μmax） and fermentation ability of this yeast recombinant strain were indistinguishable as compared to parent strain in anaerobic batch fermentations.Second, although the materials used for ethanol production contain relatively high protein content （corn: 10-13%; feeding barley: 15-18%）, the yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. To improve the substrate utilization and ethanol yield, the gene PEP4, encoding a vacuolar aspartyl protease in S. cerevisiae, and the gene Asp, encoding aspartic protease in Neurospora crassa, was cloned and expressed in industrial ethanol yeast, respectively. The obtained two recombinant strains APB2 (P_PGK1-PEP4-AG1), SA3 (P_PGK1-Asp-AG1) were studied under ethanol fermentation conditions in corn mash fermentations. The ethanol yields of APB2 and SA3 increased by 6.5±0.2% and 5.7±0.1% （relative to the amount of substrate consumed） compared with parent strain. The recombinant strains APB2 and AS3 produced 126.0 g/L and 125.0 g/L ethanol, while the parent strain produced 118.2 g/L ethanol at the end of fermentation. Since the optimal reactive temperature and pH value of PEP4 was coincided with that of SSF （Simultaneous Saccharification and Fermentation）, it was chosen and expressed in the recombinant strain AG1A1 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2) whose G418 resistance gene was deleted by Cre/loxp system. The resultant strain was named AGS1 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2-PEP4-AG1). Third, cellobiose is one of the sugars that cannot be effectively used by S. cerevisiae in the distillate of ethanol fermentation. To engineer the yeast with the ability of assimilation of cellobiose will improve the substrate utilization efficiency and promote the development of cellulose-ethanol fermentation. In our study, the BGL1 gene, encodingβ-glucosidase in Saccharomycopsis fibuligera, was expressed in industrial ethanol-producing strain of S. cerevisiae in three different patterns—in vivo, in vitro and on cell surface. The obtained recombinant strains SBA1 (P_PGK1-BGL1) expression of intracellularβ-glucosidase, SBB1 (P_PGK1-αF-BGL1) expression of extracellularβ-glucosidase and SBC2 (P_PGK1-αF-BGL1-AG1) expression of cell-wall anchoredβ-glucosidase were studied under aerobic and anaerobic conditions in medium supplemented with cellobiose. The results indicated that the parent S. cerevisiae used in industrial ethanol production is likely deficient in cellobiose transporter. However, when theβ-glucoside permease andβ-glucosidase were co-expressed in this strain, it began to uptake cellobiose and the overall performance of this strain in cellobiose-fermentation was improved. The recombinant strain consumed 10 g/L cellobiose and produced 4.4 g/L ethanol in 96 h. After the G418 resistance gene of the recombinant strain AGS1 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2-PEP4-AG1) was deleted by Cre/loxp system, theβ-glucoside permease andβ-glucosidase were co-expressed in this strain and the resultant strain AGPB3 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2-PEP4-AG1-bglP-BGL1) was studied in cassava mash fermentations. Not only the recombinant strain AGPB3 can use cellobiose to produce ethanol, but also this strain showed fast growth rate and glucose consumption rate, as well as ethanol production rate. Finally, the recombinant strain AGPB3 exhibited a 76.8% （relative to the amount of substrate consumed） decrease in glycerol production. The ethanol yield was increased from 118.5 g/L to 129.3 g/L, corresponding to 97% of theoretical yield and a 7.5% （relative to the amount of substrate consumed） increase in ethanol yield was achieved compared with the parent strain.更多还原