Progress of 3-ketosteroid Δ1-dehydrogenases for steroid production

Zhang, Bo; Zhou, Deng-Feng; Li, Mohan; Lan, Jingyu; Li, Hui; Shao, Ming; Liu, Zhi‐Qiang; Zheng, Yu‐Guo

doi:10.1007/s43393-023-00190-3

Cited by 4 publications

(1 citation statement)

References 95 publications

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“…13A). 103 In recent years, various KstDs with high activities have been discovered and applied. 104–112 For instance, Guo and coworkers used Nocardioides for the Δ 1,2 -dehydrogenation of 71 to produce 72 , which is an important intermediate for the synthesis of fluocinolone acetonide (Fig.…”

Section: Dehydrogenationmentioning

confidence: 99%

Recent developments in the enzymatic modifications of steroid scaffolds

Wang,

Abe

2024

Org. Biomol. Chem.

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Section: Dehydrogenationmentioning

confidence: 99%

Recent developments in the enzymatic modifications of steroid scaffolds

Wang,

Abe

2024

Org. Biomol. Chem.

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High‐efficiency bioconversion of phytosterol to bisnoralcohol by metabolically engineered Mycobacterium neoaurum in a micro‐emulsion system

Wang,

Ke,

Dong

et al. 2024

Biotechnology Journal

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Abstract21‐Hydroxy‐20‐methylpregn‐4‐en‐3‐one (4‐HBC, bisnoralcohol) is a crucial intermediate for the synthesis of steroidal drugs. Significant challenges including by‐products formation and poor substrate solubility were still confronted in its main synthetic route by microbial conversion from phytosterol. Construction of a direct bioconversion pathway to 4‐HBC and an efficient substrate emulsion system is therefore urgently required. In this study, three novel isoenzymes of 3‐ketosteroid‐Δ1‐dehydrogenase (KstD) and 3‐ketosteroid 9α‐hydroxylase (KsH) in Mycobacterium neoaurum were excavated and identified as KstD4, KstD5, and KsHA3. A strain capable of fully directing the synthesis of 4‐HBC was metabolically engineered via serial genetic deletion combined with enhanced expression of cholesterol oxidase (ChOx2) and enoyl‐CoA hydratase (EchA19). Moreover, a micro‐emulsion system combined with soybean oil and hydroxypropyl‐β‐cyclodextrin improved substrate solubility and bioavailability. In batch fermentation, molar yield of 96.7% with 39.5 g L−1 4‐HBC was obtained from 50 g L−1 phytosterol. Our findings demonstrate the potential for industrial‐scale biosynthesis of 4‐HBC.

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Highly Efficient Enzymatic Synthesis of C6-Substituted Δ¹-3-Ketosteroids Enabled by Finely Tuning the Substrate Binding of 3-Ketosteroid-Δ¹-Dehydrogenase

Wang,

Zhang,

Chen

et al. 2024

ACS Sustainable Chem. Eng.

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Enzymatic Δ1-dehydrogenation mediated by 3-ketosteroid-Δ1-dehydrogenases (Δ1-KstDs, EC 1.3.99.4) offers an attractive method to access pharmaceutically important Δ1-3-ketosteroids by avoiding the multistep chemical reactions and use of toxic reagents. However, Δ1-KstDs have low or no activity toward C6-substituted and other bulky 3-ketosteroids, which limits their applications in the synthesis of the corresponding dehydrogenated products that are widely used for the treatment of different diseases. Herein, structure-based site-directed saturation mutagenesis of KstD from Propionibacterium sp. (PrKstD) was performed to tune its substrate specificity. Amino acid residues potentially responsible for substrate recognition were site-directedly mutated, and the results showed that the residues H135, A356, and S422 played important roles in fine-tuning the substrate specificity. Especially, the release of the steric effect of H135 provided extra space for accommodating the substrate with C6 methyl for a higher efficiency of transferring hydride at the active site. A double variant of PrKstD (H135T/A356N) exhibited 16.7-fold increased catalytic efficiency compared to that of the wild-type enzyme toward 6α-methyl-11β,17α-dihydroxy-4-pregnene-3,20-dione (1g). Molecular dynamics simulations provide some insights into the roles of the key mutations in the enhanced activity. Furthermore, Δ1-dehydrogenation of 6α-methyl-11β,17α-dihydroxy-4-pregnene-3,20-dione (1g) was scaled from gram to kilogram scale with high substrate loading (60.0 g·L–1) at a space–time yield of 4.08 g·L–1·h–1, much higher than the previously reported results. This work offers not only an effective method for the Δ1-dehydrogenation of C6-substituted 3-ketosteroids to furnish the corresponding bulky Δ1-3-ketosteroids but also guidance for tuning the substrate profile of 3-ketosteroid-Δ1-dehydrogenases to access other pharmaceutically relevant Δ1-3-ketosteroids in a green way.

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Progress of 3-ketosteroid Δ1-dehydrogenases for steroid production

Cited by 4 publications

References 95 publications

Recent developments in the enzymatic modifications of steroid scaffolds

Recent developments in the enzymatic modifications of steroid scaffolds

High‐efficiency bioconversion of phytosterol to bisnoralcohol by metabolically engineered Mycobacterium neoaurum in a micro‐emulsion system

Highly Efficient Enzymatic Synthesis of C6-Substituted Δ¹-3-Ketosteroids Enabled by Finely Tuning the Substrate Binding of 3-Ketosteroid-Δ¹-Dehydrogenase

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Progress of 3-ketosteroid Δ1-dehydrogenases for steroid production

Cited by 4 publications

References 95 publications

Recent developments in the enzymatic modifications of steroid scaffolds

Recent developments in the enzymatic modifications of steroid scaffolds

High‐efficiency bioconversion of phytosterol to bisnoralcohol by metabolically engineered Mycobacterium neoaurum in a micro‐emulsion system

Highly Efficient Enzymatic Synthesis of C6-Substituted Δ1-3-Ketosteroids Enabled by Finely Tuning the Substrate Binding of 3-Ketosteroid-Δ1-Dehydrogenase

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Highly Efficient Enzymatic Synthesis of C6-Substituted Δ¹-3-Ketosteroids Enabled by Finely Tuning the Substrate Binding of 3-Ketosteroid-Δ¹-Dehydrogenase