Genetic engineering of Bacillus megaterium for high-yield production of the major teleost progestogens 17α,20β-di- and 17α,20β,21α-trihydroxy-4-pregnen-3-one

17α,20β-Dihydroxy-4-pregnen-3-one (17α,20βDiOH-P) and 17α,20β,21α-trihydroxy-4-pregnen-3-one (20βOH-RSS) are the critical hormones required for oocyte maturation in fish. We utilized B. megaterium's endogenous 20β-hydroxysteroid dehydrogenase (20βHSD) for the efficient production of both progestogens after genetically modifying the microorganism to reduce side-product formation. First, the gene encoding the autologous cytochrome P450 CYP106A1 was deleted, resulting in a strain devoid of any steroid hydroxylation activity. Cultivation of this strain in the presence of 17α-hydroxyprogesterone (17αOH-P) led to the formation of 17α,20α-dihydroxy-4-pregnen-3-one (17α,20αDiOH-P) as a major and 17α,20βDiOH-P as a minor product. Four enzymes were identified as 20αHSDs and their genes deleted to yield a strain with no 20αHSD activity. The 3-oxoacyl-(acyl-carrier-protein) reductase FabG was found to exhibit 20βHSD-activity and overexpressed to create a biocatalyst yielding 0.22 g/L 17α,20βDiOH-P and 0.34 g/L 20βOH-RSS after 8 h using shake-flask cultivation, thus obtaining products that are at least a thousand times more expensive than their substrates.