Generation of Useful Insertionally Blocked Sterol Degradation Pathway Mutants of Fast-Growing Mycobacteria and Cloning, Characterization, and Expression of the Terminal Oxygenase of the 3-Ketosteroid 9α-Hydroxylase in
            <i>Mycobacterium smegmatis</i>
            mc
            <sup>2</sup>
            155

Andor, Attila; Jekkel, Antónia; Hopwood, David A.; Jeanplong, Ferenc; Ilkőy, Éva; Kónya, Attila; Kurucz, István; Ambrus, Gábor

doi:10.1128/aem.00941-06

Cited by 42 publications

(38 citation statements)

References 23 publications

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Galán

Uhía

García-Fernández

et al. 2016

Microbial Biotechnology

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SummaryA 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 mc2155 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 mc2155 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 mc2155 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.

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Section: Introductionmentioning

confidence: 99%

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

Galán

Uhía

García-Fernández

et al. 2016

Microbial Biotechnology

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“…The latter metallocenter mediates the oxygenation reaction in ROs. Gene disruption studies in Rhodococcus erythropolis SQ1 (6) and Mycobacterium smegmatis (11) have established that KshAB catalyzes the 9␣-hydroxylation of 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) to 9␣-hydroxy-4-androstene-3,17-dione (9-OHAD) and 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (HSA), respectively. However, the substrate specificity of KshAB is unclear, as is the physiological sequence of the reactions catalyzed by this enzyme and KstD.…”

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Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis

Capyk

D’Angelo

Strynadka

et al. 2009

Journal of Biological Chemistry

145

175

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KshAB (3-Ketosteroid 9␣-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O 2 . Nevertheless, the reactivity of KshAB with O 2 was low in the presence of 1,4-androstadiene-3,17-dione, with a k cat / K mO 2 of 2450 ؎ 80 M ؊1 s ؊1. The crystallographic structure of KshA, determined to 2.3 Å , revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroidtransforming enzyme of biotechnological and medical importance.Mycobacterium tuberculosis, arguably the world's most successful pathogen, infects one-third of the human population and has again become a global threat due in part to the emergence of extensively drug-resistant strains (XDR-TB) that are virtually untreatable with current medicines (1). Despite this alarming development, a surprising amount of the pathogen's physiology remains unknown. One recently discovered aspect of the physiology of M. tuberculosis is its cholesterol catabolic pathway (2). Studies of mutants in cholesterol uptake (3) and degradation (4) in various animal models have indicated that cholesterol catabolism is most important during the chronic phase of infection, although the latter study also provided evidence that it occurs from an early stage and contributes to dissemination of the pathogen in the host. Further study of cholesterol catabolism and the pathway enzymes are required to elucidate the precise role of cholesterol catabolism in infection.The cholesterol catabolic pathway of M. tuberculosis involves degradation of the branched alkyl side chain and the four-ringed steroid nucleus, as occurs in Rhodococcus jostii RHA1, a nonpathogenic, mycolic acid-producing actinomycete (2), although it is unclear whether the order of this degradation is obligatory. Side-chain degradation proceeds via a ␤-oxidative type process. Degradation of the steroid nucleus is initiated by 3␤-hydroxysteroi...

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“…In fact, knowledge of KSH at the biochemical level has been extremely limited. Steroid 9␣-hydroxylation has been reported to occur in cell extracts of Nocardia restricta (7), and a partly purified three-component enzyme system of Nocardia species M117 has been described (35 (1,3). Upon purification, however, KSH activities were lost or became too low for biochemical characterization of the enzyme.…”

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confidence: 99%

“…Steroid 9␣-hydroxylation has been reported to occur in cell extracts of Nocardia restricta (7), and a partly purified three-component enzyme system of Nocardia species M117 has been described (35). Heterologous expression of kshA of M. smegmatis mc 2 155 in Escherichia coli has recently been reported (1,3). Upon purification, however, KSH activities were lost or became too low for biochemical characterization of the enzyme.…”

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confidence: 99%

“…Subsequent ⌬1-dehydrogenation of 9␣-hydroxy-AD (9OHAD) by 3-ketosteroid ⌬1-dehydrogenase (⌬1-KSTD) initiates the opening of the B-ring through formation of a chemically unstable intermediate that spontaneously hydrolyzes, forming 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione (3-HSA). KSH activity has been observed in various actinobacterial genera, e.g., Mycobacterium (1,3,6), Nocardia (35), Arthrobacter (11), and Rhodococcus (38). In view of their amino acid sequences, KSH enzymes are predicted to belong to the class IA monooxygenases (5,38).…”

