耐高渗压是产甘油假丝酵母甘油高产的基础*

运用化学诱变法从产甘油假丝酵母获得１９株渗透压突变株。对其生理及发酵性能的研究结果显示,所有渗透压敏感突变株的甘油产率皆显著降低并巨大多数渗透压敏感突变株的糖利用速度减慢．耐渗透压性能提高突变株的糖利用率几乎与亲株相同而甘油发酵性能改变呈现多样性,突变株如ＷＬ－ＯｓｍＤ、ＷＬ－ＯｓｍＦ以及ＷＬ-ＯｓｍＨ甘油转化率极度降低而突变株ＷＬ－ＯｓｍＢ的甘油耗糖转化率接近亲株。     

原材料对甘油发酵影响的研究

以木薯干片,木薯淀粉,玉米淀粉的水解糖为原料,采用好氧深层发酵法进行甘油发酵。以木薯干片为原料时,甘油产率最高为１１－１２％,全糖转化率为４７－４９％,发酵周期为６０－６６ｈ;而以玉米淀粉为原料,甘油产率仅８－９％,全糖转化率３８－４０％,发酵周期却需７２小时以上     

耐高渗压高压甘油的一个假丝酵母新种：产甘油假丝酵母

从自然标本中分离获得一高产甘油的菌株ＷＬ２００２－５,仅发酵葡萄糖及微发酵蔗糖,能利用葡萄糖、蔗糖、乙醇生产、微利用甘油和柠檬酸,不利用肌醇,硝酸盐、与ＤＢＢ显色反应为阴性。     

甘油代谢中甘油激酶的研究进展

甘油作为重要的化工原料 ,其生产一直受到国内外的广泛关注。发酵法生产甘油是除皂化法和化学合成法外生产甘油的第三条途径。江南大学 (原无锡轻工大学 )在 90年代已成功地应用产甘油假丝酵母实现了工业化生产甘油 ,经江南大学研究人员多年不断努力 ,产甘油假丝酵母ＷＬ - 2 0 0 2 - 5发酵甘油可达 1 2 %以上 ,总糖转化率超过 5 1 % ,产率 3 0ｇ·Ｌ- 1ｄ- 1,发酵时间在 72～ 96ｈ。迄今为止 ,该菌株及专利技术已被多家企业运用于实际生产中[1,2 ] 。研究人员继续进行了多方面的研究 ,如运用穿梭载体建立产甘油假丝酵母质粒基因文库[3 ] 、…     

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。     

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．     

大肠杆菌甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)的敲除及其对甘油产量的影响

利用Red重组系统构建了大肠杆菌JM109甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)缺失的双突变菌株JM109B,然后将表达酿酒酵母3-磷酸甘油脱氢酶基因(GPD1)和3-磷酸甘油酯酶基因(HOR2)的质粒pSE-gpd1-hor2转化到JM109B突变菌株中,在含1%葡萄糖的摇瓶发酵培养基中37℃发酵24 h,甘油的最高产量为5.61 g/L,是原始菌株JM109/pSE-gpd1-hor2甘油产量的1.59倍;在30 L发酵罐中发酵28 h,甘油的最高产量为103.12 g/L,是原始菌株JM109/pSE-gpd1-hor2甘油产量的1.59倍,是原始菌株BL21/pSE-gpd1-hor2甘油产量的1.41倍,葡萄糖转化率为50.39%。     

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     

二酰甘油—蛋白激酶C信使系统在LA90细胞转化中的作用

以ＬＡ９０细胞（ＲＳＶ温度敏感突变株ＬＡ９０转化的小鼠在成纤维细胞）为模型,研究了二酰甘油（ＤＧ）－蛋白激酶Ｃ（ＰＫＣ）信使系统在细胞转化中的作用。通过免疫沉淀法观察到ＬＡ９０细胞允许温度（３３℃）时具有很高的ＰＰ０＾－ｓｒｏ激酶活性,远高于非允许温度（３９℃）,当从３９℃转至３３℃１０分钟,激酶活性就已显著升高。同时运用＾３Ｈ－甘油掺入并组合板层析分离方法和酶活性测定,发现ＬＡ９０细胞中ＤＧ含量     

渗透压在葡萄酒酵母代谢中的调控作用

以磷酸丙糖异构酶部分缺失突变株做对比,研究了渗透压对葡萄酒酵母发酵过程中甘油合成与挥发酸生成的调节作用.结果表明:渗透压对野生型葡萄酒酵母中存在的磷酸二羟丙酮(DHAP)与3-磷酸甘油醛(GA3P)平衡具有调节作用,能使平衡向磷酸二羟丙酮方向迁移以合成更多的甘油,而当磷酸丙糖异构酶部分缺失时渗透压对这一平衡基本不起作用...     

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 .     

Isolation and Characterization of a Mutant of a Methanol Yeast,Candida boidinii S2, with Higher Formaldehyde Productivity

We isolated a mutant strain of a methanol-utilizing yeast, Candida boidinii S2, which shows improved formaldehyde productivity. The procedure for mutant screening consisted of; 1) induction of alcohol oxidase on a methanol-plate, 2) catabolite inactivation of alcohol oxidase on a glucose-plate, and 3) visualization of alcohol oxidase activity in a colony. One of the mutants, strain AOU-1, showed 1.7 times higher formaldehyde productivity and a higher growth rate on methanol than the parent strain. The high formaldehyde productivity was proved to be due to the high alcohol oxidase activity. No qualitative change of the enzyme was detected between the parent strain and mutant strain AOU-1. The high activity of mutant strain AOU-1 could be attributed to a quantitative change and a change in the rate of enzyme synthesis. Catabolite repression and inactivation of alcohol oxidase in the mutant were also discussed.     

