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1.
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。  相似文献   

2.
采用基因敲除手段降低啤酒酵母蛋白酶A表达的研究   总被引:3,自引:1,他引:2  
采用醋酸锂转化法,将一段目的基因转入到酵母体内,破坏酵母PEP4基因的表达。通过对目的基因的扩增和羧肽酶Y酶活检测验证基因敲除情况,并通过传代抗性试验与传代发酵稳定性实验验证突变株遗传稳定性良好。  相似文献   

3.
通过PCR方法克隆得到树干毕赤氏酵母木糖醇脱氢酶(XDH)基因XYL2.将该基因连入酵母表达载体pYX212的强启动子磷酸丙糖异构酶(TPI)启动子下,得到融合表达载体pYX-XYL2.通过电转化方法将pYX-XYL2转入酿酒酵母Saccharomyces cerevisiae W303-1A中,酶活测定表明在酿酒酵母中树干毕赤氏酵母木糖醇脱氢酶基因XYL2得到活性表达,酿酒酵母转化子粗酶液中木糖醇脱氢酶比活为每毫克蛋白0.6 U左右,约为供体菌的2.4倍.与基因供体菌不同,木糖醇脱氢酶基因在酿酒酵母中表达不需木糖诱导,为组成型表达.  相似文献   

4.
酿酒酵母的基因改良   总被引:2,自引:0,他引:2  
周世水 《酿酒科技》2005,(7):29-31,35
酿酒酵母的发酵性能直接影响到酒的质量与生产成本。利用基因工程改良酿酒酵母可提高其生产性能。基因改良酿酒酵母的研究和应用有:①增加酿酒酵母发酵性能的基因改良,如:构建含α-乙酰乳酸脱羧酶基因的低双乙酰工程酵母;含乙醇乙酰酶基因的高生香工程酵母;含糖化酶、葡聚糖酶等基因的高发酵度工程酵母;高絮凝性工程酵母和嗜杀酵母。②增强或缺失酵母自身基因的菌种改良,如构建高级醇低生成量的工程酵母和构建双乙酰低生成量的工程酵母。  相似文献   

5.
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。  相似文献   

6.
为了从工业生产菌株耐高渗产甘油假丝酵母(Candida glycerinogenes)克隆甘油合成的限速酶编码基因胞浆NAD -3-磷酸甘油脱氢酶基因(ctGPD),对不同酵母和其他真核生物的NAD -3-磷酸甘油脱氢酶进行比对,分析氨基酸和核苷酸的保守序列,设计了4对简并引物用于扩增C.glycerinogenes的NAD -3-磷酸甘油脱氢酶(GPD)基因片段,经过优化PCR反应条件,利用其中一对中等简并度的引物扩增出CgGPI)基因中约600 bp的保守核心片段.DNA序列及推绎的氨基酸序列进行比对分析表明,该基因片段与其他酵母的胞浆NAD -3-磷酸甘油脱氢酶基因的对应区域具有典型的保守区域,并且与安格斯毕赤酵母的GPI)基因相似性较高.  相似文献   

7.
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。  相似文献   

8.
乙醛是啤酒中的主要风味物质,其代谢主要来自酵母细胞。酵母中乙醇脱氢酶及乙醛脱氢酶是乙醛代谢的关键酶,对乙醛变化起着重要作用。跟踪啤酒酵母发酵过程中相对酶活力及乙醛变化,发现两种乙醇脱氢酶和乙醛脱氢酶的相对酶活力与发酵过程乙醛含量变化具有一定相关性。同时对低产乙醛啤酒酿酒酵母kb2-4与出发菌株啤酒酵母kb进行发酵试验,跟踪检测相对酶活力及乙醛含量,其乙醇脱氢酶Ⅰ和乙醇脱氢酶Ⅱ及乙醛脱氢酶相对酶活力均高于出发菌株,平均增幅分别为15.5%,11.6%和5%。3种酶活性的变化协同作用可以使乙醛含量降幅最大为33.8%。  相似文献   

