Author/Editor		Petric, Š; Hakki, T; Bernhardt, R; Žigon, D; Črešnar, B
Title		Discovery of a steroid 11alpha-hydroxylase from Rhizopus oryzae and its biotechnological application
Type		članek
Source		J Biotechnol
Vol. and No.		Letnik 150, št. 3
Publication year		2010
Volume		str. 428-37
Language		eng
Abstract		To overcome the chemically laborious stereo- and regioselective hydroxylation steps in the pharmaceutical production of corticosteroids and progestogens, certain fungal species, e.g. Rhizopus spp. and Aspergillus spp., are employed to perform the 11alpha-hydroxylation of the steroid skeleton, thereby significantly simplifying steroid drug production. Here we report for the first time the identification and expression of a fungal 11alpha-steroid hydroxylase, CYP509C12. The newly identified cytochrome P450, which is one of the 48 putative CYP genes in Rhizopus oryzae, was induced in the fungus by progesterone. By functionally expressing CYP509C12 in recombinant fission yeast, we were able to determine that its substrate spectrum includes progesterone as well as testosterone, 11-deoxycorticosterone, and 11-deoxycortisol, with the hydroxylations taking place predominantly at 11alpha and 6beta positions of the steroid ring system. To increase the 11alpha-hydroxylation activity of CYP509C12 in recombinant fission yeast, its natural redox partner, the R. oryzae NAD(P)H-dependent reductase, was coexpressed. The coexpression improved electron transfer to CYP509C12 and thus an increase in productivity from 246 to 300muMhydroxyPgd(-1) was observed, as well as a 7-fold increase of rate of hydroxyprogesterone formation within the linear phase of transformation. This newly developed strain displayed total bioconversion of progesterone into 11alpha-hydroxyprogesterone and small amounts of 6beta-hydroxyprogesterone within the first 6h of incubation with progesterone as substrate, hence demonstrating its potential for biotechnological application.
Descriptors		RHIZOPUS STEROID HYDROXYLASES CYTOCHROME P-450 SCHIZOSACCHAROMYCES HYDROXYLATION ADRENAL CORTEX HORMONES PROGESTERONE HYDROXYPROGESTERONES SEQUENCE ANALYSIS PHYLOGENY POLYMERASE CHAIN REACTION OLIGONUCLEOTIDE PROBES DNA, COMPLEMENTARY GENE LIBRARY BIOTRANSFORMATION