Abstract: Pseudomonas sp. M1 is able to mineralize highly hydrophobic and recalcitrant compounds such as benzene, phenol and their methylated/halogenated derivatives, as well as the backbone of several monoterpenes. The ability to use such spectrum of compounds as sole carbon source is, most probably, associated with a genetic background evolved under different environmental constraints. The outstanding performance of M1 strain regarding β-myrcene catabolism was elucidated in this work, with focus on the biocatalytical potential of β-myrcene-associated core-code, comprised in a 28-kb genomic island (GI), predicted to be organized in 8 transcriptional units.

Functional characterization of this locus with promoter-probes and analytical approaches validated the genetic organization predicted in silico and associated the β-myrcene-induced promoter activity to the production of β-myrcene derivatives. Notably, by using a whole-genome mutagenesis strategy, different genotypes of the 28-kb GI were generated, resulting in the identification of a novel putative β-myrcene hydroxylase, responsible for the initial oxidation of β-myrcene into myrcen-8-ol, and a sensor-like regulatory protein, whose inactivation abolished the myr+ trait of M1 cells.

Moreover, it was demonstrated that the range of monoterpene substrates of M1 enzymatic repertoire, besides β-myrcene, also includes other acyclic (e.g. β-linalool) and cyclic molecules (e.g. R-(+)-limonene and (-)-β-pinene). Our findings are the cornerstone for following metabolic engineering approaches and hint a major role of the 28-kb GI in the biotransformation of a broad monoterpene-backbone spectrum for its future biotechnological applications.

Keywords: Pseudomonas, Biotransformation, Monoterpenes, Genomic island, Promoter probes, β-myrcene hydroxylase