Identification and sequencing of beta-myrcene catabolism genes from Pseudomonas sp strain M1

The M1 strain, able to grow on beta-myrcene as the sole carbon and energy s ource, was isolated by an enrichment culture and identified as a Pseudomona s sp. One beta-myrcene-negative mutant, called N22, obtained by transposon mutagenesis, accumulated (E)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrc en-8-ol) as a unique beta-myrcene biotransformation product. This compound was identified by gas chromatography-mass spectrometry. We cloned and seque nced the DNA regions flanking the transposon and used these fragments to id entify the M1 genomic library clones containing the wild-type copy of the i nterrupted gene. One of the selected cosmids, containing a 22-kb genomic in sert, was able to complement the N22 mutant for growth on beta-myrcene. A 5 ,370-bp-long sequence spanning the region interrupted by the transposon in the mutant was determined. We identified four open reading frames, named my rA, myrB, myrC, and myrD, which can potentially code for an aldehyde dehydr ogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA hydratase, respectively. myrA, myrB, and myrC are likely orga nized in an operon, since they are separated by only 19 and 36 nucleotides (nt), respectively, and no promoter-like sequences have been found in these regions. The myrD gene starts 224 nt upstream of myrA and is divergently t ranscribed. The myrB sequence was found to be completely identical to the o ne flanking the transposon in the mutant. Therefore, we could ascertain tha t the transposon had been inserted inside the myrB gene, in complete agreem ent with the accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by t he mutant. Based on sequence and biotransformation data, we propose a pathw ay for beta-myrcene catabolism in Pseudomonas sp. strain M1.