Steroid Metabolism In Thermophilic Actinobacteria Saccharopolyspora Hirsuta Subsp. Hirsuta VKM Ac-666T

Application of thermophile microorganisms opens new prospects in steroid biotechnology, however little is known on steroid catabolism by the thermophile strains. The thermophilic Saccharopolyspora hirsuta subsp. hirsuta strain VKM Ac-666 T is capable of structural modication of different steroids, and fully degrades cholesterol. The intermediates of the cholesterol degradation pathway were identied as 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 by MS, and H 1 - and C 13 -NMR analyses. The data evidence sterol degradation by the strain occurs simultaneously through the aliphatic side chain hydroxylation at C26 and the A-ring modication that are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively. The genes orthologous to those related to the sterol side chain degradation, steroid core rings A/B and C/D disruption and the steroid uptake were revealed. Most of the genes related to steroid degradation are grouped in three clusters. The sets of the genes putatively involved in steroid catabolism and peculiarities of their organization in S. hirsuta are discussed. Only seven


Introduction
Sterols (e.g., cholesterol, ergosterol, phytosterols) are steroid 3β-alcohols with the alkyl side chain consisting of 8-10 carbon atoms. In vertebrates, the bile acids and other bioactive steroids such as corticosteroids, sex hormones and vitamin D originate from cholesterol via structural modi cation of the steroid core and decomposition of the aliphatic side chain. Structurally, bile acids differ from sterols by cis-A/B-ring juncture, α-orientation of hydroxyl at C3, saturated steroid core and a C5 acyl side chain. Actually, degradation of sterols (such as cholesterol, or phytosterols) by actinobacteria is in the focus due to its exclusive role in pathogenicity of Mycobacterium tuberculosis, as well as application of the nonpathogenic actinobacterial species and engineered strains in biotechnology for production of the valueadded steroids for the pharmaceutical industry, biomedicine and veterinary.
Sterol catabolism is a multi-step process that included cascade reactions of the side chain degradation and decomposition of the steroid core rings A/B and C/D. This pathway was reported to be controlled by two TetR-type transcriptional repressors, KstR and KstR2 (Kendall et al. 2007(Kendall et al. , 2010; Uhía et al. 2011Uhía et al. , 2012. Cholesterol catabolism has been intensively studied in mycolic acid-containing actinobacteria comprising pathogenic species  (Drzyzga et al. 2011), and also in the not-containing mycolic acid bacterium Nocardioides simplex ). Aerobic cholate degradation has been mainly studied for Rhodococcus strains (e.g. R. jostii RHA1) (Mohn et al. 2012) and gammaproteobacteria Pseudomonas sp. strain Chol1 (Philipp et al. 2006). Noteworthy, the molecular mechanisms of steroid catabolism have been studied mainly for mesophilic actinobacteria species, while little is known on the features of thermophilic actinobacteria capable of steroid oxidation. Meanwhile, application of thermophilic strains for steroid bioconversion is of great interest since it may provide economically feasible biotechnologies due to decrease the production costs for the bioreactor cooling, especially in the countries with hot climate. Besides, higher steroid solubility at the elevated temperatures is favorable for bioprocess performance.
Thermophilic microorganisms and their enzymes are widely used in the production of food products, detergents, the pulp and paper, textile and mining industries (Gallo et al. 2021). The impressive example is a Taq-polymerase (named after Thermus aquaticus) that is indispensable for PCR techniques in medicine and biology (Ishino and Ishino 2014). However, despite of steroid abundancy in the environments, little is known on structural modi cations of steroid compounds by thermophilic bacteria.
An example for this is a conversion of progesterone by the moderately thermophilic bacterium Bacillus thermoglucosidasius (syn. Parageobacillus thermoglucosidasius) resulting in 6α-/6β-hydroxy-derivatives, as well as androstenedione and testosterone (Sideso et al. 1998). Another thermophilic bacterium was reported to perform reduction of the progesterone C20 carbonyl group to 20α-or 20β-hydroxy group along with the hydroxylation at C6 (Smith et al. 1992). Regio-and stereospeci c reduction of the 3-keto group as well as the Δ4-double bond in various steroid ketones of the androstane and pregnane series was carried out by the extremely thermophilic bacterium Calderiella acidophila (Sodano et al. 1982). The strain of Geobacillus kaustophilus hydroxylated progesterone and testosterone (Al-Tamimi et al. 2010). However, the data on sterol catabolism in thermophilic bacteria are scarce (Holert et al. 2018).
In this work, cholesterol degradation by S. hirsuta VKM Ac-666 T was studied and the main intermediates were identi ed. The set of the genes putatively involved in sterol metabolism and cholate catabolism pathways, as well as their organization and clustering were revealed. The presence of the genes coding for the key enzymes accounting for steroid degradation was estimated in the genomes of thermophilic bacteria of different taxa and potent microbial steroid degraders were predicted that might function at the elevated temperatures.

