Abstract

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 DOC21ΔstdH and P. putida DOC21ΔstdJ). 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 DOC21ΔstdH and P. putida DOC21ΔstdJ were unable to degrade testosterone or 4-androstene-3,17-dione (AD), P. putida DOC21ΔstdJ was also unable to assimilate androsta-1,4-diene-3,17-dione (ADD). When cultured in medium containing lithocholate and succinate, P. putida DOC21ΔstdH and P. putida DOC21ΔstdJ accumulated AD and ADD, respectively. Genetic and bioinformatics analyses revealed that: (i) stdH encodes a 3-ketosteroid-Δ1-dehydrogenase; (ii) StdJ is the reductase component of a 3-ketosteroid 9α-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 DOC21ΔstdH 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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