Influence of temperature on nucleus degradation of 4-androstene-3, 17-dione in phytosterol biotransformation by <i>Mycobacterium</i>
 sp.

Xu, Xiangning; Gao, X.Q.; Feng, J.-X.; Wang, X.D.; Wei, Dongzhi

doi:10.1111/lam.12428

Cited by 16 publications

(12 citation statements)

References 24 publications

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“…Even more, it has been shown recently that mutations in the KstDs provokes a different ADD/AD molar ratio [71] and that environmental factors such as an increase of temperature can inhibit the KstD/Ksh action [72] on phytosterol in Mycobacterium sp. These multiplicity and versatility give to these enzymes a substantial role in the catalytic dehydrogenation of several related steroid molecules and pose many difficulties to clarify the particular role and way of acting of every single KstD.…”

Section: Resultsmentioning

confidence: 99%

Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

et al. 2017

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BackgroundThe Rhodococcus ruber strain Chol-4 genome contains at least three putative 3-ketosteroid Δ1-dehydrogenase ORFs (kstD1, kstD2 and kstD3) that code for flavoenzymes involved in the steroid ring degradation. The aim of this work is the functional characterization of these enzymes prior to the developing of different biotechnological applications.ResultsThe three R. ruber KstD enzymes have different substrate profiles. KstD1 shows preference for 9OHAD and testosterone, followed by progesterone, deoxy corticosterone AD and, finally, 4-BNC, corticosterone and 19OHAD. KstD2 shows maximum preference for progesterone followed by 5α-Tes, DOC, AD testosterone, 4-BNC and lastly 19OHAD, corticosterone and 9OHAD. KstD3 preference is for saturated steroid substrates (5α-Tes) followed by progesterone and DOC. A preliminary attempt to model the catalytic pocket of the KstD proteins revealed some structural differences probably related to their catalytic differences. The expression of kstD genes has been studied by RT-PCR and RT-qPCR. All the kstD genes are transcribed under all the conditions assayed, although an additional induction in cholesterol and AD could be observed for kstD1 and in cholesterol for kstD3. Co-transcription of some correlative genes could be stated. The transcription initiation signals have been searched, both in silico and in vivo. Putative promoters in the intergenic regions upstream the kstD1, kstD2 and kstD3 genes were identified and probed in an apramycin-promoter-test vector, leading to the functional evidence of those R. ruber kstD promoters.ConclusionsAt least three putative 3-ketosteroid Δ1-dehydrogenase ORFs (kstD1, kstD2 and kstD3) have been identified and functionally confirmed in R. ruber strain Chol-4. KstD1 and KstD2 display a wide range of substrate preferences regarding to well-known intermediaries of the cholesterol degradation pathway (9OHAD and AD) and other steroid compounds. KstD3 shows a narrower substrate range with a preference for saturated substrates. KstDs differences in their catalytic properties was somehow related to structural differences revealed by a preliminary structural modelling. Transcription of R. ruber kstD genes is driven from specific promoters. The three genes are constitutively transcribed, although an additional induction is observed in kstD1 and kstD3. These enzymes have a wide versatility and allow a fine tuning-up of the KstD cellular activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0657-1) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

et al. 2017

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“…(2015) have described a double (Δ kshA1 , Δ kstD1 ) mutant of Mycobacterium sp. (apparently a derivative of M. neoaurum NwIB‐02 ( ΔkstD1 ) (Wei et al ., 2010) that accumulates AD, ADD, 1,4‐HBC and 4‐HBC at different concentrations depending of fermentation temperature when cultured on phytosterols.…”

Section: Discussionmentioning

confidence: 99%

“…This blockage can be caused not only by specific mutations, but also by retro-inhibitions of the enzymes due to the accumulation of certain intermediates when the cells are cultured with high concentrations of sterols. During the course of this work, Xu et al (2015) have described a double (DkshA1, DkstD1) mutant of Mycobacterium sp. (apparently a derivative of M. neoaurum NwIB-02 (DkstD1) (Wei et al, 2010) that accumulates AD, ADD, 1,4-HBC and 4-HBC at different concentrations depending of fermentation temperature when cultured on phytosterols.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, they have also demonstrated that the residual KstD activity is not active on 9OH-AD (Xu et al, 2015) suggesting that a D 1 -dehydrogenation by KstD precedes Ksh hydroxylation. Remarkably, in contrast with our MS6039-5941 (DkshB1-DkstD1) double mutant, this M. neoaurum mutant still retains other Ksh and KstD active isoenzymes, because this mutant completely metabolized high amounts 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14. of the sterol nucleus (39.8% by moles), which considerably reduces the final conversion yield of the process (Xu et al, 2015). This result strongly reinforces our proposal that M. smegmatis can be considered as a good cell factory to produce steroid synthons by metabolic engineering.…”

Section: Discussionmentioning

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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“…C19 steroids should retain the steroidal nucleus for their effective functions. However, the steroidal nucleus is sensitive to a temperature leading to its degradation [7]. Steroidogenic enzymes were also sensitive to endocrine-disrupting chemicals (EDCs) [8].…”

Section: Chemical Synthesismentioning

confidence: 99%

Microbial Biotransformation for the Production of Steroid Medicament

Rokade¹,

Ravindran²,

Singh³

et al. 2018

Secondary Metabolites - Sources and Applications

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Androstenedione (AD) is a steroid intermediate valuable for the production of steroid medicaments. Microbial biotransformation of phytosterol to produce AD is a wellresearched area. However, low substrate solubility of phytosterol in aqueous media and nucleus degradation of AD to androstadienedione (ADD) or 9-hydroxy-AD are the major obstacles for AD production leading to detailed research for optimization of biotransformation process. In this review, microbial transformation of AD with respect to the existing methods of chemical or biochemical synthesis of AD are extensively discussed. This review examines the microbial biotransformation process and limitations for enhanced AD production. Factors affecting the effective biotransformation process to obtain AD are discussed and limitations are highlighted. The main content of this review focuses on the recent and futuristic biotechnological advances and strategies in techniques to enhance AD bioprocess.

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Influence of temperature on nucleus degradation of 4-androstene-3, 17-dione in phytosterol biotransformation by Mycobacterium sp.

Cited by 16 publications

References 24 publications

Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

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

Microbial Biotransformation for the Production of Steroid Medicament

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