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![Complementation experiments with the ORF23 and ORF27 mutants. HPLC chromatograms and MS chromatograms of the culture of each mutant incubated with 0.1% ADD for 7 days are shown. Mutants are the ORF27 null mutant harboring broad-host-range plasmid pMFY42 (ORF27– strain with pMFY42) (A) or harboring pMFY42 carrying ORF27 (ORF27– strain with pMFYORF27) (B); ORF23- and 27- double null mutant harboring pMFY42 (ORF23– ORF27– strain with pMFY42) (C) or harboring MFYORF27 (ORF23– ORF27– strain with pMFYORF27) (D); and ORF23 null mutant harboring pMFY42 (ORF23– strain with pMFY42) (E) or harboring pMFY42 carrying ORF23 (ORF23– strain with pMFYORF23) (F). Each panel indicates m/z 253 (V), m/z 235 (XIII), m/z 255 (IV and IIa [7α]), m/z 237 (XV, I, and III), and m/z 211 (VI). ORF25 and ORF26 are disrupted in the ORF23– ORF27– strain, but this does not affect steroid degradation. In the UPLC chromatogram, the vertical axis indicates wavelength (nm), the horizontal axis indicates RT (min), and the absorbance is plotted in a contour. In the mass chromatogram, the vertical axis indicates intensity (count/s) and the horizontal axis indicates RT (min).](publication/334898452/figure/fig5/AS:11431281220141622@1706279156492/Complementation-experiments-with-the-ORF23-and-ORF27-mutants-HPLC-chromatograms-and-MS.jpeg)
Complementation experiments with the ORF23 and ORF27 mutants. HPLC chromatograms and MS chromatograms of the culture of each mutant incubated with 0.1% ADD for 7 days are shown. Mutants are the ORF27 null mutant harboring broad-host-range plasmid pMFY42 (ORF27– strain with pMFY42) (A) or harboring pMFY42 carrying ORF27 (ORF27– strain with pMFYORF27) (B); ORF23- and 27- double null mutant harboring pMFY42 (ORF23– ORF27– strain with pMFY42) (C) or harboring MFYORF27 (ORF23– ORF27– strain with pMFYORF27) (D); and ORF23 null mutant harboring pMFY42 (ORF23– strain with pMFY42) (E) or harboring pMFY42 carrying ORF23 (ORF23– strain with pMFYORF23) (F). Each panel indicates m/z 253 (V), m/z 235 (XIII), m/z 255 (IV and IIa [7α]), m/z 237 (XV, I, and III), and m/z 211 (VI). ORF25 and ORF26 are disrupted in the ORF23– ORF27– strain, but this does not affect steroid degradation. In the UPLC chromatogram, the vertical axis indicates wavelength (nm), the horizontal axis indicates RT (min), and the absorbance is plotted in a contour. In the mass chromatogram, the vertical axis indicates intensity (count/s) and the horizontal axis indicates RT (min).
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Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in relation to human health is attracting attention. The overall aerobic degradation of the f...
Citations
... When a hydroxyl group at C7 is present, XXV-CoA ester is pro duced in ScdC1C2cultures, suggesting a bypass route (presumably via dehydration) to produce XXV-CoA ester (Fig. 5: XXIV-CoA ester (R2 = OH) → XXXIV-CoA ester → XXV-CoA ester) (47). XXV-CoA ester undergoes β-oxidation to VI-CoA ester via ScdD hydratase, ScdE dehydrogenase, and ScdF CoA-transferase (48). Throughout the β-oxidation of the cleaved B-ring to the cleavage of the C-ring, compounds with "C9 ketone and a double bond at C8(14)" and "C9 hydroxyl group and a single bond at C8 (14)" are major in most of the mutant cultures. ...
