Green diatom mutants reveal an intricate biosynthetic pathway of fucoxanthin

Abstract

Fucoxanthin is a major light-harvesting pigment in ecologically important algae such as diatoms, haptophytes, and brown algae (Phaeophyceae). Therefore, it is a major driver of global primary productivity. Species of these algal groups are brown colored because the high amounts of fucoxanthin bound to the proteins of their photosynthetic machineries enable efficient absorption of green light. While the structure of these fucoxanthin-chlorophyll proteins has recently been resolved, the biosynthetic pathway of fucoxanthin is still unknown. Here, we identified two enzymes central to this pathway by generating corresponding knockout mutants of the diatom Phaeodactylum tricornutum that are green due to the lack of fucoxanthin. Complementation of the mutants with the native genes or orthologs from haptophytes restored fucoxanthin biosynthesis. We propose a complete biosynthetic path to fucoxanthin in diatoms and haptophytes based on the carotenoid intermediates identified in the mutants and in vitro biochemical assays. It is substantially more complex than anticipated and reveals diadinoxanthin metabolism as the central regulatory hub connecting the photoprotective xanthophyll cycle and the formation of fucoxanthin. Moreover, our data show that the pathway evolved by repeated duplication and neofunctionalization of genes for the xanthophyll cycle enzymes violaxanthin de-epoxidase and zeaxanthin epoxidase. Brown algae lack diadinoxanthin and the genes described here and instead use an alternative pathway predicted to involve fewer enzymes. Our work represents a major step forward in elucidating the biosynthesis of fucoxanthin and understanding the evolution, biogenesis, and regulation of the photosynthetic machinery in algae.

Keywords: biosynthesis; diatoms; fucoxanthin; haptophytes; xanthophyll cycle.

Fig. 1.

Molecular characterization and pigment phenotypes of vdl2- and zep1-knockout (KO) mutants from P. tricornutum. (A and B) Schemes showing insertion sites of the 1.4-kB zeocin resistance cassette (Ble) in the target genes; PCR primer binding sites for differentiation of wild-type (VDL2/ZEP1) and mutants (VDL2/ZEP1 + Ble) indicated by blue open triangles, for detection of the inserted Ble genes (Ble + VDL2/ZEP1) by red filled triangles; photographs show cuvettes with cultures of wild-type, mutants, and mutants complemented with randomly integrated native genes, and agarose gels of PCR products from genomic DNA confirming biallelic KOs (product colors correspond to colors of primers in the top schemes; amplification of fragments of reintroduced native genes was strongly favored over genes with Ble insertion; DNA controls amplified a fragment of the plastome-encoded 23S rRNA gene). (C) HPLC traces of pigment extracts from wild-type and the KO mutants. (D) Chemical structures of the pigments that show compensatory accumulation in the mutants.

Fig. 2.

Proposed pathways of fucoxanthin biosynthesis in algae. The fucoxanthin biosynthetic pathway and pathway-specific enzymes in algae with diadinoxanthin cycle such as diatoms and haptophytes are labeled in brown, those in algae with violaxanthin cycle such as brown algae are labeled in khaki; question marks denote the steps catalyzed by unknown enzymes. Intermediates labeled in green accumulate in the respective KO mutants of the diatom P. tricornutum (see Fig. 1C). Note that the acetylation steps (Ac) in both pathways are predicted to occur on opposite rings (A or B) of their last common precursor neoxanthin. Dashed red arrows denote high light-induced xanthophyll cycle activity, dotted blue arrows denote back reactions catalyzed in low light. VDE, violaxanthin de-epoxidase; VDL, violaxanthin de-epoxidase-like; ZEP, zeaxanthin epoxidase.

Fig. 3.

