Transformation of the Green Alga Haematococcus pluvialis with a Phytoene Desaturase for Accelerated Astaxanthin Biosynthesis

Abstract

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to -carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.

Astaxanthin in microalgae: pathways, functions and biotechnological implications

“…Recently, a stable nuclear transformation system has been established in H. pluvialis (Steinbrenner and Sandmann 2006) and in C. zofingiensis . A modified version of PDS gene in H. pluvialis and C. zofingiensis has been developed as a dominant selectable marker and was transformed into these two species through biolistic transformation (Steinbrenner and Sandmann 2006, Liu et al 2009). Agrobacterium-mediated transformation has also been reported toexpress foreign genes in Haematococcus (Kathiresan et al 2009).…”

Genetic Engineering of Algae for Enhanced Biofuel Production

“…Members of the chlorophyte group that have been transformed include C. reinhardtii, which has been transformed using a variety of methods (44,87,88,93,170); Chlorella ellipsoidea (22, 83); Chlorella saccharophila (108); C. vulgaris (30, 69); Haematococcus pluvialis (177,187); V. carteri (81,163); Chlorella sorokiniana (30); Chlorella kessleri (49); Ulva lactuca (76); Dunaliella viridis (180); and D. salina (181,183). Heterokontophytes that have reportedly been transformed include Nannochloropsis oculata (21); diatoms such as T. pseudonana (147), P. tricornutum (2,210,211), Navicula saprophila (45), Cylindrotheca fusiformis (52,148), Cyclotella cryptica (45), and Thalassiosira weissflogii (51); and phaeophytes, such as Laminaria japonica (150) and Undaria pinnatifada (151).…”

Genetic Engineering for Microalgae Strain Improvement in Relation to Biocrude Production Systems

“…using a herbicide resistance cassette (Lapidot et al 2002) RNAi has also been used to engineer nuclear genes in the chlorophyte Dunaliella salina (Sun et al 2008) and in the diatom Phaeodactylum tricornutum (De Riso et al 2009). Applicable genetic modifications of green algae for industry are the transformation of Haematococcus pluvialis (Steinbrenner and Sandmann 2006;Teng et al 2002), an important Volvox carteri Hallmann and Rappel (1999) and Hallmann and Sumper (1994) Hallmann and Sumper (1996) and Jakobiak et al …”