*Article* **Testosterone Degradative Pathway of** *Novosphingobium tardaugens*

#### **Juan Ibero, Beatriz Galán, Eduardo Díaz and José L. García \***

Department of Microbial and Plant Biotechnology, Centro de Investigaciones Biológicas, Agencia Estatal Consejo Superior de Investigaciones Científicas. Ramiro de Maeztu 9, 28040 Madrid, Spain; jicaballero@cib.csic.es (J.I.); bgalan@cib.csic.es (B.G.); ediaz@cib.csic.es (E.D.)

**\*** Correspondence: jlgarcia@cib.csic.es

Received: 4 September 2019; Accepted: 28 October 2019; Published: 31 October 2019

**Abstract:** In this work, we have shown that *Novosphingobium tardaugens* NBRC 16725 (strain ARI-1), a bacterial strain that was isolated due to its capacity to mineralize the estrogenic endocrine compound 17β-estradiol, is also able to mineralize testosterone, the androgenic endocrine compound. Using in silico analysis, we predicted a new putative steroid degradation (SD) gene cluster in strain ARI-1, which resembles genes involved in testosterone degradation in *Comamonas testosteroni* and other testosterone degrading bacteria like Actinobacteria (like *Rhodococcus* and *Mycobacteria* genera) although with significant differences in gene organization. A whole transcriptomic analysis of *N. tardaugens* revealed that testosterone produces a limited induction of the genes of the SD cluster that show a high basal expression in its absence. The 3β/17β-hydroxysteroid dehydrogenase involved in the first metabolic step of testosterone degradation was identified by using genetic and biochemical approaches. The construction of knockout mutant strains in the genes of the SD cluster together with in silico analyses suggests the existence of gene redundancy in the genome of *N. tardaugens.* This work will expand the knowledge about the metabolic pathways and biotransformation capabilities of a Gram-negative bacterium that could become a new model system in the bacterial steroid degradation field.

**Keywords:** testosterone; steroid; catabolism; transcriptomic; biodegradation