The VDL protein from the cryptophyte G. theta (GtVDL) is closely related to the VDL2 protein from P. tricornutum and other diatoms and catalyzes the isomerization of allenoxanthin and diadinoxanthin in vitro. (A) Maximum likelihood tree showing the close phylogenetic relation of GtVDL (red asterisk) with VDL1 and VDL2 from diatoms and haptophytes (species names are color-coded according to their taxonomic affiliation: Viridiplantae, different shades of green; ochrophytes with Vx cycle, khaki; ochrophytes with fucoxanthin and Ddx cycle, light brown; Dinophyta, dark brown; Haptophyta, magenta; Cryptophyta, blue; bootstrap support (100 replicates) for nodes indicated by black dots if higher than 90%, by white dots if higher than 75%. (B) Reaction scheme showing saponification of haptoxanthin (Hpx) to allenoxanthin (Anx) by cleavage of the acetyl ester, followed by enzymatic isomerization of Anx to diadinoxanthin (Ddx) by GtVDL. (C) HPLC chromatograms of Hpx purified from the zep1 mutant of P. tricornutum (black trace) and of Anx resulting from saponification of Hpx (blue trace). Incubation of Anx for 2 h with recombinant GtVDL yielded a mixture of Anx and its tautomer Ddx (Anx + GtVDL, red trace); incubation of Ddx purified from wild type cells of P. tricornutum with GtVDL also resulted in a mixture of Anx and Ddx (Ddx + GtVDL, purple trace). The experiments were completed twice, with similar results.

Fig. 4.

ZEP1 from P. tricornutum catalyzes the 5,6-epoxidation of haptoxanthin to phaneroxanthin, and requires FAD as prosthetic group and NADPH and molecular oxygen as cosubstrates. (A) Reaction scheme showing the epoxidation of Hpx to phaneroxanthin (Phx) and some characteristic MS fragments of the latter. (B) In the in vitro assays, Hpx was incubated in reaction buffer for 2 h at room temperature with different additives, followed by pigment extraction and HPLC analysis. The resulting chromatograms showed no enzymatic activity when only NADPH and FAD (black trace), only ZEP1 (yellow), or ZEP1 and FAD (light orange) were added. Addition of ZEP1 and NADPH (dark orange) resulted in partial conversion of Hpx to Phx, indicating that a fraction of the enzyme prepared from E. coli already contained FAD produced by the bacteria. Addition of ZEP1, NADPH, and FAD (red trace) led to almost complete conversion. Lowering dissolved oxygen in the assay by addition of catalase, glucose oxidase and glucose (CGG) resulted in a reduced conversion (blue trace), indicating that molecular oxygen is the donor of the epoxy group in Phx. When this experiment was repeated with heat-inactivated glucose oxidase (CiGG), the conversion was almost complete again (purple trace). (C) APCI-MS scan (positive ion mode) of Phx resulting from in vitro epoxidation of Hpx by ZEP1(48 spectra averaged; calculated mass of 641.4201 for [M+H]⁺); the differences between expected and measured masses were below 2 ppm (below 0.001 U) for all fragment peaks analyzed). (D) Transient expression of ZEP1 in leaves of the ZEP-deficient aba2 mutant of Nicotiana plumbaginifolia resulted in the formation of antheraxanthin (Ax) and lutein epoxide (LutE); other pigments: Car, carotene; Chl, chlorophyll; Lut, lutein; Zx, zeaxanthin.

Fig. 5.

Comparative genomics of enzymes involved in fucoxanthin biosynthesis. (A) Occurrence of VDL2 and ZEP1 in publicly available sequence data from 194 species of photosynthetic eukaryotes (see Dataset S3 for species and sequence accessions). For each phylum/class, the number of examined species (in brackets), major light-harvesting xanthophyll(s) (Main Xan), and type of xanthophyll cycle (Xan cycle) are indicated. Branches indicate phylogenetic relations between taxa; dotted arrows in red and dashed arrows in blue indicate putative plastid acquisitions by endosymbioses. Allo, alloxanthin; cNx, cis-neoxanthin; Ddx, diadinoxanthin; IsoFx, isofucoxanthin; Lut, lutein; Peri, peridinin; Vaux, vaucheriaxanthin ester; Vx, violaxanthin; Zx, zeaxanthin. (B) Midpoint-rooted radial maximum-likelihood tree of the ZEP family from land plants and algae inferred from a protein alignment (324 amino acid positions) of 239 sequences from 121 species and using the same taxonomic color code as in (A) (sequences from Kareniaceae and dinotoms labeled with asterisks; see Dataset S4 for species and sequence accessions). Numbered circles indicate basal nodes of the main ZEP paralog clusters in ochrophytes and haptophytes. The paralogs from P. tricornutum (PtZEP1-3) and the haptophyte Prymnesium parvum (PpZEP1-5) and the single sequences from Arabidopsis thaliana (AtZEP) and the phaeophyte Ectocarpus siliculosus (EsZEP) are labeled. Bootstrap support (100 replicates) for major nodes indicated by black dots if higher than 90%, by white dots if higher than 75%.