利用荧光蛋白研究产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因CgGPD启动子

[目的]克隆产甘油假丝酵母(Candida glycerinogenes)胞浆3-磷酸甘油脱氢酶基因CgGPD的启动子(PCggpd),并通过报告基因gfp的差异表达来研究葡萄糖浓度对PCggpd在酿酒酵母(Saccharomyces cerevisiae)中的诱导特性.[方法]采用PCR扩增的方法分别从产甘油假丝酵母基因组和pCAMBIA1302载体中克隆出CgGPD的启动序列PCggpd和绿色荧光蛋白基因gfp.将两个基因同时构建到酿酒酵母表达载体pYX212-zeocin中,构建时将绿色荧光蛋白基因gfp置于CgGPD的启动序列下游,获得重组质粒pYX212-zeocin-PCggpd-gfp.通过电击转化酿酒酵母W303-lA.将重组酿酒酵母S.cerevisiae W303-1A-GFP置于不同葡萄糖浓度培养基中进行培养,利用荧光显微技术对其进行荧光检测.[结果]重组酿酒酵母能产生稳定的荧光,当葡萄糖浓度为2%时,重组酿酒酵母在YEPD培养基中产生较弱的荧光,随着葡萄糖浓度的升高,荧光强度有明显的增强.[结论]PCggpd属于环境胁迫诱导型启动子,高浓度的葡萄糖能诱导PCggpd启动绿色荧光蛋白的高水平表达,这对完善产甘油假丝酵母的遗传背景研究,阐明其高产甘油的机理具有重要意义.     

产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因（CgGPD）功能分析

【目的】从高产甘油生产菌株产甘油假丝酵母（Candida glycerinogenes）基因组中克隆了NAD+依赖3-磷酸甘油脱氢酶编码基因（CgGPD）,但是该基因及其上游调控序列具体的功能还是未知的。本文研究了CgGPD基因及其上游调控序列的功能。【方法】本文以酿酒酵母（Saccharomyces cerevisiae）及其渗透压敏感型突变株为宿主,构建3种不同的酵母表达载体导入酵母细胞,研究了不同酵母转化子在渗透压胁迫条件下CgGPD基因表达对细胞的耐高渗透压胁迫应答及其细胞的甘油合成能力的影响。【结果】实验结果表明无论是以来源于S. cerevisiae 的TPI启动子还是来源于CgGPD基因的启动子,过量表达CgGPD基因的转化子均能够显著加速葡萄糖消耗速度和提高甘油合成能力,在gpd1/gpd2突变株中表达CgGPD基因能够消除细胞对外界高渗透压的敏感性,同时转化子胞内甘油大量积累。【结论】CgGPD基因在野生型酵母S. cerevisiae W303-1A表达显著提高细胞的甘油合成能力,在gpd/1gpd2突变株中能够互补GPD1基因的功能,CgGPD基因表达受渗透压诱导 调控。     

一步法产1,3-丙二醇酿酒酵母基因工程菌的构建

1,3-丙二醇(1,3-PD)是一种重要的化工原料,发酵法生产1,3-PD是一条新颖且具有潜在竞争力的生产途径。本研究在前期工作的基础上,将分别来源于大肠杆菌和肺炎克雷伯氏菌的基因片段yqhD和dhaB串联表达,构建重组表达载体pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;并得到重组酿酒酵母(Saccharomyces cerevisiae)W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB。该重组菌和对照S.cerevisiae分别以葡萄糖为底物摇瓶发酵72h后,重组酿酒酵母发酵液中1,3-PD含量约为1.5g/L;而对照菌株不产1,3-PD。以上结果表明本研究在国内首次成功构建了直接以葡萄糖为底物发酵生产1,3-PD的酿酒酵母基因工程菌。为进一步将dhaB、yqhD基因导入其他以葡萄糖为底物高产甘油的酵母宿主中表达,获得以葡萄糖为底物一步法发酵高产1,3-丙二醇工程菌打下了坚实的基础。     

