Transformation of Mucor circinelloides with autoreplicative vectors containing homologous and heterologous ARS elements and the dominant Cbx(r) carboxine-resistance gene.

Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu(+) Cbx(r)) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbx(r)(carboxine-resistance) gene of Ustilago maydis and ARS sequences from this basidiomycete (plasmid pGG37) or from the 2 mu plasmid of Saccharomyces cerevisiae (plasmid pGG43). The presence in the same plasmid molecule of the M. circinelloides leuA gene and adjacent ARS element together with heterologous ARS elements produced an increase in the transformation frequency of about 65-120%. The presence of autoreplicating plasmid molecules in the transformants was demonstrated by mitotic stability experiments, by Southern analysis, and by the rescue of plasmids from transformed bacterial cells.

Molecular analysis of an NAD-dependent alcohol dehydrogenase from the zygomycete Mucor circinelloides.

NAD-dependent alcohol dehydrogenase (ADH) activity was detected mainly in the cytosol of aerobically cultured mycelium and in anaerobically grown yeast cells of Mucor circinelloides. ADH levels were about 2.5-fold higher in yeast cells than in mycelium; zymogram analysis suggested that the same ADH enzyme is produced in both developmental stages. The enzyme, named ADH1, was purified to homogeneity from yeast cells, using ion-exchange and affinity chromatography. The active ADH1 appears to be a homomeric tetramer of 37,500-kDa subunits. Km values obtained for acetaldehyde, ethanol, NADH and NAD+ indicated that in vivo the enzyme mainly serves to reduce acetaldehyde to ethanol. Amino acid sequences of internal peptides obtained from the purified ADH1 were used to design oligonucleotides that allowed the cloning of the corresponding cDNA by RT-PCR, and the characterization of the genomic DNA sequence. The adh1 ORF is interrupted by two small introns located towards the 5'-end. M. circinelloides adh1 encodes a protein of 348 amino acids, which display moderate to high overall identity to several hypothetical ADH enzymes from the related zygomycete Rhizopus oryzae. adh1 mRNA is expressed at similar levels in aerobic mycelium and anaerobic yeast cells. During exponential growth under aerobic conditions, the level of adh1 transcript was correlated with the glucose concentration in the growth medium.