Molecular and functional characterization of kshA and kshB, encoding two components of 3‐ketosteroid 9α‐hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1

Geize, Robert van der; Hessels, G.I.; Gerwen, Rick van; Meijden, Peter van der; Dijkhuizen, Lubbert

doi:10.1046/j.1365-2958.2002.03069.x

Cited by 112 publications

(59 citation statements)

References 43 publications

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“…In contrast to the frequent observation that the components of bacterial multicomponent oxygenases are encoded in a single operon, the kshA and kshB genes encoding Ksh activity in Rhodococcus (van der Geize et al ., 2002a) or in M. tuberculosis (Cole et al ., 1998) are located far from each other in the genome. Nevertheless, using the sequence of the annotated kshA and kshB genes from Rhodococcus (van der Geize et al ., 2002a, 2008) and M. tuberculosis (Capyk et al ., 2009) as probes, we have localized the corresponding orthologues in M. smegmatis (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…the best characterized cholesterol‐degrading organism) to produce AD, ADD and 9α‐hydroxy‐4‐androstene‐3,17‐dione (9OH‐AD) from natural sterols (e.g. cholesterol or phytosterols), but these mutants have not been used at industrial scale yet (van der Geize et al ., 2000, 2001a,b, 2002a,b, 2008; Wilbrink et al ., 2011; Yeh et al ., 2014). …”

Section: Introductionmentioning

confidence: 99%

“…The 3‐ketosteroid 9α‐hydroxylase (Ksh) has been proposed as the key enzyme for ring‐B opening, and therefore, the removal of this activity should render ADD as end‐product. The Ksh enzymes of Rhodococcus and Mycobacterium have been characterized as two‐component monooxygenases, composed of an oxygenase (KshA) and a ferredoxin reductase (KshB) (van der Geize et al ., 2002a; Capyk et al ., 2009, 2011; Petrusma et al ., 2009, 2012, 2014; Hu et al ., 2010; Bragin et al ., 2013; Penfield et al ., 2014). Therefore, theoretically, a deletion mutant of one of the Ksh encoding genes, i.e.…”

Section: Introductionmentioning

confidence: 99%

“…(2002a, 2008) have demonstrated that Ksh mutants of R. erythropolis SQ1 deleted in kshA or kshB genes can produce ADD from AD, but surprisingly they do not accumulate AD, ADD or other metabolites during sterol conversion. Interestingly, the degradation of phytosterols was not impaired in a kshA ‐ mutant and rates of degradation were comparable to those of the parent strain, suggesting that Rhodococcus has alternative enzymes to catabolize phytosterols.…”

Section: Introductionmentioning

confidence: 99%

“…Interestingly, the degradation of phytosterols was not impaired in a kshA ‐ mutant and rates of degradation were comparable to those of the parent strain, suggesting that Rhodococcus has alternative enzymes to catabolize phytosterols. Moreover, the kshB ‐ mutant failed to cleave the side‐chain of sterols and, although phytosterols were oxidized to their stenone derivatives, they were not metabolized further, suggesting that kshB could be also involved in the degradation of sterol side‐chain in Rhodococcus (van der Geize et al ., 2002a). The situation can be more complex taking into account that some strains of Rhodococcus may contain up to five KshA homologous proteins, each displaying unique steroid induction patterns and substrate ranges, confirmed that the 9α‐hydroxylation can take place at different steps of steroid oxidation (Petrusma et al ., 2011).…”

Section: Introductionmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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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3‐Ketosteroid‐9α‐Hydroxylase

Capyk

Strynadka

Eltis

2004

Handbook of Metalloproteins

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KshAB is a Rieske oxygenase (RO) in the aerobic steroid degradation pathways of bacteria. It is a monooxygenase responsible for the 9α‐hydroxylation of the nucleus of 3‐keto‐4‐ene steroids bearing short side chains at C17. It is composed of two domains arranged in a head‐to‐tail fashion to form the functional trimer. The N‐terminal Rieske domain harbors a Fe 2 S 2 cluster in which the two irons are coordinated by two cysteine and two histidine residues, respectively. The larger, C‐terminal catalytic domain contains a mononuclear nonheme iron coordinated by a His‐His‐Asp facial triad. Purified KshAB from Mycobacterium tuberculosis exhibits substrate specificities for 1,4‐androstadien‐3,17‐one and dioxygen 2‐3 orders of magnitude lower than those measured for other ROs and their best substrates. In agreement with the large fused‐ring substrate, the structure of KshA incorporates an active site channel and substrate binding pocket that are longer than those observed in other ROs, and positioned in a different relative orientation within the catalytic domain. KshA also possesses a C‐terminal helix which occupies a position not observed in other RO structures and which likely stabilizes trimeric quaternary structure. Finally, KshA exhibits a minimal core catalytic domain with respect to secondary structural elements that may prove archetypical for ROs.

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