Creation and Characterization of Mycolicibacterium Smegmatis mc²155 with Deletions in Genes Encoding Sterol Oxidation Enzymes

¹ G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”, Pushchino, Russian Federation
² Pushchino State Natural Science Institute, Pushchino, Russian Federation

^* Corresponding author: artyom.byakov.wrk@yandex.ru

Abstract

The fast-growing saprotrophic strain Mycolicibacterium smegmatis mc²155 is capable of utilizing plant and animal sterols and can be used for creation of genetically engineered strains producing biologically active steroids. Oxidation of the 3β-hydroxyl group and Δ⁵⁽⁶⁾→Δ⁴⁽⁵⁾ double bond isomerization followed by formation of stenones from sterols are considered as the initial stage of steroid catabolism in some actinobacteria. The study of the mechanism of steroid nucleus 3β-hydroxyl group oxidation is relevant for the creation of a method of the microbiological production of valuable 3β-hydroxy-5-en-steroids. A mutant strain of M. smegmatis with deletions in three genes (MSMEG_1604, MSMEG_5228 and MSMEG_5233) encoding known enzymes exhibiting 3β-hydroxysteroid dehydrogenase activity was constructed by homologous recombination coupled with double selection. The resulting mutant retained macromorphological properties and the ability to convert cholesterol. 3-Keto-4-en-steroids were found among the sterol catabolism intermediates. Experimentally obtained data indicate the presence of a previously undetected intracellular enzyme that performs the function of 3β-hydroxysteroid dehydrogenase/Δ⁵⁽⁶⁾→Δ⁴⁽⁵⁾ isomerase.