CRISPR/Cas9介导的酿酒酵母ADH2基因中断及反义RNA干扰GPD1的表达

CRISPR/Cas9是一个简单、高效的用于靶向目的基因和无标记的基因组工程的工具。本文通过构建酿酒酵母沉默组件PGK-SGPD1-CYC1,使甘油-3-磷酸脱氢酶I(Glycerol-3-phosphate dehydrogenase,GPD1)基因在PGK强启动子、CYC1终止子在特定区域内进行干扰和表达。应用CRISPR/Cas9基因编辑技术,在中断乙醇脱氢酶Ⅱ(alcohol dehydrogenase Ⅱ,ADH2)基因的同时,定点敲入GPD1基因的反义干扰组件,从而特定地干扰GPD1的表达。采用高效的酵母化学转化法将反应组件敲入酿酒酵母Y1H中,CRISPR/Cas9介导的同源重组效率达43.48%,由此获得了ADH2基因中断和GPD1反义干扰的酿酒酵母突变株。发酵实验结果表明,酿酒酵母突变菌株SG1-1与出发菌株Y1H相比,乙醇产率提高了9.07%,甘油产率下降了12.05%,乙酸产率下降了12.30%,结果表明通过中断ADH2基因及插入GPD1反义干扰组件,既能够中断ADH2基因的功能,减少乙醇转化为乙醛,同时也能在一定程度上干扰GPD1基因的表达,提高乙醇产率。     

乙醇脱氢酶Ⅰ基因敲除的酿酒酵母重组菌构建的初步研究

本实验根据酿酒酵母乙醇代谢途径,构建一株低乙醇产量的酿酒酵母基因工程菌株,以满足人们对低醇啤酒的需要.利用抗性基因筛选基因敲除突变体的方法,通过引物L1和L2扩增潮霉素B基因(两翼与酿酒酵母同源),按常规醋酸锂法转化酵母细胞后,筛选标记与酵母adh Ⅰ基因发生同源重组,得到一株ADH Ⅰ酶活性降低的工程菌株.发酵实验结果表明,转化菌株乙醇含量平均值为1.8%(V/V),较原始菌株低了65%.说明转化菌株体内乙醇生成途径受到干扰.     

乙醇脱氢酶I基因敲除的酿酒酵母重组菌构建的初步研究

本实验根据酿酒酵母乙醇代谢途径,构建一株低乙醇产量的酿酒酵母基因工程菌株,以满足人们对低醇啤酒的需要。利用抗性基因筛选基因敲除突变体的方法,通过引物L1和L2扩增潮霉素B基因(两翼与酿酒酵母同源),按常规醋酸锂法转化酵母细胞后,筛选标记与酵母adhI基因发生同源重组,得到一株ADHI酶活性降低的工程菌株。发酵实验结果表明,转化菌株乙醇含量平均值为1.8%(V/V),较原始菌株低了65%。说明转化菌株体内乙醇生成途径受到干扰。     

啤酒酵母HD34-1乙醇脱氢酶活性的研究

菌株HD34-1是将B.sub的乙醇脱氢酶(ADH)基因转入啤酒酵母HD34而构建的无醇啤酒酵母工程菌株,该菌株的ADH基因受Gal启动子的控制,因此为了考察该菌株ADH基因的表达情况,本研究设计了半乳糖和乙醇的浓度梯度进行发酵试验并测定了相应条件下的的ADH活性,结果表明2%的半乳糖浓度,诱导16h最适宜ADH基因的表达。     

酿酒酵母Y3401产乙醇条件优化及其产香特性

以酿酒酵母Y3401为出发菌株,对其产乙醇条件进行优化,并探究其发酵产香特性。在乙醇发酵培养基的基础上,采用单因素Step-by-step和正交试验方法确定酿酒酵母Y3401高产乙醇的发酵条件是:葡萄糖300 g/L,酵母浸粉37.8 g/L,初始pH 5.2,活化24 h,接种量3%,装液量30 mL/250 mL,在30℃,100 r/min条件下培养32 h。在此条件下,酵母Y3401产乙醇质量浓度高达117.2 g/L,相比初期产量(52.4 g/L)提高了1.24倍。采用顶空固相微萃取-气质联用仪分析该酵母的高粱浸出液在30℃,100 r/min培养32 h后的挥发性风味物质,结果表明,该酵母能产苯乙醇、乙酸乙酯、乙酸苯乙酯等40种对白酒品质有重要贡献的风味物质。这说明酿酒酵母Y3401不仅高产乙醇,而且产生多种改善白酒品质的风味物质,这些特性有利于其在白酒酿造中的应用。     

