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 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.

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