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confidence: 99%

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Rhodococcus rhodochrous DSM 43269 3-Ketosteroid 9α-Hydroxylase, a Two-Component Iron-Sulfur-Containing Monooxygenase with Subtle Steroid Substrate Specificity

Petrusma

Dijkhuizen

Geize

2009

Appl Environ Microbiol

115

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This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9␣-hydroxylase (KSH). KSH of Rhodococcus rhodochrous DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in Escherichia coli. E. coli cell cultures, expressing both KshA and KshB, converted 4-androstene-3,17-dione (AD) into 9␣-hydroxy-4-AD (9OHAD) with a >60% molar yield over 48 h of incubation. Coexpression and copurification were critical to successfully obtain pure and active KSH. Biochemical analysis revealed that the flavoprotein KshB is an NADH-dependent reductase using flavin adenine dinucleotide as a cofactor. Reconstitution experiments confirmed that KshA, KshB, and NADH are essential for KSH activity with steroid substrates. KSH hydroxylation activity was inhibited by several divalent metal ions, especially by zinc. The reconstituted KSH displayed subtle steroid substrate specificity; a range of 3-ketosteroids, i.e., 5␣-⌯, 5␤-⌯, ⌬1, and ⌬4 steroids, could act as KSH substrates, provided that they had a short side chain. The formation of 9OHAD from AD by KSH was confirmed by liquid chromatography-mass spectrometry analysis and by the specific enzymatic conversion of 9OHAD into 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione using 3-ketosteroid ⌬1-dehydrogenase. Only a single KSH is encoded in the genome of the human pathogen Mycobacterium tuberculosis H37Rv, shown to be important for survival in macrophages. Since no human KSH homolog exists, the M. tuberculosis enzyme may provide a novel target for treatment of tuberculosis. Detailed knowledge about the biochemical properties of KSH thus is highly relevant in the research fields of biotechnology and medicine.Hydroxylated steroids are pharmaceutically very interesting bioactive compounds. 9␣-Hydroxylated steroids are of particular importance for the synthesis of corticoids such as 9␣-fluorohydrocortisone. Microorganisms are widely used for the stereo-specific hydroxylation of steroids, but little is known about the enzymes involved, and current processes suffer from low conversion rates and yields (12,18,23).Rhodococcus species are well known for their broad catabolic potential and ability to degrade sterols and steroids (14,21,25,39). In this paper, we focus on 3-ketosteroid 9␣-hydroxylase (KSH), which is essential for the growth of Rhodococcus strains on steroids (38). KSH acts on the B-ring of 3-keto-⌬4 steroids, e.g., 4-androstene-3,17-dione (AD), introducing a 9␣-hydroxyl moiety (Fig. 1). Subsequent ⌬1-dehydrogenation of 9␣-hydroxy-AD (9OHAD) by 3-ketosteroid ⌬1-dehydrogenase (⌬1-KSTD) initiates the opening of the B-ring through formation of a chemically unstable intermediate that spontaneously hydrolyzes, forming 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione (3-HSA). KSH activity has been observed in various actinobacterial genera, e.g., Mycobacterium (1,3,6), Nocardia (35), Arthrobacter (11), and Rhodococcus (38). In view of their amino acid sequences, KSH ...

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Generation of Useful Insertionally Blocked Sterol Degradation Pathway Mutants of Fast-Growing Mycobacteria and Cloning, Characterization, and Expression of the Terminal Oxygenase of the 3-Ketosteroid 9α-Hydroxylase in Mycobacterium smegmatis mc ² 155

Cited by 42 publications

References 23 publications

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

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

Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis

Rhodococcus rhodochrous DSM 43269 3-Ketosteroid 9α-Hydroxylase, a Two-Component Iron-Sulfur-Containing Monooxygenase with Subtle Steroid Substrate Specificity

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Generation of Useful Insertionally Blocked Sterol Degradation Pathway Mutants of Fast-Growing Mycobacteria and Cloning, Characterization, and Expression of the Terminal Oxygenase of the 3-Ketosteroid 9α-Hydroxylase in Mycobacterium smegmatis mc 2 155

Cited by 42 publications

References 23 publications

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

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

Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis

Rhodococcus rhodochrous DSM 43269 3-Ketosteroid 9α-Hydroxylase, a Two-Component Iron-Sulfur-Containing Monooxygenase with Subtle Steroid Substrate Specificity

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Generation of Useful Insertionally Blocked Sterol Degradation Pathway Mutants of Fast-Growing Mycobacteria and Cloning, Characterization, and Expression of the Terminal Oxygenase of the 3-Ketosteroid 9α-Hydroxylase in Mycobacterium smegmatis mc ² 155