**3. Results**

#### *3.1. Catabolism of C-19 Compounds in N. tardaugens*

Although the ability of *N. tardaugens* strain ARI-1 to grow in E2 as a sole carbon and energy source has been described [24], nothing was known regarding its capacity to mineralize other C-19 steroids, such as TES, AD, ADD and DHEA. Figure 2a shows that *N. tardaugens* is able to mineralize these compounds. Moreover, we were unable to detect by TLC any of these steroids after 18 h cultivation (39 h in case of DHEA) (Figure 2b). Interestingly, strain ARI-1 was also able to grow in the xenobiotic steroid compound TES-Ac generating TES as transient intermediate (Figure 2a,b). Taken together all these results reveal that *N. tardaugens* contains an e fficient aerobic degradative pathway for C-19 steroids.

**Figure 2.** (**a**) Bacterial growth (Log2OD600) of *N. tardaugens* NBRC 16725 when cultured in M63 minimal medium containing 1.89 mM AD (purple), 1.89 mM ADD (yellow), 1.89 mM TES (red), 1.89 mM DHEA (light blue), 1.71 mM TES-Ac (green), 2 mM E2 (dark blue) and 13.33 mM CDX (grey) and (**b**) TLC analysis of the organic extraction of the culture of *N. tardaugens* along time growing in: (**i**) AD, (**ii**) ADD, (**iii**) TES, (**iv**) DHEA and (**v**) TES-Ac. The AD, ADD, TES, DHEA and TES-Ac standards (Std.) (1 mM) are also shown.

#### *3.2. In Silico Identification of N. tardaugens Genes for Catabolism of C-19 Compounds*

In silico analysis of the assembled *N. tardaugens* genome (4,358,096 bp) [29] using as query the amino acid sequences of the coding genes responsible for TES degradation in *C. testosteroni* TA441 revealed the existence of a putative steroid degradation gene cluster covering a 26.4 kb region (*EGO55\_13795*-*EGO55\_13670* (Figure 3). The predicted SD cluster is organized in two regions that are transcribed divergently, i.e., *EGO55\_13690*-*EGO55\_13670* and *EGO55\_13695*-*EGO55\_13795* (Figure 3b). In contrast to *C. testosteroni*, where the TES degradative genes are organized in two di fferent clusters (Figure 3a), *N. tardaugens* shows a more compact gene organization.

Remarkably, the four genes essential for the three initial steps of C-19 steroid catabolism, i.e., the *hsd* gene encoding the 3β/17β-hydroxysteroid dehydrogenase (3β/17β-HSD), the *kstD* gene encoding the ketosteroid dehydrogenase, and the *kshA* and *kshB* genes encoding the oxidase and reductase subunits of the 9α-ketosteroid hydroxylase, respectively (Figure 1), are not contained within this degradation cluster. Interestingly, several genes homologous to *kstD*, *kshA* and *hsd* from *C. testosteroni* have been found distributed along the genome of *N. tardaugens* (Table S5). However, only one gene, *EGO55\_04915*, encoded a protein showing identity (33%) with the reductase subunit of the ketosteroid hydroxylase *ORF17* (*kshB*) of *C. testosteroni*. Finally, a TeiR-like regulatory protein which is a positive regulator that induces the expression of the TES cluster in *C. testosteroni* [39], is not present in the *N. tardaugens* genome. Furthermore, the absence of other putative regulatory genes located near the SD cluster sugges<sup>t</sup> either the absence of a specific regulation by TES or at least a different transcriptional regulation in this bacterium.

We have tried to demonstrate the involvement of the SD cluster in the degradation of C-19 compounds by constructing two *N. tardaugens* knockout mutant strains in two representative genes of the SD cluster, i.e., the *EGO55\_13795* (*fadD3*) and *EGO55\_13685* (*tesD*) genes. When both mutants were grown in TES as the sole carbon and energy sources their growth was not impaired (Figure S2). Interestingly, the analysis of the *N. tardaugens* genome revealed the existence of several homologous genes of the SD cluster located along the chromosome (Table S5) suggesting that some reactions involved in C19 steroid degradation could be replaced by such homologous genes. Thus, the analysis of the *N. tardaugens* genome revealed the existence of 9 *fadD3* and 3 *tesD* homologous genes (Table S5) that would explain the observed growth phenotype of the *fadD3* and *tesD* mutant strains.

