Pseudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The ΔstdA1 and ΔstdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione (AD). In contrast, a ΔstdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ(1/4) -3-ketocholate and Δ(1,4) -3-ketocholate, whereas ΔstdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC). When incubated with testosterone or bile acids, ΔstdA3 accumulated 3aα-H-4α(3'propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ(1/4) -3-ketocholate, and Δ(1,4) -3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria.

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Pseudomonas putida DOC21 assimilates a large variety of steroids, including bile acids, via a single 9, 10-seco pathway. Two specific mutants knocked down in stdH and stdJ were obtained by deletion (strains P. putida DOC21stdH and P. putida DOC21stdJ). Analysis of these mutants revealed that both had lost the ability to fully degrade bile acids and that the genes stdH and stdJ are involved in oxidation of the A and B rings of the polycyclic steroid structure. Moreover, whereas P. putida DOC21stdH and P. putida DOC21stdJ were unable to degrade testosterone or 4-androstene-3,17-dione (AD), P. putida DOC21stdJ was also unable to assimilate androsta-1,4-diene-3,17-dione (ADD). When cultured in medium containing lithocholate and succinate, P. putida DOC21stdH and P. putida DOC21stdJ accumulated AD and ADD, respectively. Genetic and bioinformatics analyses revealed that: (i) stdH encodes a 3-ketosteroid- 1 -dehydrogenase; (ii) StdJ is the reductase component of a 3-ketosteroid 9-hydroxylase; (iii) the trans-expression of stdH and stdJ in the corresponding mutant restored the lost catabolic function(s), and (iv) full steroid metabolism by P. putida DOC21stdH was restored by its expression of kstD2, but not kstD1 or kstD3, of Rhodococcus ruber Chol-4. Our results shed light on the systems used by bacteria to oxidize the A and B rings of steroid compounds. In addition, as the mutants described herein were able to synthesize two pharmaceutically important synthons, AD and ADD, they may be of value in industrial applications.

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