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。     

无醇啤酒酵母菌株HD34-1的生产性实验

从构建的乙醇脱氢酶(ADH)工程菌株中筛选到ADH活性最强的菌株HD34-1,以其为发酵菌株进行1吨发酵罐发酵实验,经过生产性发酵实验与理化指标的测定,证明HD34-1菌株的发酵酒精度低于1%(W/W),满足无醇啤酒的要求,并确定了该菌株啤酒发酵的发酵条件为1吨发酵罐密闭发酵,温度选择11℃,接种量为2%,糖度降为5%时终止发酵,降温至4℃封存。     

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。     

压力耐受啤酒酵母的筛选及其RT-PCR研究

为了提高啤酒酵母对发酵环境中存在的多种压力的耐受性,采用紫外线对啤酒工业生产菌株Y-1进行诱变。经过米卡芬净平板的初筛和压力平板的复筛,获得了2株压力耐受性明显增强的啤酒酵母MR0-8和MR1-2。与出发菌株Y-1相比,突变菌株MR0-8和MR1-2发酵结束细胞活力分别提高了19%和34%。进一步对出发菌株Y-1和突变菌株MR1-2的压力耐受途径的基因进行了RT-PCR分析。结果显示:在正常条件下,MR1-2的压力应答基因及细胞完整性基因的表达量比出发菌株Y-1都有不同程度的提高;在乙醇压力和高渗透压压力下,菌株Y-1的大部分相关基因都上调,而MR1-2的相关基因则表现出复杂的应答反应。     

酿酒酵母乙醇脱氢酶Ⅱ基因反义干扰及对乙醇发酵的影响

通过构建酿酒酵母沉默表达载体PURH-ADH2,使ADH2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。采用Bam HI和Xmal I限制性内切酶对SADH2基因和PURH质粒进行双酶切,构建反义重组表达质粒PURH-SADH2,通过高效酵母转化法和电转法将重组质粒组件转化至酿酒酵母SY01细胞中,获得阳性克隆菌株JY01。酿酒酵母JY01突变菌株与出发菌株SY01和Y01发酵试验结果相比,JY01甘油脱氢酶酶活比出发菌株Y01与SY01分别下降了16.31%和13.5%;当酿酒酵母Y01、SY01和JY01菌株发酵36~60 h时,JY01菌株甘油含量相比Y01分别下降了16.34%、14.25%、14.89%;当酿酒酵母突变菌株发酵48 h时,Y01、SY01和JY01的乙醇浓度分别为6.243 g/100 m L、7.145 g/100 m L和7.288 g/100 m L,酿酒酵母JY01发酵液乙醇量比比原始菌株Y01乙醇含量提高了14.33%。结果表明通过反义干扰ADH2基因5’UTR序列,能有效干扰酵母工程菌株ADH2转录与表达,削弱甘油积累途径,促进乙醇代谢途径。     

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.     

酿酒酵母关键节点基因缺损对法尼烯合成的影响

以法尼烯为评价效应物,研究了缺损乙醇合成途径、甘油合成途径、胞质乙酰辅酶A转运途径和法尼基焦磷酸消耗支路关键基因对酿酒酵母WHE4菌株合成法尼烯的影响。通过CRISPR-cas9基因编辑技术,获得8株关键基因缺损菌株。结果表明,与WHE4菌株相比,缺损乙醇脱氢酶基因ADH3-6对乙醇和法尼烯产量没有影响;单独缺损甘油三磷酸脱氢酶基因GPD1和GPD2使甘油积累量分别降低了15%和34%,缺损半乳糖激酶基因GAL1、GAL7、GAL10下调了甲羟戊酸途径所有基因转录水平,它们的缺损均不能提高菌株的法尼烯产量;缺损香叶基香叶基焦磷酸合酶基因BTS1和二酰基甘油二磷酸磷酸酶基因DPP1,法尼烯产量提高了29%,在5 L发酵罐补料分批发酵,菌株WHE4-33(WHE4 Δbts1,Δdpp1)的法尼烯产量达到1 578.91 mg/L。该研究对甲羟戊酸途径上游和下游关键节点基因进行了缺损影响法尼烯合成研究,为构建酿酒酵母萜类化合物高效平台提供了参考价值。     

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。     

乙醛法筛选抗乙醛啤酒酵母菌株的应用研究

乙醛是使啤酒产生硬纸板味、烂苹果味的主要来源,对啤酒品质有较大影响。本实验采用紫外诱变与高浓乙醛平板选育结合法,将筛选得到的菌株(共20株)与出发菌株进行低温发酵实验,然后利用气相色谱法测定菌株发酵液乙醛含量,并将其与出发菌株乙醛含量相比较。对具有较大乙醛降幅的菌株(共3株)进行乙醇脱氢酶活性的验证及稳定性实验,由此筛选出抗乙醛啤酒酵母菌株。该菌株可抵抗高浓度乙醛环境,从而快速还原代谢产生的乙醛,其乙醛含量为4.42 mg/L±0.14 mg/L,较出发菌株降低了71.13%;乙醇脱氢酶(ADH1、ADH3与ADH4)活性显著高于出发菌株(增幅为88.80%),且遗传稳定性表现良好。     

