Functional analysis of structural genes for NAD⁺‐dependent formate dehydrogenase in Saccharomyces cerevisiae

Abstract

Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate. In this respect, this strain differed from two previously investigated S. cerevisiae strains, in which formate oxidation did not lead to an increased biomass yield on glucose. Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD + -dependent formate dehydrogenase.To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1D null mutant was constructed. This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate. The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S. cerevisiae CEN.PK 113-7D. FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S. cerevisiae genome databases. Sequence analysis confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D. In the latter strain background an fdh1Dfdh2D double mutant lacked formate dehydrogenase activity and was unable to co-consume formate. Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose-formate mixtures. These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate.

Multiple Pathways Are Co-regulated by the Protein Kinase Snf1 and the Transcription Factors Adr1 and Cat8

“…able to support yeast growth, can be co-metabolized with growth-limiting amounts of glucose to produce NADH, thus providing reducing equivalents that increase the growth potential of the cell (36,37). Table III lists the ADR1-dependent genes of known function, their degree of ADR1 dependence, and their biochemical or physiological function based on information available on the SGD or the MIPS web sites.…”

“…These two recently diverged ORFs are 94% identical and are not distinguishable by the array hybridization conditions. In some strains, including W303 studied here, FDH2 is inactive due to a frameshift mutation (36). The primary role of formate dehydrogenase in yeast metabolism is unknown, but formate can be co-metabolized with growth-limiting amounts of glucose to increase the yield of yeast biomass.…”

“…The primary role of formate dehydrogenase in yeast metabolism is unknown, but formate can be co-metabolized with growth-limiting amounts of glucose to increase the yield of yeast biomass. This is thought to result from oxidation of formate to CO 2 , which increases the amount of cytosolic NADH available for the production of ATP (36). A putative formate/ nitrite transporter encoded by YHL008C (Table III) is also expressed in an ADR1-dependent manner.…”

The ORF YNL274c (GOR1) codes for glyoxylate reductase in Saccharomyces cerevisiae

“…We used these known sequences as query sequences for a BLAST search against the genome of S. cerevisiae. The best homologues were the following seven ORFs: (a) YNL274c, which had been assigned as a potential hydroxyisocaproate dehydrogenase (Dickinson et al, 1997); (b) YER081w (SER3 ) and (c) YIL074c (SER33 ), identified as phosphoglycerate dehydrogenases (Albers et al, 2003); (d) YOR388c, shown to be a formate dehydrogenase, FDH1 (Overkamp et al, 2002) and (e) YPL275w/YPL276w, which together are highly homologous to YOR388c, but are separated by a stop codon in the genomic reference strain S288C, but not in some other strains, and thus have been denoted as FDH2 (Overkamp et al, 2002); (f) YPL113c and (g) YGL185c, which have similarity to hydroxyacid dehydrogenases. All these ORFs, except the two ORFs encoding FDH2 with a stop codon in the S288C strain (YPL275w/YPL276w) were investigated further.…”

Expression inEscherichia coliof an Unnamed Protein Gene fromAspergillus oryzaeRIB40 and Cofactor Analyses of the Gene Product as Formate Oxidase

“…There are many reports on characterization of FDH from various microorganisms and higher plants and on the cloning and expression of their genes. [1][2][3][4][5] On the other hand, only two FODs have been purified from Debaryomyces vanrijiae MH201 and Aspergillus nomius IRI013 and characterized. [6][7][8] These FODs show similar UV-visible spectra, suggesting the presence of similar cofactors in their molecules.…”