啤酒酵母HD34-1乙醇脱氢酶浮眭的研究

菌株HD34-1是将B．sub的乙醇脱氢酶（ADH）基因转入啤酒酵母HD34而构建的无醇啤酒酵母工程菌株,该菌株的ADH基因受Gal启动子的控制,因此为了考察该菌株ADH基因的表达情况,本研究设计了半乳糖和乙醇的浓度梯度进行发酵试验并测定了相应条件下的的ADH活性,结果表明2％的半乳糖浓度,诱导16h最适宜ADH基因的表达。     

乙醇胁迫下酿酒酵母(Sc131)生长状态和基因的表达

为了探究果酒发酵酵母菌混合培养条件下酿酒酵母菌(Sc131)细胞行为变化的分子机制,考察乙醇胁迫条件下菌株Sc131细胞生长状态和基因的表达,建立3%,6%和10%乙醇胁迫酿酒酵母体系。通过分光光度法检测Sc131生长状态,SYBR GREEN实时荧光定量PCR检测醇酰基转移酶EHT1和EEB1基因,转醛醇酶TAL1和NQM1基因,乙醇脱氢酶ADH1基因和乙醛脱氢酶ALD2基因表达量。结果表明,低浓度乙醇胁迫下Sc131细胞生长状态差异不显著。然而,醇酰基转移酶EEB1基因、乙醇脱氢酶ADH1基因和乙醛脱氢酶ALD2基因在乙醇胁迫下的相对表达量都有显著变化。例如:乙醇脱氢酶ADH1基因在10%乙醇胁迫下的相对表达量达到对照组的9 000倍。     

构建ADH2和THI3基因敲除酿酒酵母用于降低糯米酒中高级醇的研究

为了降低糯米酒高级醇含量,以酿酒酵母（Saccharomyces cerevisiae）菌株XF1的单倍体XF1a7和XF1α6为原始菌,采用Cre/loxP同源重组系统构建乙醇脱氢酶基因ADH2和类丙酮酸脱羧酶基因THI3缺失的单倍体酵母,再通过单倍体的杂交构建ADH2单基因缺失双倍体酵母XF1-A和ADH2与THI3双基因缺失的双倍体酵母XF1-AT。结果表明,重组菌XF1-A、XF1-AT与原始菌XF1的生长性能相似,菌株XF1-A和XF1-AT的基本发酵性能与菌株XF1无显著差异,菌株XF1-A酿造糯米酒中高级醇含量为522.16 mg/L,比菌株XF1低11.16%;菌株XF1-AT的高级醇含量为462.03 mg/L,比菌株XF1低21.39%。综上,ADH2和THI3基因敲除酿酒酵母能够有效降低糯米酒中高级醇生成量。     

安全酵母Candida glycerinogenesis食品级甘油合成关键基因的研究

为了研究酵母甘油合成关键酶基因在食品级甘油合成中的重要作用,以产甘油假丝酵母基因组为模板,设计引物,克隆获得甘油合成关键酶基因3-磷酸-甘油脱氢酶基因(CgGPD),并在大肠杆菌中通过tac启动子串联表达来自酿酒酵母的3-磷酸甘油酯酶基因(ScGPP2)。经IPTG诱导培养后,3-磷酸-甘油脱氢酶(CgGPD)的比酶活达到了60mU/mg。在30%的葡萄糖浓度下,经过48h的摇瓶发酵,可合成甘油2.52g/L。表明3-磷酸甘油酯酶在甘油合成过程中起了关键作用,过表达CgGPD基因有助于食品级甘油大量合成,从而降低下游分离纯化的难度,可从分子水平进一步提高甘油的产量。     

通气量对酿酒酵母GGSF16高浓度乙醇发酵的影响

在5 L发酵罐中,研究了以260 g/L葡萄糖为底物,不同通气量对酿酒酵母GGSF16高浓度乙醇发酵的影响。结果表明,与厌氧条件相比,通入适当的空气是高浓度乙醇发酵过程中重要的控制参数,能够缩短发酵时间,增加酵母细胞量,提高酵母细胞的存活率和乙醇耐受性。然而,乙醇产率与单位质量的酵母细胞消耗葡萄糖和生成乙醇的能力降低。最适通气量为80 m L/min,酵母细胞干重为14.16 g/L,发酵强度为3.93 g/(L·h),比厌氧分别提高104%和70.1%,终点乙醇浓度为117.9 g/L,乙醇产率为0.452 g/g(发酵效率87.8%)。     

