title: 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.
abstract: 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 0.22g/L 17α,20βDiOH-P and 0.34g/L 20βOH-RSS after 8h using shake-flask cultivation, thus obtaining products that are at least a thousand times more expensive than their substrates.
authors.affiliation: Saarland University, Institute of Biochemistry, Campus B2.2, 66123 Saarbrücken, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Gerber
authors.affiliation: Saarland University, Institute of Biochemistry, Campus B2.2, 66123 Saarbrücken, Germany.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Milhim
authors.affiliation: Saarland University, Institute of Biochemistry, Campus B2.2, 66123 Saarbrücken, Germany.
authors.forename: Philip
authors.initials: P
authors.lastname: Hartz
authors.affiliation: Saarland University, Institute of Pharmaceutical Biology, Campus C2.3, 66123 Saarbrücken, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Zapp
authors.affiliation: Saarland University, Institute of Biochemistry, Campus B2.2, 66123 Saarbrücken, Germany. Electronic address: ritabern@mx.uni-saarland.de.
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000818:Animals; D001410:Bacillus megaterium / Q000502:physiology*; D053898:Biosynthetic Pathways / Q000502:physiology*; D005399:Fishes / Q000254:growth & development / Q000378:metabolism; D024861:Genetic Enhancement / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology*; D011372:Progestins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011372:Progestins; D011994:Recombinant Proteins
keywords: 17α,20β,21α-Trihydroxy-4-pregnen-3-one; 17α,20β-Dihydroxy-4-pregnen-3-one; Bacillus megaterium; Hydroxysteroid dehydrogenase; MIH; Steroid hormones