Comamonas testosteroni is one of the representative aerobic steroid-degrading bacteria. We previously revealed the mechanism of steroidal A,B,C,D-ring degradation by C. testosteroni TA441. The corresponding genes are located in two clusters at both ends of a mega-cluster of steroid degradation genes. ORF7 and ORF6 are the only two genes in these clusters, whose function has not been determined. Here, we characterized ORF7 as encoding the dehydrase responsible for converting the C12β hydroxyl group to the C10(12) double bond on the C-ring (SteC), and ORF6 as encoding the hydrogenase responsible for converting the C10(12) double bond to a single bond (SteD). SteA and SteB, encoded just upstream of SteC and SteD, are in charge of oxidizing the C12α hydroxyl group to a ketone group and of reducing the latter to the C12β hydroxyl group, respectively. Therefore, the C12α hydroxyl group in steroids is removed with SteABCD via the C12 ketone and C12β hydroxyl groups. Given the functional characterization of ORF6 and ORF7, we disclose the entire pathway of steroidal A,B,C,D-ring breakdown by C. testosteroni TA441.
IMPORTANCE
Studies on bacterial steroid degradation were initiated more than 50 years ago, primarily to obtain materials for steroid drugs. Now, their implications for the environment and humans, especially in relation to the infection and the brain-gut-microbiota axis, are attracting increasing attention. Comamonas testosteroni TA441 is the leading model of bacterial aerobic steroid degradation with the ability to break down cholic acid, the main component of bile acids. Bile acids are known for their variety of physiological activities according to their substituent group(s). In this study, we identified and functionally characterized the genes for the removal of C12 hydroxyl groups and provided a comprehensive summary of the entire A,B,C,D-ring degradation pathway by C. testosteroni TA441 as the representable bacterial aerobic degradation process of the steroid core structure.
... In contrast, the microbial metabolic pathway for sapogenin degradation has not been uncovered. The steroid (but not sapogenin)-degradation enzymes that are present in Comamonas testosteroni TA441 have been well studied; moreover, their orthologs in the Novosphingobium tardaugens strain ARI-1 (NBRC 16725) have also been identified via in silico analysis (39,40). In the present study, we sequenced the genome of Sphingobium spp. ...
... Microbial steroid-degrading enzymes have been well studied in several soil-borne microorganisms, and degradation pathways have been proposed (39). Metagenomics analyses of the genes encoding steroid-catabolizing enzymes from various environments revealed that those isolated from Alphaproteobacteria and Actinobacteria are predomi nant in the rhizosphere, and that the former mainly consist of Sphingomonadaceae and Rhizobiales (60). ...
Plant roots exude various organic compounds, including plant specialized metabolites (PSMs), into the rhizosphere. The secreted PSMs enrich specific microbial taxa to shape the rhizosphere microbiome, which is crucial for the healthy growth of the host plants. PSMs often exhibit biological activities; in turn, some microorganisms possess the capability to either resist or detoxify them. Saponins are structurally diverse triterpene-type PSMs that are mainly produced by angiosperms. They are generally considered as plant defense compounds. We have revealed that α-tomatine, a steroid-type saponin secreted from tomato ( Solanum lycopersicum ) roots, increases the abundance of Sphingobium bacteria. To elucidate the mechanisms underlying the α-tomatine-mediated enrichment of Sphingobium , we isolated Sphingobium spp. from tomato roots and characterized their saponin-catabolizing abilities. We obtained the whole-genome sequence of Sphingobium sp. RC1, which degrades steroid-type saponins but not oleanane-type ones, and performed a gene cluster analysis together with a transcriptome analysis of α-tomatine degradation. The in vitro characterization of candidate genes identified six enzymes that hydrolyzed the different sugar moieties of steroid-type saponins at different positions. In addition, the enzymes involved in the early steps of the degradation of sapogenins (i.e., aglycones of saponins) were identified, suggesting that orthologs of the known bacterial steroid catabolic enzymes can metabolize sapogenins. Furthermore, a comparative genomic analysis revealed that the saponin-degrading enzymes were present exclusively in certain strains of Sphingobium spp., most of which were isolated from tomato roots or α-tomatine-treated soil. Taken together, these results suggest a catabolic pathway for highly bioactive steroid-type saponins in the rhizosphere.
