Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides

Abstract

Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.

Genetic Modification of Mucor circinelloides for Canthaxanthin Production by Heterologous Expression of β-carotene Ketolase Gene

“…For example, high light requirement, slow growth at room temperature, as well as vulnerable to contamination by other algae (26). Over the past two decades, M. circinelloides has been successfully engineered for various useful metabolites by either overexpressing indigenous genes or by introducing the exogenous biosynthetic genes (11,(27)(28)(29)(30). In the present study, bkt gene from H. pluvialis was successfully overexpressed in M. circinelloides under the control of strong promoter zrt1 to construct the canthaxanthin biosynthesis pathway.…”

“…Csernetics et al also introduced two genes crtR and crtS from the yeast Xanthophyllomyces dendrorhous in M. circinelloides , which are responsible for the conversion of β-carotene to astaxanthin. After laborious genetic modification, a maximum of 190 μg/g of canthaxanthin was produced in a mutant strain of M. circinelloides ( 29 ). So, the higher production of canthaxanthin could be attributed to the higher catalytic efficiency of bkt from an algal source in comparison to its bacterial and yeast counterpart.…”

Identification and functional characterization of the CYP51 gene from the yeast Xanthophyllomyces dendrorhous that is involved in ergosterol biosynthesis

“…Recent findings suggested that astaxanthin supplementation could be beneficial as a treatment for several degenerative diseases in addition to other potential benefits for human health [ 6 - 9 ]. To date, X. dendrorhous is the only known organism that synthesizes astaxanthin from β-carotene through a cytochrome P450 enzyme system [ 10 - 13 ], suggesting that this yeast has developed a unique P450 system specialized for the production of this carotenoid.…”

Evidence for hybrid breakdown in production of red carotenoids in the marine invertebrate Tigriopus californicus

“…Complex I uses NADH as its primary electron donor, producing NAD+ as a result, while Complex II uses FADH as an electron donor, producing FAD+. It is possible that carotenoid ketolase enzymes, probably members of the Cytochrome P450 family or CrtS-like enzymes [ 18 , 77 ], also operate using NADH/NAD+ or NADPH/NADP+ systems [ 22 , 26 , 78 , 79 ]. Perhaps the stronger relationship between astaxanthin bioconversion and Complex I ATP production could be explained by a shared pool of electron donors [ 79 ].…”