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Identification and Expression of the 11Β‐Steroid Hydroxylase From bs_bs_banner Identification and expression of the 11b-steroid hydroxylase from Cochliobolus lunatus in Corynebacterium glutamicum 1 1 Carmen Felpeto-Santero, Beatriz Galan, Jose Introduction M. Luengo,2 Jose M. Fernandez-Ca non,~ 2 Carlos del Cerro,1 Francisco J. Medrano3 and Jose L. Garcıa1,4* Microbial steroid transformation has been used for years 1Department of Environmental Biology, Centro de as a powerful tool to generate novel steroidal drugs and Investigaciones Biologicas, CSIC, Madrid, Spain . key synthons of pharmaceutical interest (Donova and 2Department of Molecular Biology, University of Leon, Egorova, 2012). Many of these microbial transformations Leon, Spain . are carried out by P450 cytochromes (CYP) (Bernhardt, 3Department of Chemical and Physical Biology, Centro 2006). The reactions carried out by CYPs are extremely de Investigaciones Biologicas, CSIC, Madrid, Spain . diverse and contribute to the biotransformation of drugs, 4Department of Applied Biotechnology, Institute for the bioconversion of xenobiotics and the biosynthesis of Integrative Systems Biology (I2SysBio), (Universidad de physiologically important compounds such as steroids Valencia-CSIC), Valencia, Spain . among others (Ortiz de Montellano and De Voss, 2002; Hannemann et al., 2006). Cytochromes are hemopro- teins encoded by a superfamily of genes nearly ubiqui- Summary tously distributed in many different organisms from all biological kingdoms but especially in fungi, where some Hydroxylation of steroids has acquired special rele- fungal genomes have over 150 CYPs, representing over vance for the pharmaceutical industries. Particularly, 1% of all genes (Doddapaneni et al., 2005). Although the 11b-hydroxylation of steroids is a reaction of fungal CYPs have been used for different steroid bio- biotechnological importance currently carried out at transformations, hydroxylation is one of the most studied industrial scale by the fungus Cochliobolus lunatus. and used processes at industrial scale (Zakelj-Mavric In this work, we have identified the genes encoding and Belic, 1987; Vita et al., 1994; Fernandes et al., the cytochrome CYP103168 and the reductase 2003; Kristan and Rizner, 2012). Fungal steroid hydroxy- CPR64795 of C. lunatus responsible for the 11b-hy- lation is usually carried out by two-component systems droxylase activity in this fungus, which is the key consisting of a NAD(P)H-cytochrome P450 reductase step for the preparative synthesis of cortisol in (CPR) and a cytochrome P450 monooxygenase which industry. A recombinant Corynebacterium glutam- are frequently attached to the membrane of the endo- icum strain harbouring a plasmid expressing both plasmic reticulum (Kristan and Rizner, 2012). Hydroxyla- genes forming a synthetic bacterial operon was able tion performed by fungi has acquired special relevance to 11b-hydroxylate several steroids as substrates. for the pharmaceutical industries. Among them, the This is a new example to show that the industrial hydroxyl group in position 11b of the cortisol molecule strain C. glutamicum can be used as a suitable chas- and its synthetic derivatives are the key functionalization sis to perform steroid biotransformation expressing that provides their glucocorticoid effects (Mahato and eukaryotic cytochromes. Garai, 1997; Schiffer et al., 2015). Currently, the 11b-hy- droxylation in the industrial production of corticosteroid precursors is catalysed by fungal cultures of Cochliobo- lus lunatus (Paraszkiewicz and Długon, 1998; Lu et al., 2007). Moreover, 11b-hydroxylation of progesterone is Received 18 February, 2019; revised 26 April, 2019; accepted 30 another reaction of biotechnological importance carried April, 2019. out by C. lunatus (anamorph Curvularia lunata) (Vita *For correspondence. E-mail [email protected]; et al., 1994). Although there are many data concerning Tel. +34 918373112; Fax +34 915360432. b Microbial Biotechnology (2019) 0(0), 1–13 the CYP encoding genes responsible for 11 -hydroxyla- doi:10.1111/1751-7915.13428 tion in mammals (Estabrook, 2005), so far, none of the Funding Information This work was supported by grants from the fungal genes encoding the CYPs having the 11b-hydrox- Ministry of Science and Innovation (BFU2006-15214-C03-01, fi BFU2009-11545-C03-03) and Ministry of Economy and ylating activity have been identi ed and cloned. Only the Competitiveness (BIO2012-39695-C02-01). 