9.
酿酒酵母ADR1基因是编码过氧化物酶蛋白质转录的正向调节基因,对乙醇代谢有正向调节作用。本文采用PCR技术首次在贝酵母基因组DNA中对ADR1基因序列进行全长克隆,利用在线分析工具Prot Param、Prot Scale、TMHMM、Predict Protein、Swiss-Model等软件对其编码蛋白质的基本理化性质进行分析,同时预测了该基因所编码蛋白质的二级结构和三级结构。结果表明:该核苷酸序列含有一个长3960 bp的开放阅读框,可编码1319个氨基酸。编码的蛋白质为在细胞核中行使调控功能的亲水蛋白,含有17个丝氨酸(S)激酶潜在磷酸化位点、四个coil区和2个锌指结构域,与酿酒酵母ADR1基因所编码的蛋白质结构和性质极为相似,可初步认为贝酵母ADR1基因是乙醇脱氢酶的调控基因。  相似文献   

10.
渗透压对酿酒酵母胞内代谢关键酶活性的影响   总被引:1,自引:0,他引:1  
对耐高渗酵母与普通酿酒酵母在丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、苹果酸脱氢酶、异柠檬酸脱氢酶、乙醇脱氢酶的酶活性特征的差异进行比较分析,建立酶活力测定方法.发现耐高渗酵母在高渗环境的诱导下,其EMP途径、磷酸戊糖途径的关键酶活力都高于普通酿酒酵母.耐高渗酵母具有高活力的乙醇脱氢酶,其能高效地将丙酮酸转化成乙醇.并且三羧酸循环中的关键酶异柠檬酸脱氢酶和苹果酸脱氢酶的活性也得到加强.这些酶活力的增强维持了耐高渗酵母在高渗、高酒精环境下的生长需要与能量代谢的平衡.  相似文献   

11.
The relationship between quinoprotein alcohol dehydrogenase (ADH) and NAD-dependent ADH was studied by constructing quinoprotein ADH-deficient mutants. Quinoprotein ADH-deficient mutants were successfully constructed from Acetobacter pasteurianus SKU1108 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and also by adhA gene disruption with a kanamycin cassette. The NTG mutant exhibited a complete loss of its acetate-producing ability and acetic acid resistance, while the disruptant also exhibited a loss of its acetic acid resistance but retained a weak ADH activity. The immunoblot analysis of quinoprotein ADH indicated that there are no appreciable ADH subunits in the membranes of both mutant strains. The NTG mutant grew better than the wild-type strain in ethanol-containing medium, despite the absence of quinoprotein ADH. In the mutant, the activities of two NAD-dependent ADHs, present in a small amount in the wild-type strain, markedly increased in the cytoplasm when cultured in a medium containing ethanol, concomitant to the increase in the activities of the key enzymes in TCA and glyoxylate cycles. The disruptant showed a poorer growth than the wild-type strain, producing a lower amount of acetic acid in ethanol culture, and it induced one of the two NAD-dependent ADHs and some of the acetate-assimilating enzymes induced in the NTG mutant. This study clearly showed that quinoprotein ADH is extensively involved in acetic acid production, while NAD-dependent ADH only in ethanol assimilation through the TCA and glyoxylate cycles in acetic acid bacteria. The differences between the NTG mutant and the disruptant are also discussed.  相似文献   

12.
The cytosolic NAD?-dependent alcohol dehydrogenases (NAD?-ADHs) are induced in the quinoprotein ADH-(PQQ-ADH) defective Acetobacter pasteurianus SKU1108 mutant during growth in an ethanol medium. The adhI and adhII genes, which encode NAD?-ADH I and ADH II, respectively, of this strain have been cloned and characterized. Sequence analyses have revealed that the adhI gene consists of 1029 bp coding for 342 amino acids, which share 99.71% identity with the same protein from A. pasteurianus IFO 3283. Conversely, the adhII gene is composed of 762 bp encoding for a polypeptide of 253 amino acids, which exhibit 99.60% identity with the A. pasteurianus IFO 3283 protein. ADH I is a member of the group I Zn-dependent long-chain ADHs, while the ADH II belongs to the group II short-chain dehydrogenase/reductase NAD?-ADHs. The NAD?-adh gene disruptants exhibited a growth reduction when grown in an ethanol medium. In Escherichia coli, ethanol induced adhI and adhII promoter activities by approximately 1.5 and 2.0 times, respectively, and the promoter activity of the adhII gene exceeded that of the adhI gene by approximately 3.5 times. The possible promoter regions of the adhI and adhII genes are located at approximately 81-105 bp and 74-92 bp, respectively, from their respective ATG start codons. Their repressor regions might be located in proximity to these promoters and may repress gene expression in the wild-type, where the membrane-bound ADH effectively functions.  相似文献   

