1887

Abstract

Dihydroxyacetone synthase (DHAS) is a key enzyme involved in the assimilation of methanol in sp. strain JC1 DSM 3803. The structural gene encoding DHAS in sp. strain JC1 was cloned using random-primed probes synthesized after PCR with synthetic primers based on the amino acid sequences conserved in two yeast DHASs and several transketolases. The cloned gene, , had an ORF of 2193 nt, encoding a protein with a calculated molecular mass of 78 197 Da. The deduced amino acid sequence of contained an internal sequence of sp. strain JC1 DHAS and exhibited 29.2 and 27.3 % identity with those of and enzymes, respectively. transformed with the cloned gene produced a novel protein with a molecular mass of ∼78 kDa, which cross-reacted with anti-DHAS antiserum and exhibited DHAS activity. Primer-extension analysis revealed that the transcriptional start site of the gene was the nucleotide A located 31 bp upstream from the start codon. RT-PCR showed that was transcribed as a monocistronic message. Northern hybridization and -galactosidase assay with the putative promoter region of revealed that the gene was transcribed only in cells growing on methanol. The expression of in sp. strain JC1 was free from catabolite repression.

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2007-12-01
2020-08-12
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