Abstract:Androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) are valuable steroid pharmaceutical intermediates obtained by soybean phytosterol biotransformation by Mycobacterium. Cyclodextrins (CDs) are generally believed to be carriers for phytosterol delivery and can improve the production of AD and ADD due to their effects on steroid solubilization and alteration in cell wall permeability for steroids. To better understand the mechanisms of CD promotion, we performed proteomic quantification of the … Show more
“…However, the purity of 9-OH-4-HP remained stable. Previous research has reported that phytosterols and their metabolites could be noxious to cells during bioconversion [ 9 , 14 – 16 ], for the poor performance of Δ kstD Δ hsd4A Δ fadA5 under high concentration phytosterols bioconversion. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Some surfactants like Tween 80 can reduce the tendency of Mycobacterium to aggregate [ 25 ] and thus promote the formation of stable suspensions of phytosterol by increasing phytosterol solubility and decreasing dynamic interfacial tension [ 23 ].These above cause the improvement of phytosterol bioconversion. Sterol-solubilizing agents like cyclodextrins could form inclusion complexes with steroids and make them as the effective carriers to deliver of hydrophobic steroids to cells, which enhances the biotransformation of steroid compounds [ 26 ]. Phytosterols and their derivatives are also reported to be toxic and inhibitory to cell growth [ 26 , 27 ], which also hinder the bioconversion and result in low molar yields.…”
Section: Discussionmentioning
confidence: 99%
“…Sterol-solubilizing agents like cyclodextrins could form inclusion complexes with steroids and make them as the effective carriers to deliver of hydrophobic steroids to cells, which enhances the biotransformation of steroid compounds [ 26 ]. Phytosterols and their derivatives are also reported to be toxic and inhibitory to cell growth [ 26 , 27 ], which also hinder the bioconversion and result in low molar yields. Balancing the intracellular environment is another promising method to improve phytosterols bioconversion.…”
Section: Discussionmentioning
confidence: 99%
“…Phytosterols are reported to inhibit the cell growth by reducing the utilization of carbon sources, which hinder the steroids biosynthesis progress with phytosterols as a substrate [ 9 ]. A steady-state intracellular environment could be beneficial for phytosterol degradation by Mycobacterium .…”
Background
Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain.
Results
The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP.
Conclusion
KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.
“…However, the purity of 9-OH-4-HP remained stable. Previous research has reported that phytosterols and their metabolites could be noxious to cells during bioconversion [ 9 , 14 – 16 ], for the poor performance of Δ kstD Δ hsd4A Δ fadA5 under high concentration phytosterols bioconversion. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Some surfactants like Tween 80 can reduce the tendency of Mycobacterium to aggregate [ 25 ] and thus promote the formation of stable suspensions of phytosterol by increasing phytosterol solubility and decreasing dynamic interfacial tension [ 23 ].These above cause the improvement of phytosterol bioconversion. Sterol-solubilizing agents like cyclodextrins could form inclusion complexes with steroids and make them as the effective carriers to deliver of hydrophobic steroids to cells, which enhances the biotransformation of steroid compounds [ 26 ]. Phytosterols and their derivatives are also reported to be toxic and inhibitory to cell growth [ 26 , 27 ], which also hinder the bioconversion and result in low molar yields.…”
Section: Discussionmentioning
confidence: 99%
“…Sterol-solubilizing agents like cyclodextrins could form inclusion complexes with steroids and make them as the effective carriers to deliver of hydrophobic steroids to cells, which enhances the biotransformation of steroid compounds [ 26 ]. Phytosterols and their derivatives are also reported to be toxic and inhibitory to cell growth [ 26 , 27 ], which also hinder the bioconversion and result in low molar yields. Balancing the intracellular environment is another promising method to improve phytosterols bioconversion.…”
Section: Discussionmentioning
confidence: 99%
“…Phytosterols are reported to inhibit the cell growth by reducing the utilization of carbon sources, which hinder the steroids biosynthesis progress with phytosterols as a substrate [ 9 ]. A steady-state intracellular environment could be beneficial for phytosterol degradation by Mycobacterium .…”
Background
Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain.
Results
The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP.
Conclusion
KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.
“…Yao et al reported an engineered Mycobacterium-transformed PS to 9a-OH-AD. 5 In addition, diverse sterol-solubilizing reaction systems, including a cyclodextrin complexing system, 4,6 biocompatible ionic liquid system, 7 microdispersion/emulsication system, and surfactant addition, 8,9 were established to improve the poor water solubility of PS. Among them, the cyclodextrin-resting cell system is an ideal method that has been successfully used for the production of a variety of steroid intermediates.…”
We identified an inhibitory intermediate, 4-ene-3-keto steroids, that limits the bioconversion rate and provided a solution based on resin adsorption for improving 9α-OH-AD production efficiency in a commercial-scale process.
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%.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