11b-hydroxylating CYP from C. lunatus has been ª 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 2 C. Felpeto-Santero et al. biochemically characterized (Zuidweg, 1968; Janig et al., In this work, we have identified the gene encoding the 1992; Suzuki et al., 1993), but all attempts to identify the CYP responsible for the 11b-hydroxylase activity of C. lunatus 11b-hydroxylase coding gene have failed C. lunatus among the 112 putative CYP encoding genes (Berne et al., 2008). The identification and cloning of this present in this fungus. The gene encoding its auxiliary gene are fundamental to develop more efficient biotrans- CPR was also identified. We have cloned and expressed formation processes since, apart from its limited regiose- both genes forming a synthetic operon in C. glutamicum lectivity, 11b-hydroxylation is generally accompanied by R31. The resulting recombinant strain was able to syn- 14a- and 7a-hydroxylations (Janig et al., 1992; Vita thesize 11b-hydroxysteroids from different steroid sub- et al., 1994), and the fungus produces other steroid strates. derivatives. The recombinant expression of these CYP systems in heterologous hosts appears as a promising Results alternative for the development of cleaner and more effi- In silico analyses of the CYPome and CPRome from cient introduction of the 11b-hydroxyl group. However, C. lunatus only few examples of steroid hydroxylation have been described using recombinant heterologous hosts. For The genes from C. lunatus that code for putative P450 example, Schizosaccharomyces pombe has been used cytochromes (CYPome) were extracted from its genome as host in the bioconversion of 11-deoxycorticosterone annotation at JGI, and 112 non-redundant putative CYP to aldosterone and deoxycorticosterone (DOC) to sequences were identified containing the Pfam PF00067 hydroxycortisone using the human mitochondrial cyto- and InterPro IPR002238 domains (Table S1). The 11b- chromes CYP11B2 and CYP11B1 respectively (Bureik hydroxylase was purified and biochemically character- et al., 2002; Drǎgan et al., 2005; Hakki et al., 2008). The ized proposing a molecular weight (MW) of 60 kDa for 11a-hydroxylase CYPs from Aspergillus ochraceus and the CYP protein (Zuidweg, 1968; Janig et al., 1992). Rhizopus oryzae together with their corresponding oxi- Therefore, the sequences in the CYPome rendering doreductases have been expressed in Spodoptera frugi- MWs lower than 50 kDa or higher than 70 kDa were perda (Sf-9) insect cells (Bolten et al., 2006) and in excluded as 11b-hydroxylase candidates. These criteria S. pombe (Bernhardt et al., 2009) respectively. Recently, eliminated 20 CYPs from the list (Table S1). The other the 11a-hydroxylase from the fungus Absidia coerulea 92 CYPs candidates were compared using local Blast has been identified and expressed in Pichia cells (Wang with 11a-hydroxylase CYP from A. ochraceus et al., 2017). Although the human 11a-or11b-hydroxy- (USP7033807). This comparison rendered eight putative lating CYPs have been expressed in bacteria (Durairaj CYPs with scores higher than 100, named CYP116182 et al., 2016), to the best of our knowledge, the fungal (score 335, 37% identity), CYP51519 (score 215, 29% 11a-or11b-hydroxylating CYPs have not been identity), CYP115117 (score 207, 27% identity), expressed in bacteria yet. CYP103168 (score 170, 26% identity), CYP135200 In this sense, we propose here to use the bacterium (score 152, 24% identity), CYP34615 (score 114, 24% Corynebacterium glutamicum as a host, because it been identity), CYP31052 (score 113, 23% identity) and widely used for industrial purposes, and the publication CYP56034 (score 105, 24% identity). of its complete genome, (Ikeda and Nakagawa, 2003; Analogously, the genes from C. lunatus encoding Kalinowski et al., 2003) has provided the basis for an putative CPRs (CPRome) were extracted from the gen- enormous progress in the use of this microorganism for ome annotation at JGI and five putative non-redundant other biotechnological applications placing it as an ideal CPR sequences were identified containing the PF00258 chassis for cell factories (De Lorenzo, 2015). Moreover, (flavodoxin domain), PF00667 (FAD-binding domain) we have demonstrated that a recombinant strain of and PF00175 (NAD binding domain) Pfam domains. C. glutamicum carrying the 3b-hydroxysteroid dehydro- Two of the selected sequences, i.e. CPR112637 and genase of Mycobacterium smegmatis was able to catal- CPR152579, were type III CYPs and were discarded. yse the biotransformation of short chain (C19 and C21) The three remaining CPRs, i.e. CPR59830, CPR64795 steroids indicating that these compounds are efficiently and CPR128465, were selected for further analysis. transported across the cytoplasmic membrane by C. glu- tamicum (Garcia-Fernandez et al., 2017). In addition, Identification of CYP and CPR transcripts induced by this work showed that C. glutamicum
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