Steroid Metabolism in Thermophilic Actinobacterium Saccharopolyspora hirsuta VKM Ac-666T

Abstract

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-666^T 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.

Keywords: Saccharopolyspora hirsuta; bioconversion; cholate; steroids; sterol catabolism; thermophilic actinobacteria.

Figure 1

S. hirsuta growth at 20–60 °C for 24 h.

Figure 2

Cholesterol bioconversion by S. hirsuta VKM Ac-666^T. Thin-layer chromatography (TLC) chromatogram of 3-keto-4-ene steroids (A), visualization under ultraviolet (UV) light (254 nm), cholest-4-en-3-one as a reference compound; TLC chromatogram of 3β-hydroxycholest-5-ene steroids (B), visualization after phosphomolybdic acid staining, cholesterol as a reference compound; time course of cholesterol consumption (C); time course of the intermediates/metabolites of cholesterol bioconversion (D). The data are the averages of triplicates. I, cholesterol (cholest-5-ene-3β-ol); II, cholest-4-en-3-one; III, cholesta-1,4-dien-3-one; IV, 26-hydroxycholest-4-en-3-one; V, 3-oxo-cholest-4-en-26-oic acid; VI, 3-oxo-cholesta-1,4-dien-26-oic acid; VII, 26-hydroxycholesterol (cholest-5-ene-3β,26-diol); VIII, 3β-hydroxy-cholest-5-en-26-oic acid.

Figure 3

Scheme of cholesterol bioconversion by S. hirsuta VKM Ac-666^T. Compounds: I, cholesterol; II, cholest-4-en-3-one; III, cholesta-1,4-dien-3-one; IV, 26-hydroxycholest-4-en-3-one; V, 3-oxo-cholest-4-en-26-oic acid; VI, 3-oxo-cholesta-1,4-dien-26-oic acid; VII, 26-hydroxycholesterol; VIII, 3β-hydroxycholest-5-en-26-oic acid. Biochemical reactions: 1, 3β-hydroxyl group dehydrogenation and ∆⁵→∆⁴-isomerization; 2, 3-oxo-4-ene-steroid 1(2)-dehydrogenation; 3, C26(27)-hydroxylation; 4, C26-alcohol hydroxylation; 5, oxidative side-chain degradation.

Figure 4

Organization of the S. hirsuta VKM Ac-666^T genes putatively involved in cholesterol and cholic acid catabolism. For comparison, the organization of the corresponding genes of Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 [5] is shown. Genes related to cholesterol or bile acid side chain degradation are shown in green; genes related to A/B-rings degradation are shown red (cholesterol catabolism) and orange (cholic acid catabolism); genes coding for C/D-ring degradation are shown purple; blue color indicates genes coding for transport systems; regulatory elements are indicated yellow. I, II, and III are the S. hirsuta gene clusters discussed in the text.

Figure 5

Biochemical scheme proposed for cholesterol catabolism in S. hirsuta VKM Ac-666^T. Genes encoding respective proteins are denoted. (A) Modification of 3β-ol-5-ene to 3-keto-4-ene moiety in the A-ring of the steroid core and degradation of the sterol side chain to C₁₉-steroids. (B) Steroid core modifications. (C) Steroid core degradation via the 9(10)-seco pathway. 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-4,24-dien-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-4-en-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-4-en-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, 4,5-9,10-diseco-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-7aβ-methylhexahydro-1-indanone (5-OH-HIP); XXX, 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oyl-CoA (5-OH-HIP-CoA); XXXI, 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-ene-5-oyl-CoA (5-OH-HIPE-CoA); XXXII, 7,9-Dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oyl-CoA; XXXIII, 9-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oyl-CoA; 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, 9,17-dioxo- 1,2,3,4,5,6,10,19-octa-norandrostan-7-oyl-CoA; XXXVI, 9-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octa-norandrost-8(14)-en-7-oyl-CoA; XXXVII, 9,17-dioxo-1,2,3,4,5,6,10,19-octa-norandrost-8(14)-en-7-oyl-CoA or 7a-methyl-1,5-dioxo-2,3,5,6,7,7a-hexahydro-1H-indene-4-carboxylic acid (HIEC-CoA); XXXVIII, 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid-CoA-ester or (R)-2-(2-carboxyethyl)-3-methyl-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].

Figure 6

Dendrogram showing the phylogeny of KstD homologs. The tree was drawn to scale, with branch lengths measured in substitutions per site. Bootstrap values (based on 1000 replications) are indicated at the branch points.