Steroid degradation by Comamonas testosteroni and Nocardia restrictus have been intensively studied for the purpose of obtaining materials for steroid drug synthesis. C. testosteroni degrades side chains and converts single/double bonds of certain steroid compounds to produce androsta-1,4-diene 3,17-dione or the derivative. Following 9-hydroxylation leads to aromatization of the A-ring accompanied by cleavage of the B-ring, and aromatized A-ring is hydroxylated at C-4 position, cleaved at ∆4 by meta-cleavage, and divided into 2-hydroxyhexa-2,4-dienoic acid (A-ring) and 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (B,C,D-ring) by hydrolysis.Reactions and the genes involved in the cleavage and the following degradation of the A-ring are similar to those for bacterial biphenyl degradation, and 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid degradation is suggested to be mainly -oxidation. Genes involved in A-ring aromatization and degradation form a gene cluster, and the genes involved in -oxidation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid also comprise a large cluster of more than 10 genes. The DNA region between these two main steroid degradation gene clusters contain 3-hydroxysteroid dehydrogenase gene, ∆5,3-ketosteroid isomerase gene, genes for inversion of an -oriented-hydroxyl group to a -oriented-hydroxyl group at C-12 position of cholic acid, and genes possibly involved in the degradation of a side chain at C-17 position of cholic acid, indicating this DNA region of more than 100kb to be a steroid degradation gene hot spot of C. testosteroni.

In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-⌬ 1 -dehydrogenase and 3-ketosteroid-⌬ 4 (5␣)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9␣-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.Comamonas testosteroni is able to utilize certain C 19 and C 21 steroids, as well as a number of aromatic compounds, as sole carbon and energy sources via a complex metabolic pathway involving many steroid-inducible enzymes. The mechanism by which testosterone is degraded in Nocardia restrictus and C. testosteroni was proposed in the 1960s (4,8,14). However, the genes involved in this degradation pathway in C. testosteroni have yet to be identified, except for the initial 17␤-dehydrogenation (1, 3, 7) and the following ⌬ 1 -dehydrogenation (13). In previous studies, we identified the testosterone degradation genes tesB and tesDEFG among some open reading frames (ORFs) which are involved in testosterone degradation in C. testosteroni TA441 (9, 10). tesB encodes the meta-cleavage enzyme for 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3,4-DHSA) (10), and tesD encodes the hydrolase for 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid (4,9-DSHA) (9). TesEFG are probably the enzymes for degradation of 2-oxohex-4-enoic acid, which is one of the two products of the hydrolysis of 4,9-DSHA by TesD.Studies on these genes have shown the probable testosterone degradation pathway of C. testosteroni TA441 to be that presented in Fig. 1. The process is initiated by dehydrogenation of the 17␤-hydroxyl group on testosterone to 4-androstene-3,17-dione (4-AD) (Fig. 1, reaction 1), which then undergoes ⌬ 1 -dehydrogenation to 1,4-androstadiene-3,17-dione (ADD) (reaction 2), followed by 9␣-hydroxylation to produce 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3-HSA) (reaction 3 and the following spontaneous cleavage). The C-4 of 3-HSA is hydroxylated to yield 3,4-DHSA (Fig. 1, reaction 4), which is cleaved between C-4 and C-5 via a meta-cleavage re...

Comamonas testosteroni metabolizes testosterone as the sole carbon source via a meta-cleavage reaction. A meta-cleavage enzyme gene, tesB, was cloned from C. testosteroni TA441. The deduced N-terminal amino acid sequence of tesB matched that of the purified meta-cleavage enzyme which is induced in TA441 during growth on testosterone as the sole carbon source. The tesBdisrupted mutant did not show growth on testosterone, suggesting that tesB is necessary for TA441 to grow on testosterone. Downstream from tesB, three putative ORFs which encode products also necessary for growth of TA441 on testosterone were identified. The usual substrate of TesB is probably 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione. Although this compound was not identified in the gene disrupted mutants, accumulation of upstream metabolites of testosterone degradation, 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione, was shown by TLC analysis.

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