IMPORTANCE
Saponins are a group of plant specialized metabolites with various bioactive properties, both for human health and soil microorganisms. Our previous works demonstrated that Sphingobium is enriched in both soils treated with a steroid-type saponin, such as tomatine, and in the tomato rhizosphere. Despite the importance of saponins in plant–microbe interactions in the rhizosphere, the genes involved in the catabolism of saponins and their aglycones (sapogenins) remain largely unknown. Here we identified several enzymes that catalyzed the degradation of steroid-type saponins in a Sphingobium isolate from tomato roots, RC1. A comparative genomic analysis of Sphingobium revealed the limited distribution of genes for saponin degradation in our saponin-degrading isolates and several other isolates, suggesting the possible involvement of the saponin degradation pathway in the root colonization of Sphingobium spp. The genes that participate in the catabolism of sapogenins could be applied to the development of new industrially valuable sapogenin molecules.
... Microbial bile acids are important hormones that modulate host cholesterol metabolism and energy balance through nuclear receptors and G-protein-coupled receptors (Wahlström et al., 2016). Aerobic degradation of bile acids has primarily been studied in soil bacteria such as Pseudomonas and Comamonas (Barrientos et al., 2015;Horinouchi et al., 2019;Feller et al., 2021). The bacterivore C. elegans can grow and reproduce on a variety of bacterial diets. ...
Microbiota consist of microorganisms that provide essential health benefits and contribute to the animal’s physiological homeostasis. Microbiota-derived metabolites are crucial mediators in regulating host development, system homeostasis, and overall fitness. In this review, by focusing on the animal model Caenorhabditis elegans, we summarize key microbial metabolites and their molecular mechanisms that affect animal development. We also provide, from a bacterial perspective, an overview of host-microbiota interaction networks used for maintaining host physiological homeostasis. Moreover, we discuss applicable methodologies for profiling new bacterial metabolites that modulate host developmental signaling pathways. Microbiota-derived metabolites have the potential to be diagnostic biomarkers for diseases, as well as promising targets for engineering therapeutic interventions against animal developmental or health-related defects.
... The following genes were selected for the RT-qPCR expression analysis: cyp125, which is associated with the initial step in cholesterol side chain degradation [22,23]; kstD3 and kshA, which encode enzymes responsible for key degradation steps of the steroid core [24,25]; and fadE30 and ipdF, which are associated with the C/D ring degradation pathway [26][27][28]. Cyp125 is responsible for C26-hydroxylation of the terminal methyl group of cholesterol/cholestenone; KstDs and KshAs are the key enzymes for the ring B opening in the so-called 9(10)-seco pathway; IpdF is responsible for reduction of the C5-oxo-group to hydroxyl of the KstR2 inducer molecule HIP-CoA [1]; and FadE30 is involved in dehydrogenation of 5-OH-HIP-CoA [29] ( Figure 5). The 16S rRNA gene was used as a reference gene. ...
Steroids are abundant molecules in nature, and various microorganisms evolved to utilize steroids. Thermophilic actinobacteria play an important role in such processes. However, very few thermophiles have so far been reported capable of degrading or modifying natural sterols. Recently, genes putatively involved in the sterol catabolic pathway have been revealed in the moderately thermophilic actinobacterium Saccharopolyspora hirsuta VKM Ac-666T, but peculiarities of strain activity toward sterols are still poorly understood. S. hirsuta catalyzed cholesterol bioconversion at a rate significantly inferior to that observed for mesophilic actinobacteria (mycobacteria and rhodococci). Several genes related to different stages of steroid catabolism increased their expression in response to cholesterol as was shown by transcriptomic studies and verified by RT–qPCR. Sequential activation of genes related to the initial step of cholesterol side chain oxidation (cyp125) and later steps of steroid core degradation (kstD3, kshA, ipdF, and fadE30) was demonstrated for the first time. The activation correlates with a low cholesterol conversion rate and intermediate accumulation by the strain. The transcriptomic analyses revealed that the genes involved in sterol catabolism are linked functionally, but not transcriptionally. The results contribute to the knowledge on steroid catabolism in thermophilic actinobacteria and could be used at the engineering of microbial catalysts.
