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     

Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress.

The Saccharomyces cerevisiae FPS1 gene, which encodes a channel protein belonging to the MIP family, has been isolated previously as a multicopy suppressor of the growth defect of the fdp1 mutant (allelic to GGS1/TPS1) on fermentable sugars. Here we show that overexpression of FPS1 enhances glycerol production. Enhanced glycerol production caused by overexpression of GPD1 encoding glycerol-3-phosphate dehydrogenase also suppressed the growth defect of ggs1/tps1 delta mutants, suggesting a novel role for glycerol production in the control of glycolysis. The suppression of ggs1/tps1 delta mutants by GPD1 depends on the presence of Fps1. Mutants lacking Fps1 accumulate a greater part of the glycerol intracellularly, indicating that Fps1 is involved in glycerol efflux. Glycerol-uptake experiments showed that the permeability of the yeast plasma membrane for glycerol consists of an Fps1-independent component probably due to simple diffusion and of an Fps1-dependent component representing facilitated diffusion. The Escherichia coli glycerol facilitator expressed in a yeast fps1 delta mutant can restore the characteristics of glycerol uptake, production and distribution fully, but restores only partially growth of a ggs1/tps1 delta fps1 delta double mutant on glucose. Fps1 appears to be closed under hyperosmotic stress when survival depends on intracellular accumulation of glycerol and apparently opens rapidly when osmostress is lifted. The osmostress-induced High Osmolarity Glycerol (HOG) response pathway is not required for inactivation of Fps1. We conclude that Fps1 is a regulated yeast glycerol facilitator controlling glycerol production and cytosolic concentration, and might have additional functions.     

Conditional osmotic stress in yeast: a system to study transport through aquaglyceroporins and osmostress signaling

The accumulation and transport of solutes are hallmarks of osmoadaptation. In this study we have employed the inability of the Saccharomyces cerevisiae gpd1Delta gpd2Delta mutant both to produce glycerol and to adapt to high osmolarity to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling. High levels of different polyols, including glycerol, inhibited growth of the gpd1Delta gpd2Delta mutant. This growth inhibition was suppressed by expression of the hyperactive allele Fps1-Delta1 of the osmogated yeast aquaglyceroporin, Fps1. The degree of suppression correlated with the relative rate of transport of the different polyols tested. Transport studies in secretory vesicles confirmed that Fps1-Delta1 transports polyols at increased rates compared with wild type Fps1. Importantly, wild type Fps1 and Fps1-Delta1 showed similarly low permeability for water. The growth defect on polyols in the gpd1Delta gpd2Delta mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9. We surmised that this suppression was due to polyol influx, causing the cells to passively adapt to the stress. Indeed, when aquaglyceroporin-expressing gpd1Delta gpd2Delta mutants were treated with glycerol, xylitol, or sorbitol, the osmosensing HOG pathway was activated, and the period of activation correlated with the apparent rate of polyol uptake. This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation. Taken together, our "conditional" osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system.     

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.     

Osmoregulation of fission yeast: cloning of two distinct genes encoding glycerol-3-phosphate dehydrogenase, one of which is responsible for osmotolerance for growth

Many types of microorganisms, including both prokaryotes and eukaryotes, have developed mechanisms to adapt to severe osmotic stress. In this study, we isolated multicopy suppressor genes for a Schizosaccharomyces pombe mutant, which exhibited the clear phenotype of being osmosensitive for growth (Osm^s) on agar plates containing high concentrations of either non-ionic or ionic osmotic solutes. Two genes were thus identified, and each was suggested to encode an NADH-dependent glycerol-3-phosphate dehydrogenase (GPD), which is required for glycerol synthesis. The nucleotide sequences, determined for these genes (named gpd1 ⁺ and gpd2 ⁺, respectively), revealed that S. pombe has two distinct GPD isozymes. They are only 60% identical to each other in their amino acid sequences. One such isozyme, GPD1, was shown to be directly involved in osmoregulation, based on the following observations. (i) Expression of gpd1 ⁺ was regulated at the mRNA level in response to osmotic upshift, (ii) It was demonstrated that wild-type cells markedly accumulated internal glycerol under high-osmolarity growth conditions. (iii) Δ gpd1 mutants, however, failed to do so even in a high-osmolarity medium, and thus exhibited an Osm^s phenotype. On the other hand, the gpd2 ⁺ gene was constitutively expressed at a particular low level, regardless of the osmolarity of the medium.     

Hog1 mitogen-activated protein kinase plays conserved and distinct roles in the osmotolerant yeast Torulaspora delbrueckii

Torulaspora delbrueckii has emerged during evolution as one of the most osmotolerant yeasts. However, the molecular mechanisms underlying this unusual stress resistance are poorly understood. In this study, we have characterized the functional role of the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway in mediating the osmotic stress response, among others, in T. delbrueckii. We show that the T. delbrueckii Hog1p homologue TdHog1p is phosphorylated after cell transfer to NaCl- or sorbitol-containing medium. However, TdHog1p plays a minor role in tolerance to conditions of moderate osmotic stress, a trait related mainly with the osmotic balance. In consonance with this, the absence of TdHog1p produced only a weak defect in the timing of the osmostress-induced glycerol and GPD1 mRNA overaccumulation. Tdhog1Delta mutants also failed to display aberrant morphology changes in response to osmotic stress. Furthermore, our data indicate that the T. delbrueckii HOG pathway has evolved to respond to specific environmental conditions and to play a pivotal role in the stress cross-protection mechanism.     

