expression of a bacterial β-carotene hydroxylase in canthaxanthin producing mutant Mucor circinelloides strains

Xanthophylls, primarily hydroxyland keto-derivatives of β-carotene (such as zeaxanthin, β-cryptoxanthin, canthaxanthin, echinenone and astaxanthin) have powerful antioxidant activity. Due to several positive effects on human and animal health, industrial application of xanthophylls shows an increasing tendency. In our previous study, carotenoid biosynthesis of the β-carotene producing zygomycetes, Mucor circinelloides was modified by integrating the β-carotene ketolase encoding gene (crtW) of Paracoccus sp. N81106 into the genome. The isolated mutants accumulated mainly canthaxanthin and echinenone. Although, the fungus has β-carotene hydroxylase activity the astaxanthin production remained low even under different cultivation conditions, whereby canthaxanthin accumulation was further enhanced. In this study, the β-carotene hydroxylase encoding gene (crtZ) of the same bacterium was introduced into these canthaxanthin producing mutants of M. circinelloides either on autonomously replicating plasmids or by integrating it into the genome using the REMI method. Increased astaxanthin accumulation was observed in the transformants, which together with zeaxanthin and β-cryptoxanthin content could be further enhanced several fold by the modification of the cultivation conditions. An effective method for the extraction of carotenoids from Mucor mycelia grown in liquid culture was also developed. Acta Biol Szeged 58(2):139-146 (2014) Key WordS Paracoccus sp. N81106 β-carotene hydroxylase heterologous expression carotenoid extraction astaxanthin Accepted Dec 11, 2014 *Corresponding author. E-mail: [email protected] 139 Mucor circinelloides (Mucoromycotina) is an intensively studied carotenoid producing zygomycetes; well-developed transformation systems are available for the fungus and most of the genes involved in the carotenoid biosynthesis have been isolated and characterized (Velayos et al. 2000a, 2000b, 2003, 2004; Papp et al. 2006, 2010; Csernetics et al. 2011). Carotenoids are tetraterpene molecules and synthesized via the mevalonate – isoprenoid pathway in fungi. Condensation of two molecules of geranylgeranyl pyrophosphate, several dehydrogenation and two cyclisation steps lead to the formation of β-carotene in M. circinelloides (Fig. 1.), which is the major carotenoid compound in the fungus, but intermediates (such as lycopene and γ-carotene) are also accumulated (Velayos et al. 2000a, 2000b; Csernetics et al. 2011; Papp et al. 2013). Furthermore, M. circinelloides shows a weak β-carotene hydroxylase activity, therefore it is able to synthesize zeaxanthin (3,3′-dihydroxy-β,β-carotene) and β-cryptoxanthin (3-hydroxy-β,β-carotene) (Papp et al. 2006, 2013; Csernetics et al. 2011). At the same time, several microorganisms, such as Paracoccus sp. N81106 able to accumulate canthaxanthin (β,β-carotene-4,4’-dione), echinenone (β,β-carotene-4-on) and astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) as well; formation of astaxanthin from β-carotene requires a β-carotene hydroxylase and a ketolase activity (Misawa et al. 1995). These xanthophylls have powerful antioxidant properties and their preventive action against different types of cancer, age-related macular degeneration, heartor bone disease makes them dietary important (Chew et al. 1999; Nishino et al. 2002; Mares-Perlman et al. 2002; Guerin et al. 2003; Beatty et al. 2004; Yamaguchi 2004; Kumaresan et al. 2008). Besides, they are frequently used in pharmaceutical and cosmetic industry and also in feed additives (Bhosale and Bernstein 2005). Today, most of the commercially available xanthophylls are synthesized chemically. Biosynthesis of astaxanthin is limited to microorganisms and along with other carotenoids an increasing demand can be observed to replace chemical synthesis with natural sources (Bhosale and Bernstein 2005). In our previous studies, crtW and crtZ genes of the Paracoccus sp. N81106, encoding β-carotene ketolase and hydroxylase, respectively, were expressed in M. circinelloides