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。     

The alcohol dehydrogenase system in the yeast, Kluyveromyces lactis

We have studied the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis. Southern hybridization to the Saccharomyces cerevisiae ADH2 gene indicates four probable structural ADH genes in K. lactis. Two of these genes have been isolated from a genomic bank by hybridization to ADH2. The nucleotide sequence of one of these genes shows 80% and 50% sequence identity to the ADH genes of S. cerevisiae and Schizosaccharomyces pombe respectively. One K. lactis ADH gene is preferentially expressed in glucose-grown cells and, in analogy to S. cerevisiae, was named K1ADH1. The other gene, homologous to K1ADH1 in sequence, shows an amino-terminal extension which displays all of the characteristics of a mitochondrial targeting presequence. We named this gene K1ADH3. The two genes have been localized on different chromosomes by Southern hybridization to an orthogonal-field-alternation gel electrophoresis-resolved K. lactis genome. ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K. lactis cells depending on the carbon source.     

低频静电场辐射对酿酒酵母生长代谢的影响

为了探究低频静电场辐射对酿酒酵母生长代谢的影响和作用机理。本实验运用60 Hz低频静电场对酿酒酵母进行辐射处理,通过扫描电子显微镜、流式细胞仪、酶标仪和紫外分光光度计等仪器对酿酒酵母的生长趋势、形态特征、葡萄糖代谢、胞内蛋白总量和胞内乙醇脱氢酶的活性进行测定分析。结果表明:低频静电场辐射条件下生长的酿酒酵母缩短了生长迟滞期,加快了其对数生长期,且其生长速率相比自然生长提高了14.5%。辐射条件下生长的酿酒酵母对葡萄糖的消耗量在时间为16 h时比自然生长提高了15.64%,其胞内乙醇脱氢酶(ADH)的比活力比自然生长提高了7.25%,但胞内蛋白的总量无显著变化。因此,低频静电场辐射可影响酿酒酵母原有的代谢机制,对其生长代谢过程具有明显的促进效应。     

Two genes encoding putative mitochondrial alcohol dehydrogenases are present in the yeast Kluyveromyces lactis

Four structural genes encoding isozymes of the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis have been identified by hybridization to ADH2 DNA probes from Saccharomyces cerevisiae. In this paper we report on the isolation of KlADH4 and the complete sequencing of KlADH3 and KlADH4, two genes which show high homology to KlADH1, the ADH gene previously isolated in K. lactis, and to the ADH genes of S. cerevisiae. When compared with KlADH1, both KlADH3 and KlADH4 encode amino-terminal extensions which show the characteristics of the mitochondrial targeting sequences. These extensions are poorly conserved both at the nucleotide and the amino acid level. Surprisingly, the KlADH4 extension shows a higher identity at the amino acid level to the one encoded by ADH3 of S. cerevisiae than to the KlADH3 presequence. KlADH3 and KlADH4, in contrast to the ADH3 gene of S. cerevisiae, show a strong bias in the choice of codons.     

Identification of an NADH-dependent 5-hydroxymethylfurfural-reducing alcohol dehydrogenase in Saccharomyces cerevisiae

We report on the identification and characterization of a mutated alcohol dehydrogenase 1 from the industrial Saccharomyces cerevisiae strain TMB3000 that mediates the NADH-dependent reduction of 5-hydroxymethylfurfural (HMF) to 2,5-bis-hydroxymethylfuran. The co-factor preference distinguished this alcohol dehydrogenase from the previously reported NADPH-dependent S. cerevisiae HMF alcohol dehydrogenase Adh6. The amino acid sequence revealed three novel mutations (S109P, L116S and Y294C) that were all predicted at the vicinity of the substrate binding site, which could explain the unusual substrate specificity. Increased biomass production and HMF conversion rate were achieved in a CEN.PK S. cerevisiae strain overexpressing the mutated ADH1 gene.     

Stable disruption of ethanol production by deletion of the genes encoding alcohol dehydrogenase isozymes in Saccharomyces cerevisiae

We analyzed the effects of the deletions of genes encoding alcohol dehydrogenase (ADH) isozymes of Saccharomyces cerevisiae. The decrease in ethanol production by ADH1 deletion alone could be partially compensated by the upregulation of other isozyme genes, while the deletion of all known ADH isozyme genes stably disrupted ethanol production.