Highlight on Engineering <i>Mycobacterium smegmatis</i> for testosterone production

Sood, Utkarsh; Singh, Yogendra; Shakarad, Mallikarjun; Lal, Rup

doi:10.1111/1751-7915.12466

Cited by 8 publications

(5 citation statements)

References 28 publications

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“…As shown in Figure a, with P450 monooxygenase and 17β-ketosteroid reductase-catalyzed reactions in two one-pot two biocatalytic pathways in parallel could be envisioned via first hydroxylation/reduction to generate 7 (in an 11α or an 11β form, designated as 7a or 7b ) or 8 as an intermediate followed by further reduction/hydroxylation to give the desired product 2 (in an 11α or an 11β form, designated as 2a or 2b ). Since only 17β-ketosteroid reductases with high activity toward 1 and 7 have been reported, a P450 monooxygenase with high C11-hydroxylation activity for both 1 and 8 would be highly desirable. To start with, we screened the P450-BM3 mutant libraries stored in our lab using 1 as the model substrate .…”

Section: Results and Discussionmentioning

confidence: 99%

“…To achieve the biotransformation of 1 to 11α-OH-9(10)-dehydronandrolone ( 2a ) by parallel C11-hydroxylation/17β-ketoreduction in a one-pot–one-step manner (Figure b), we considered the 17-ketosteroid reductase (17β-HSDcl) from Cochliobolus lunatus because it is known to catalyze the structurally related reduction of testosterone derivatives with formation of the corresponding 17β-alcohols . With both efficient enzymes in hand, the whole cells coexpressing P450-BM3 LG-23/T438S and 17β-HSDcl were then constructed.…”

Section: Results and Discussionmentioning

confidence: 99%

“…Since chemocatalysis could be easily conducted with high yield, the key to success of this proposed route would be the development of a one-pot biocatalytic system for converting 1 to 2 . To achieve this goal, several 17β-ketosteroid reductase candidates reported with high activity could in principle be employed in the reduction step . The major problem was developing an efficient enzymatic procedure for selective C11-hydroxylation.…”

Section: Introductionmentioning

confidence: 99%

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A Chemoenzymatic Strategy for the Synthesis of Steroid Drugs Enabled by P450 Monooxygenase-Mediated Steroidal Core Modification

et al. 2022

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The synthesis of steroid drugs by multistage modifications of the steroidal core is challenging since site-specific and selective modification is essentially required, which is often difficult or complicated for chemocatalysis. For example, the synthesis of Trenbolone (3), a versatile anabolic−androgenic steroid, relies on a four-step chemical procedure on its core modifications of estra-4,9-diene-3,17-dione (1). Here, we have designed a two-step chemoenzymatic strategy that includes a biocatalytic one-pot C11-hydroxylation/17β-ketoreduction of 1 with a computationally designed P450 monooxygenase and an appropriate 17-ketosteroid reductase to generate 11α-OH-9(10)dehydronandrolone (2a) as an intermediate followed by chemical dehydration to introduce the double bond at carbons 11 and 12 with the formation of Trenbolone (3). To obtain a highly active and C11-selective enzyme, molecular dynamics simulations were performed, uncovering a crucial role of water molecules for substrate recognition and targeted hydroxylation of steroids. Moreover, Trenbolone is further subjected to esterification to produce Trenbolone acetate (9) that has been widely used in veterinary medicine. Finally, our approach enables the regio-and stereoselective synthesis of both steroid drugs 3 and 9 on a (nearly) gram scale with 83−91% isolated yields, showing great potential for industrial applications.

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Section: Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Chemoenzymatic Strategy for the Synthesis of Steroid Drugs Enabled by P450 Monooxygenase-Mediated Steroidal Core Modification

et al. 2022

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“…The 17β-HSD from fungi and bacteria has been reported for the enzymatic reduction in 4-AD to TS. In 2017, Fernandez-Cabezon et al [14,15] cloned the genes encoding 17β-HSDs from the bacterium Comamonas testosteroni and the fungus Cochliobolus lunatus, and the engineered strains of Mycobacterium smegmatis produced high yields of TS from sterols or androst-4-ene-3,17-dione (AD). In 2019, Govinda Guevara et al [16] cloned 17β-HSD from Cochliobolus lunatus into Rhodococcus ruber Chol-4, and TS was synthesized from 4-AD, with a molar conversion rate of 61% using glucose for co-factor regeneration.…”

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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“…Industrially, testosterone is synthesized chemically (1) from AD using a four-step process (Imada et al 1981 ) or (2) from sterols in a multi-step process (Hoberman and Yesalis 1995 ). However, in addition to other disadvantages, such as environmental risks, chemically synthesized testosterone may cause undesirable side effects (Sood et al 2017 ).…”

Section: Introductionmentioning

confidence: 99%

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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Highlight on Engineering Mycobacterium smegmatis for testosterone production

Cited by 8 publications

References 28 publications

A Chemoenzymatic Strategy for the Synthesis of Steroid Drugs Enabled by P450 Monooxygenase-Mediated Steroidal Core Modification

A Chemoenzymatic Strategy for the Synthesis of Steroid Drugs Enabled by P450 Monooxygenase-Mediated Steroidal Core Modification

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

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

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