Microorganism
The strain Saccharopolyspora hirsuta subsp. hirsuta VKM Ac-666 T was obtained from the All-Russian Collection of Microorganisms (VKM).

Microorganism cultivation and cholesterol conversion
The strain was cultivated on the medium GSMY (Park et al. 2005) containing (g/L): glucose -7, soluble starch -10, malt extract -5, yeast extract -4.5, CaCO 3 -0.05 (pH 7.0-7.2) aerobically (200 rpm) at 45°C for 48 h. The seed culture (5 ml) was added into 750-ml shake asks containing 50 ml GSMY medium. Cultivation was carried out aerobically on a rotary shaker (200 rpm) at 45°C for 24 h. After 24 h of cultivation, aqueous solution of cholesterol (0.5 g/l) with MCD (8.9 g/l) was added aseptically and cultivation continued at the same conditions for 144 h. The experiments were performed in triplicates.

Steroid metabolite isolation and identi cation
After 48 and 144 h of cholesterol conversion, the biomass was separated from the broth (500 mL) by centrifuge (8000× g, for 30 min). Isolation and puri cation of the steroid intermediates and products were performed as described earlier ). HPLC analyses were performed using reversedphase HPLC on Agilent In nity 1200 system (Agilent Technologies, Germany SA) with Symmetry column (250 × 4.6 mm, 5 µm) with precolumn Symmetry C18 (5 µm, 3.9 x 20 mm) (Waters, USA) at 50°C and ow rate 1 ml/min. For assays of steroids two mobile phases (acetonitrile:water:acetic acid (60:40:0.01 v/v/v/) and acetonitrile: 2-propanol: water (50:45:5, v/v/v)) with UV-detection at 200 nm (for compounds with 3β-ol-5-ene con guration) and 240 nm (for compounds with 3-oxo-4-ene con guration) were used. MS spectra of II, III and IV were recorded on a tandem mass spectrometer LCQ Advantage MAX (Thermo between the genes of the S. hirsuta subsp. hirsuta VKM Ac-666 T , Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 genomes were found using OrthoFinder 2.5.1 (Emms and Kelly 2015, 2019) with in ation parameter 1.5. Additional analysis was performed using BLAST search (Altschul et al. 1990) against the non-redundant protein database. Reciprocal BLAST was used in several cases for searching genes which corresponded to the known genes one-to-one. Conservative domains in the protein sequences were determined using a CDD database (https://www.ncbi.nlm.nih.gov/cdd/) (Lu et al. 2020). The pairwise similarity between the gene and protein sequences was determined using TaxonDC 1.3.1 (Tarlachkov and Starodumova 2017).
Search for the key genes of the steroid catabolic 9,10-seco-pathway: kstD and kshAB was carried out against several dozen available genomes of thermophilic strains using the BLAST + program (Camacho et al. 2009). The protein sequences of KstD (NP_218054.1) and KshA (NP_218043.1), KshAB (NP_218088.1) of M. tuberculosis H37Rv were used as the reference ones. The list of bacteria to be screened (Supplementary Table S1) was compiled on the basis of the literature data (Shivlata and Satyanarayana 2015) on having complete genomes or annotated contigs thermophilic and thermotolerant actinobacteria, and from available sources on other known thermophilic bacteria of diverse phylogenetic positions.
The complete genomes of Geobacillus kaustophilus and Parageobacillus thermoglucosidasius strains capable of performing some modi cations of steroid compounds were screened for the steroid catabolism genes (Supplementary Table S2) using the BLAST + program (Camacho et al. 2009).

