The biosynthetic pathway for myxol-2' fucoside (myxoxanthophyll) in the cyanobacterium Synechococcus sp. strain PCC 7002

Abstract

Synechococcus sp. strain PCC 7002 produces a variety of carotenoids, which comprise predominantly dicylic beta-carotene and two dicyclic xanthophylls, zeaxanthin and synechoxanthin. However, this cyanobacterium also produces a monocyclic myxoxanthophyll, which was identified as myxol-2' fucoside. Compared to the carotenoid glycosides produced by diverse microorganisms, cyanobacterial myxoxanthophyll and closely related compounds are unusual because they are glycosylated on the 2'-OH rather than on the 1'-OH position of the psi end of the molecule. In this study, the genes encoding two enzymes that modify the psi end of myxoxanthophyll in Synechococcus sp. strain PCC 7002 were identified. Mutational and biochemical studies showed that open reading frame SynPCC7002_A2032, renamed cruF, encodes a 1',2'-hydroxylase [corrected] and that open reading frame SynPCC7002_A2031, renamed cruG, encodes a 2'-O-glycosyltransferase. The enzymatic activity of CruF was verified by chemical characterization of the carotenoid products synthesized when cruF was expressed in a lycopene-producing strain of Escherichia coli. Database searches showed that homologs of cruF and cruG occur in the genomes of all sequenced cyanobacterial strains that are known to produce myxol or the acylic xanthophyll oscillaxanthin. The genomes of many other bacteria that produce hydroxylated carotenoids but do not contain crtC homologs also contain cruF orthologs. Based upon observable intermediates, a complete biosynthetic pathway for myxoxanthophyll is proposed. This study expands the suite of enzymes available for metabolic engineering of carotenoid biosynthetic pathways for biotechnological applications.

FIG. 1.

HPLC elution profiles for pigments from two cyanobacteria. (A) HPLC elution profiles (obtained by the jegpsu method) for pigments extracted from the wild type (solid line) and the slr1293 mutant (dotted line) of Synechocystis sp. strain PCC 6803. (B) HPLC elution profiles (obtained by the jegpsu method) for pigments extracted from the wild type (solid line) and SynPCC7002_A1623 mutant (dotted line) of Synechococcus sp. strain PCC 7002. Peak identities: s, synechoxanthin; md, myxol-2′ dimethylfucoside; mf, myxol-2′ fucoside; z, zeaxanthin; c, cryptoxanthin; e, echinenone; and b, β-carotene.

FIG. 2.

Neighbor-joining distance tree showing relationships among homologs of CruF found in cyanobacteria and other bacteria.

FIG. 3.

Restriction map and PCR verification of the cruF mutant of Synechococcus sp. strain PCC 7002. (A) Restriction map showing the construction of the cruF mutant of Synechococcus sp. strain PCC 7002. (B) Agarose gel electrophoresis analysis of amplicons from the cruF loci of the wild type (lane 1) and cruF::aacC1 mutant (lane 2). The data show that the cruF and cruF::aacC1 alleles in the mutant strain are fully segregated. Selected sizes of markers (lane M) in kilobases are indicated to the left.

FIG. 4.

HPLC elution profiles (obtained by the jegpsu method) of pigments extracted from cells of the wild type (solid line) and the cruF mutant (dotted line) of Synechococcus sp. strain PCC 7002. Peak identities: s, synechoxanthin; mf, myxol-2′ fucoside; z, zeaxanthin; he, hydroxy-echinenone; i3, 18-hydroxyrenierapurpurin; c, cryptoxanthin; e, echinenone; and b, β-carotene.

FIG. 5.

HPLC elution profiles (obtained by the Ecolicar method) of pigments extracted from E. coli BL21(DE3) strains harboring pAClyc, along with empty vector pET3atr (solid line) or plasmid pSLF (dotted line). Peak identities: L, lycopene; L2, 1-hydroxylycopene; and L3, 1,1′-dihydroxylycopene.

FIG. 6.

Restriction map and PCR verification of the cruG mutant of Synechococcus sp. strain PCC 7002. (A) Restriction map showing the construction of the cruG mutant of Synechococcus sp. strain PCC 7002. (B) Agarose gel electrophoresis analysis of amplicons from the cruG loci of the wild type (lane 1) and the cruG::aadA mutant (lane 2). The data show that the cruG and cruG::aadA alleles in the mutant strain are fully segregated. The sizes of selected marker fragments (lane M) in kilobases are indicated to the left.

FIG. 7.

HPLC elution profiles (obtained by the jegpsu method) of pigments extracted from cells of the wild type (solid line) and the cruG mutant (dotted line) of Synechococcus sp. strain PCC 7002. Peak identities: s, synechoxanthin; mf, myxol-2′ fucoside; mx, myxol; z, zeaxanthin; he, hydroxy-echinenone; z-cis, cis isomer of zeaxanthin; p, plectaniaxanthin; c, cryptoxanthin; e, echinenone; and b, β-carotene.

FIG. 8.

Proposed pathway for the biosynthesis of myxol-2′ fucoside. The roles of CruF, CruA, CrtR, and CruG are indicated. The order of cyclization and 1′ hydroxylation may be reversed (see Fig. S7 in the supplemental material). Compound identities: 1, lycopene; 2, 1-hydroxylycopene; 3, 1′-hydroxy-γ-carotene; 4, plectaniaxanthin; 5, myxol; and 6, myxol-2′ fucoside. The enzyme(s) for the introduction of the 3′,4′ double bond and the 2′-OH group is currently unknown.