Cloning, characterization and expression of a gene encoding dihydroxyacetone synthase in Mycobacterium sp. strain JC1 DSM 3803

Abstract

Dihydroxyacetone synthase (DHAS) is a key enzyme involved in the assimilation of methanol in Mycobacterium sp. strain JC1 DSM 3803. The structural gene encoding DHAS in Mycobacterium 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, dasS, had an ORF of 2193 nt, encoding a protein with a calculated molecular mass of 78,197 Da. The deduced amino acid sequence of dasS contained an internal sequence of Mycobacterium sp. strain JC1 DHAS and exhibited 29.2 and 27.3 % identity with those of Candida boidinii and Hansenula polymorpha enzymes, respectively. Escherichia coli transformed with the cloned gene produced a novel protein with a molecular mass of approximately 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 dasS start codon. RT-PCR showed that dasS was transcribed as a monocistronic message. Northern hybridization and beta-galactosidase assay with the putative promoter region of dasS revealed that the gene was transcribed only in cells growing on methanol. The expression of dasS in Mycobacterium sp. strain JC1 was free from catabolite repression.