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.     

Proline over-production results in enhanced osmotolerance in Salmonella typhimurium

Summary Mutants of S. typhimurium with enhanced osmotolerance were isolated. These mutants were obtained as strains which over-produced proline due to regulatory mutations affecting proline biosynthesis. The mutations are located on FproBA and upon transfer to other S. typhimurium strains, they confer enhanced osmotolerance on the recipients. The osmotolerant mutants not only have higher intracellular proline levels than the osmosensitive parental strain, but the proline levels in the osmotolerant mutants are regulated such that they increase in response to osmotic stress. Possible reasons why elevated proline levels lead to enhanced osmotolerance are discussed.     

Physiological and genetic characterisation of osmosensitive mutants of Saccharomyes cerevisiae

The screening of 20,000 Saccharomyces cerevisiae random mutants to identify genes involved in the osmotic stress response yielded 14 mutants whose growth was poor in the presence of elevated concentrations of NaCl and glucose. Most of the mutant strains were more sensitive to NaCl than to glucose at the equivalent water activity (a_w) and were classified as salt-sensitive rather than osmosensitive. These mutants fell into 11 genetic complementation groups and were designated osr1–osr11 (osmotic stress response). All mutations were recessive and showed a clear 2⁺ : 2^– segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain. The complementation groups osr1, osr5 and osr11 were allelic to the genes PBS2, GPD1 and KAR3, respectively. Whereas intracellular and extracellular levels of glycerol increased in the wild-type strains when exposed to NaCl, all mutants demonstrated some increase in extracellular glycerol production upon salt stress, but a number of the mutants showed little or no increase in intracellular glycerol concentrations. The mutants had levels of glycerol-3-phosphate dehydrogenase, an enzyme induced by osmotic stress, either lower than or similar to those of the parent wild-type strain in the absence of osmotic stress. In the presence of NaCl, the increase in glycerol-3-phosphate dehydrogenase activity in the mutants did not match that of the parent wild-type strain. None of the mutants had defective ATPases or were sensitive to heat stress. It is evident from this study and from others that a wide spectrum of genes is involved in the osmotic stress response in S. cerevisiae. Received: 5 January 1998 / Accepted: 24 March 1998     

A <Emphasis Type="Italic">Candida guilliermondii</Emphasis> lysine hyperproducer capable of elevated citric acid production

A mutant of the yeast Candida guilliermondii ATCC 9058 exhibiting elevated citric acid production was isolated based upon its ability to overproduce lysine. This method involved the use of a solid medium containing a combination of lysine analogues to identify a mutant that produced a several-fold higher lysine level compared to its parent strain using glucose or glycerol as a carbon source. The mutant strain was also capable of producing more than a fivefold higher citric acid level on glycerol as a carbon source compared to its parent strain. It was concluded that the screening of yeast lysine hyperproducer strains could provide a rapid approach to isolate yeast citric acid hyperproducer strains.     

Erythritol production with minimum By-product using Candida magnoliae mutant

In order to enhance erythritol production, mutants of Candida magnoliae DSM70638 were generated by ultraviolet and chemical mutagenesis. Erythritol productivity of samples was analyzed by TLC and HPLC with the refractive index detector. One of the mutants named mutant 12-2 gave a 2.4-fold increase in erythritol (20.32 g/L) and a 5.5-fold decrease in glycerol production compared to the wild strain. A sequence-based map of erythrose reductase gene in this mutant showed a replacement of the A³²¹ by G³²¹ that did not cause any amino acid exchange in protein structure. Therefore, the reason of higher erythritol production in C. magnoliae mutant 12-2 is probably the increase in expression of the open reading frame gene. This study revealed that a mutation or minor change in the sequence of genes involved in a production pathway can lead to a significant increase in protein translation.     

1,3-Propanediol formation with product-tolerant mutants of Clostridium butyricum DSM 5431 in continuous culture: productivity, carbon and electron flow

Glycerol fermentation and product formation of two product-tolerant mutants of Clostridium butyricum DSM 5431 were investigated in continuous culture at increasing glycerol feed concentrations. Under conditions of glycerol excess (above 55 g l⁻¹ at D = 0·15 h⁻¹), the mutants maintained a constant level of glycerol consumption and product formation, whereas the parent strain exhibited a substantial decrease in substrate conversion, 1,3-propanediol and butyrate formation, and an increase in acetate formation. The activities of the glycerol dehydrogenase, the glycerol dehydratase and the 1,3-propanediol dehydrogenase showed only slight changes with glycerol concentrations in the mutants, but dropped markedly at high concentrations in the wild type. Intracellular concentrations of NADH, NAD ⁺ and acetyl-CoA remained at a relatively constant level in the mutants, but increased sharply with the wild type strain. The NADH content was always higher than the NAD ⁺ content in the mutants as well as in the wild type.     

A novel <Emphasis Type="Italic">Candida glycerinogenes</Emphasis> mutant with high glycerol productivity in high phosphate concentration medium

A novel Candida glycerinogenes mutant, which possesses high glycerol productivity in a high phosphate concentration medium, was obtained by mutagenesis of an industrial glycerol producer. The mutant accumulated a total biomass of 11.5 g l⁻¹, which is less than the 15 g l⁻¹of the wild-type strain, but it consumed glucose faster than the wild-type strain did. The mutant reached its maximal glycerol concentration of 129 g l⁻¹ in 84 h compared to 96 h for the wild-type strain. High cytoplasmic glycerol-3-phosphate dehydrogenase activity of the mutant in the early glycerol formation phase, leading to a rapid glycerol synthesis and accumulation, may be the main reason for the short fermentation process.