Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis

Abstract

The isolation and characterization of the phytoene synthase gene from the green microalga Chlorella zofingiensis (CzPSY), involved in the first step of the carotenoids biosynthetic pathway, have been performed. CzPSY gene encodes a polypeptide of 420 amino acids. A single copy of CzPSY has been found in C. zofingiensis by Southern blot analysis. Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to catalyze the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to form phytoene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the phytoene synthases (PSY) of the chlorophycean microalgae studied, being very closely related to PSY of plants. This new isolated gene has been adequately inserted in a vector and expressed in Chlamydomonas reinhardtii. The overexpression of CzPSY in C. reinhardtii, by nuclear transformation, has led to an increase in the corresponding CzPSY transcript level as well as in the content of the carotenoids violaxanthin and lutein which were 2.0- and 2.2-fold higher than in untransformed cells. This is an example of manipulation of the carotenogenic pathway in eukaryotic microalgae, which can open up the possibility of enhancing the productivity of commercial carotenoids by molecular engineering.

Fig. 1

Schematic diagram of the carotenoid biosynthetic pathway in plants and microalgae. Phytoene synthase (PSY) catalyses the first step in the carotenoid specific pathway, which leads the carbon flux towards carotenes and xantophylls production. IPP isopentenyl pyrophosphate, DMAPP dimethylallyl pyrophosphate, GGPP geranylgeranyl pyrophosphate, GGPPS geranylgeranyl pyrophosphate synthase, PDS phytoene desaturase, Z-ISO 15-cis-ζ-carotene isomerase, ZDS ζ-carotene desaturase, CRTISO carotene isomerase, LCYb lycopene β-cyclase, LCYe lycopene ε-cyclase, P450b-CHY cytochrome P450 β-hydroxylase, P450e-CHY cytochrome P450 ε-hydroxylase, CHYb carotene β-hydroxylase, BKT β-carotene oxygenase, ZEP zeaxanthin epoxidase, VDE violaxanthin de-epoxidase

Fig. 2

CzPSY gene organization. The diagram shows exons (I–IV) and introns (1–3) location. The 5′ UTR and 3′ UTR sequences are indicated with arrows. Numbers indicate cDNA sequence coordinates (bp)

Fig. 3

Southern blot analysis of genomic DNA from C. zofingiensis. a DNA was digested with HincII (lane 1) and HindIII (lane 2), electrophoretically separated on a 0.8% agarose gel, blotted and hybridized at high stringency with a probe of 752 bp of the PSY gene amplified by PCR. A plasmid containing the PSY gene was used as a positive control (lane 3). b HincII and HindIII restriction sites present in the CzPSY gene. The black bar indicates the probe location

Fig. 4

UPGMA tree analysis of the indicated plant, algal, cyanobacterial, and bacterial PSY amino acid sequences. Analysis was performed in MEGA4 (Tamura et al. 2007). The GenBank accession numbers for other species are as follows: Dunaliella salina, AAT46069; Dunaliella sp., ABE97388.1; Dunaliella bardawil, AAB51287.1; Haematococcus pluvialis, AAW28851.1; Chamydomonas reinhardtii, XP_001701192.1; Triticum turgidum, ACQ59152.1; Sorghum bicolor, ACY70869; Salicornia europaea, AAX19898.1; Solanum lycopersicum, ABM45873.1; Tagetes erecta, AAG10427.1; Synechocystis sp. PCC 6803, BAA17848.1; Anabaena variabilis PCC 29413, YP_325286.1; Nostoc sp. PCC 7120, BAB73532.1; Gloeobacter violaceus PCC 7421, NP_924690.1; Synechococcus sp. JA-3-3-Ab, YP_473801.1; Brevibacterium linens, AAF65581.1; Corynebacterium glutamicum, AAK64298.1. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances correspond to the number of amino acid substitutions per site and were computed using the Poisson correction method. Numbers at nodes indicate bootstrap values calculated over 500 replicates

Fig. 5

HPLC elution profile of carotenoids extracted from cultures of E. coli carrying plasmids pAC-85b+pQE-CzPSY. The absorption spectrum of β-carotene is also shown. E. coli BL21 (DE3) colonies transformed with the indicated plasmids were isolated in the presence of chloramphenicol + ampicillin. Peak identification, β-carotene

Fig. 6

Verification of the insertion of the plasmid pSI105-CzPSY in the genome of C. reinhardtii by PCR. C. reinhardtii cells transformed with plasmid pSI105-CzPSY were grown in TAP medium with paromomycin (30 μg mL⁻¹), and paromomycin-resistant colonies were tested by PCR. Line 6 is the 2-log DNA ladder (0.1–10 kb, Biolabs). Lines 1–5 and 7–11 are transformants analysed. Lines 12 and 13 are wild strain from C. zofingiensis and C. reinhardtii, respectively

Fig. 7

Carotenoids content (a) and mRNA relative abundance of endogenous CrPSY and foreign CzPSY (b) in cells of C. reinhardtii wild type (wt) and six selected transformants (T1, T2, T3, T4, T10, and T11). a Violaxanthin (white bar), lutein (light-grey bar), α-carotene (dark-grey bar), and β-carotene (black bar). b Levels of PSY transcripts were normalized respect to the housekeeping control gene (CBLP). Endogenous CrPSY (dark-grey bar); foreign CzPSY (light-grey bar). Error bars indicate the standard deviations of four independent measurements