Cholesterol bioconversion
As shown in Fig. 1, the S. hirsuta strain fully utilized cholesterol for 144 h.
Cholestenone (II) was observed as a major intermediate to reach a maximum of ~ 20 mol% for 24 h. Its abrupt decrease after 48 h was accompanied with rough accumulation of 3-oxo-cholest-4-en-26-oic acid (V) which reached highest level of ~ 17 mol% to 72 h and then drastically decreased to 2-3 mol% (Fig.  1b). 26-Hydroxycholesterol (cholest-5-ene-3β,26-diol) (VII) accumulated to achieve about 10 mol% during 72 h and then decreased. Its decrease after 72 h accompanied with the elevation in the rate of VIII (Fig.  1b). Minor amounts of cholesta-1,4-dien-3-one (III) were observed with maximum level less than 4 mol% for 24 h conversion. Noteworthy, the total steroid content after 144 h incubation was low, thus con rming degradation of the steroid core.
Based on the structures and the time-courses of the steroids detected, the following scheme of cholesterol bioconversion with S. hirsuta VKM Ac-666 T was proposed (Fig. 2).
A candidate gene encoding FadD3 lies alone in the Ac-666 T genome out of clusters. The genes choD and choE presumably encoding cholesterol oxidases were found out of clusters in Ac-666 T (Fig. 3, Table S3).
In the Ac-666 T genome, clusters 2 and 3 ( Fig. 3) contain candidate genes related to the cholate degradation pathway, namely orthologs of kshAB subunits; two orthologs of kstDs: kstD2 and kstD1; the A/B-rings opening operon hsaEGF and orthologs of hsaD3 and hsaB3; steroid delta-isomerase ksdI; a predicted transcriptional regulator kstR3; an operon that contain orthologs of genes encoding degradation of the cholate side chain casACEHI (Table S3).  Table S4).

Discussion
Several thermophilic bacteria species have been reported to catalyze distinct reactions of steroid structural modi cations, while sterol degradation by thermophilic microorganisms has not been studied so far. As shown in this research, the strain of S. hirsuta fully utilized cholesterol (Fig. 1), and the degradation pathway was predicted (Fig. 2) based on the time-courses of the intermediates (Fig. 1) and genome-wide bioinformatics analysis (Fig. 3).
The set and the order of the genes putatively involved in steroid catabolism in the clusters of S. hirsuta genome are similar to the clusters described for the reference actinobacteria: M. tuberculosis H37Rv and R. jostii RHA1 (Olivera and Luengo 2019) (Fig. 3). In general, cluster 1 in S. hirsuta is similar to the cluster of the sterol catabolic pathway in M. tuberculosis H37Rv (Fig. 3). The differences are that hsa genes in S. hirsuta are represented as a "complete" operon hsaBCDAFGE, while in M. tuberculosis H37Rv (as well as in R. jostii RHA1) the genes of this block are divided into two parts hsaFGE and hsaBCDA within the cluster genes (Fig. 3). The orthologs of kshAB in cluster 1 in the S. hirsuta genome locate close to each other (Fig. 3, Table S3); there is only one gene of an unde ned function between them, in contrast to kshA and kshB from M. tuberculosis H37Rv, which are very far from each other in mycobacterial cluster of cholesterol catabolism (Fig. 3).
Together, clusters 2 and 3 of the S. hirsuta contain the vast majority of orthologs of the cholate cluster genes from R. jostii RHA1 participating in the bile acids rings A/B degradation, but not all orthologs of the genes from R. jostii RHA1 coding for the enzymes of the bile acids side chain degradation (Fig. 3, Table  S3).
Cholesterol oxidases are most likely involved in the 3β-hydroxyl group dehydrogenation and ∆ 5 →∆ 4isomerization (I→II, VII→IV) in S. hirsuta since no candidate genes responsible for the 3β-hydroxysteroid Most of the actinobacterial ChOs are of the dual functions: catalyze both oxidation of the 3β-hydroxy group and Δ 5→4 isomerization of 3β-hydroxy-5-ene steroids. A separate Δ 5 -3-ketosteroid isomerase, encoded by the gene ksi in Comamonas testosteroni was shown to be responsible for the Δ 5→4 isomerization (Horinouchi et al. 2012). The genome of S. hirsuta contains two candidate genes ksi (ksdI). KsdI F1721_32675 is located between cyp125 F1721_32680 and ltp3-ltp4 F1721_32665-F1721_32660 in cluster 1, and another ksdI F1721_00740 is situated among the genes putatively involved in the A/B-rings oxidation: kshB3 F1721_00735 and hsaB3 F1721_00745 in cluster 2 (Fig. 3, Table S3). In the genome of N. simplex, only one of two ksdI genes, -KR76_23530 with an unclear function was up-regulated in the presence of phytosterol (Shtratnikova et al. 2021).

