Isolation and characterization of canthaxanthin biosynthesis genes from the photosynthetic bacterium Bradyrhizobium sp. strain ORS278

Abstract

A carotenoid biosynthesis gene cluster involved in canthaxanthin production was isolated from the photosynthetic Bradyrhizobium sp. strain ORS278. This cluster includes five genes identified as crtE, crtY, crtI, crtB, and crtW that are organized in at least two operons. The functional assignment of each open reading frame was confirmed by complementation studies.

FIG. 1

Scheme of the canthaxanthin (15) and spirilloxanthin (20) biosynthesis pathways.

FIG. 2

Organization of the canthaxanthin biosynthesis gene cluster of Bradyrhizobium sp. strain ORS278 and locations of various subcloned fragments. The restriction fragments are inserted into pUC18 (pSTM108, pSTM107, and pSTM51) or pUC19 (pSTM462), the crt genes are transcribed from the lac promoter of the vector. In the plasmid pSTM78, the insert was obtained by Long PCR using the primers Crt.canta.f (5′-GCAACCGGTACCCGAGTTAATTCGCTCGGAATG-3′) and Crt.canta.r (5′-ATGGTGAAGCTTATGCGGCAGCGGGTTTAGTC-3′) and was cloned into pGEM-T (Promega). In pSTM78, the crtY, crtI, crtB, and crtW genes are under lac promoter control.

FIG. 3

Comparison of the organization of the cyclic carotenoid gene clusters of Bradyrhizobium sp. strain ORS278, A. aurantiacum (15), Flavobacterium sp. strain R1534 (21), E. uredovora (14), and E. herbicola (9). Arrows represent the orientations of ORFs. The percentage values below the genes indicate the percentages of amino acid identity compared to Bradyrhizobium sp. strain ORS278.

FIG. 4

Phylogenetic tree based on the CrtB sequences and constructed by using the neighbor-joining method (22). Bootstrap values (5), expressed as percentages of 1,000 replications, are given at the branching points. P, Proteobacteria; F, Flavobacteria; a, bicyclic carotenoid; b, acyclic carotenoid; c, monocyclic carotenoid. GenBank accession numbers are as follows: AF218415, Bradyrhizobium sp. strain ORS278; D58420, A. aurantiacum; Y15112, Paracoccus marcusii; U62808, Flavobacterium sp.; M87280, E. herbicola EHO10; M90698, E. herbicola 557; D90087, E. uredovora; AF195122, R. sphaeroides; X52291, Rhodobacter capsulatus; U87626, Rubrivivax gelatinosus; Z211955, Myxococcus xanthus.