... I, cholesterol; II, cholest-4-en-3-one; IV, 26-hydroxy-cholest-4-en-3-one; V, 3-oxo-cholest-4-en-26-oic acid; VII, cholest-5-ene-3β,26-diol; VIII, 3β-hydroxy-cholest-5-en-26-oic acid; IX, 3-oxo-cholest-4-en-26-oyl-CoA; X, 3-oxo-cholesta-4,24dien-26-oyl-CoA; XI, 24-hydroxy-3-oxo-cholest-4-en-26-oyl-CoA; XII, 3,24-dioxo-cholest-4-en-26-oyl-CoA; XIII, 3-Oxo-chol-4en-24-oyl-CoA; XIV, 3-oxo-chola-4,22-dien-24-oyl-CoA; XV, 22-hydroxy-3-oxo-chol-4-en-24-oyl-CoA; XVI, 3,22-dioxo-chol-4en-24-oyl-CoA; XVII, 3-oxo-4-pregnene-20-carboxyl-CoA; XVIII, androst-4-ene-3,17-dione (AD); XIX, androsta-1,4-diene-3,17-dione (ADD); XX, 9α-hydroxy-AD; XXI, unstable 9α-hydroxy-ADD; XXII, 3β-hydroxy-9,10-seco-androsta-1,3,5(10)triene-9,17-dione (3βHSA); XXIII, 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3,4-DHSA); XXIV, 4,5-9,10diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-diene-4-oic acid (4,9-DSHA); XXV, 2-hydroxyhexa-2,4-dienoic acid (2-HHD); XXVI, 4-hydroxy-2-oxohexanoic acid; XXVII, 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (DOHNAA) or 3aα-H-4α-(3 -propanoate)-7aβ-methylhexahydro-1,5-indadione (HIP); XXVIII, 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oyl-CoA (HIP-CoA); XXIX, 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid or 3aα-H-4α(3 -propanoate)-5α-hydroxy-XXXIV,-9-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octa-norandrostan-7-oyl-CoA or 3aα-H-4α(carboxylCoA)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH-HIC-CoA); XXXV, 19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid-CoA-ester or (R)-2-(2-carboxyethyl)-3methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA); XXXIX, 6-methyl-3,7-dioxo-decane-1,10-dioic acid-CoA ester; XL, 4-methyl-5-oxo-octane-1,8-dioic acid-CoA ester; XLI, 4-methyl-5-oxo-oct-2-ene-1,8-dioic acid-CoA ester (MOODA-CoA); XLII, 3-hydroxy-4-methyl-5-oxo-octane-1,8-dioic acid-CoA ester; XLIII, 4-methyl-3,5-dioxo-octane-1,8-dioic acid-CoA ester; XLIV, 2-methyl-3-oxo-hexane-1,6-dioic acid-CoA ester; XLV, succinyl-CoA; XLVI, propionyl-CoA. Adopted from:[8,16,[38][39][40][41][42][43][44][45][46]. ...
The application of thermophilic microorganisms opens new prospects in steroid biotechnology, but little is known to date on steroid catabolism by thermophilic strains. The thermophilic strain Saccharopolyspora hirsuta VKM Ac-666T has been shown to convert various steroids and to fully degrade cholesterol. Cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3β-hydroxy-cholest-5-en-26-oic acid were identified as intermediates in cholesterol oxidation. The structures were confirmed by 1H and 13C-NMR analyses. Aliphatic side chain hydroxylation at C26 and the A-ring modification at C3, which are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively, occur simultaneously in the strain and are followed by cascade reactions of aliphatic sidechain degradation and steroid core destruction via the known 9(10)-seco-pathway. The genes putatively related to the sterol and bile acid degradation pathways form three major clusters in the S. hirsuta genome. The sets of the genes include the orthologs of those involved in steroid catabolism in Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 and related actinobacteria. Bioinformatics analysis of 52 publicly available genomes of thermophilic bacteria revealed only seven candidate strains that possess the key genes related to the 9(10)-seco pathway of steroid degradation, thus demonstrating that the ability to degrade steroids is not widespread among thermophilic bacteria.
