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、乙醇生长，微利用甘油和柠檬酸，不利用肌醇、硝酸盐、赤藓醇、阿拉伯醇、甘露醇，与DBB显色反应为阴性，可在含500g/L葡萄糖或100mL/L醋酸的培养基中40℃下生长，可在水活度为0.890-0.900的培养基中生长，出芽生殖，易形成“假丝酵母菌型”假菌丝，不进行有性生殖，线粒体DNA的分子量为20kb，是假丝酵母属的一个新种，定名为产甘油假丝酵母（Candida glycerol genesis Zhuge sp. nov.）。

利用PCR方法从运动发酵单孢菌染色体DNA扩增出乙醇合成途径的关键酶基因pdc、adhB，分别用tac启动子控制表达，构建了可以在Escherichia coli JMI 09中表达的重组质粒pKK-PA、pEtac-PA。初步的乙醇发酵结果表明，在 E. coli中只引入 adhB基因不能拓宽其中的产乙醇途径，引入pdc基因可以与宿主自身的ADH酶协同作用，使碳流有效导向产乙醇方向。同时引入pdc、adhB基因可以在宿主E. coli中成功建立产乙醇途径。

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。

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。GPDI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（Candida glycerolo-genesis）染色体DNA经Sau3 AI部分酶解后的5-10kbDNA片段与经Bam HI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DH5a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Saccbaro myces cervisiae642菌株由于其GPD1，GAPD2两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。

The tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (IDH) and the glyoxylate bypass enzyme isocitrate lyase are involved in catabolism of isocitrate and play a key role in controlling the metabolic flux between the TCA cycle and the glyoxylate shunt. Two IDH genes icd1 and icd2 of Ralstonia eutropha HF39, encoding isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), were identified and characterized. Icd1 was functionally expressed in Escherichia coli, whereas icd2 was expressed in E. coli but no activity was obtained. Interposon-mutants of icd1 (HF39vicd1) and icd2 (HF39vicd2) of R. eutropha HF39 were constructed and their phenotypes were investigated. HF39vicd1 retained 43% of IDH activity, which was not induced by a cetate, and HF39vicd2 expressed 74% of acetate-induced IDH activity. Both HF39vicd1and HF39vicd2 kept the same growth rate on acetate as the wild-type. These data suggested that IDH1 is induced by acetate. The interposon-mutants HF39vicd1 and HF39vicd2 accumulated the same amount of poly(3-hydroxybutyric acid) as the wild-type.

The glyoxylate bypass genes aceA1 (isocitrate lyase 1, ICL1), aceA2 (isocitrate lyase 2, ICL2) and aceB1 (malate synthase, MS1) of Ralstonia eutropha HF39 were cloned, sequenced and functionally expressed in Escherichia coli. Interposon-mutants of all three genes (vaceA1, vaceA2 and vaceB1) were constructed, and the phenotypes of the respective mutants were investigated. Whereas R. eutropha HF39vaceA1 retained only 19% of ICL activity and failed to grow on acetate, R. eutropha HF39vaceA2 retained 84% of acetate-inducible ICL activity, and growth on acetate was not retarded. These data suggested that ICL1 is in contrast to ICL2 induced by acetate and specific for the glyoxylate cycle. R. eutropha HF39vaceB1 retained on acetate as well as on gluconate about 41-42% of MS activity and exhibited retarded growth on acetate, indicating the presence of a second hitherto not identified MS in R. eutropha HF39. Whereas in R. eutropha HF39vaceA1 and R. eutropha HF39vaceA2 the yields of poly(3-hydroxybutyric acid), using gluconate as carbon source, were significantly reduced, R. eutropha HF39vaceB1 accumulated the same amount of this polyester from gluconate as well as from acetate as R. eutropha HF39.

Candida glycerinogenes, an osmotolerant yeast isolated from a natural sample in an environment of high osmotic pressure, had a modest sugar-tolerance and an extremely high glycerol productivity. The optimum conditions for glycerol formation by C. glycerinogenes were a temperature of 29-33℃ and a pH of 4-6. The optimum medium for glycerol production consisted of 230-250g glucose/l, 2g urea/l and 5ml corn steep liquor/l (55-65mg phosphates/l); the pH was not adjusted. The highest yield of glycerol was 64.5% (w/w) based on consumed glucose from 240g glucose/l, and the highest concentration of glycerol was 137g/l from 260g glucose/l. These results were obtained by using a 30-l agitated fermentor under optimal fermentation conditions. In ten batch-fermentations carried out in a 50,000-l airlift fermentor, an average yield of glycerol of 50.67% (w/w) and an average glycerol concentration of 121.9g/l were obtained from an average 240.6g glucose/l.

The dihydroxyacetone pathway, an alternative pathway for the dissimilation of glycerol via reduction by glycerol dehydrogenase and subsequent phosphorylation by dihydroxyacetone (DHA) kinase, is activated in the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii during osmotic stress. In experiments aimed at investigating the physiological function of the DHA pathway in Z. rouxii, a typical osmotolerant yeast, we cloned and characterized a DAK gene encoding dihydroxyacetone kinase from Z. rouxii NRRL 2547. Sequence analysis revealed a 1761 bp open reading frame, encoding a peptide composed of 587 deduced amino acids with the predicted molecular weight of 61 664 Da. As the amino acid sequence was most closely homologous (68% identity) to the S. cerevisiae Dak1p, we named the gene and protein ZrDAK1 and ZrDak1p, respectively. A putative ATP binding site was also found but no consensus element associated with osmoregulation was found in the upstream region of the ZrDAK1 gene. The ZrDAK1 gene complemented a S. cerevisiae W303-1A dak1 dak2 strain by improving the growth of the mutant on 50mmol/l dihydroxyacetone and by increasing the tolerance to dihydroxyacetone in a medium containing 5% sodium chloride, suggesting that it is a functional homologue of the S. cerevisiae DAK1. However, expression of the ZrDAK1 gene in the S. cerevisiae dak1 dak2 strain had no significant effect on glycerol levels during osmotic stress. The ZrDAK1 sequence has been deposited in the public data bases under Accession No.AJ294719; regions upstream and downstream of ZrDAK1are deposited as Accession Nos AJ294739 and AJ294720, respectively. Copyright 2002 John Wiley & Sons, Ltd. Copyright