The Biosynthetic pathway for synechoxanthin, an aromatic carotenoid synthesized by the euryhaline, unicellular cyanobacterium Synechococcus sp. strain PCC 7002

Abstract

The euryhaline, unicellular cyanobacterium Synechococcus sp. strain PCC 7002 produces the dicyclic aromatic carotenoid synechoxanthin (chi,chi-caroten-18,18'-dioic acid) as a major pigment (>15% of total carotenoid) and when grown to stationary phase also accumulates small amounts of renierapurpurin (chi,chi-carotene) (J. E. Graham, J. T. J. Lecomte, and D. A. Bryant, J. Nat. Prod. 71:1647-1650, 2008). Two genes that were predicted to encode enzymes involved in the biosynthesis of synechoxanthin were identified by comparative genomics, and these genes were insertionally inactivated in Synechococcus sp. strain PCC 7002 to verify their function. The cruE gene (SYNPCC7002_A1248) encodes beta-carotene desaturase/methyltransferase, which converts beta-carotene to renierapurpurin. The cruH gene (SYNPCC7002_A2246) encodes an enzyme that is minimally responsible for the hydroxylation/oxidation of the C-18 and C-18' methyl groups of renierapurpurin. Based on observed and biochemically characterized intermediates, a complete pathway for synechoxanthin biosynthesis is proposed.

FIG. 1.

Neighbor-joining distance tree of aryl-carotenoid-forming β-ring carotene desaturase/methyltransferase enzymes, CrtU and CruE. The CrtP sequences of Arabidopsis thaliana and Synechocystis sp. strain PCC 6803 are included as an outgroup. The bold lines indicate biochemically or genetically characterized enzymes.

FIG. 2.

HPLC elution profiles for pigments for two cyanobacteria. (A) HPLC elution profile for pigments extracted from the wild-type (solid line) and cruE (dotted line) mutant strains of Synechococcus sp. strain PCC 7002. (B) HPLC elution profile for pigments extracted from wild-type (solid line) and sll0254/cruE (dotted line) mutant strains of Synechocystis sp. strain PCC 6803. s, synechoxanthin; m1, myxol-2′-fucoside; m2, myxol-2′-dimethylfucoside; z, zeaxanthin; he, 3′-hydroxy-echinenone; z-cis, cis-zeaxanthin; c, cryptoxanthin; e, echinenone; b, β-carotene.

FIG. 3.

Restriction maps and PCR verification of cruE mutants of two cyanobacteria. (A and C) Restriction maps showing construction of disruption mutations of the cruE gene of Synechococcus sp. strain PCC 7002 (A) and the sll0254/cruE gene of Synechocystis sp. strain PCC 6803 (C). (B and D) Agarose gel electrophoresis of PCR amplicons from the wild-type (WT) and cruE mutant strains of Synechococcus sp. strain PCC 7002 (B) and wild-type and sll0254/cruE mutant strains of Synechocystis sp. strain PCC 6803 (D). In panel B the PCR amplicons were both digested with SphI to demonstrate the difference, because the inserted drug resistance cartridge was nearly identical in size to the DNA fragment that had been deleted. The data show that both mutant strains are completely segregated. Size markers are indicated at the left of panels B and D, and selected sizes in kb are indicated.

FIG. 4.

HPLC elution profile of pigments extracted from stationary-phase cells of the wild-type (solid line) and cruE mutant (dotted line) strains of Synechococcus sp. strain PCC 7002. I1, I2, I3, and I4 indicate intermediates in the synthesis of synechoxanthin (s) (see text for details). r, renierapurpurin. The abbreviations used to identify other carotenoids are the same as in Fig. 2.

FIG. 5.

Absorption spectra of methanolic extracts of spent growth media recovered from stationary-phase cultures of the wild-type (solid line) and cruE mutant (dotted line) strains of Synechococcus sp. strain PCC 7002.

FIG. 6.

Restriction map and PCR verification of the cruH mutant of Synechococcus sp. strain PCC 7002. (A) Restriction map showing the construction of the cruH mutant of Synechococcus sp. strain PCC 7002. (B) Agarose gel electrophoresis of amplicons from the cruH locus for the wild type (WT) and a cruH::aadA mutant (lane 1). The data show that the cruH and cruH::aadA alleles are fully segregated in the mutant strain.

FIG. 7.

HPLC elution profile of pigments extracted from stationary-phase cells of the wild-type (solid line) and cruH mutant (dotted line) strains of Synechococcus sp. strain PCC 7002. s, synechoxanthin; r, renierapurpurin. The designations of other compounds is the same as for Fig. 2.

FIG. 8.

Proposed pathway for the conversion of β-carotene into synechoxanthin. The roles of CruE and CruH are indicated. For additional details, see the text. It should be noted that it is possible that CruH also carries out the hydroxylation/oxidation steps indicated by the two question marks.