Estudio de la ruta catabólica responsable de la degradación aeróbica del desoxicolato sódico y de otros compuestos esteroideos en Pseudomonas putida DOC21

Abstract

The presence of steroids into the environment is increasing as result of the spillage of industrial byproducts, pharmacological industry wastes and bulk sewage sludges. These compounds (or their derivatives) could have a hazardous effect over life cycle of different animals and humans. Thus, it is becoming evident the need to characterize and use bacteria steroid-degradative potential for the design of biotechnological strategies to alleviate the environmental accumulation of these compounds. Moreover, the knowledge of the genes and enzymes used by microorganisms to degrade (or to transform) certain steroids could be potentially useful for the development of new pharmacologically interesting molecules. The strain Pseudomonas putida DOC21 was isolated from a soil sample by using a selective media containing deoxycholate as the only carbon source. This bacterium is able to use deoxycholate, cholate, chenodeoxycholate¸ lithocholate, ursodeoxycholate, dehydrocholate, testosterone, androstanolone and 4-androsten-3,17-dione (AD) as the only carbon sources. By contrast, this strain is unable to metabolize cholesterol, β-sitosterol, stigmasterol, ergosterol, 5β-cholanate, progesterone, prednisone, 5-pregnen-3β-ol-20-one, 17-α-methyltestosterone, 1-dehydro-17α-methyltestosterone, β-estradiol, estrone, transdehydroandrosterone and trans-androsterone. This catabolic behaviour suggests: (1) the inability of P. putida DOC21 to degrade compounds containing side chains in position 17 without an oxidized C-terminus; (2) the critical importance of a hydroxy- or a keto-group in the position 3 of the molecule; and (3) that processing of the hydroxyl-group in position 3 and the degradation of the side chain seem to be required for the degradation of the rings A and B of the steroid. The sequencing of the genome of P. putida DOC21 and proteome study allowed us to identify genes and regulatory enzymes required for the degradation of steroid compounds. Using a mutational strategy with the Tn5 transposon, 50 mutants unable to metabolize deoxycholate were obtained. These mutants have been classified into two different groups according to their differential catabolic use of steroids: mutants handicapped for the use of deoxycholate but able to metabolize testosterone or 4- androsten-3,17-dione, and mutants unable to use deoxycholate, testosterone and 4- androsten-3,17-dione as the only carbon source. The collection of these two classes of mutants strongly suggests that the degradation of deoxycholate, testosterone and 4- androsten-3,17-dione occurs throughout different peripheral catabolic routes that converge X onto a central pathway which transforms a common intermediate into general metabolites. All these routes belong to the cholesterol catabolon. Taking into account both the gene affected by the Tn5 insertion in the different mutants and the metabolic manifestation, they can be classified into three groups: (i) mutants impaired in genes encoding functions necessary to carry out the degradation of the side chain of deoxycholate, represented by strains unable to grow in deoxycholate, but are still able to degrade testosterone and AD (mutants 8, 9, 12, 14, 15, 17, 20, 24, 26, 27, 30, 31, 36, 37, 38, 39, 40, 41, 43, 45, 46, 47, 48, 49, 52 and 53); (ii) mutants affected in common functions for using deoxycholate, testosterone and AD and, therefore, affected in some processes of a central path of degradation in the converging common metabolites of steroid compounds catabolism (mutants 1, 3, 4, 5, 6, 11, 13, 16, 18, 19, 22, 23, 25, 29, 32, 35, 44, 50 and 51); and (iii) mutants affected in processes that concern the general metabolism of the bacteria (glyoxylate cycle, the methylcitrate cycle) (mutants 7, 28, 33, 34 and 42). The group of mutants affected in steroid degradation pathway (ii) was analyzed by gas chromatography (GC-MS) to determine if it accumulated HIP. The results allow to distinguish two different types: (a) those mutants involved in the degradation of central metabolites to obtain HIP (mutants 1, 3, 4 and 50), and (b) those other mutants that accumulate HIP or some of hydroxylated derivatives, and thus involved in the degradation of this intermediate (mutants 6, 13, 16, 18, 19, 22, 23, 25 and 51). The analysis of the mutants revealed that mutants 1, 4 and 50 are involved in central steroid degradation pathway (oxidation of the rings A and B). Mutant 1 is concerned in the gene encoding the protein StdF, one opening goal dioxygenase that opens the ring of the molecule 3,4-dihydroxy-9,10-secoandrosta-1,3,5 (10) -trien-9,17-dione (3,4- DHSA), and mutants 4 and 50 are involved in a gene encoding an acetyl CoA acetyltransferase catalyzing the unknown enzymatic reaction in the pathway. The other mutants (mutants 6, 11, 13, 16, 18, 19, 22, 23, 25, 32, 35 and 51) are related to oxidation of the rings C and D (low steroid degradation pathway). Specifically, the mutant 35 has Tn5 inserted into the gene stdP2 encoding β subunit of a CoA-transferase; mutants 6, 13, 16, 18, 19, 22, 23, 25 and 51 are affected in the gene stdQ encoding an enoyl-CoA hydratase/isomerase; the mutant 11 has interrupted the gene stdS encoding an enoyl-ACP reductase; and mutant 32 associated with an acetyl-CoA acetyltransferase encoded in the gene stdT. All them participate in the transformation of HIP-CoA in general catabolites. XI The results reported in this Thesis suggest that deoxycholate, cholate, lithocholate, chenodeoxycholate, testosterone and other steroid compounds are degraded into androst- 1,4-diene-3,17-dione (ADD), intermediate which is then turned into HIP and 2- hidroxihexa-2,4-dienoate. These compounds are catabolized to general metabolites. The catabolic process responsible of the degradation of HIP is still unknown although it has been established that HIP is activated to HIP-CoA by the action of enzyme StdA3. The 2- hidroxihexa 2,4-dienoic acid, generated as indicated above, is degraded to general metabolites by the enzymes StdMNO, as revealed the studies performed with the mutant ΔtesG. Furthermore, we have reported the isolation of mutants affected in the glyoxylate cycle (mutants 28, 33, 34 and 42); in the methylcitrate cycle (mutant 7 and ΔacnB) and in the genes related with the metabolism of pyruvate (mutant 44) and electron transfer (mutant 9). The existence of these mutants indicates that, in P. putida DOC21, the steroidal compounds are decomposed into acetyl-CoA and propionyl-CoA which are later degraded throughout general metabolic pathway.