Biosensors for Analysis of Ethanol in Foods

An amperometric ethanol sensor was constructed by co-immobilization of alcohol dehydrogenase (ADH) and bovine serum albumin (BSA) in carbon paste containing entrapped NAD⁺. Conditions for optimum electrode response were determined by manipulation of experimental variables. Incorporation of both ADH and NAD⁺ in carbon paste resulted in better relationships between the enzymatic reaction and the electrochemical process. The surface of such an electrode could be renewed by polishing and smoothing on a clean paper. The sensor showed rapid response time and high sensitivity.     

Ginsenoside‐Free Molecules from Steam‐Dried Ginseng Berry Promote Ethanol Metabolism: An Alternative Choice for an Alcohol Hangover

Ethanol metabolism produces harmful compounds that contribute to liver damage and cause an alcohol hangover. The intermediate metabolite acetaldehyde is responsible for alcohol hangover and CYP2E1‐induced reactive oxygen species damage liver tissues. In this study, we examined whether ginsenoside‐free molecules (GFMs) from steam‐dried ginseng berries promote ethanol metabolism and scavenge free radicals by stimulating primary enzymes (alcohol dehydrogenase, aldehyde dehydrogenase, CYP2E1, and catalase) and antioxidant effects using in vitro and in vivo models. The results revealed that GFM effectively scavenged 2,2‐diphenyl‐1‐picrylhydrazyl hydrate radicals and hydroxyl radicals. Notably, GFM significantly enhanced the expression of primary enzymes within 2 h in HepG2 cells. GFM clearly removed the consumed ethanol and significantly reduced the level of acetaldehyde as well as enhancement of primary gene expression in BALB/c mice. Moreover, GFM successfully protected HepG2 cells from ethanol attack. Of the major components identified in GFM, it was believed that linoleic acid was the most active ingredient. Based on these findings, we conclude that GFM holds promise for use as a new candidate for ethanol metabolism and as an antihangover agent.     

Modulation of Glycerol and Ethanol Yields During Alcoholic Fermentation in Saccharomyces cerevisiae Strains Overexpressed or Disrupted for GPD1 Encoding Glycerol 3-Phosphate Dehydrogenase

The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3-phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol-3-phosphatase were found when low and high glycerol-forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose-rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1Δ mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production (×4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by-products such as pyruvate, acetate, acetoin, 2,3 butane-diol and succinate was observed, with a marked increase in acetoin production. © 1997 John Wiley & Sons, Ltd.     

耐高浓度酒精酵母菌与酒精敏感酵母菌中海藻糖含量与耐酒精的关系

对存在葡萄糖和无葡萄糖存在情况下的耐酒精酵母菌Saccharomycescerevisiae 12 0 0菌株和酒精敏感酵母菌SaccharomycescerevisiaeK5 5A菌株的耐酒精能力进行了比较。同时分析了它们在高浓度酒精冲击过程中的海藻糖含量变化。结果发现 ,这 2株酒精酵母菌的海藻糖含量与它们的耐酒精能力、保护线粒体膜完整性、防止线粒体丢失DNA有很大的关系     

酒精与化妆品

简要介绍了酒精的生产方法、生产原料及分类,并从化妆品的角度介绍了酒精的应用情况,同时指出了酒精是化妆品的必要原料,只要正确使用,对人体无害。     

美国的燃料酒精工业

美国是世界第二大酒精生产国 ,年产酒精500万吨 ,占世界总产量的24%。自1979年以来 ,在美国已经建立起一个强大的由国产再生资源为原料的燃料酒精工业。该工业体系包括了法律体系、政府决策体系、工业生产体系、研发体系和公共认识体系几大部分 ,动员了社会各个部门的参与。     

酒精复合酶在玉米酒精浓醪发酵中的应用

探讨了添加酒精复合酶对玉米酒精浓醪发酵的影响.结果表明,添加酒精复合酶对酒精发酵有明显的促进作用.与时照相比,添加0.75 g/100 g的酒精复合酶.发酵酒精浓度提高2.62%vol,相对原料出酒率提高4.37%.