Preparation of androsta-1,4-diene-3,17-dione from sterols using <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis> VKPM Ac-1656 strain

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM Ac-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI₃ in situ.     

Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) in Mycobacterium neoaurum

Abstract Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 μg/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 μg/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 μg/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.     

A Keto Reductase Involved in Steroid Degradation in Mycolicibacterium neoaurum

Phytosterols can be used by microorganisms as carbon and energy sources and completely degraded into CO₂ and H₂O. The catabolic pathway of phytosterols was well characterized in many microorganisms. Blocking the steroid core ring degradation by deletions of fadE30 and fadD3 genes, two important steroid intermediates, 3aα-H-4α-(3’-Propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone-δ-lactone (sitolactone, or HIL) and 3aα-H-4α-(3’-propionic acid)-7aβ-methylhexahydro-1,5-indanedione (HIP) can be accumulated. They are currently used to synthesize nor-steroid drugs with an α-methyl group or without the methyl group at the C₁₀-position, such as estrone and norethindrone. In this study, a key gene involved in the bioconversion of HIP to HIL was identified in Mycolicibacterium neoaurum. Through heterologous expression, gene hipR was found to be involved in the reduction of the C₅ keto group of HIP to a hydroxy group, leading to spontaneously lactonization into HIL in vitro. Through gene complementation and knockout, HipR functions were verified and two HIP degradation pathways in vivo were elucidated. The finding of this research facilitated the understanding of the metabolic pathway of sterols, and was directly applied to engineering robust production strains by overexpression or knockout of related genes.