Mycobacterium sp. mutant strain producing 9α-hydroxyandrostenedione from sitosterol

Abstract

Mycobacterium sp. VKM Ac-1815D and its derivatives with altered resistance to antibacterial agents were able to produce androst-4-ene-3,17-dione (AD) as a major product from sitosterol. In this study, those strains were subjected to subsequent mutagenization by chemical agents and UV irradiation in combination with sitosterol selection pressure. The mutant Mycobacterium sp. 2-4 M was selected, being capable of producing 9alpha-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol, with a 50% molar yield. Along with 9-OH-AD, both AD and 9alpha-hydroxylated metabolites with a partially degraded side-chain were formed from sitosterol by the mutant strain. The strain was unable to degrade 9-OH-AD, but degraded androsta-1,4-diene-3,17-dione (ADD), thus indicating a deficiency in steroid 1(2)-dehydrogenase and the presence of 9alpha-hydroxylase activity.

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

“…These synthons can be produced by microbial side‐chain cleavage of cholesterol or phytosterols as an alternative to multi‐step chemical synthesis based on digoxigenin, a steroid found exclusively in the flowers and leaves of Digitalis plants, as a starting material. Industrially, AD and ADD have been produced through fermentative processes using wild microorganisms that have been subsequently modified and optimized by conventional mutagenic procedures (Donova et al ., 2005; Andor et al ., 2006; Donova and Egorova, 2012; García et al ., 2012). Mycobacterium spp.…”

“…Mycobacterium spp. NRRL 3805B and 3683B capable of forming AD and ADD from sterols, respectively, are examples of these mutants used at industrial scale (Donova et al ., 2005; Donova and Egorova, 2012). A drawback of the AD and ADD industrial production based on these wild strains is the usual and concomitant accumulation of unwanted by‐products which hinder downstream processes.…”

From Soybean Phytosterols to Steroid Hormones

“…Traditional mutagenesisscreening techniques have long been employed as a practical means of producing C19-steroid-producing strains (Donova, et al, 2005c;Huang, et al, 2006;Gulla, et al, 2010). Nevertheless, C19 steroids such as AD, ADD, and testosterone are known to coexist in products (Donova, et al, 2005c;Huang, et al, 2006). This is one of the more significant deficiencies between the strains selected from random mutations; the subtle differences in structure between C19 steroids make them difficult to separate in industrial applications.…”

“…Most phytosterol-transforming strains can transform phytosterols into many structurally similar products, such as the C19 steroids AD, ADD, and their derivatives, at the same time (Egorova, et al, 2002;Donova, et al, 2005aDonova, et al, , 2005bDonova, et al, , 2005cMolchanova, et al, 2007). Because the differences in the structures of these products are very subtle, it is difficult to separate and purify them.…”

“…In theory, the production of AD can be achieved via the inactivation of both Kshs and KstDs in a microbial cell, and the accumulation of ADD or 9 -OHAD can be achieved by disrupting the Ksh or KstD genes. Traditional mutagenesisscreening techniques have long been employed as a practical means of producing C19-steroid-producing strains (Donova, et al, 2005c;Huang, et al, 2006;Gulla, et al, 2010). Nevertheless, C19 steroids such as AD, ADD, and testosterone are known to coexist in products (Donova, et al, 2005c;Huang, et al, 2006).…”