Biosynthesis of fucoxanthin and diadinoxanthin and function of initial pathway genes in Phaeodactylum tricornutum

Abstract

The biosynthesis pathway to diadinoxanthin and fucoxanthin was elucidated in Phaeodactylum tricornutum by a combined approach involving metabolite analysis identification of gene function. For the initial steps leading to β-carotene, putative genes were selected from the genomic database and the function of several of them identified by genetic pathway complementation in Escherichia coli. They included genes encoding a phytoene synthase, a phytoene desaturase, a ζ-carotene desaturase, and a lycopene β-cyclase. Intermediates of the pathway beyond β-carotene, present in trace amounts, were separated by TLC and identified as violaxanthin and neoxanthin in the enriched fraction. Neoxanthin is a branching point for the synthesis of both diadinoxanthin and fucoxanthin and the mechanisms for their formation were proposed. A single isomerization of one of the allenic double bounds in neoxanthin yields diadinoxanhin. Two reactions, hydroxylation at C8 in combination with a keto-enol tautomerization and acetylation of the 3'-HO group results in the formation of fucoxanthin.

Fig. 1.

HPLC separation of carotenoids from Escherichia coli with different carotenoid backgrounds co-transformed with plasmids containing carotenogenic genes from Phaeodactylum tricornutum, as follows: (A) phytoene synthase gene Pt56881; (B) phytoene background and a phytoene desaturase gene Pt45735; (C) ζ-carotene background with a ζ-carotene desaturase gene Pt53794; (D) lycopene background with a lycopene β-cyclase gene Pt56484. Peak names: bC, β-carotene; gC, γ-carotene; L’, non-specified lycopene cis isomer; N, neurosporene; P, phytoene; proL, prolycopene (7,9,7′,9′-tetra-cis lycopene); tL, all-trans lycopene; Z, ζ-carotene.

Fig. 2.

Phylogenetic tree of the related Pds and Zds proteins. It includes the gene products from Phaeodactylum tricornutum including the non-active P. tricornutum Pds (indicated by X) together with the gene products from another diatom (Thalassiosira pseudodona), two red algae (Galdieria sulfuraria and Cyanidionschizon merolae), a brown alga (Ectocarpus siliculosus), and several green algae. The algal genes that have been functionally assigned are boxed. Bar, 0.1 substitutions per amino acid.

Fig. 3

. The carotenoid biosynthesis pathway in Phaeodactylum tricornutum from the synthesis of phytoene to β-carotene. The functionally assigned carotenogenic genes from P. tricornutum are arranged with their numbers from the genome sequence next to the reaction which is catalysed by their products. Lcy-b, lycopene β-cyclase; Pds, phytoene desaturaser; Psy, phytoene synthase; Zds, ζ-carotene desaturase. A gene for the isomerization of prolycopene to all-trans lycopene was not found.

Fig. 4

. Identification of carotenoids from Phaeodactylum tricornutum by HPLC in extracts (A) or enriched fraction (B) together with reference carotenoids (C). Peaks: 1, fucoxanthin;1′, a fucoxanthin cis isomer; 2, diadinoxanthin; 3, neoxanthin(N); 4, violaxanthin (V); 5, zeaxanthin (Z).

Fig. 5

. Proposed carotenoid biosynthesis pathway in Phaeodactylum tricornutum from β-carotene to the end products diadinoxanthin and fucoxanthin. Neoxanthin as the branch point is boxes. The mechanisms for the formation of an acetylenic bond, of allenic double bonds and the formation of the C8 keto group is indicated.