The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3‐phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol‐3‐phosphatase were found when low and high glycerol‐forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose‐rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1Δ mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production (×4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by‐products such as pyruvate, acetate, acetoin, 2,3 butane‐diol and succinate was observed, with a marked increase in acetoin production. © 1997 John Wiley & Sons, Ltd.

Six commercial wine yeast strains and three nonindustrial strains (two laboratory strains and one haploid strain derived from a wine yeast strain) were engineered to produce large amounts of glycerol with a lower ethanol yield. Overexpression of the GPD1 gene, encoding a glycerol-3-phosphate dehydrogenase, resulted in a 1.5- to 2.5-fold increase in glycerol production and a slight decrease in ethanol formation under conditions simulating wine fermentation. All the strains overexpressing GPD1 produced a larger amount of succinate and acetate, with marked differences in the level of these compounds between industrial and nonindustrial engineered strains. Acetoin and 2,3-butanediol formation was enhanced with significant variation between strains and in relation to the level of glycerol produced. Wine strains overproducing glycerol at moderate levels (12 to 18 g/liter) reduced acetoin almost completely to 2,3-butanediol. A lower biomass concentration was attained byGPD1-overexpressing strains, probably due to high acetaldehyde production during the growth phase. Despite the reduction in cell numbers, complete sugar exhaustion was achieved during fermentation in a sugar-rich medium. Surprisingly, the engineered wine yeast strains exhibited a significant increase in the fermentation rate in the stationary phase, which reduced the time of fermentation.

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A new mesophilic strain of Methanothrix, strain FE, was highly purified from the sludge of an anaerobic digester after enrichment on sodium acetate and is described. Strain FE was compared with other strains of Methanothrix, Methanothrix soehngenii strain OpfikonT (= DSM 2139T) (T = type strain) and Methanothrix concilii strain GP6T (= DSM 3671T). The differences within the strains were mainly related to their requirement for yeast extract. The three strains were found to be similar in their deoxyribonucleic acid guanine-plus-cytosine contents (50.2 to 52.6 mol %) and showed 100 % deoxyribonucleic acid-deoxyribonucleic acid homology. For these reasons we propose to recognize synonymy, reject the name Methanothrix concilii Patel 1985, 35223, and assign this organism to the species Methanothrix soehngenii Huser, Wuhrmann and Zehnder 1983, 33: 439.Methanothrix soehngenii was first isolated by serial dilution from a continuous culture on acetate starting from anaerobically digested sludge in Switzerland (17, 40; B. A.Huser, thesis, Eidgenossischen Teschnischen Hochschule, Zurich, Switzerland, 1981). This bacterium was unique at that time among the methane bacteria because of its ability tlo split acetate into methane and carbon dioxide and its inability to use other methanogenic substrates, especially H,-C02. It was first described as a slow-growing bacterium with doubling times ranging from 5 to 8 days. This bacterium was positioned in the family Methanosarcinaceae by 16s ribosomal ribonucleic acid oligonucleotide sequencing (33). In 1984 (27), Patel described the isolation of Methanothrix concilii, which differed from Methanothrix soehngenii mainly by deoxyribonucleic acid (DNA) base composition and nutritional requirements. In this paper we characterize a new strain of Methanothrix, strain FE, and compare it with the two mesophilic type strains of the genus. MATERIALS AND METHODSSources of strains. Strain FE was initially purified from the sludge of an industrial mesophilic anaerobic contact digester treating wastewaters from the vegetable-canning industry (39). Methanothrix soehngenii strain OpfikonT (= DSM 2139T) (T = type strain) and Methanothrix concilii strain CiP6T (= DSM 3671T) were obtained from the Deutsche Slammlung von Mikroorganismen, Gottingen, West Germany. All of the strains used for immunological studies were also obtained from the Deutsche Sammlung von Mikroorganismen, except for Methanosarcina sp. strains CHTI55, MST-A1, and MC3, which were isolated in our laboratory, and Methanobrevibacter arboriphilicus AZ and Methanosarcina barkeri MST, which were gifts from J. G. Zeikus.Media and culture conditions. Cultivation of methanogens was done on BCYT medium as previously described (37). The medium was buffered with 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) or PIPES [piperazine-l,4-bis(2-ethanesulfonic acid)] unless otherwise indicated. The anaerobic gas was an N,-CO, mixture (85:15). * Corresponding author.The sterile media were inoculated with 2% (vol/vol) portions of previo...

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A gene encoding an extracellular lipase (CaLIP4) from Candida albicans was successfully expressed in Saccharomyces cerevisiae after mutagenesis of its unusual CUG serine codon into a universal one. The ability of this lipase, which shares 60% sequence homology with the lipase/acyltransferase from Candida parapsilosis, to synthesise esters was investigated. CaLIP4 behaved as a true lipase, displaying activity towards insoluble triglycerides and having no activity in the presence of short-chain fatty acid (FA) esters and phosphatidylcholine. Methyl, ethyl and propyl esters were efficiently used. The lipase exhibited highest selectivity for unsaturated FA. With saturated FAs, C14-C16 acyl chains were preferred. In a biphasic aqueous/lipid system, CaLIP4 displayed a high alcoholysis activity with a range of alcohols (e.g. methanol, ethanol, propanol and isopropanol) as acyl acceptor. During the course of the alcoholysis reaction, new esters are produced at concentrations above the thermodynamic equilibrium of the esterification reaction, indicating that ester synthesis does not proceed by esterification but mainly by direct acyltransfer. Ester synthesis is under kinetic control due to the high rate of alcoholysis. Unwanted hydrolysis is limited by competition between the acyl acceptor (alcohol) and water for the acyltransfer reaction, favouring the alcohol.