... This gene synteny is also known for other ACADs involved in steroid metabolism, which are heterotetramers of two ACAD subunits (29). In a reciprocal BLASTp analysis, Nov2c221 and Nov2c222 were indeed annotated as the two subunits of the HIP-ACAD, Scd3A (20) (also called ScdD1 [26]) and Scd3B (ScdD2), with 58% and 40% identity to the homologs of P. stutzeri Chol1, respectively (12). This is also reflected in a phylogenetic tree of steroid-degradation ACADs (Fig. S2), in which Nov2c221 and Nov2c222 cluster with the aand b-subunits, respectively, of HIP-ACADs. ...
The reaction sequence for aerobic degradation of bile salts by environmental bacteria resembles degradation of other steroid compounds. Recent findings show that bacteria belonging to the Sphingomonadaceae use a pathway variant for bile-salt degradation. This study addresses this so-called Δ 4,6 -variant by comparative analysis of unknown degradation steps in Sphingobium sp. strain Chol11 with known reactions found in Pseudomonas stutzeri Chol1. Investigations with strain Chol11 revealed an essential function of the acyl-CoA dehydrogenase Scd4AB for growth with bile salts. Growth of the scd4AB deletion mutant was restored with a metabolite containing a double bond within the side chain which was produced by the Δ ²² -acyl-CoA dehydrogenase Scd1AB from P. stutzeri Chol1. Expression of scd1AB in the scd4AB deletion mutant fully restored growth with bile salts while expression of scd4AB only enabled constricted growth in P. stutzeri Chol1 scd1A or scd1B deletion mutants. Strain Chol11 Δ scd4A accumulated hydroxylated steroid metabolites which were degraded and activated with coenzyme A by the wild type. Activities of five Rieske type monooxygenases of strain Chol11 were screened by heterologous expression and compared to the B-ring cleaving KshAB Chol1 from P. stutzeri Chol1. Three of the Chol11 enzymes catalyzed B-ring cleavage of only Δ 4,6 -steroids while KshAB Chol1 was more versatile. Expression of a fourth KshA homolog, Nov2c228 led to production of metabolites with hydroxylations at an unknown position. These results indicate functional diversity of β-proteobacterial enzymes for bile-salt degradation and suggest a novel side-chain degradation pathway involving an essential ACAD reaction and a steroid hydroxylation step.
Importance
This study highlights the biochemical diversity of bacterial degradation of steroid compounds in different aspects. First, it further elucidates an unexplored variant in the degradation of bile-salt side chains by Sphingomonads, a group of environmental bacteria that is well-known for their broad metabolic capabilities. Moreover, it adds a so-far unknown hydroxylation of steroids to the reactions Rieske monooxygenases can catalyze with steroids. Additionally, it analyzes a proteobacterial ketosteroid-9α-hydroxylase and shows that this enzyme is able to catalyze side reactions with non-native substrates.
... In this process, the former 12-OH is removed and a hydroxy group at former C 7 is introduced into 7-deoxy bile salt derivatives during b-oxidation of the former B-ring. Further degradation of HIPs proceeds via b-oxidation of the former Bring and hydrolytic cleavages of rings C and D (26,27). ...
... Phase 4: Complete degradation of the 9,10-seco intermediates (orange section in Fig. 3). Most proteins required for the degradation of the 9,10-seco degradation intermediates, derived from both cholate and deoxycholate, and the respective HIP intermediates (27) are encoded in gene cluster 3 (Fig. 7A). All of these proteins are increased in abundance at least 1.5-fold in steroid-grown cells (Fig. 4). ...
... Furthermore, C. testosteroni KF-1 was reported to degrade taurocholate (62), and its genome encodes two Bsa homologs (identity for both, 44%). The respective homologs ORF25 and ORF26 from model organism C. testosteroni TA441 are encoded in its steroid-degradation megacluster (14,27). The function of Nov2c229 remains unknown so far. ...