产甘油假丝酵母甘油合成关键酶编码基因的克隆

产甘油假丝酵母(Candida glycerinogenes)作为优良的甘油生产菌株已经成功应用于工业化生产。但相对于酿酒酵母, 该菌株的耐高渗机理和甘油代谢的分子机制还不甚清楚。本文根据已公布的3-磷酸甘油脱氢酶基因的序列信息, 设计出一组寡核苷酸, 再运用简并PCR结合反向PCR技术从C. glycerinogenes的基因组DNA中获得了4 900 bp的核苷酸序列, 递交GenBank (No. EU186536)。该序列包含完整的编码胞浆3-磷酸甘油脱氢酶编码基因(CgGPD)开放阅读框及其上、下游调控序列。1 167 bp的开放阅读框编码388个氨基酸残基的蛋白。所演绎出氨基酸序列分析比对结果表明该基因产物的序列具有典型的胞浆3-磷酸甘油脱氢酶结构特征, 但与已鉴定的相关基因存在中等程度的同源性并在相应的辅酶催化位点和底物结合位点区域具有高度的保守性, 在氨基酸水平上与安格斯毕赤酵母的相似性最高, 达到70.9%。该基因在Saccharomyces cerevisiae W303A中异源表达能够显著提高细胞的甘油合成能力。     

产甘油假丝酵母甘油代谢关键酶的研究

本文对产甘油假丝酵母的甘油代谢关键酶进行了研究,发现产甘油假丝酵母同化甘油能力极弱,少量葡萄糖明显改善其同化甘油的能力;线粒体3磷酸甘油脱氢酶受3磷酸甘油的强烈诱导,受葡萄糖代谢的阻遏。在甘油发酵过程中,产甘油假丝酵母胞浆3磷酸甘油脱氢酶酶活处于较高水平并在36h和60h时出现两次酶活高峰,其中第一次酶活峰值水平决定产甘油假丝酵母的甘油合成和积累水平,成为甘油高速积累期(18～48h)甘油合成的关键性的限速酶。在甘油发酵18～48h内,3磷酸甘油酯酶的酶活处于高水平,并在36h时出现酶活峰值;处于缓慢甘油积累阶段的48～72h间,3磷酸甘油酯酶已处于低水平表达,此时,3磷酸甘油酯酶则成为甘油合成的限速酶。产甘油假丝酵母稳定并高表达其胞浆3磷酸甘油脱氢酶基因并且其所表达的3磷酸甘油酯酶酶活远高于胞浆3磷酸甘油脱氢酶这一特征是其高产甘油根本所在。     

酿酒酵母gpd1和hor2基因在大肠杆菌中的共表达

利用途径工程的方法,在大肠杆菌中构建一条新的产甘油的代谢途径。从酿酒酵母(Saccharomyces cerevisiae)克隆3-磷酸甘油脱氢酶基因(gpd1)和3-磷酸甘油酯酶基因(hor2),并将两个基因串连到启动子trc的下游,构建由trc启动子控制的能高效表达的多顺反子重组质粒pSE-gpd1-hor2,将重组质粒导入大肠杆菌BL21菌株中,构建得到的重组菌株GxB-gh能将葡萄糖转化为甘油。结果表明重组菌株GxB-gh以葡萄糖为底物进行发酵,甘油产量为46．67g／L,葡萄糖的转化率为42.87％。这为利用工程菌绿色生产甘油进行了前期的探索,也为进一步构建能生产1,3-丙二醇的工程菌打下了良好的基础。     

胆固醇7,8位脱氢酶的表达及催化活性研究

果蝇的neverland基因是胆固醇7,8位脱氢的重要酶基因。为了探究其在胆固醇脱氢反应中的催化机制,将neverland分别克隆至表达载体p IEx-6及p XY212,再转染导入S2细胞并电转化到S.cerevisiae W303-1A中表达。Western blot结果证实NVD蛋白在重组S2细胞及S.cerevisiae W303-1A中实现了表达。胆固醇转化实验经HPLC分析发现,重组S2细胞可以将胆固醇转化为7-脱氢胆固醇,而重组S.cerevisiae W303-1A并不能实现胆固醇的7,8位脱氢。此外,在重组S.cerevisiae W303-1A和S2细胞的破碎液共同转化胆固醇及NVD体外转化实验中也未发现产物7-脱氢胆固醇的生成。实验结果显示,neverland基因在S2细胞中具有生物活性而在S.cerevisiae中没有生物活性,表明它在胆固醇脱氢时需要其它的伴侣蛋白协助,实验结果为进一步研究其催化机制提供了理论基础。     

