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 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

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.