Cloning, DNA sequencing and expression of (3–17)β hydroxysteroid dehydrogenase from Pseudomonas testosteroni

Engineering Mycobacterium smegmatis for testosterone production

“…As the genes encoding 17β‐HSD enzymes from mycobacterial species have not been identified and these proteins have been only partially purified and characterized (Goren et al ., 1983; Egorova et al ., 2002a, 2005), we initially selected as enzyme candidates for metabolic engineering the well‐described 17β‐HSDs from the bacterium C. testosteroni (Schultz et al ., 1977; Lefebvre et al .,1979; Minard et al ., 1985 ; Genti‐Raimondi et al ., 1991; Yin et al ., 1991; Abalain et al ., 1993; Benach et al ., 1996, 2002; Oppermann et al ., 1997; Cabrera et al ., 2000) and the fungus C. lunatus (Plemenitas et al ., 1988; Rižner et al ., 1996, 1999, 2000, 2001a,b; Rižner and Zakelj‐Mavric, 2000; Zorko et al ., 2000; Kristan et al ., 2003, 2005, 2007a,b; Cassetta et al ., 2005; Ulrih and Lanisnik Rižner, 2006; Brunskole et al ., 2009; Svegelj et al ., 2012), because both enzymes present some relevant differences. Although they catalyse a reversible reaction and display similar reaction mechanisms, the reaction equilibrium of the fungal 17β‐HSD is shifted towards reduction, whereas the bacterial enzyme is shifted towards oxidation, as this enzyme is mainly involved into the TS catabolism in C. testosteroni (Genti‐Raimondi et al., 1990; Cabrera et al ., 2000).…”

“…Although several microbial 17β‐HSD enzymes have been cloned and characterized (Abalain et al ., 1993; Rižner et al ., 1999; Chang et al ., 2010), none of them were used to develop genetically engineered bacteria to improve the biotechnological production of TS. These genes have been only expressed in Escherichia coli , a bacterium unable to efficiently transport sterols or AD, impairing the development of an industrial biotransformation processes.…”

“…phytosterols or cholesterol) or AD into TS in order to compete with the current chemical synthesis of TS by using a new biotechnological process. To fulfil this goal, we have cloned and overexpressed the genes encoding two microbial 17β‐HSDs, from the bacterium Comamonas testosteroni (Abalain et al ., 1993) and from the fungus Cochliobolus lunatus (Rižner et al ., 1999), using as hosts the wild‐type M. smegmatis and an AD‐producing mutant of this bacterium. The performances of the new created recombinant bacterial strains have been tested both in growing and resting‐cell conditions using sterols and AD as substrates respectively (Fig.…”

Meta-cleavage enzyme gene tesB is necessary for testosterone degradation in Comamonas testosteroni TA441

“…Degradation of testosterone in C. testosteroni is considered to be initiated by dehydrogenation of the 17β-hydroxyl group to 4-androstene-3,17-dione (reaction 1 in Fig. 1) (Abalain et al, 1993 ;Genti-Raimondi et al, 1991), followed by desaturation of the A ring (Fig. 1).…”

“…The gene for the 17β-dehydrogenase, which catalyses the initial dehydrogenation reaction (reaction 1 in Fig. 1), has been cloned and characterized (Abalain et al, 1993 ;Genti- (Coulter & Talalay, 1968). Compound I, 9α-hydroxy-4-androstene-3,17-dione ; II, 9α-hydroxy-1,4-androstadiene-3,17-dione ; III, 2-oxo-cis-4-hexenoic acid ; IV, 3aα,4β,5,6,7,7a-hexahydro-7aβ-methyl-1,5-dioxo-4-indanpropionic acid.…”