13.
The alcohol dehydrogenase system in the yeast, Kluyveromyces lactis   总被引:5,自引:0,他引:5  
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.  相似文献   

14.
15.
为探究葛花粉体粒径与功效释放速度的相关性及其醒酒机理,通过比较4种不同粉碎方法处理得到的植物本体、常规粉体、Ⅰ级粉体、Ⅱ级粉体在总物质溶出度、黄酮溶出量及乙醇脱氢酶(ADH)体外活性实验方面的差异性,发现4种粉体均可在体外激活乙醇脱氢酶,但在总溶出度上Ⅱ级粉体优于Ⅰ级粉体,其次是常规粉体和植物本体,其瞬时溶出度可达到植物本体的8.6倍。在黄酮溶出量和ADH激活率上也呈现相同趋势,Ⅱ级粉体黄酮溶出含量较植物本体提高57.98%,激活率为本体的2倍。实验表明,采用高效球磨技术制备的葛花Ⅱ级粉体较其他粉体均呈现出最优实验结果,且其醒酒作用机制与其能激活乙醇脱氢酶有关,为开发新型醒酒功能食品提供理论依据。  相似文献   

16.
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。  相似文献   

17.
Four genes coding for alcohol dehydrogenase (ADH) activities were identified in Kluyveromyces lactis. Due to the presence in this yeast of multiple ADH isozymes, mutants in the individual genes constructed by gene replacement yielded no clear phenotype. We crossed these mutants and developed a screening procedure which allowed us to identify strains lacking several ADH activities. The analysis of the adh triple mutants revealed that each activity confers to the cell the ability to grow on ethanol as the sole carbon source. On the contrary, adh null strains failed to grow on this substrate, indicating that no other important ADH activities are present in K. lactis cells. In the adh null mutants we also found a residual production of ethanol, as has been reported to be the case in Saccharomyces cerevisiae. This production showed a ten-fold increase when the K1ADHI activity was reintroduced in the null mutant and cells were cultivated under oxygen-limiting conditions. Differently from S. cerevisiae, glycerol is poorly accumulated in K. lactis adh null mutants.  相似文献   

18.
The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely used in recombinant protein production. The widespread use of a methanol-regulated alcohol oxidase 1 (AOX1) promoter for recombinant protein production has directed studies particularly about methanol metabolism in this yeast. Although there is comprehensive knowledge about methanol metabolism, there are other mechanisms in P. pastoris that have not been investigated yet, such as ethanol metabolism. The gene responsible for the consumption of ethanol ADH2 (XM_002491337, known as ADH3) was identified and characterized in our previous study. In this study, the ADH genes (XM_002489969, XM_002491163, XM_002493969) in P. pastoris genome were investigated to determine their roles in ethanol production by gene disruption analysis. We report that the ADH900 (XM_002491163) is the main gene responsible for ethanol production in P. pastoris. The ADH2 gene, previously identified as the only gene responsible for ethanol consumption, also plays a minor role in ethanol production in the absence of the ADH900 gene. The investigation of the carbon source regulation mechanism has also revealed that the ADH2 gene exhibit similar expression behaviours with ADH900 on glucose, glycerol, and methanol, however, it is strongly induced by ethanol.  相似文献   

19.
Scheffersomyces stipitis PJH was mutagenized by random integrative mutagenesis and the integrants were screened for lacking the ability to grow with glutamate as sole carbon source. One of the two isolated mutants was damaged in the COX5 gene, which encodes a subunit of the cytochrome c oxidase. BLAST searches in the genome of Sc. stipitis revealed that only one singular COX5 gene exists in Sc. stipitis, in contrast to Saccharomyces cerevisiae, where two homologous genes are present. Mutant cells had lost the ability to grow with the amino acids glutamate, proline or aspartate and other non-fermentable carbon sources, such as acetic acid and ethanol, as sole carbon sources. Biomass formation of the mutant cells in medium containing glucose or xylose as carbon source was lower compared with the wild-type cells. However, yields and specific ethanol formation of the mutant were much higher, especially under conditions of higher aeration. The mutant cells lacked both cytochrome c oxidase activity and cyanide-sensitive respiration, whereas ADH and PDC activities were distinctly enhanced. SHAM-sensitive respiration was obviously essential for the fermentative metabolism, because SHAM completely abolished growth of the mutant cells with both glucose or xylose as carbon source.  相似文献   

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