鲁氏酵母3-磷酸甘油脱氢酶基因（ZrGPD1）在粉状毕赤酵母中的表达

为探索在野生型粉状毕赤酵母（Pichia farinosa）中整合表达来源于耐高渗鲁氏酵母（Zygosacharomyces rouxii）的3-磷酸甘油脱氢酶基因（ZrGPD1）以提高产甘油能力的可行性,应用PCR方法从P. farinosa的染色体中扩增出乳清苷酸脱羧酶基因（URA3）片段,以此作为同源整合的靶序列,构建了整合型表达载体pUR-ZG。电击转化粉状毕赤酵母,以抗生素Zeocin为筛选标记,获得转化子pfa-gu,摇瓶发酵结果表明：以P. farinosa作为对照菌株,发酵72h后,转化子pfa-gu的生物量和甘油含量均高于对照菌株,其中甘油含量达到37g/L,比对照提高了30%。结论：在P. farinosa中异源表达ZrGPD1能够提高细胞的产甘油能力和对渗透压的调节能力。     

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。     

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.     

Cloning and expression of the 3-isopropylmalate dehydrogenase gene from Candida albicans

Abstract A variety of Saccharomyces cerevisiae genes e.g. HIS3, LEU2, TRP1, URA3 , are expressed in Escherichia coli and have been isolated by complementation of mutations in the corresponding E. coli genes [1]. The LEU2 gene was one of the first S. cerevisiae genes to be isolated in this way [2], and its isolation led to the development of transformation systems for S. cerevisiae [3,4]. The leuB gene in E. coli [5] and the LEU2 gene in S. cerevisiae [6] both code for 3-isopropylmalate dehydrogenase (3-IMDH; EC 1.1.1.85) which is essential for the biosynthesis of leucine in both organisms. This paper describes the cloning of a fragment of C. albicans DNA carrying the gene for 3-IMDH which will be useful in the development of transformation methods in C. albicans .     

Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina

A cDNA encoding a nicotinamide adenine dinucleotide (NAD+) -dependent glycerol 3-phosphate dehydrogenase (GPDH) has been cloned by rapid amplification of cDNA ends from Dunaliella salina. The cDNA is 3032 base pairs long with an open reading frame encoding a polypeptide of 701 amino acids. The polypeptide shows high homology with published NAD+ -dependent GPDHs and has at its N-terminal a chloroplast targeting sequence. RNA gel blot analysis was performed to study GPDH gene expression under different conditions, and changes of the glycerol content were monitored. The results indicate that the cDNA may encode an osmoregulated isoform primarily involved in glycerol synthesis. The 701-amino-acid polypeptide is about 300 amino acids longer than previously reported plant NAD+ -dependent GPDHs. This 300-amino-acid fragment has a phosphoserine phosphatase domain. We suggest that the phosphoserine phosphatase domain functions as glycerol 3-phosphatase and that, consequently, NAD+ -dependent GPDH from D. salina can catalyze the step from dihydroxyacetone phosphate to glycerol directly. This is unique and a possible explanation for the fast glycerol synthesis found in D. salina.     

Overexpressing <Emphasis Type="Italic">GLT1</Emphasis> in <Emphasis Type="Italic">gpd1</Emphasis>Δ mutant to improve the production of ethanol of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant, KAM-4, the GPD1 gene, which encodes a glycerol 3-phosphate dehydrogenase of S. cerevisiae to synthesize glycerol, was deleted. The mutant KAM-12 had the GLT1 gene (encodes glutamate synthase) placed under the PGK1 promoter while harboring the GPD1 deletion. Notably, overexpression of GLT1 by the PGK1 promoter along with GPD1 deletion resulted in a 10.8% higher ethanol production and a 25.0% lower glycerol formation compared to the wild type in anaerobic fermentations. The growth rate of KAM-4 was slightly lower than that of the wild type under the exponential phase whereas KAM-12 and the wild type were indistinguishable in the biomass concentration at the end of growth period. Meanwhile, dramatic reduction of formation of acetate and pyruvic acid was observed in all the mutants compared to the wild type.     

