Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae: I. Isolation and genetic analysis of adh mutants

On the basis of allyalcohol resistance, Saccharomyces cerevisiae mutanta were isolated that were deficient in alcohol dehydrogenase (ADH). The mutants were divided into three classes by their different ADH isozyme pattern obtained after starch-gel electrophoresis: adc mutants that did not produce the constitutive ADH, adr mutants from which the glucose repressible enzyme (ADHII) was absent, and adm mutants deficient in ADH activity associated with the mitochondria.Genetic analysis showed that two genes control synthesis of the glucose repressible enzyme ADHII, one gene the constitutive ADHI and a fourth nuclear gene the mitochondrial ADH. None of these four genes showed any linkage.The various mutant types did not show drastic effects on yeast growth on media containing glucose or ethanol as sole carbon sources.     

Cultivation of the hyperthermophilic archaeonSulfolobus solfataricus in low-salt media

Two low-salt complex media, bactopeptone and desalted yeast extract, were used for high density cultivation of the hyperthermophilic archaeonSulfolobus solfataricus (DSM 1617). Bactopeptone, which has low mineral ion content among various complex media, was good for cell growth in batch cultures; the maximal cell density in bactopeptone was comparable to that in yeast extract. Howver, cell growth was rather poor when bactopeptone was added by the fed-batch procedure. Since several vitamins are deficient in bactopeptone, the effect of vitamins on cell growth was examined. Among the vitamins tested, pyridoxine was found to improve the growth rate ofS. solfataricus. To reduce the growth inhibition caused by mineral ions, yeast extract was dialyzed against distilled water and then fed-batch cultures were carried out using a feed medium containing desalted yeast extract. Although the concentrations of mineral ions in yeast extract were significantly lowered by the dialysis procedure, fed-batch cultivation with desalted yeast extract was unsatisfactory. To examine whether low molecular weight solutes in yeast extract are crucial for cell growth, we investigated the effect of trehalose, a most abundant compatible solute in yeast extract, on the growth pattern. Cell densities were increased and the length of the lag phase was markedly shortened by the presence of trehalose, indicating that trehalose plays an important role in the growth ofS. solfataricus.     

Transformation of Rhodosporidium toruloides

Rhodosporidium toruloides protoplasts could be transformed, in the presence of polyethylene glycol (PEG), at frequencies of approx. 1 X 10(3) transformants/micrograms of DNA. The plasmid used, pHG2, which contains the phenylalanine ammonia-lyase (PAL)-coding gene (PAL) of R. toruloides, could replicate as an unstable plasmid in the yeast, or could integrate at the PAL locus to give stable transformants. Plasmids that function in R. toruloides were constructed using either the PAL gene or LEU2 gene of Saccharomyces cerevisiae as dominant selectable markers. R. toruloides transformed with pHG8, which contains both genes, coinherited the two markers. It is also shown that the 2mu replicon of S. cerevisiae does not function in R. toruloides; neither is the PAL gene expressed in S. cerevisiae.