SUMMARY: Transposon mutagenesis and antibiotic enrichment were employed to isolate a mutant of Si4 designated strain M22, that had lost the ability to grow on D-mannitol and to produce the enzyme mannitol dehydrogenase (MDH). DNA flanking the transposon in the mutant strain was used as a probe for the identification and cloning of the MDH gene . A 5.5 kb RI//II fragment from Si4 was isolated and shown to complement the mutation in M22. Successful complementation required that a promoter of the vector-plasmid pRK415 be present, suggesting that the gene is part of a larger operon. Using oligonucleotides derived from the N-terminal sequence of MDH as probes was located on the complementing fragment and the gene was sequenced. The open reading frame encodes a protein of 51 404 Da with an N-terminal sequence identical to that obtained from amino acid analysis of the purified MDH. The MDH of Si4 exhibits distant similarity to the mannitol-1-phosphate dehydrogenases from and , with 28.1% and 26.3% identity, respectively. Mutant strains deficient in MtlK displayed substantial levels of sorbitol dehydrogenase activity, originally thought to be only a minor activity associated with the MDH enzyme. It is likely that we have uncovered an additional polyol dehydrogenase with activity for sorbitol. The gene can be used for overexpression of MDH in in order to obtain sufficient amounts of enzyme for further investigations and applications.


Article metrics loading...

Loading full text...

Full text loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error