Carotenoid hydroxylase from Haematococcus pluvialis: cDNA sequence, regulation and functional complementation

Abstract

A cDNA homologous to β-carotene hydroxylase from Arabidopsis thaliana was isolated from the green alga Haematococcus pluvialis. The predicted amino acid sequence for this enzyme shows homology to the three known plant β-carotene hydroxylases from Arabidopsis thaliana and from Capsicum annuum (38% identity) and to prokaryote carotenoid hydroxylases (32–34% identities). Heterologous complementation using E. coli strains which were genetically engineered to produce carotenoids indicated that the H. pluvialis β-carotene hydroxylase was able to catalyse not only the conversion of β-carotene to zeaxanthin but also the conversion of canthaxanthin to astaxanthin. Furthermore, Northern blot analysis revealed increased β-carotene hydroxylase mRNA steady state levels after induction of astaxanthin biosynthesis. In accordance with the latter results, it is proposed that the carotenoid hydroxylase characterized in the present publication is involved in the biosynthesis of astaxanthin during cyst cell formation of H. pluvialis.

Introduction

In some green algae such as Dunaliella bardawil and Haematococcus pluvialis, accumulation of carotenoids is induced by various environmental stress factors including high light, high salinity and nutrient deprivation [1], [2]. Thus, large amounts of the keto carotenoid astaxanthin (3,3′-dihydroxy-β-carotene-4,4′-dione) are produced in the green alga Haematococcus pluvialis under unfavourable culture conditions such as nitrogen or phosphate deficiency and increased light intensities [3], [4]. This massive production of astaxanthin in H. pluvialis (up to 3% of the dry weight [4]) is normally but not necessarily accompanied by morphological changes of biflagellate vegetative cells into non-mobile cyst cells. In contrast to the situation in higher plants where the carotenoids are produced in chloroplasts and chromoplasts, the accumulation of astaxanthin in H. pluvialis seems to occur in the cytoplasm [5]. During this extraplastidic accumulation of astaxanthin, the general structure of the chloroplast remains intact as shown by electron microscopy. It is not clear at present whether astaxanthin (or the precursors for astaxanthin biosynthesis) are synthesized in the chloroplast and then transported to the cytoplasm or whether biosynthesis occurs both in the chloroplast and in the cytoplasm. The induction and regulation of astaxanthin biosynthesis in H. pluvialis has recently received considerable attention due to the increasing use of astaxanthin as a source for pigmentation for fish aquacultures and due to a putative function in cancer prevention and as free radical quencher [4].

Carotenoids are synthesized by all photosynthetic organisms. They play an important role as light harvesting pigments and protect the photosynthetic apparatus from photooxidative damage under excess light conditions (reviewed in [6]). The early steps of carotenoid biosynthesis which consist of the biosynthesis of phytoene from isoprenoid precursors as well as the desaturation of phytoene to lycopene, followed by two cyclization reactions converting lycopene into β-carotene have been extensively studied and the corresponding genes have been isolated from bacteria and plants [6], [7]. Several of the enzymes involved in xanthophyll biosynthesis, carotenoids modified with oxygen containing groups, have also been characterized, e.g., the β-carotene hydroxylase which converts β-carotene into β-cryptoxanthin and zeaxanthin from bacteria [8], [9], [10] and higher plants [11], [12]. Moreover, some genes encoding β-carotene ketolase which catalyses the conversion of β-carotene to canthaxanthin via echinenone have been isolated not only from bacteria but also from H. pluvialis [13], [14], [15]. In enzymatic studies both in vitro and in vivo in Escherichia coli using the Haematococcus β-carotene ketolase, it was shown that the latter enzyme essentially converted β-carotene into canthaxanthin [14], [16], [17], [18]. In contrast, the presence of hydroxy groups (β-cryptoxanthin and zeaxanthin) either reduced or prevented the formation of keto groups. These findings led to a putative biosynthetic pathway with the most favourable route to astaxanthin being via echinenone, canthaxanthin and adonirubin as outlined in Fig. 1. The same route has also been proposed previously after the detection of the latter intermediates following the inhibition of astaxanthin biosynthesis by diphenylamine [19]. Consequently, the existence of a carotenoid hydroxylase in H. pluvialis has been predicted which is capable of converting canthaxanthin into astaxanthin and which is active during the induction of astaxanthin biosynthesis in response to environmental stress situations [14].

In spite of many reports published on the accumulation of carotenoids in H. pluvialis in response to high light and unfavourable culture conditions, little research has been carried out on the molecular process of the regulation of astaxanthin biosynthesis. Only recently has the expression of two IPP isomerases as well as the expression of lycopene β-cyclase and β-carotene ketolase during the induction of astaxanthin biosynthesis by light been examined [20]. The lycopene cyclase did not show higher protein levels, whereas the mRNA steady state levels of β-carotene ketolase and of the IPP isomerases were upregulated in response to higher light intensities.

In the present publication, the isolation and characterization of a cDNA coding for a Haematococcus carotenoid hydroxylase is described. Heterologous complementation and astaxanthin production in E. coli as well as the increased mRNA steady state levels during cyst cell formation indicated that this carotenoid hydroxylase is involved in the biosynthesis of astaxanthin in H. pluvialis.