Bile salts are amphiphilic steroids chain with digestive functions in vertebrates. Upon excretion, bile salts are degraded by environmental bacteria. Degradation of the bile-salt steroid skeleton resembles the well-studied pathway for other steroids like testosterone, while specific differences occur during side-chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either Δ 1,4 - or Δ 4,6 -3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the Δ 1,4 - variant is well-known from many model organisms, the Δ 4,6 -variant involving a 7-hydroxysteroid dehydratase as key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic and functional analyses of the Δ 4,6 -variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to the Δ 1,4 -variant except for the elimination of the 7-OH as a key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster degradation of the C 5 carboxylic side chain encoding a CoA-ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase, but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the Δ 4,6 -variant to be widespread among the Sphingomonadaceae , which was verified for three type strains which also have the predicted side-chain degradation cluster. A second amidase in the side-chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile-salt hydrolases. This study signifies members of the Sphingomonadaceae remarkably well-adapted to the utilization of bile salts via a partially distinct metabolic pathway.
Importance
This study highlights the biochemical diversity of bacterial degradation of steroid compounds, in particular bile salts. Furthermore, it substantiates and advances knowledge of a variant pathway for degradation of steroids by sphingomonads, a group of environmental bacteria that are well-known for their broad metabolic capabilities. Biodegradation of bile salts is a critical process due to the high input of these compounds from manure into agricultural soils and wastewater treatment plants. In addition, these results may also be relevant for the biotechnological production of bile salts or other steroid compounds with pharmaceutical functions.
... 7a,9adihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid in cholic acid degradation) via aromatization of the A-ring and the subsequent ring cleavage and hydrolysis (28)(29)(30)(31)(32)(33)(34)(35). Then, coenzyme A (CoA) is incorporated into compound VII by ScdA (36), and the resulting VII-CoA ester is further degraded, mainly by two cycles of b-oxidation on the cleaved B-ring and on the cleaved C,D-rings (37)(38)(39)(40)(41)(42). The first cycle of b-oxidation involves the removal of two carbons of cleaved B-ring from VII-CoA ester to generate 9a-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid (compound XII)-CoA ester. ...
... Based on these compounds, XV-CoA ester, XVI-CoA ester, and XVII-CoA ester were proposed as intermediate compounds with only the C-ring remaining unaffected in steroidal degradation because it was isolated in much greater amounts than the other two compounds (41); however, the substrate of the ring cleavage by ScdL1L2 is not entirely clear. 4-Methyl-5-oxo-octane-1,8-dioic acid (compound XX) and 4-methyl-5-oxo-oct-3-ene-1,8dioic acid (compound XXa) are identified as compounds produced after C-ring cleavage (42), suggesting that 6-methyl-3,7-dioxo-decane-1,10-dioic acid (compound XIX)-CoA ester is the C-ring cleavage product. XIX-CoA ester is presumed to be produced by C-ring cleavage of 4-hydroxy-9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrostane-7,17-dioic acid (compound XVIII)-CoA ester, but compound XVIII has not been isolated in the previous studies. ...
... However, the decrease in amount was small and the product, XXI, was scarcely detected (Fig. 4D m/z 155). The product is often detected in trace quantities in the complemented mutants, as all the steroidal degradation enzymes are expressed in the mutants (39)(40)(41)(42). ...