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.     

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。     

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 .     

The mitogen-activated protein kinase homolog HOG1 gene controls glycerol accumulation in the pathogenic fungus Candida albicans.

The Candida albicans HOG1 gene (HOG1CA) was cloned by functional complementation of the osmosensitive phenotype associated with Saccharomyces cerevisiae hog1 delta mutants. HOG1CA codes for a 377-amino-acid protein, 78% identical to S. cerevisiae Hog1p. A C. albicans hog1 null mutant was found to be sensitive to osmotic stress and failed to accumulate glycerol on high-osmolarity media.     

Gpd1 and Gpd2 fine-tuning for sustainable reduction of glycerol formation in Saccharomyces cerevisiae

Gpd1 and Gpd2 are the two isoforms of glycerol 3-phosphate dehydrogenase (GPDH), which is the rate-controlling enzyme of glycerol formation in Saccharomyces cerevisiae. The two isoenzymes play crucial roles in osmoregulation and redox balancing. Past approaches to increase ethanol yield at the cost of reduced glycerol yield have most often been based on deletion of either one or two isogenes (GPD1 and GPD2). While single deletions of GPD1 or GPD2 reduced glycerol formation only slightly, the gpd1Δ gpd2Δ double deletion strain produced zero glycerol but showed an osmosensitive phenotype and abolished anaerobic growth. Our current approach has sought to generate "intermediate" phenotypes by reducing both isoenzyme activities without abolishing them. To this end, the GPD1 promoter was replaced in a gpd2Δ background by two lower-strength TEF1 promoter mutants. In the same manner, the activity of the GPD2 promoter was reduced in a gpd1Δ background. The resulting strains were crossed to obtain different combinations of residual GPD1 and GPD2 expression levels. Among our engineered strains we identified four candidates showing improved ethanol yields compared to the wild type. In contrast to a gpd1Δ gpd2Δ double-deletion strain, these strains were able to completely ferment the sugars under quasi-anaerobic conditions in both minimal medium and during simultaneous saccharification and fermentation (SSF) of liquefied wheat mash (wheat liquefact). This result implies that our strains can tolerate the ethanol concentration at the end of the wheat liquefact SSF (up to 90 g liter(-1)). Moreover, a few of these strains showed no significant reduction in osmotic stress tolerance compared to the wild type.     

The control of intracellular glycerol in Saccharomyces cerevisiae influences osmotic stress response and resistance to increased temperature

Glycerol has been demonstrated to serve as the major osmolyte of Saccharomyces cerevisiae. Consistently, mutant strains gpd1gpd2 and gpp1gpp2, which are devoid of the main glycerol biosynthesis pathway, have been shown to be osmosensitive. In addition, the primary hyperosmotic stress response is affected in these strains. Hog1p phosphorylation turned out to be prolonged and osmostress-induced gene expression is delayed compared with the kinetics observed in wild-type cells. A hog1 deletion strain was previously found to contain lower internal glycerol and therefore displays an osmosensitive phenotype. Here, we show that the osmosensitivity of hog1 is suppressed by growth at 37 degrees C. We reasoned that this temperature-remedial osmoresistance might be caused by a higher intracellular glycerol level at the elevated temperature. This hypothesis was confirmed by measurement of the glycerol concentration, which was shown to be similar for wild type and hog1 cells only at elevated growth temperatures. In agreement with this finding, hog1 cells containing an fps1 allele, encoding a constitutively open glycerol channel, have lost their temperature-remedial osmoresistance. Furthermore, gpd1gpd2 and gpp1gpp2 strains were found to be temperature sensitive. The growth defect of these strains could be suppressed by adding external glycerol. In conclusion, the ability to control glycerol levels influences proper osmostress-induced signalling and the cellular potential to grow at elevated temperatures. These data point to an important, as yet unidentified, role of glycerol in cellular functioning.     

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．     

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.     

Gene expression profiles and intracellular contents of stress protectants in Saccharomyces cerevisiae under ethanol and sorbitol stresses

In response to osmotic stress, proline is accumulated in many bacterial and plant cells. During various stresses, the yeast Saccharomyces cerevisiae induces glycerol or trehalose synthesis, but the fluctuations in gene expression and intracellular levels of proline in yeast are not yet well understood. We previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. In this study, we examined the relationships between the gene expression profiles and intracellular contents of glycerol, trehalose, and proline under stress conditions. When yeast cells were exposed to 1 M sorbitol stress, the expression of GPD1 encoding glycerol-3-phosphate dehydrogenase is induced, leading to glycerol accumulation. In contrast, in the presence of 9% ethanol, the rapid induction of TPS2 encoding trehalose-6-phosphate phosphatase resulted in trehalose accumulation. We found that intracellular proline levels did not increase immediately after addition of sorbitol or ethanol. However, the expressions of genes involved in proline synthesis and degradation did not change during exposure to these stresses. It appears that the elevated proline levels are due primarily to an increase in proline uptake from a nutrient medium caused by the induction of PUT4. These results suggest that S. cerevisiae cells do not accumulate proline in response to sorbitol or ethanol stress different from other organisms.