#### *3.3. Whole Transcriptomic Analysis of N. tardaugens Grown in Testosterone*

To determine the expression of the genes involved in the degradation of C-19 steroids we performed RNA-seq analyses in *N. tardaugens* cultured using pyruvate (control condition) or TES as carbon sources. Differential expression analysis yielded 2046 differentially expressed genes (DEGs) (from 3980 total genes in genome), where 863 were up regulated and 1183 were down regulated in TES condition compared to pyruvate (being FC 2 or −2, respectively, the cut-off value) (Table S6), showing a noticeable contrast in differential expression pattern (Figure 4). The functional classification of DEGs in different GO terms is shown in Figure 5. The highest level of up regulation (fold change (FC) > 10) was observed in 49 genes (Table S6) but other 111 genes were notably up regulated (5 < FC < 10) (Table S6).

**Figure 4.** Heatmap diagram of cluster analysis showing the log2 mean normalize expression in each experimental growth condition for those genes where a FC > 2 and FC < −2 was observed.

**Figure 5.** GO enrichment bar chart of upregulated genes (DEG) representing the number of DEGs enriched in biological process, cellular component and molecular function. Colors represent different GO types: biological process (green) and molecular function (orange). The term with a star "\*" is significantly enriched term (corrected *p*value < 0.05).

The data obtained from the transcriptomic analysis show a slight di fferential induction of the SD cluster in the presence of TES with respect to the pyruvate control condition (Table S6). Nevertheless, it is important to notice that the basal expression level of these genes in pyruvate condition is already high when compared to that of housekeeping genes. The level of induction of the genes included in the *EGO55\_13695*-*EGO55\_13795* genes is slightly higher than those of the *EGO55\_13690*-*EGO55\_13670* genes (Table S6). Several genes showed a fourfold increase in expression levels: *EGO55\_13735*, *EGO55\_13740*, *EGO55\_13745* and *EGO55\_13750*. FC values of previously identified genes homologous to *kstD* and *kshA* from *C. testosteroni* TA441 (Table S5), allowed us to propose *EGO55\_13510* (3.5-fold induction) and *EGO55\_13445* (7.8-fold induction), as the KstD and KshA involved in TES degradation pathway in *N. tardaugens*, respectively (Figure 1). These *kstD* and *kshA* genes are located approximately 30 and 50 kb away from the SD cluster, respectively. Additionally, closed to the predicted kshA coding gene (*EGO55\_13445*) there is a predicted TesA2 coding gene (*EGO55\_13440*) that is induced 7.2-fold, which is a higher value than the one located within the SD cluster. Also the gene *EGO55\_15045*, encoding a putative TesD homolog, showed higher induction than the corresponding gene in the SD cluster. These sugges<sup>t</sup> that *EGO55\_13440* and *EGO55\_15045* genes could be involved in TES degradation in *N. tardaugens*.

Interestingly, *EGO55\_01995*, *EGO55\_02005*, *EGO55\_02015* and *EGO55\_02020* genes, encoding a putative propionyl-CoA carboxylase biotin-containing subunit, a methylmalonyl-CoA mutase, a methylmalonyl-CoA epimerase, and a propionyl-CoA carboxylase carboxyl transferase subunit, respectively, are found among the most upregulated genes in TES condition (Table S6). This result allowed us to identify a cluster (*EGO55\_01990*-*EGO55\_02025*) containing genes showing a high level of identity to those experimentally described as responsible of the methylmalonyl degradation pathway for propionic acid degradation [40–44] (Table S7). Furthermore, this cluster also contains a putative biotin synthase (*EGO55\_02000*) that is upregulated, in agreemen<sup>t</sup> with the requirement of B7 cofactor for propionyl-CoA carboxylase activity [45] needed to metabolize propionyl-CoA, presumably generated in TES degradation (Figure 1).

