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