Creation and Functional Analysis of Mycolicibacterium smegmatis Recombinant Strains Carrying the Bacillary Cytochromes CYP106A1 and CYP106A2 Genes

Karpov, Mikhail V.; Николаева, В. М.; Фокина, В. В.; Шутов, А. А.; Kazantsev, A. V.; Strizhov, N.; Donova, Marina V.

doi:10.1134/s0003683822090058

Cited by 7 publications

(7 citation statements)

References 29 publications

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Section: Discussionmentioning

confidence: 99%

“…The activity of recombinant P450 cytochromes depended on the recipient strain used and the presence or absence of suitable redox partners proteins in host cells [ 14 , 16 , 28 ]. For the manifestation of the activity of bacillary CYP106A1 and CYP106A2 in M. smegmatis mc 2 155 cells, the host’s own proteins were sufficient to ensure electron transport to recombinant cytochromes P450 [ 28 ]. However, to ensure the synthesis of pregnenolone in recombinant cells of Saccharomyces cerevisiae [ 18 ], E. coli [ 14 , 21 ] and Bacillus megaterium [ 16 ] the presence of all the components of the mammalian CH/L (P450scc/Adx/AdR) is required.…”

Section: Discussionmentioning

confidence: 99%

“…A strain of Mycolicibacterium smegmatis mc 2 155 was kindly provided by Dr. E. Noens (European Molecular Biology Laboratory (EMBL), Hamburg Outstation, Hamburg, Germany). The M. smegmatis cultures were maintained on M3 medium [ 28 ] at 37 °C. E. coli strain DH5α (Thermo Fisher Scientific, USA) were grown at 37 °C in LB medium [ 37 ].…”

Section: Methodsmentioning

confidence: 99%

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Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system

Karpov,

Strizhov,

Novikova

et al. 2024

Microb Cell Fact

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Background Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. Results Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. Conclusion The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones—progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system

Karpov,

Strizhov,

Novikova

et al. 2024

Microb Cell Fact

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“…When the genes coding for marker fluorescent proteins and the genes kshA and kshB encoding 3-ketosteroid-9α-hydroxylase were expressed in M. smegmatis, the hsp60 promoter was inferior in strength only to the artificial CP6 promoter [49]. Previously, the inducible acetamidase promoter from M. smegmatis mc 2 155 [61] was successfully used for heterologous expression of genes of steroidogenesis in closely related strains [39]. In the current work, a direct comparison of the strengths of the acetamidase and hsp60 promoters was performed for the first time.…”

Section: Discussionmentioning

confidence: 99%

“…As model recipient organisms having endogenous steroid catabolism, the two strains M. neoaurum NRRL B-3805∆kstD [38] and M. smegmatis BD [39] were applied. These strains convert sterols into 1(2)-saturated 3-keto-4-en-steroids-androst-4-en-3,17-dione (AD) and 20-hydroxymethyl-pregn-1-en-3-one (HMP).…”

Section: Construction Of Recombinant Mycolicibacterium Strainsmentioning

confidence: 99%

Reconstruction of the Steroid 1(2)-Dehydrogenation System from Nocardioides simplex VKM Ac-2033D in Mycolicibacterium Hosts

Fufaeva,

Dovbnya,

Ivashina

et al. 2023

Microorganisms

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Microbial 1(2)-dehydrogenation of 3-ketosteroids is an important basis for the production of many steroid pharmaceuticals and synthons. When using the wild-type strains for whole cell catalysis, the undesirable reduction of the 20-carbonyl group, or 1(2)-hydrogenation, was observed. In this work, the recombinant strains of Mycolicibacterium neoaurum and Mycolicibacterium smegmatis were constructed with blocked endogenous activity of 3-ketosteroid-9α-hydroxylase, 3-ketosteroid-1(2)-dehydrogenase (3-KSD), and expressing 3-KSD encoded by the gene KR76_27125 (kstD2NS) from Nocardioides simplex VKM Ac-2033D. The in vivo activity of the obtained recombinant strains against phytosterol, 6α-methyl-hydrocortisone, and hydrocortisone was studied. When using M. smegmatis as the host strain, the 1(2)-dehydrogenation activity of the constructed recombinant cells towards hydrocortisone was noticeably higher compared to those on the platform of M. neoaurum. A comparison of the strengths of inducible acetamidase and constitutive hsp60 promoters in M. smegmatis provided comparable results. Hydrocortisone biotransformation by M. smegmatis BD/pMhsp_k expressing kstD2NS resulted in 95.4% prednisolone yield, and the selectivity preferred that for N. simplex. Mycolicibacteria showed increased hydrocortisone degradation at 35 °C compared to 30 °C. The presence of endogenous steroid catabolism in Mycolicibacterium hosts does not seem to confer an advantage for the functioning of KstD2NS. The results allow for the evaluation of the prospects for the development of simple technological methods for the selective 1(2)-dehydrogenation of 3-ketosteroids by growing bacterial cells.

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Current Trends and Perspectives in Microbial Bioconversions of Steroids

Donova

2023

Methods in Molecular Biology

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Creation and Functional Analysis of Mycolicibacterium smegmatis Recombinant Strains Carrying the Bacillary Cytochromes CYP106A1 and CYP106A2 Genes

Cited by 7 publications

References 29 publications

Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system

Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system

Reconstruction of the Steroid 1(2)-Dehydrogenation System from Nocardioides simplex VKM Ac-2033D in Mycolicibacterium Hosts

Current Trends and Perspectives in Microbial Bioconversions of Steroids

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