Genetic differences in <i>ksdD</i> influence on the ADD/AD ratio of <i>Mycobacterium neoaurum</i>

Xie, Rili; Shen, Yanbing; Qin, Ning; Wang, Yibo; Su, Liqiu; Wang, Min

doi:10.1007/s10295-014-1577-2

Cited by 29 publications

(11 citation statements)

References 22 publications

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“…Microorganisms and culture conditions. M. neoaurum M3C2 is the ksdD gene replacement strain M. neoaurum M3ΔksdD::ksdD-MNR constructed by homologous recombination (52). M. neoaurum M3C2 possesses an improved and stable phenotype that is more suitable for ADD accumulation than that of M. neoaurum M3.…”

Section: Methodsmentioning

confidence: 99%

The Sterol Carrier Hydroxypropyl-β-Cyclodextrin Enhances the Metabolism of Phytosterols by Mycobacterium neoaurum

Shen

et al. 2020

Appl Environ Microbiol

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Androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) are valuable steroid pharmaceutical intermediates obtained by soybean phytosterol biotransformation by Mycobacterium. Cyclodextrins (CDs) are generally believed to be carriers for phytosterol delivery and can improve the production of AD and ADD due to their effects on steroid solubilization and alteration in cell wall permeability for steroids. To better understand the mechanisms of CD promotion, we performed proteomic quantification of the effects of hydroxypropyl-β-CD (HP-β-CD) on phytosterol metabolism in Mycobacterium neoaurum TCCC 11978 C2. Perturbations are observed in steroid catabolism and glucose metabolism by adding HP-β-CD in a phytosterol bioconversion system. AD and ADD, as metabolic products of phytosterol, are toxic to cells, with inhibited cell growth and biocatalytic activity. Treatment of mycobacteria with HP-β-CD relieves the inhibitory effect of AD(D) on the electron transfer chain and cell growth. These results demonstrate the positive relationship between HP-β-CD and phytosterol metabolism and give insight into the complex functions of CDs as mediators of the regulation of sterol metabolism. IMPORTANCE Phytosterols from soybean are low-cost by-products of soybean oil production and, owing to their good bioavailability in mycobacteria, are preferred as the substrates for steroid drug production via biotransformation by Mycobacterium. However, the low level of production of steroid hormone drugs due to the low aqueous solubility (below 0.1 mmol/liter) of phytosterols limits the commercial use of sterol-transformed strains. To improve the bioconversion of steroids, cyclodextrins (CDs) are generally used as an effective carrier for the delivery of hydrophobic steroids to the bacterium. CDs improve the biotransformation of steroids due to their effects on steroid solubilization and alterations in cell wall permeability for steroids. However, studies have rarely reported the effects of CDs on cell metabolic pathways related to sterols. In this study, the effects of hydroxypropyl-β-CD (HP-β-CD) on the expression of enzymes related to steroid catabolic pathways in Mycobacterium neoaurum were systematically investigated. These findings will improve our understanding of the complex functions of CDs in the regulation of sterol metabolism and guide the application of CDs to sterol production.

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Section: Methodsmentioning

confidence: 99%

The Sterol Carrier Hydroxypropyl-β-Cyclodextrin Enhances the Metabolism of Phytosterols by Mycobacterium neoaurum

Shen

et al. 2020

Appl Environ Microbiol

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“…The mechanism of substrate recognition and selectivity are poor understood. Xie reported that S138 played an important role in maintaining the active center via hydrogen bond network of Mycobacterium neoaurum KsdD [ 43 ]. However, this residue is partial conserved in this family enzyme, which is H134 in A. simplex and S131 in R. erythropolis SQ1 (Additional file 1 : Fig.…”

Section: Resultsmentioning

confidence: 99%

Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates

et al. 2018

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BackgroundBiosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor.ResultsRecombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively).ConclusionsThe successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity. Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0981-0) contains supplementary material, which is available to authorized users.

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“…Nevertheless, mutations of other sites of KstD enzymes also have been shown to influence their activity. For example, p.S138L decreased the activity of KstD1 of M. neoaurum , whereas a p.V366S increased its activity [ 46 , 47 ]. Furthermore, a p.Y125H substitution of the KstD1 from M. neoaurum ATCC 25795 had only a relatively small effect [ 10 ].…”

Section: Discussionmentioning

confidence: 99%

Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

et al. 2018

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Background3-Ketosteroid-Δ1-dehydrogenase (KstD) is a key enzyme in the metabolic pathway for chemical modifications of steroid hormones. Only a few KstDs have thus far been characterized biochemically and applied for the production of steroidal pharmaceutical intermediates. Three KstDs, KstD1, KstD2, and KstD3, were identified in Mycobacterium neoaurum DSM 1381, and they shared up to 99, 85 and 97% amino acid identity with previously reported KstDs, respectively. In this paper, KstDs from M. neoaurum DSM 1381 were investigated and exemplified their potential application for industrial steroid transformation.ResultsThe recombinant KstD2 from Bacillus subtilis exhibited higher enzymatic activity when 4-androstene-3,17-dione (AD) and 22-hydroxy-23, 24-bisnorchol-4-ene-3-one (4HP) were used as the substrates, and resulted in specific activities of 22.40 and 19.19 U mg−1, respectively. However, the specific activities of recombinant KstD2 from Escherichia coli, recombinant KstD1 from B. subtilis and E. coli, and recombinant KstD3, also fed with AD and 4HP, had significantly lower specific activities. We achieved up to 99% bioconversion rate of 1,4-androstadiene-3,17-dione (ADD) from 8 g L−1 AD after 15 h of fermentation using E. coli transformant BL21-kstD2. And in vivo transcriptional analysis revealed that the expression of kstD1 in M. neoaurum DSM 1381 increased by 60.5-fold with phytosterols as the substrate, while the mRNA levels of kstD2 and kstD3 were bearly affected by the phytosterols. Therefore, we attempted to create a 4HP producing strain without kstD1, which could covert 20 g L−1 phytosterols to 14.18 g L−1 4HP.ConclusionsIn vitro assay employing the recombinant enzymes revealed that KstD2 was the most promising candidate for biocatalysis in biotransformation of AD. However, in vivo analysis showed that the cellular regulation of kstD1 was much more active than those of the other kstDs in response to the presence of phytosterols. Based on the findings above, we successfully constructed E. coli transformant BL21-kstD2 for ADD production from AD and M. neoaurum DSM 1381 ΔkstD1 strain for 4HP production using phytosterols as the substrate.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0916-9) contains supplementary material, which is available to authorized users.

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Genetic differences in ksdD influence on the ADD/AD ratio of Mycobacterium neoaurum

Cited by 29 publications

References 22 publications

The Sterol Carrier Hydroxypropyl-β-Cyclodextrin Enhances the Metabolism of Phytosterols by Mycobacterium neoaurum

The Sterol Carrier Hydroxypropyl-β-Cyclodextrin Enhances the Metabolism of Phytosterols by Mycobacterium neoaurum

Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates

Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

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