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Abstract
11α-hydroxylated steroid synthons are one of the most important commercially pharmaceutical intermediates used for the production of contraceptive drugs and glucocorticoids. These compounds are currently produced by biotransformation using fungal strains in two sequential fermentation steps. In this work, we have developed by a rational design new recombinant bacteria able to produce 11α-hydroxylated synthons in a single fermentation step using cholesterol (CHO) or phytosterols (PHYTO) as feedstock. We have designed a synthetic operon expressing the 11α-hydroxylating enzymes from the fungus Rhizopus oryzae that was cloned into engineered mutant strains of Mycolicibacterium smegmatis that were previously created to produce 4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD) from sterols. The introduction of the fungal synthetic operon in these modified bacterial chassis has allowed producing for the first time 11αOH-AD and 11αOH-ADD with high yields directly from sterols in a single fermentation step. Remarkably, the enzymes of sterol catabolic pathway from M. smegmatis recognized the 11α-hydroxylated intermediates as alternative substrates and were able to efficiently funnel sterols to the desired hydroxylated end-products.
Highlights
Steroids represent one of the most widely used drugs for multiple clinical purposes (Fernandez-Cabezon et al, 2018)
We have developed several mutant strains of Mycolicibacterium smegmatis able to produce a la carte a large number steroid synthons (Galan et al, 2017; Garcıa et al, 2017)
The synthetic operon was cloned into the mycobacterial replicative plasmid pMV261, creating the pMVFUN plasmid, that was transformed into the M. smegmatis MS6039 mutant strain
Summary
Steroids represent one of the most widely used drugs for multiple clinical purposes (e.g. anti-inflammatory, immunosuppressive, anti-allergic, anti-cancer) (Fernandez-Cabezon et al, 2018). Fungi have been reported to carry out hydroxylations at almost all stereogenic centres of the steroid molecule (Kristan and Rizner, 2012), and they have been widely applied at industrial scale for the production of hydroxylated steroids with a broad range of biological activities (Donova, 2017). This oxyfunctionalization of steroids is mainly catalysed by cytochromes P450 (CYPs) acting as monooxygenases by inserting a single oxygen atom into a non-activated C–H bond of the substrate with a concomitant reduction of other oxygen atom to water (Bernhardt, 2006). Fungal hydroxylations are usually carried out by two-component enzyme systems consisting of a CYP monooxygenase and a NAD(P)H CYP-reductase (CPR) (Cresnar and Petric, 2011; Kristan and Rizner, 2012)
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