Transformation of phytosterols into pregnatetraenedione by a combined microbial and chemical process

Liu, Yongjun; Ji, Weiting; Lu, Song; Tao, Xinyi; Zhao, Ming; Gao, Bei; Meng, Hua; Wang, Fengqing; Wei, Dongzhi

doi:10.1039/d1gc04819h

Cited by 11 publications

(8 citation statements)

References 47 publications

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“…The microbial catabolism of sterols is a sophisticated process, and some mechanisms remain obscure [ 14 ]. Although the main gene cluster for sterols degradation has been identified and considerable progress has been made in the use of metabolic engineering techniques to develop high-yielding strains for the conversion of phytosterols to valuable steroids [ 7 , 21 ], the development of industrial strains faces some challenges, such as inevitable byproduct formation and low yields.…”

Section: Resultsmentioning

confidence: 99%

“…This phenomenon benefited from the significant progress of modification technology of Mycolicibacteria by metabolic engineering along with the catabolic pathway of sterols [ 13 ]. Moreover, the complex catabolic pathway of sterols has some obvious deficiencies in the strains used in industry, which significantly affect their application performance [ 14 , 15 ]. One of the most prominent problems is the generation of byproducts, especially the co-generation of the C-19 and C-22 steroids in the engineered C-19 or C-22 steroid-producing strains [ 5 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

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Unravelling and engineering an operon involved in the side-chain degradation of sterols in Mycolicibacterium neoaurum for the production of steroid synthons

Zhao

Liu

et al. 2023

Biotechnol Biofuels

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Background Harnessing engineered Mycolicibacteria to convert cheap phytosterols into valuable steroid synthons is a basic way in the industry for the production of steroid hormones. Thus, C-19 and C-22 steroids are the two main types of commercial synthons and the products of C17 side chain degradation of phytosterols. During the conversion process of sterols, C-19 and C-22 steroids are often produced together, although one may be the main product and the other a minor byproduct. This is a major drawback of the engineered Mycolicibacteria for industrial application, which could be attributed to the co-existence of androstene-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (HBC) sub-pathways in the degradation of the sterol C17 side chain. Since the key mechanism underlying the HBC sub-pathway has not yet been clarified, the above shortcoming has not been resolved so far. Results The key gene involved in the putative HBC sub-pathway was excavated from the genome of M. neoaurum by comparative genomic analysis. Interestingly, an aldolase- encoding gene, atf1, was identified to be responsible for the first reaction of the HBC sub-pathway, and it exists as a conserved operon along with a DUF35-type gene chsH4, a reductase gene chsE6, and a transcriptional regulation gene kstR3 in the genome. Subsequently, atf1 and chsH4 were identified as the key genes involved in the HBC sub-pathway. Therefore, an updated strategy was proposed to develop engineered C-19 or C-22 steroid-producing strains by simultaneously modifying the AD and HBC sub-pathways. Taking the development of 4-HBC and 9-OHAD-producing strains as examples, the improved 4-HBC-producing strain achieved a 20.7 g/L production titer with a 92.5% molar yield and a 56.4% reduction in byproducts, and the improved 9-OHAD producing strain achieved a 19.87 g/L production titer with a 94.6% molar yield and a 43.7% reduction in byproduct production. Conclusions The excellent performances of these strains demonstrated that the primary operon involved in the HBC sub-pathway improves the industrial strains in the conversion of phytosterols to steroid synthons.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Unravelling and engineering an operon involved in the side-chain degradation of sterols in Mycolicibacterium neoaurum for the production of steroid synthons

Zhao

Liu

et al. 2023

Biotechnol Biofuels

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“…Earlier studies suggested the possible existence of microorganisms in nature that could degrade diosgenin (Kondo & Mitsugi, 1966 , 1973 ). It has been known that the degradation of substances by microbial cells is usually conducted in a specific sequence of steps (Liu et al., 2019 , 2022 ). Therefore, microorganisms with the ability to preferentially remove 5,6‐spiroketals rather than to disrupt the parent nucleus of diosgenin can be further utilized as cell factories for the production of steroid precursors by applying genetic and metabolic engineering strategies.…”

Section: Introductionmentioning

confidence: 99%

Microbial metabolism of diosgenin by a novel isolated Mycolicibacterium sp. HK‐90: A promising biosynthetic platform to produce 19‐carbon and 21‐carbon steroids