产甘油假丝酵母产甘油关键酶基因在酿酒酵母中的表达

甘油是一种极其理想的耐高渗透压介质。利用PCR方法,从产甘油假丝酵母WL2002-5中扩增出了2个产甘油的关键酶基因GPD和GPP,分别编码3-磷酸甘油脱氢酶（glycerol 3-phosphate dehydrogenase, GPD）和3-磷酸甘油磷酸酶（glycerol 3-phosphate phosphatase, GPP）。利用T-Vector在Escherichia coli JM109中克隆得到大量的GPD和GPP基因,并成功构建了重组质粒pYX212-GPD和pYX212-GPP;通过LiAc转化法将重组质粒导入酿酒酵母Saccharomyces cerevisiae W303-1A。初步实验结果表明：发酵过程中pYX212-GPD/S. cerevisiae W303-1A的生物量高于pYX212-GPP/S. cerevisiae W303-1A和野生型S. cerevisiae W303-1A;发酵72h后,pYX212 GPD/S. cerevisiae W303-1A发酵液中甘油含量大约为12mmol/L,明显高于野生型S. cerevisiae W303-1A的甘油含量,而pYX212-GPP/S. cerevisiae W303-1A与野生型S. cerevisiae W303-1A在甘油含量上相差不大,均只有4mmol/L 左右。     

Engineering of the metabolism of Saccharomyces cerevisiae for anaerobic production of mannitol

Under anaerobic conditions, Saccharomyces cerevisiae uses NADH-dependent glycerol-3-phosphate dehydrogenase (Gpd1p and Gpd2p) to re-oxidize excess NADH, yielding substantial amounts of glycerol. In a Deltagpd1 Deltagpd2 double-null mutant, the necessary NAD+ regeneration through glycerol production is no longer possible, and this mutant does not grow under anaerobic conditions. The excess NADH formed can potentially be used to drive other NADH-dependent reactions or pathways. To investigate this possibility, a double-null mutant was transformed with a heterologous gene (mtlD) from Escherichia coli, coding for NADH-dependent mannitol-1-phosphate dehydrogenase. Expression of this gene in S. cerevisiae should result in NADH oxidation by the NADH-requiring formation of mannitol-1-phosphate from fructose-6-phosphate. The strain was characterized using step-change experiments, in which, during the exponential growth phase, the inlet gas was changed from air to nitrogen. It was found that the mutant produced mannitol only under anaerobic conditions. However, anaerobic growth was not regained, which was probably due to the excessive accumulation of mannitol in the cells.     

Distribution of glycerol dehydrogenase and glycerol kinase activity in halophilic archaea

Abstract A constitutive NAD⁺-dependent glycerol dehydrogenase activity was detected in Halobacterium salinarium and Halobacterium cutirubrum . Optimal activity was found at 3 M KCl and pH 8–10. No glycerol dehydrogenase activity could be demonstrated in representatives of the genera Haloferax and Haloarcula , even when grown in the presence of glycerol, or in Halobacterium saccharovorum and Halobacterium sodomense . Glycerol kinase activity was shown to be present constitutively in all halophilic archaea examined. The finding that glycerol dehydrogenase is found only in part of the halophilic arachaea makes dihydroxyacetone an improbable candidate as the precursor for the glycerol moiety of halobacterial lipids.     

Physiological and genetic engineering of cytosolic redox metabolism in Saccharomyces cerevisiae for improved glycerol production

Previous metabolic engineering strategies for improving glycerol production by Saccharomyces cerevisiae were constrained to a maximum theoretical glycerol yield of 1 mol.(molglucose)(-1) due to the introduction of rigid carbon, ATP or redox stoichiometries. In the present study, we sought to circumvent these constraints by (i) maintaining flexibility at fructose-1,6-bisphosphatase and triosephosphate isomerase, while (ii) eliminating reactions that compete with glycerol formation for cytosolic NADH and (iii) enabling oxidative catabolism within the mitochondrial matrix. In aerobic, glucose-grown batch cultures a S. cerevisiae strain, in which the pyruvate decarboxylases the external NADH dehydrogenases and the respiratory chain-linked glycerol-3-phosphate dehydrogenase were deleted for this purpose, produced glycerol at a yield of 0.90 mol.(molglucose)(-1). In aerobic glucose-limited chemostat cultures, the glycerol yield was ca. 25% lower, suggesting the involvement of an alternative glucose-sensitive mechanism for oxidation of cytosolic NADH. Nevertheless, in vivo generation of additional cytosolic NADH by co-feeding of formate to aerobic, glucose-limited chemostat cultures increased the glycerol yield on glucose to 1.08 mol mol(-1). To our knowledge, this is the highest glycerol yield reported for S. cerevisiae.     

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 .     

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.     

Enhanced glycerol 3-phosphate dehydrogenase activity in adipose tissue of obese humans