产甘油假丝酵母与酿酒酵母胞浆3-磷酸甘油脱氢酶基因的功能比较

胞浆3-磷酸甘油脱氢酶(GPD)是酿酒酵母细胞甘油合成过程中的关键限速酶．尽管高产甘油菌株产甘油假丝酵母基因组中编码该酶的基因CgGPD已经被克隆出来,但是具体的功能,特别是与酿酒酵母GPD1和GPD2基因的功能比较值得进一步研究．以酿酒酵母渗透压敏感型的gpd1/gpd2和gpd1突变株为宿主,分别导入CgGPD、GPD1和GPD2基因,比较分析了CgGPD、GPD1和GPD2基因在高渗透压胁迫条件下和厌氧环境中的表达调控,及其对细胞甘油合成能力的影响．研究发现,GPD1基因受到渗透压诱导表达,GPD2基因在细胞厌氧条件下起着氧化还原平衡调节作用,而CgGPD基因不仅能够在渗透压胁迫条件下通过过量快速合成甘油调节渗透压平衡,而且能够在厌氧培养环境中互补GPD2基因的缺失,使gpd1/gpd2缺失突变株能够正常生长,同时提高了突变株的甘油合成能力．结果表明,CgGPD基因在gpd1/gpd2缺失突变株中既具有GPD1基因的功能,又能发挥GPD2基因的功能．     

利用酿酒酵母稳定完整表达融合蛋白hPTH(1-34)ab-HSA

为稳定完整表达人甲状旁腺激素(hPTH)(1-34)二联体与人血清白蛋白(HSA)融合蛋白,以酿酒酵母为宿主,利用重叠PCR技术,将α-factor基因拼接到hPTH(1-34)ab-HSA基因片段上,获得α-hPTH(1-34)ab-HSA,并插入穿梭表达质粒pYX212,构建了基因工程菌S.cerevisiae W303-1A/pYX-α-hPTH (1-34)ab-HSA.经Western blot分析及N端氨基酸测序表明,该工程菌表达分泌了具有HSA和hPTH抗原性的完整hPTH(1-34)ab-HSA融合蛋白,解决了毕赤酵母不能稳定完整表达融合蛋白hPTH(1-34) ab-HSA的问题.     

新一代高产甘油重组菌的构建及其初步发酵

产甘油假丝酵母(Candida glycerinogenes WL2002-5)是一株发酵生产甘油的工业化菌株。为进一步提高其产甘油能力,本研究利用前期研究中成功克隆的产甘油假丝酵母中甘油合成关键酶3-磷酸甘油脱氢酶基因CgGPD1,构建根癌农杆菌双元载体pCAM3300-zeocin-CgGPD1后,电击转化根癌农杆菌LBA4404,通过根癌农杆菌介导法(ATMT)转化产甘油假丝酵母,构建了产甘油假丝酵母重组菌。并从中筛选出一株酶活力和产甘油性能较好的产甘油假丝酵母重组菌株C.g-G8。以葡萄糖为底物摇瓶发酵96h后,重组菌C.g-G8的甘油产量比野生型菌株Candida glycerinogene提高18.06%,平均耗糖速率提高12.97%,平均酶活力提高27.55%。本研究成功利用ATMT法转化产甘油假丝酵母构建新一代高产甘油菌株。     

(R)-、(S)-羰基还原酶在酿酒酵母中的表达和亚细胞定位

【目的】将增强型荧光蛋白标记的(R)-和(S)-羰基还原酶于酿酒酵母(Saccharomyces cerevisiae W303-1A)细胞中表达,分析荧光蛋白表达谱,确定两种酶在细胞中的功能分布和亚细胞定位。【方法】采用SOE-PCR法克隆出增强型荧光蛋白与(R)-和(S)-羰基还原酶的融合基因,构建到真核表达载体pYX212中,电击转化酵母细胞,以荧光蛋白为筛选标志,观察两种酶在酵母细胞中的表达和分布。【结果】激光扫描共聚焦显微观察表明(R)-和(S)-羰基还原酶多定位于细胞内膜和细胞质中稳定表达,少数成点状分布于细胞中央。根据荧光强度可知(S)-羰基还原酶的表达水平明显高于(R)-羰基还原酶。生物转化结果显示融合型(R)-和(S)-羰基还原酶催化底物2-羟基苯乙酮,分别获得(R)-和(S)-苯基乙二醇,前者产物的光学纯度和产率为86.6%和70.4%,后者产物的光学纯度和产率分别为92.3%和81.8%。【讨论】荧光蛋白与酶的融合没有改变靶蛋白的分子构象与生物活性,酿酒酵母工程菌较重组大肠杆菌具有更明显的生物功能优势,该研究为羰基还原酶蛋白的功能表达调控与亚细胞定位的可视化研究奠定了坚实的基础。     