Comamonas testosteroni TA441 degrades steroids aerobically via aromatization of the A-ring accompanied by B-ring cleavage, followed by D- and C-ring cleavage. We previously revealed major enzymes and intermediate compounds in A,B-ring cleavage, β-oxidation cycle of the cleaved B-ring, and partial C,D-ring cleavage process. Here, we elucidated the C-ring cleavage and the β-oxidation cycle that follows. ScdL1L2, a 3-ketoacid Coenzyme A (CoA) transferase which belongs to the SugarP_isomerase superfamily, was thought to cleave the C-ring of 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid-CoA ester, the key intermediate compound in the degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (3aα- H -4α [3′-propionic acid]-7aβ-methylhexahydro-1,5-indanedione; HIP)-CoA ester in the previous study; however, this study suggested that ScdL1L2 is the isomerase of the derivative with a hydroxyl group at C-14 which cleaves C ring. The subsequent ring-cleaved product was indicated to be converted to 4-methyl-5-oxo-octane-1,8-dioic acid-CoA ester mainly by ORF33-encoded CoA-transferase (named ScdJ), followed by dehydrogenation by ORF21 and 22-encoded acyl-CoA dehydrogenase (named ScdM1M2). Then a water molecule is added by ScdN for further degradation by β-oxidation. ScdN is considered to catalyze the last reaction in C,D-ring degradation by the enzymes encoded in the steroid degradation gene cluster tesB to tesR .
IMPORTANCE
Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in human is attracting attention. The overall degradation of steroidal four rings is proposed, however there are still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in C. testosteroni , which is one of the most studied representative steroid degrading bacteria and is suitable for exploring the degradation pathway because the involvement of degradation-related genes can be determined by gene disruption.
... Transcriptomic studies in a bile acid and sterol degrading Actinobacterium suggested that lithocholic acid is degraded by a mix of cholesterol and bile acid degradationspecific reactions [30]. Aerobic degradation of cholic acid and other bile acids has predominantly been studied in Pseudomonas stutzeri Chol1 [31,33,34], Pseudomonas putida DOC21 [17,35], Comamonas testosteroni [24,[36][37][38][39], Sphingobium sp. strain Chol11 [18,23,[40][41][42], and Rhodococcus jostii RHA1 [28,43]. ...
... Bile acid degradation can be divided into four phases ( Figure 2B): (1) partial oxidation of the A-ring (Figure 3), (2) stepwise removal of the C5 carboxylic side chain, including Aerobic degradation of cholic acid and other bile acids has predominantly been studied in Pseudomonas stutzeri Chol1 [31,33,34], Pseudomonas putida DOC21 [17,35], Comamonas testosteroni [24,[36][37][38][39], Sphingobium sp. strain Chol11 [18,23,[40][41][42], and Rhodococcus jostii RHA1 [28,43]. ...
... In general, bile acid degradation genes are organized in large gene clusters [24,27,28,32,51], which often contain sub-clusters for individual reaction steps such as side chain and ring degradation ( Table 1). The genomes of many bile acid-degrading Proteobacteria have a single gene cluster for bile acid degradation as found in P. stutzeri Chol1 [51] and C. testosteroni TA441 [39,52]. Others have multiple distinct gene clusters, such as Pseudoalteromonas haloplanktis [27] or Azoarcus sp. ...
Bile acids are surface-active steroid compounds with a C5 carboxylic side chain at the steroid nucleus. They are produced by vertebrates, mainly functioning as emulsifiers for lipophilic nutrients, as signaling compounds, and as an antimicrobial barrier in the duodenum. Upon excretion into soil and water, bile acids serve as carbon- and energy-rich growth substrates for diverse heterotrophic bacteria. Metabolic pathways for the degradation of bile acids are predominantly studied in individual strains of the genera Pseudomonas, Comamonas, Sphingobium, Azoarcus, and Rhodococcus. Bile acid degradation is initiated by oxidative reactions of the steroid skeleton at ring A and degradation of the carboxylic side chain before the steroid nucleus is broken down into central metabolic intermediates for biomass and energy production. This review summarizes the current biochemical and genetic knowledge on aerobic and anaerobic degradation of bile acids by soil and water bacteria. In addition, ecological and applied aspects are addressed, including resistance mechanisms against the toxic effects of bile acids.