The primary purpose of this investigation was to determine whether adipose tissue glycerol 3-phosphate dehydrogenase activity is associated with human obesity. The data presented in this paper indicate that the glycerol 3-phosphate dehydrogenase activity in adipose tissue from morbidly obese subjects is approximately 2-fold higher than from lean individuals. Moreover, positive correlation between adipose tissue glycerol 3-phosphate dehydrogenase activity and body mass index (BMI) (r = 0.5; p < 0.01) was found. In contrast, the adipose tissue fatty acid synthase (FAS) and ATP-citrate lyase (ACL) activities in morbidly obese patients are significantly lower than in lean subjects. Furthermore, negative correlation between adipose tissue FAS activity and BMI (r = –0.3; p < 0.05) as well as between ACL activity and BMI (r = –0.3; p < 0.05) was found.These data indicate that elevated glycerol 3-phosphate dehydrogenase might contribute to the increase of triacylglycerol (TAG) synthesis in obese subjects, however, fatty acids necessary for glycerol 3-phosphate esterification must be derived (because of lower FAS and ACL activities) mainly from TAG in circulating lipoproteins formed in liver (VLDL), and/or from the intake with food (chylomicrons).The conclusion is, that the enhanced activity of glycerol 3-phosphate dehydrogenase, and hence the generation of more glycerol 3-phosphate in adipose tissue offers a novel explanation for increased TAG production in adipose tissue of obese subjects.     

Implications of FPS1 deletion and membrane ergosterol content for glycerol efflux from Saccharomyces cerevisiae

The deletion of the gene encoding the glycerol facilitator Fps1p was associated with an altered plasma membrane lipid composition in Saccharomyces cerevisiae. The S. cerevisiae fps1delta strain respectively contained 18 and 26% less ergosterol than the wild-type strain, at the whole-cell level and at the plasma membrane level. Other mutants with deficiencies in glycerol metabolism were studied to investigate any possible link between membrane ergosterol content and intracellular glycerol accumulation. In these mutants a modification in intracellular glycerol concentration, or in intra- to extracellular glycerol ratio was accompanied by a reduction in plasma membrane ergosterol content. However, there was no direct correlation between ergosterol content and intracellular glycerol concentration. Lipid composition influences the membrane permeability for solutes during adaptation of yeast cells to osmotic stress. In this study, ergosterol supplementation was shown to partially suppress the hypo-osmotic sensitivity phenotype of the fps1delta strain, leading to more efficient glycerol efflux, and improved survival. The erg-1 disruption mutant, which is unable to synthesise ergosterol, survived and recovered from the hypo-osmotic shock more successfully when the concentration of exogenously supplied ergosterol was increased. The results obtained suggest that a higher ergosterol content facilitates the flux of glycerol across the plasma membrane of S. cerevisiae cells.     

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.     

The Alcaligenes eutrophus ldh structural gene encodes a novel type of lactate dehydrogenase

Abstract The lactate dehydrogenase gene, ldh , of Alcaligenes eutrophus H16 was identified on a 14-kbp Eco RI restriction fragment of a genomic library in the cosmid pHC79 by hybridization with a 50-mer synthetic oligonucleotide which was derived from the N-terminal amino acid sequence of the purified enzyme. Recombinant strains of Escherichia coli JM83, which harboured a 2.0-kbp Pst I subfragment in pUC9-1, expressed LDH at a high level, if ldh was downstream from and colinear to the E. coli lac promoter. The nucleotide sequence of a region of 4245 bp revealed several open reading frames which might represent coding regions. One represented the ldh gene. The amino acid sequence deduced from ldh exhibited 29% and 36% identity to the L-malate dehydrogenase of Methanothermus fervidus and to the putative translation product of an E. coli sequence of unknown function, respectively. The ldh was separated by short intergenic regions from two other open reading frames: ORF5 was located downstream of and colinear to ldh , and its putative translational product revealed 38 to 56% amino acid identity to penicillin-binding proteins. ORF3 was located upstream of and colinear to ldh , and its putative gene translational product represented a hydrophobic protein. A sequence, which resembled the A. eutrophus alcohol dehydrogenase promoter, was detected upstream of ORF3, which most probably represents the first transcribed gene of an operon consisting of ORF3, ldh and ORF5.     

Ycf1p-dependent Hg(II) detoxification in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, disruption of the YCF1 gene increases the sensitivity of cell growth to mercury. Transformation of the resulting ycf1 null mutant with a plasmid harbouring YCF1 under the control of the GAL promoter largely restores the wild-type resistance to the metal ion. The protective effect of Ycf1p against the toxicity of mercury is especially pronounced when yeast cells are grown in rich medium or in minimal medium supplemented with glutathione. Secretory vesicles from S. cerevisiae cells overproducing Ycf1p are shown to exhibit ATP-dependent transport of bis(glutathionato)mercury. Moreover, using beta-galactosidase as a reporter protein, a relationship between mercury addition and the activity of the YCF1 promoter can be shown. Altogether, these observations indicate a defence mechanism involving an induction of the expression of Ycf1p and transport by this protein of mercury-glutathione adducts into the vacuole. Finally, possible coparticipation in mercury tolerance of other ABC proteins sharing close homology with Ycf1p was investigated. Gene disruption experiments enable us to conclude that neither Bpt1p, Yor1p, Ybt1p nor YHL035p plays a major role in the detoxification of mercury.