We have cloned and characterized a homologue of the previously isolated GPD1 gene, encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae. This second gene, called GPD2, encodes a protein of 384 amino acids that shares 69% sequence identity with GPD1. Like GPD1 it has an amino-terminal extension of unknown function. GPD2 is located on chromosome VII and cross-hybridizes with GPD1 at chromosome IV as well as with an unknown homologue at chromosome XV. Disruption of the GPD2 gene did not reveal any observable phenotypic effects, whereas overexpression resulted in a slight, but significant, increase of GPD enzyme activity in wild-type cells. Analysis of gene transcription by a CAT-reporter gene fused to the GPD promoters revealed decreased transcriptional activity of the GPD2 promoter in cells grown on nonfermentable as opposed to fermentable carbon sources, and no induction in cells exposed to high osmolarity or heat shock. Similar analysis of GPD1 demonstrated an 8-17-fold higher basal level of transcription compared to GPD2. Furthermore, such analysis revealed that the GPD1 promoter was induced by increased osmolarity essentially independent of the type of stress solute used, the level of GPD1 transcription being increased about sevenfold in cells growing at 1.4 M NaCl.

Production of glycerol and a key enzyme in glycerol production, glycerol 3-phosphate dehydrogenase (NAD+) (GPD), was studied in Saccharomyces cerevisiue cultured in basal media or media of high salinity, with glucose, raffinose or ethanol as the sole carbon source. At high salinity, glycerol production was stimulated with all carbon sources and glycerol was accumu!ated to high intracellular concentration in cells grown on glucose and raflinose. Cells grown on ethanol accumulated glycerol to a lower level but showed an increased content of trehalose at high salinity. Wowever, the trehalose concentration corresponded only to about 20% of the glycerol level, and did not compensate for the shortfall in intracellular osmolyte content. Immunoblot analysis demonstrated an increased production of GPD at high salinity. This increase was osmotically mediated but was lower when glycerol was substituted for NaCI or sorbitol as the stress-solute. The enzyme also appeared to be subject to glucose repression; the specific activity of GPD was signiticantly lower in cells grown on glucose, than on raffinose OP ethanol.

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The cDNA copies of the two endo-beta-1,4-glucanase genes, egl1 and egl3, from the filamentous fungus Trichoderma reesei were expressed in yeast Saccharomyces cerevisiae under the control of the yeast phosphoglycerate kinase gene promoter. Active EGI and EGIII enzyme was produced and secreted by yeast into the growth medium. The recombinant EGI enzyme was larger and more heterogeneous in size than the native enzyme secreted by Trichoderma, due to differences in the extent of N-glycosylation between these two organisms. The morphology of the yeast cells producing EGI or EGIII was clearly different from control strain.