Side chain degradation
As well-established for many actinobacteria, the aliphatic side chain of sterols is degraded through a cascade of reactions similar to the β-oxidation of fatty acids. The C26-oic acid is activated by the coenzyme A (CoA) and then the side chain is shortened with release of two propionyl-CoAs and one acetyl-CoA (Fig. 4).
The steroid metabolite with the C5 carbon side chain is ligated further by the steroid-24-oyl-coenzyme A synthetase. The phylogenetic analysis of more than 70 acyl-CoA synthetases aimed on the elucidation of their physiological role revealed four different types of the acyl-CoA synthetases from R. jostii RHA1 and M. tuberculosis H37Rv which were speci c to the chain length of steroids (Casabon et al. 2014). FadD19 from M. tuberculosis H37Rv activated cholesterol metabolites with the C8 steroid side chain, whilst FadD17 from M. tuberculosis H37Rv the C5-or longer side chain, and CasG from R. jostii RHA1 the C5 cholate side chain. The metabolites with the C3 side chain accumulated during the cholate oxidation by R. jostii RHA1 were activated by the steroid-22-oyl-CoA synthetase CasI (Casabon et al. 2014). The orthologs of fadD19 (F1721_32635), fadD17 (F1721_32615), сasG (F1721_02405) and сasI (F1721_28770) encoding isofunctional acyl-coenzyme A synthases were revealed in the genome of S. hirsuta (Fig. 3). Probably, the presence of the homologous genes encoding various acyl-coenzyme A synthases of cholesterol and cholate catabolic pathways in S. hirsuta contributes to the adaptation of the thermophile microorganism in nature.
A special function of acyl-CoA synthetase FadD19 was reported to consist in its participation in the degradation of C24-branched sterols (sitosterol, stigmasterol, and others) as it was shown for R. rhodochrous (Wilbrink et al. 2011). The same function can be assumed for the fadD19 ortholog in the S. hirsuta (Fig. 4).
As shown for R. rhodochrous RG32, the oxidative decomposition of the phytosterols with the branched βsitosterol-like side chain is mediated by the aldol lyases encoded by ltp3 and ltp4 ). The candidate genes ltp3 (F1721_32665) and ltp4 (F1721_32660) putatively involved in the degradation of sterols with the C 24 -branched side chain have been identi ed also in S. hirsuta (Figs. 3, 4).  (Fig.  3). Interestingly, both hsd4A genes were detected in cluster 1; and each hsd4A gene is located at the beginning or at the end of a group of genes encoding the sterol aliphatic side chain cleavage in the cluster. Due to distant homology of two Hsd4As (coding by F1721_32600 and F1721_33680) with a.a. identity of the proteins about 49%, one can assume different substrate speci city for these S. hirsuta isoenzymes towards the steroids with distinct side chain length.
The role of thiolase FadA5 at the last cycle of the cholesterol side chain β-oxidation was demonstrated for M. tuberculosis H37Rv (Schaefer et al. 2015). Orthologous fadA5 (F1721_32685) is present in the genome of S. hirsuta (Fig. 3).
In the present study, detection of the intermediates with a 3-keto-1,4-diene structure such as cholesta-1,4dien-3-one (III) and 3-oxo-cholesta-1,4-diene-26-oic acid (VI) evidenced that the 1(2)-dehydrogenation can take place at the early stages of sterol catabolism in S. hirsuta (Fig. 2) Several KshAs with different substrate speci city have also been found in R. rhodochrous DSM 43269: KshA1 was shown to participate only in the cholic acid catabolism while KshA5 could hydroxylate several substrates ). The number of the genes coding for the kshA or kshB subunits depends on the strain species (Bragin et al. 2013;Shtratnikova et al. 2016). Herein, two orthologs of kshA and two orthologs of kshB were revealed in the genome of S. hirsuta (Fig. 3). One pair of the genes (kshA F1721_32745, kshB F1721_32755) is located far from the second pair (kshA F1721_00725, kshB F1721_00735). Most likely, the two KshABs might differ on their substrate speci city in S. hirsuta.
It should be noted that no any C19-steroid intermediates such as AD, ADD, testosterone, or 1(2)dehydrotestosterone were detected during the cholesterol transformation by S. hirsuta. It could be explained either by their rapid degradation when their concentrations are below the level of detection, or ring A/B disruption of the "earlier" intermediate steroids (with preserved side chain at C17). For instance, at the bile acid transformation with Rhodococcus strains, the 9,10-seco-steroid intermediates with the partially degraded side chains were formed, evidencing that side chain degradation and opening of the ring B occurred simultaneously (Costa et al. 2013a, b). The order of 9α-hydroxylation and 1(2)dehydrogenation of 3-oxo-4-ene-steroids resulting in the formation of the unstable 9α-hydroxy-3-oxo-1,4diene-intermediates depends on the substrate speci city of the corresponding enzymes.
Degradation of the C/D rings begins with the action of FadD3 which physiological role has been studied in M. tuberculosis . Unlike mycolic acid rich M. tuberculosis H37Rv and R. jostii RHA1, as well as not-containing mycolic acids N. simplex, in which genomes fadD3 encoding the HIP-CoA synthetase lies in the corresponding cluster, ortholog of fadD3 in the S. hirsuta genome is located out of clusters (Fig. 3).
The fadE30 gene encoding the acyl-CoA dehydrogenase was shown to be involved in dehydrogenation at C4 of 5-OH-HIP (Van der Geize et al. 2011). The intermediate with the intact rings C and D: 5-OH-HIC-CoA is produced by two reactions catalyzed by IpdC which introduces a double bond in the C ring and IpdF that oxidizes the 5-OH group in M. tuberculosis. Crotonase Ech20 is responsible for the hydrolytic cleavage of the ring C to give HIEC-CoA. IpdAB encodes an enzyme that hydrolytically cleaves the C ring in the substrate COCHEA-CoA ). The candidate genes: ipdAB F1721_33690-F1721_33695, ipdC F1721_33700, fadE30 F1721_33715, and echA20 F1721_33685 found in the cluster 1 of the S. hirsuta genome represent the genes presumably involved in the degradation of the rings C and D (Fig. 3).
The product of the opening of both the C and D rings is subjected to the action of a putative thiolase FadA6 resulting in acetyl-CoA and 4-methyl-5-oxo-octanedioyl-CoA (Fig. 3) (Olivera and Luengo 2019).
Search for the key genes of steroid catabolism in the genomes of thermophilic/thermotolerant bacteria In order to nd out whether steroid degraders are widespread among the thermophile bacteria, the BLAST search for the key genes of the steroid catabolic 9,10-seco-pathway, -kstD and kshAB has been performed using 52 publicly available genomes of the thermophilic/thermotolerant strains. Only seven actinobacterial strains were proposed to be steroid degraders (Supplementary Table S4). The rest thermophilic/thermotolerant strains do not contain enzymes similar to the reference ones (KstD and KshAB of M. tuberculosis H37Rv) by more than 35% and, most likely do not degrade steroids.
The thermophilic strains of G. kaustophilus and P. thermoglucosidasius were reported to provide separate reactions of steroid modi cations (Sideso et al. 1998; Al-Tamimi et al. 2010). The BLAST search of more than 20 enzymes related to steroid catabolism in these bacteria discovered putative proteins that are 47% and 45% similar to the reference FadA5, respectively, as well as enzymes of P. thermoglucosidasius that are similar to HsaF and HsaE of M. tuberculosis H37Rv by 48% and 41%, respectively (Supplementary  Table S5). Taking into consideration that FadA5 is known to be involved also in fatty acid β-oxidation, the corresponding proteins in G. kaustophilus and P. thermoglucosidasius may not be intended for steroid catabolism. Products of hsaE and hsaF participate in the oxidation of a fragment of the steroid nucleus, which is a hydroxydiene-derivative of hexanoic acid, that means that the similar genes do not necessarily participate in the catabolism of steroid compounds. Taking into account the absence of other genes coding for steroid oxidation enzymes, most likely, these enzymes of G. kaustophilus and P. thermoglucosidasius are not associated with steroid catabolism, and the oxidation/reduction and hydroxylation reactions performed by the strains (Sideso et al. 1998