... I -sterols; IIstenones. R = H, campesterol, campestenone; R = CH 3 , sitosterol, stigmast-4-en-3-one (β-sitostenone); XVII -androst-4-ene-3,17-dione (AD), XVIIIandrosta-1,4-diene-3,17-dione (ADD), XIX -9α-hydroxy-AD, XX -unstable 9α-hydroxy-ADD, XXI -3β-hydroxy-9,10-seco-androsta-1,3,5(10)-triene-9,17-dione (3βHSA), XXII -3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3,4-DHSA), XXIII -4,5-9,10-diseco-3-hydroxy-5,9,17trioxoandrosta-1(10),2-diene-4-oic acid (4,9-DSHA), XXIV -2-hydroxyhexa-2,4-dienoic acid (2-HHD), XXV -4-hydroxy-2-oxohexanoic acid, XXVI -9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (DOHNAA) or 3aα-H-4α-(3′-propanoate)-7aβ-methylhexahydro-1,5-indadione (HIP), XXVII -9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oyl-CoA (HIP-CoA) XXVIII -9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid or 3aα-H-4α(3′propanoate)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH-HIP), XXIX -9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oyl-CoA (5-OH-HIP-CoA), XXX 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-ene-5-oyl-CoA (HIPE-CoA), XXXI -7,9-dihydroxy-17-oxo-1,2,3,4,10,19hexanorandrostan-5-oyl-CoA, XXXII -9-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oyl-CoA, XXXIII -9-hydroxy-17-oxo-1,2,3,4,5,6,10,19octa-norandrostan-7-oyl-CoA or 3aα-H-4α(carboxylCoA)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH-HIC-CoA), XXXIV -9,17-dioxo-1,2,3,4,5,6,10,19-octa-norandrostan-7-oyl-CoA, XXXV -9-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octa-norandrost-8 (14) [6,12,14,17,20,60,67,75,76,79] might be carried out by the kstD2 KR76_27125 product. Amino acid sequence of KstD2 KR76_27125 of N. simplex differ from consensus KstD2 [86] in some residues ( Supplementary Fig. S3). ...
Background
Bacterial degradation/transformation of steroids is widely investigated to create biotechnologically relevant strains for industrial application. The strain of Nocardioides simplex VKM Ac-2033D is well known mainly for its superior 3-ketosteroid Δ ¹ -dehydrogenase activity towards various 3-oxosteroids and other important reactions of sterol degradation. However, its biocatalytic capacities and the molecular fundamentals of its activity towards natural sterols and synthetic steroids were not fully understood. In this study, a comparative investigation of the genome-wide transcriptome profiling of the N. simplex VKM Ac-2033D grown on phytosterol, or in the presence of cortisone 21-acetate was performed with RNA-seq.
Results
Although the gene patterns induced by phytosterol generally resemble the gene sets involved in phytosterol degradation pathways in mycolic acid rich actinobacteria such as Mycolicibacterium, Mycobacterium and Rhodococcus species, the differences in gene organization and previously unreported genes with high expression level were revealed. Transcription of the genes related to KstR- and KstR2-regulons was mainly enhanced in response to phytosterol, and the role in steroid catabolism is predicted for some dozens of the genes in N. simplex . New transcription factors binding motifs and new candidate transcription regulators of steroid catabolism were predicted in N. simplex .
Unlike phytosterol, cortisone 21-acetate does not provide induction of the genes with predicted KstR and KstR2 sites. Superior 3-ketosteroid-Δ ¹ -dehydrogenase activity of N. simplex VKM Ac-2033D is due to the kstDs redundancy in the genome, with the highest expression level of the gene KR76_27125 orthologous to kstD2, in response to cortisone 21-acetate. The substrate spectrum of N. simplex 3-ketosteroid-Δ ¹ -dehydrogenase was expanded in this study with progesterone and its 17α-hydroxylated and 11α,17α-dihydroxylated derivatives, that effectively were 1(2)-dehydrogenated in vivo by the whole cells of the N. simplex VKM Ac-2033D.
Conclusion
The results contribute to the knowledge of biocatalytic features and diversity of steroid modification capabilities of actinobacteria, defining targets for further bioengineering manipulations with the purpose of expansion of their biotechnological applications.