Wang,

Qiu,

Chen

et al. 2024

Microbial Biotechnology

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Green manufacture of steroid precursors from diosgenin by microbial replacing multistep chemical synthesis has been elusive. It is currently limited by the lack of strain and degradation mechanisms. Here, we demonstrated the feasibility of this process using a novel strain Mycolicibacterium sp. HK‐90 with efficiency in diosgenin degradation. Diosgenin degradation by strain HK‐90 involves the selective removal of 5,6‐spiroketal structure, followed by the oxygenolytic cleavage of steroid nuclei. Bioinformatic analyses revealed the presence of two complete steroid catabolic gene clusters, SCG‐1 and SCG‐2, in the genome of strain HK‐90. SCG‐1 cluster was found to be involved in classic phytosterols or cholesterol catabolic pathway through the deletion of key kstD1 gene, which promoted the mutant m‐∆kstD1 converting phytosterols to intermediate 9α‐hydroxyandrostenedione (9‐OHAD). Most impressively, global transcriptomics and characterization of key genes suggested SCG‐2 as a potential gene cluster encoding diosgenin degradation. The gene inactivation of kstD2 in SCG‐2 resulted in the conversion of diosgenin to 9‐OHAD and 9,16‐dihydroxy‐pregn‐4‐ene‐3,20‐dione (9,16‐(OH)2‐PG) in mutant m‐ΔkstD2. Moreover, the engineered strain mHust‐ΔkstD1,2,3 with a triple deletion of kstDs was constructed, which can stably accumulate 9‐OHAD by metabolizing phytosterols, and accumulate 9‐OHAD and 9,16‐(OH)2‐PG from diosgenin. Diosgenin catabolism in strain mHust‐ΔkstD1,2,3 was revealed as a progression through diosgenone, 9,16‐(OH)2‐PG, and 9‐OHAD to 9α‐hydroxytestosterone (9‐OHTS). So far, this work is the first report on genetically engineered strain metabolizing diosgenin to produce 21‐carbon and 19‐carbon steroids. This study presents a promising biosynthetic platform for the green production of steroid precursors, and provide insights into the complex biochemical mechanism of diosgenin catabolism.

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“…[ 11 ] Liu reported that the yields of 9‐OHPDC‐M were remarkably improved by individually overexpressing the genes hsd4A and kshA1 . [ 12 ]…”

Section: Introductionmentioning

confidence: 99%

“…[11] Liu reported that the yields of 9-OHPDC-M were remarkably improved by individually overexpressing the genes hsd4A and kshA1. [12] However, there are still problems affecting the biosynthesis efficiency of steroid intermediates, especially the accumulation of byproducts. [13] In the production of 9-OH-AD by mycobacteria, there are mainly two types of by-products accumulated, one is the incomplete degradation of the side chain, including the by-product 9,22-dihydroxy-23,24-bisnorster-4-en-3-one (DHBC) and 9,24-dihydroxycholester-4en-3-one (DHC).…”

Section: Introductionmentioning

confidence: 99%

Enhancing production and purity of 9‐OH‐AD from phytosterols by balancing metabolic flux of the side‐chain degradation and 9‐position hydroxylation in Mycobacterium neoaurum

Zhu,

Wang,

Zhang

et al. 2023

Biotechnology Journal

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Abstract9α‐Hydroxyandroster‐4‐ene‐3,17‐dione (9‐OH‐AD) is a representative steroid drug intermediate that can be prepared by phytosterols (PS) biotransformation with mycobacteria in a resting cell‐cyclodextrin system. In this study, over‐expression of 17β‐hydroxysteroid dehydrogenase (Hsd4A) was testified to enhance the side‐chain degradation of PS and to reduce the incomplete degradation by‐products. Meanwhile, the complete degradation product 4‐androstene‐3,17‐dione (AD) was increased due to the lack of 3‐Ketosteroid 9α‐Hydroxylase (KshA1) activities. To increase the production and purity of 9‐OH‐AD, the metabolic pathway of the side‐chain degradation of PS and 9‐position hydroxylation was modulated by balancing the over‐expression of Hsd4A and KshA1 in mycobacteria and reducing the bioconversion rate via lowering the ratio of PS and cyclodextrin. The production and purity of 9‐OH‐AD in broth were improved from 22.18 g L−1 and 77.13% to 28.27 g L−1 and 87.84%, with a molar yield of 78.32%.

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Transformation of phytosterols into pregnatetraenedione by a combined microbial and chemical process

Abstract: Pregna-1,4,9(11),16(17)- tetraene-3,20-dione (pregnatetraenedione) is the most useful precursor for the systhesis of corticosteroids in the industry. However, the production of pregnatetraenedione is still a complex and expensive process. Here, we...

Cited by 11 publications

References 47 publications

Unravelling and engineering an operon involved in the side-chain degradation of sterols in Mycolicibacterium neoaurum for the production of steroid synthons

Unravelling and engineering an operon involved in the side-chain degradation of sterols in Mycolicibacterium neoaurum for the production of steroid synthons

Microbial metabolism of diosgenin by a novel isolated Mycolicibacterium sp. HK‐90: A promising biosynthetic platform to produce 19‐carbon and 21‐carbon steroids

Enhancing production and purity of 9‐OH‐AD from phytosterols by balancing metabolic flux of the side‐chain degradation and 9‐position hydroxylation in Mycobacterium neoaurum

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