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

Abstract

A number of pharmaceutical steroid synthons are currently produced through the microbial side-chain cleavage of natural sterols as an alternative to multi-step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc² 155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc² 155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3-ketosteroid 9α-hydroxylase (KshAB) and a ketosteroid Δ¹ -dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, we have constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4-androstadiene-3,17-dione. In addition, we have demonstrated that a double deletion mutant M. smegmatis MS6039-5941 [ΔMSMEG_6039 (ΔkshB1) and ΔMSMEG_5941 (ΔkstD1)] transforms natural sterols into 4-androstene-3,17-dione with high yields. These findings suggest that the catabolism of cholesterol in M. smegmatis mc² 155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids.

Figure 1

Proposed pathway for cholesterol degradation in Mycobacterium smegmatis. Cholest‐4‐en‐3‐one or any of the subsequent metabolites from degradation of the side‐chain up to (and including) AD may undergo a dehydrogenation reaction to introduce a double bond in the position 1, leading to compound cholest‐1,4‐diene‐3‐one in the case of cholest‐4‐en‐3‐one, or to the corresponding 1,2‐dehydro derivatives for other molecules. The side‐chain degradation of this compounds will be identical to that of the cholest‐4‐en‐3‐one to the common intermediate 9α‐hydroxyandrosta‐1,4‐diene‐3,17‐dione (9OHAD). 3β‐hydroxysteroid dehydrogenase (3β‐HSD), 3‐ketosteroid‐Δ¹‐dehydrogenase (KstD), 3‐ketosteroid‐9α‐hydroxylase (KshAB), 3‐hydroxy‐9,10‐secoandrosta‐1,3,5(10)‐trien‐9,17‐dione (3‐HSA), 3,4‐dihydroxy‐9,10‐secoandrosta‐1,3,5(10)‐trien‐9,17‐dione (3,4‐HSA), 4,5,9,10‐diseco‐3‐hydroxy‐5,9,7‐trioxoandrosta‐1(10),2‐diene‐4‐oic acid (4,9‐DSHA), 3aα‐H‐4α(3′‐propionic acid)‐7aβ‐methylhexahydro‐1,5‐indanedione (HIP), 3aα‐H‐4α(3′‐propionic acid)‐5α‐hydroxy‐7β‐methylhexahydro‐1‐indanone (5OH‐HIP), 3‐hydroxy‐9,10‐secoandrosten‐1,3,5(10)‐trien‐9,17‐dione dyoxigenase (HsaC), 4,5,9,10‐diseco‐3‐hydroxy‐5,9,7‐trioxoandrosta‐1(10),2‐diene‐4‐oic acid hydroxylase (HsaD), 2‐hydroxy‐2,4‐hexadienoic acid hydratase (HsaE), 4‐hydroxy‐2‐hydroxy‐2‐ketovalerate aldolase (HsaF), acetaldehyde dehydrogenase (HsaG), HIP‐CoA transferase (LpdAB and FadD3), FadE30 (Acyl‐CoA dehydrogenase).

Figure 2

Growth curves of M. smegmatis mc²155, mutants and complemented strains cultured in shake flasks with different carbon sources. (A) Strains cultured with 0.4 g l⁻¹ of cholesterol (mc²155 (squares), MS6039 (circles) and MS6039‐5941 (triangles)); (B) Strains cultured with 18 mM glycerol (mc²155 (squares), MS6039 (circles) and MS6039‐5941 (triangles)); (C) Strains cultured with 0.4 g l⁻¹ of phytosterols (mc²155 (squares), MS6039 (circles) and MS6039‐5941 (triangles)); (D) Gene complementation of MS6039 mutant strain cultured with 1.8 mM of cholesterol (mc²155 (pMV261) (diamonds), MS6039 (pMV261) (squares) and MS6039 (pMV6039) (circles)). The data reported are the averages of three different assays.

Figure 3

Production of ADD by the MS6039 mutant in shake flasks. ADD is represented by triangles. (A) 9 mM glycerol + 0.4 g l⁻¹ of cholesterol (circles) used as substrates; (B) 9 mM glycerol + 0.4 g l⁻¹ of phytosterols (diamonds) used as substrates; (C) Analysis by CG‐MS of the products after 96 h of incubation on phytosterols. (1) ADD, (2) cholestenone (internal standard), and (3) 1,4‐HBC. (D) Chemical structure and fragmentation pattern of 1,4‐HBC. The data reported are the averages of three different assays.

Figure 4

Production of AD by the MS6039‐5941 mutant in shake flasks. AD is represented by squares. (A) 9 mM glycerol + 0.4 g l⁻¹ of cholesterol (circles) used as substrates; (B) 9 mM glycerol + 0.4 g l⁻¹ of phytosterols (diamonds) used as substrates; (C) Analysis by GC/MS of the products after 96 h of culture on phytosterols. (1) AD, (2) cholestenone (internal standard) and (3) 4‐HBC. (D) Chemical structure and fragmentation pattern of 4‐HBC. The data reported are the averages of three different assays.