Cascade Biotransformation of Phytosterol to Testosterone by Mycolicibacterium neoaurum VKM Ас-1815D and Nocardioides simplex VKM Ас-2033D Strains

Tekucheva, Daria N.; Фокина, В. В.; Николаева, В. М.; Шутов, А. А.; Karpov, Mikhail V.; Donova, Marina V.

doi:10.1134/s0026261722300099

Cited by 5 publications

(5 citation statements)

References 28 publications

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“…The presence of the NAD(H)-dependent 1-reductase activity towards ADD in two Mycolicibacterium strains has been recently demonstrated in vitro and in vivo [56]. The presence of 1-reductase activity in N. simplex has been evidenced earlier [11,57] and was confirmed in this study. Noteworthy, no 1(2)-hydrogenation was observed in N. simplex in the presence of EEA menadione.…”

Section: Discussionsupporting

confidence: 83%

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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Section: Discussionsupporting

confidence: 83%

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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“…As follows from the abovementioned results (“ 17β‑HSD and 1‑ene‑reductase activities and localization ”), effective regeneration of NAD(H) and decreased aeration are the two major factors influencing reduction of 17-carbonyl group of AD and ADD. These results and literature data (Llanes 1995 ; Liu and Lo 1997 ; Peltoketo et al 1999 ; Lo et al 2002 ; Tekucheva et al 2022 ) indicate that effective phytosterol bioconversion to AD/ADD and accumulation of testosterone/dTs require different intracellular Red/Ox status, i.e., cannot be combined in time.…”

Section: Resultssupporting

confidence: 60%

“…Moreover, glucose supplement also slowed down the undesirable testosterone oxidation, which contributed to its higher yield (Liu and Lo 1997 ; Egorova et al 2009 ). Supplements of glucose and limited aeration were found to be the key factors that provide a shift of the 17β-HSD activity toward reduction of AD+ADD formed by M. neoaurum VKM Ac-1815D to testosterone in whole Nocardioides simplex VKM Ac-2033D cells during the cascade bioconversion of phytosterol (Tekucheva et al 2022 ).…”

Section: Resultsmentioning

confidence: 99%

“…As mentioned above, CDs have a great influence on steroid bioconversions, and the effect depends on the concentration and the type of CDs used (Donova et al 2007 ; Shen et al 2012 ). CD-mediated intensifications of testosterone/dTs production from AD(D) by Saccharomyces cerevisiae (Singer et al 1991 ), Nocardioides simplex (Tekucheva et al 2022 ), Arthrobacter simplex CPCC 140451 and Pichia pastoris GS115 (Tang et al 2019 ), and Mycolicibacterium strains (Egorova et al 2009 ) have been reported, as well as the enhancement of cholesterol and phytosterol conversion to testosterone by Lactobacillus bulgarius (Kumar et al 2001 ), and by different strains of actinobacteria (Donova et al 2007 ; Shen et al 2012 ; Shtratnikova et al 2017 ; Tekucheva et al 2022 ).…”

Section: Resultsmentioning

confidence: 99%

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Bioproduction of testosterone from phytosterol by Mycolicibacterium neoaurum strains: “one-pot”, two modes

Tekucheva

Nikolayeva

Karpov

et al. 2022

Bioresour. Bioprocess.

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The main male hormone, testosterone is obtained from cheap and readily available phytosterol using the strains of Mycolicibacterium neoaurum VKM Ac-1815D, or Ac-1816D. During the first “oxidative” stage, phytosterol (5–10 g/L) was aerobically converted by Ac-1815D, or Ac-1816D to form 17-ketoandrostanes: androstenedione, or androstadienedione, respectively. At the same bioreactor, the 17-ketoandrostanes were further transformed to testosterone due to the presence of 17β-hydroxysteroid dehydrogenase activity in the strains (“reductive” mode). The conditions favorable for “oxidative” and “reductive” stages have been revealed to increase the final testosterone yield. Glucose supplement and microaerophilic conditions during the “reductive” mode ensured increased testosterone production by mycolicibacteria cells. Both strains effectively produced testosterone from phytosterol, but highest ever reported testosterone yield was achieved using M. neoaurum VKM Ac-1815D: 4.59 g/l testosterone was reached from 10 g/l phytosterol thus corresponding to the molar yield of over 66%. The results contribute to the knowledge on phytosterol bioconversion by mycolicibacteria, and are of significance for one-pot testosterone bioproduction from phytosterol bypassing the intermediate isolation of the 17-ketoandrostanes. Graphical Abstract

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“…In 2022, DN. Tekucheva et al [17] reported the two-stage transformation of phytosterol by the actinobacteria Mycolicibacterium neoaurum VKM Ac-1815D and Nocardioides simplex VKM Ac-2033D, which were capable of oxidizing the phytosterol side chain and reducing androstenedione at C17, respectively. A total testosterone yield of 53% was obtained using 10 g/L of phytosterol.…”

Section: Introductionmentioning

confidence: 99%

Testosterone Biosynthesis from 4-Androstene-3,17-Dione Catalyzed via Bifunctional Ketoreductase

Wei,

Mei,

Zhao

et al. 2023

Fermentation

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Testosterone (TS) is an important androgen drug and a precursor of steroid drug synthesis. Ketoreductase 2 (KR-2) (GenBank accession no. ABP64403.1) is observed to stereo-selectively catalyze the bioreduction of 4-androstene-3,17-dione (4-AD) to testosterone and contribute to the regeneration of NADH using isopropanol as a co-substrate. The Km value of KR-2 was 2.22 mmol/L with 4-AD, and the optimal pH was 6.5–7.0. The enzyme is stable and demonstrates relatively high-level enzyme activity at 40 °C. Acetone significantly inhibits this activity. This inhibition was overcome using an intermittent vacuum during the reaction process. Finally, the amount of TS reached 65.42 g/L after a 52 h reaction with 65.8 g/L 4-AD, 10% isopropanol, and 2 g/L β–NAD+ at 40 °C, with a conversion rate of 98.73%. A total of 6.15 g of TS was obtained from 6.58 g of 4-AD after the reaction and purification; the HPLC purity was 99.82%, and the overall yield was 92.81%. This enzyme provides a promising route for the green biosynthesis of testosterone for industrial applications.

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Cascade Biotransformation of Phytosterol to Testosterone by Mycolicibacterium neoaurum VKM Ас-1815D and Nocardioides simplex VKM Ас-2033D Strains

Cited by 5 publications

References 28 publications

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

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

Bioproduction of testosterone from phytosterol by Mycolicibacterium neoaurum strains: “one-pot”, two modes

Testosterone Biosynthesis from 4-Androstene-3,17-Dione Catalyzed via Bifunctional Ketoreductase

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