As shown in Table S6, up to 19 genes annotated as involved in B12 cofactor biosynthesis pathway are significantly upregulated in TES condition. Figure 5 also shows that the highest number of upregulated genes in TES condition belong to cobalamin biosynthetic and metabolic processes, water-soluble vitamin biosynthesis, vitamin biosynthetic process and vitamin and water-soluble vitamin metabolic processes GO terms. High expression levels of genes involved in cobalamin synthesis pathway correlates with the requirement of this cofactor by the methylmalonyl-CoA mutase, which is upregulated in TES condition as indicated above (Table S6).

#### *3.4. Identification of the Initial Biochemical Step of TES Degradation Pathway in N. tardaugens*

In *C. testosteroni*, the degradation of TES starts by the dehydrogenation of the 17β-hydroxyl group to render AD. This step is catalysed by a short chain dehydrogenase 3β/17β-HSD, a tetrameric NAD(H)-dependent reversible enzyme [46]. Comparative gene analyses yielded up to 16 proteins homologous to the 3β/17β-HSD of *C. testosteroni* in *N. tardaugens* genome (Table S5). Due to this gene redundancy, we looked at the induction fold of those genes in the presence of TES. Among the 16 genes the *EGO55\_02235* was slightly induced 2-fold and it is located in tandem with *EGO55\_02230* encoding also a putative 3β/17β-HSD. These isoenzymes, Hsd60 and Hsd70, show 80% amino acid sequence identity. They form a putative four-gene operon together with the *EGO55\_02225* gene coding for a putative esterase and the *EGO55\_02240* gene annotated as a permease of the major facilitator superfamily. To prove the involvement of Hsd60 and Hsd70 in TES catabolism, a double knock out mutant was produced, where *EGO\_02230* and *EGO\_02235* genes were deleted. The mutant strain was grown in TES as sole carbon and energy source showing that its growth was not impaired (Figure S2). This result is not surprising given the number of homologous genes found in *N. tardaugens* genome (Table S5) that could be replacing the enzymatic activity of the deleted genes.

To further determine the putative role of Hsd60 and Hsd70 in TES metabolism the *EGO55\_02230* and *EGO55\_02235* genes were cloned in the pET29a vector and the resulting plasmid, named pET29Hsd70-Hsd60, was transformed in *E. coli* BL21 (DE3) cells to overproduce both enzymes (Figure S1). Enzymatic assays using crude extracts from *E. coli* BL21 (DE3) (pET29Hsd70-Hsd60) cells revealed a 17β-HSD reaction converting TES to AD (Figure 6a) and E2 to E1 (Figure 6b). Same enzymatic assays using DHEA and PREG as substrates showed that the catalytic activity present in the crude extract is able to transform them (according to their mobility in TLC) into the expected keto compounds, i.e., Δ5 androstadione and isoprogesterone, respectively (Figure 6c,d). The crude extract showed a lower activity when PREG (Figure 6d) was used as substrate, suggesting that the C-17 chain of PREG impairs the recognition of substrate. In this sense, no activity was detected when cholesterol with a long C-17 side chain was used as substrate.

**Figure 6.** TLC analysis of the enzymatic reaction of crude extracts from *E. coli* BL21(DE3) cells harbouring pETHsd70-Hsd60, pETHsd70 and pETHsd60 transforming (**a**) TES, (**b**) E2, (**c**) DHEA and (**d**) PREG. In (**d**) the lower panel shows reduced products revealed with UV light. Standards of AD, TES, E2, E1, DHEA and PREG were also added. The use of inductor (IPTG) for overexpression is indicated. The molecular structure of the steroidal compounds involved in the reductive reactions are represented.

Once the 3β/17β-HSD activity was determined within these cell extracts, the genes encoding Hsd70 and Hsd60 were cloned separately into the pET29a vector to explore their individual catalytic abilities. The resulting plasmids, pETHsd70 and pET29Hsd60, were transformed in *E. coli* BL21 (DE3) cells. Enzymatic assays using crude extracts showed, interestingly, that Hsd60 enzyme catalyzes more efficiently the dehydrogenation of C17-OH (Figure 6a,b), whereas Hsd70 catalyzes more e fficiently the dehydrogenation of C3-OH (Figure 6c,d).
