Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum

Abstract

Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and β-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.

Keywords: PSY; carotenoid; marine diatom; microalgae; transformation.

Figure 1

Schematic representation of the MEP pathway and the carotenoid biosynthetic pathway in P. tricornutum. Modified from Bertrand [38], Coesel et al. [39], Dambek et al. [40], Hemmerlin [41], Lohr et al. [42], Mikami and Hosokawa [43] and Vranová et al. [44]. DXS: 1-deoxy-d-xylulose 5-phosphate synthase; DXR: 1-deoxy-d-xylulose 5-phosphate reductoisomerase; MCT: 2C-methyl-d-erythritol 4-phosphate cytidyltransferase; CMK: 4-diphosphocytidyl-2C-methyl-d-erythritol kinase; MDS: 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; HDS: 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; HDR: 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; IDI: isopentenyl diphosphate:dimethylallyl diphosphate isomerase; GGDS: geranylgeranyl diphosphate synthase; PSY: phytoene synthase; PDS: phytoene desaturase; ZDS: ζ-carotene desaturase; CRTISO: carotenoid isomerase; LCYB: lycopene β-cyclase; LUT-like: lutein deficient-like; ZEP: zeaxanthin epoxidase; VDE: violaxanthin de-epoxidase; VDL: violaxanthin de-epoxidase-like; NXS: neoxanthin synthase.

Figure 2

RNA-seq analysis of putative genes in the carotenoid biosynthetic pathway. FPKM: fragments per kilobase of exon per million mapped sequence reads; solid bar: FPKM of each gene; open bar: sum of FPKM of genes that are considered to belong to the same gene family; * the gene shows homology to the plant NXS gene, which was not available in our database of P. tricornutum strain NRIA-0065.

Figure 3

Sequence and structure of the PSY gene of P. tricornutum strain NRIA-0065. The amino acid sequence of PSY was analyzed by an NCBI-provided Conserved Domain Search. Highlighted in grey: homology to trans-isoprenyl diphosphate synthase; framed amino acids: aspartate rich regions and substrate-Mg²⁺-binding sites (DXXXD); open circle: substrate binding pocket; filled circle: catalytic residues; line: active site lid residues.

Figure 4

Structure of the transformation vector. Abbreviations used in this map: PtfcpA pro.: the promoter region of FCP gene A derived from P. tricornutum UTEX 646; Ptpsy: P. tricornutum PSY gene isolated from P. tricornutum NRIA-0065; egfp: eGFP gene; CffcpA ter.: the terminator region of FCP gene derived from Cyl. fusiformis; CffcpA pro.: the promoter region of FCP gene derived from Cyl. fusiformis; Sh ble: antibiotic Zeocin™ resistance gene isolated from a mutant strain of Streptomyces verticillus; (P-ble) denotes the destination vector produced from pCfcp-ble [15].

Figure 5

Genomic PCR analysis of the transformed P. tricornutum. (A) The illustration of transgenes. Arrows indicate the primers used for PCR determination; (B) typical electrophoretogram of genomic PCR determination of the P. tricornutum transformants. NC shows a negative control without template cells; M: DNA size maker; * indicate that transformants clones possessed the DNA fragment containing the Ptpsy::Gx5::egfp with the PtfcpA promoter (approximately 2700 base pairs); † indicates transformants clones used for the determination of total carotenoid content by the spectrophotometric analysis [64].

Figure 6

The relative abundances of the PSY mRNA in the wild-type (WT) cells and the transformants cells of P. tricornutum at the log and stationary phases determined by qRT-PCR. (A) The illustration of the introduced gene. Arrows indicate the primers used for PCR determination; (B) PCR determination of PSY mRNA expression. “Exogenous” indicates the abundance of the introduced PSY mRNA. “Total” indicates the total abundance of the intracellular PSY mRNA in transformants cells; “endogenous” indicates the abundance of the intracellular PSY mRNA in the wild-type cells; the data represent the average value of the three independent experiments with standard error; * above bars indicates a significant difference from the endogenous PSY mRNA abundance of the wild-type; ** above bars of the wild-type indicates a significant difference between the endogenous PSY mRNA abundance of the wild-type cells at the log phase and that at the stationary phase.

Figure 7

Chromatographic profiles of carotenoids in the wild-type cells and transformant (#2–12) cells of P. tricornutum at the log and stationary phases.

Figure 8

Carotenoid content in P. tricornutum in the log and stationary phases. The content of fucoxanthin per cells (A) and per mL culture medium (B). The content of β-carotene per cells (C) and per mL culture medium (D). The data represent the average values of the three independent experiments with standard error. * Above bars denotes significantly different from the carotenoid content of the wild-type (WT) cells; ** above bars of the wild-type denotes significantly different between the carotenoid content of the wild-type cells at the log phase and the stationary phase.

Figure 9

Microscopic image of wild-type and transformed P. tricornutum. Images of cells resting in the stationary phase are shown. Differential interference contrast (DIC) images, eGFP fluorescence images and endogenous chlorophyll fluorescence images are presented.