Conclusions
The thermophilic strain Saccharopolyspora hirsuta subsp. hirsuta VKM Ac-666 T is capable of effective metabolizing cholesterol and lithocholic acid, as well as transforming different exogenous steroids (Kollerov et al. 2013;Lobastova et al. 2019). As con rmed in this study, the strain completely degrade cholesterol, and the 26-alcohols both with 3β-ol-5-ene and 3-keto-4-ene structures of the ring A are the key intermediates. The genes related to sterol metabolism and cholic acid catabolism were rst identi ed in the genome of this thermophilic strain. The organization of the steroid catabolism genes is generally similar to that in other actinobacteria, with some differences related to the individual genes and their grouping. Future transcriptomic and proteomic studies are of signi cance for clearer understanding of the peculiarities of steroid catabolism in the thermophilic actinobacteria.
The presence of key enzymes accounting for steroid core disruption was identi ed only in seven of 52 thermophilic bacteria of various phylogenetic positions thus suggesting that steroid degrading activity is not common in the thermophilic species. The potent microbial degraders capable of steroid degradation under the elevated temperatures were determined.
The results contribute to the knowledge on the diversity of microbial steroid degraders, the features of steroid catabolism by the thermophilic actinobacteria and could be useful for application in the steroid pharmaceutical and environmental biotechnology.

Con icts of interest/Competing interests
The authors declare no con ict of interest in this work.
Availability of data and material Not applicable.