Glycerol Export and Glycerol-3-phosphate Dehydrogenase, but Not Glycerol Phosphatase, Are Rate Limiting for Glycerol Production in Saccharomyces cerevisiae

Glycerol, one of the most important by-products of alcoholic fermentation, has positive effects on the sensory properties of fermented beverages. It was recently shown that the most direct approach for increasing glycerol formation is to overexpress GPD1, which encodes the glycerol-3-phosphate dehydrogenase (GPDH) isoform Gpd1p. We aimed to identify other steps in glycerol synthesis or transport that limit glycerol flux during glucose fermentation. We showed that the overexpression of GPD2, encoding the other isoform of glycerol-3-phosphate dehydrogenase (Gpd2p), is equally as effective as the overexpression of GPD1 in increasing glycerol production (3.3-fold increase compared to the wild-type strain) and has similar effects on yeast metabolism. In contrast, overexpression of GPP1, encoding glycerol 3-phosphatase (Gpp1p), did not enhance glycerol production. Strains that simultaneously overexpress GPD1 and GPP1 did not produce higher amounts of glycerol than a GPD1-overexpressing strain. These results demonstrate that GPDH, but not the glycerol 3-phosphatase, is rate-limiting for glycerol production. The channel protein Fps1p mediates glycerol export. It has recently been shown that mutants lacking a region in the N-terminal domain of Fps1p constitutively release glycerol. We showed that cells producing truncated Fps1p constructs during glucose fermentation compensate for glycerol loss by increasing glycerol production. Interestingly, the strain with a deregulated Fps1 glycerol channel had a different phenotype to the strain overexpressing GPD genes and showed poor growth during fermentation. Overexpression of GPD1 in this strain increased the amount of glycerol produced but led to a pronounced growth defect.     

Effects of deletion of glycerol-3-phosphate dehydrogenase and glutamate dehydrogenase genes on glycerol and ethanol metabolism in recombinant Saccharomyces cerevisiae

Bioethanol is currently used as an alternative fuel for gasoline worldwide. For economic production of bioethanol by Saccharomyces cerevisiae, formation of a main by-product, glycerol, should be prevented or minimized in order to reduce a separation cost of ethanol from fermentation broth. In this study, S. cerevisiae was engineered to investigate the effects of the sole and double disruption of NADH-dependent glycerol-3-phosphate dehydrogenase 1 (GPD1) and NADPH-requiring glutamate dehydrogenase 1 (GDH1) on the production of glycerol and ethanol from glucose. Even though sole deletion of GPD1 or GDH1 reduced glycerol production, double deletion of GPD1 and GDH1 resulted in the lowest glycerol concentration of 2.31 g/L, which was 46.4% lower than the wild-type strain. Interestingly, the recombinant S. cerevisiae ?GPD1?GDH1 strain showed a slight improvement in ethanol yield (0.414 g/g) compared with the wild-type strain (0.406 g/g). Genetic engineering of the glycerol and glutamate metabolic pathways modified NAD(P)H-requiring metabolic pathways and exerted a positive effect on glycerol reduction without affecting ethanol production.     

产甘油假丝酵母胞浆3-磷酸甘油脱氢酶编码基因的克隆

当酵母细胞处于高渗压环境时,甘油被诱导合成以提高其胞内渗透压,这一过程受ＨＯＧ途径的调控。ＧＰＤ１基因为ＨＯＧ途径的重要靶基因,高效表达使胞内３磷酸甘油脱氢酶酶活水平提高可极大地提高甘油的产量。本研究将产甘油假丝酵母（Ｃａｎｄｉｄａｇｌｙｃｅｒｏｌｏｇｅｎｅｓｉｓ）染色体ＤＮＡ经Ｓａｕ３ＡＩ部分酶解后的５～１０ｋｂＤＮＡ片段与经ＢａｍＨＩ线性化及ＣＩＰ处理过的酵母大肠杆菌穿梭质粒ＹＥｐ５１连接,以大肠杆菌ＤＨ５α为受体,构建产甘油假丝酵母的染色体基因文库。通过遗传互补法,在含５０ｇ／Ｌ氯化钠的培养基上筛选出１５个转化子,对转化子０６０１进行了进一步鉴定,转化子０６０１所含质粒ＹＥｐ０６０１带有ＹＥｐ５１的标记并可以消除Ｓａｃｃｂａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ６４２菌株由于其ＧＰＤ１,ＧＰＤ２两基因的缺失突变而表现出的渗透压敏感性,表明已克隆到产甘油假丝酵母的编码胞浆３磷酸甘油脱氢酶的基因     

Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance

To synthesize glycerol, a major by-product during anaerobic production of ethanol, the yeast Saccharomyces cerevisiae would consume up to 4% of the sugar feedstock in typical industrial ethanol processes. The present study was dedicated to decreasing the glycerol production mostly in industrial ethanol producing yeast without affecting its desirable fermentation properties including high osmotic and ethanol tolerance, natural robustness in industrial processes. In the present study, the GPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of S. cerevisiae, was deleted. Simultaneously, a non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus was expressed in the mutant deletion of GPD1. Although the resultant strain AG1A (gpd1△ P(PGK)-gapN) exhibited a 48.7±0.3% (relative to the amount of substrate consumed) lower glycerol yield and a 7.6±0.1% (relative to the amount of substrate consumed) higher ethanol yield compared to the wild-type strain, it was sensitive to osmotic stress and failed to ferment on 25% glucose. However, when trehalose synthesis genes TPS1 and TPS2 were over-expressed in the above recombinant strain AG1A, its high osmotic stress tolerance was not only restored but also improved. In addition, this new recombinant yeast strain displayed further reduced glycerol yield, indistinguishable maximum specific growth rate (μ(max)) and fermentation ability compared to the wild type in anaerobic batch fermentations. This study provides a promising strategy to improve ethanol yields by minimization of glycerol production.     

Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer.

The osmotolerant yeast Candida glycerinogenes produces glycerol as a major metabolite on an industrial scale, but the underlying molecular mechanisms are poorly understood. We cloned and characterized a 4900-bp genomic fragment containing the CgGPD gene encoding a glycerol-3-phosphate dehydrogenase homologous to GPD genes in other yeasts using degenerate primers in conjunction with inverse PCR. Sequence analysis revealed a 1167-bp open reading frame encoding a putative peptide of 388 deduced amino acids with a molecular mass of 42 695 Da. The CgGPD gene consisted of an N-terminal NAD(+)-binding domain and a central catalytic domain, whereas seven stress response elements were found in the upstream region. Functional analysis revealed that Saccharomyces cerevisiae gpd1Delta and gpd1Delta/gpd2Delta osmosensitive mutants transformed with CgGPD were restored to the wild-type phenotype when cultured in high osmolarity media, suggesting that it is a functional GPD protein. Transformants also accumulated glycerol intracellularly and GPD-specific activity increased significantly when stressed with NaCl, whereas the S. cerevisiae mutants transformed with the empty plasmid showed only slight increases. The full-length CgGPD gene sequence including upstream and downstream regions has been deposited in GenBank under accession no. EU186536.     

Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer

The osmotolerant yeast Candida glycerinogenes produces glycerol as a major metabolite on an industrial scale, but the underlying molecular mechanisms are poorly understood. We cloned and characterized a 4900-bp genomic fragment containing the CgGPD gene encoding a glycerol-3-phosphate dehydrogenase homologous to GPD genes in other yeasts using degenerate primers in conjunction with inverse PCR. Sequence analysis revealed a 1167-bp open reading frame encoding a putative peptide of 388 deduced amino acids with a molecular mass of 42 695 Da. The CgGPD gene consisted of an N-terminal NAD⁺-binding domain and a central catalytic domain, whereas seven stress response elements were found in the upstream region. Functional analysis revealed that Saccharomyces cerevisiae gpd1 Δ and gpd1 Δ/ gpd2 Δ osmosensitive mutants transformed with CgGPD were restored to the wild-type phenotype when cultured in high osmolarity media, suggesting that it is a functional GPD protein. Transformants also accumulated glycerol intracellularly and GPD-specific activity increased significantly when stressed with NaCl, whereas the S. cerevisiae mutants transformed with the empty plasmid showed only slight increases. The full-length CgGPD gene sequence including upstream and downstream regions has been deposited in GenBank under accession no. EU186536 .