Knockout of glycerol metabolic pathways enables efficient mycolicibacterial phytosterol conversion using glycerol as cosovlent
Abstract Mycobacterial phytosterol conversion naturally occurs within a vegetable oil environment, a process that has been widely adopted in water‒oil two-phase fermentation to produce active pharmaceutical intermediates of steroids. The use of hydroxypropyl-β-CD (HP-β-CD) as a potential replacement...
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Springer
2025-07-01
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| Series: | Applied Microbiology and Biotechnology |
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| Online Access: | https://doi.org/10.1007/s00253-024-13360-7 |
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| author | Shikui Song Ye Wang Jinpeng Yu Qianqian Ma Xiyao Cheng Baoguo Zhang Zhengding Su |
| author_facet | Shikui Song Ye Wang Jinpeng Yu Qianqian Ma Xiyao Cheng Baoguo Zhang Zhengding Su |
| author_sort | Shikui Song |
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| description | Abstract Mycobacterial phytosterol conversion naturally occurs within a vegetable oil environment, a process that has been widely adopted in water‒oil two-phase fermentation to produce active pharmaceutical intermediates of steroids. The use of hydroxypropyl-β-CD (HP-β-CD) as a potential replacement for vegetable oils has been explored. However, both approaches encounter challenges, particularly the need for a high concentration of either vegetable oil or HP-β-CD in the fermentation medium, which significantly affects the efficiency of mycobacterial phytosterol conversion as well as the recovery of the resulting products. In this study, we demonstrated that the knockout of glycerol metabolic pathways enables the utilization of glycerol as cosovlent for efficient phytosterol conversion in Mycolicibacterium neoaurum HGMS6, an industrial strain known for its production of 4-androstene-3,17-dione (4-AD). Our bioinformatic analysis revealed two parallel glycerol metabolic pathways in the HGMS6 strain. These pathways are independently initiated by glycerol kinase (GLPK) and glycerol dehydrogenase (ADHC). To investigate their roles, we conducted gene knockout experiments in which both the glycerol kinase gene (glpk) and the glycerol dehydrogenase gene (adhc) were targeted in HGMS6, resulting in the creation of a glycerol metabolism-deficient mutant, denoted HGMS6 Δadhc/Δglpk . This double mutant exhibited complete inactivation of mycobacterial glycerol metabolism without any adverse effects on mycobacterial growth when cultured in conventional fermentation medium containing glucose. We then evaluated HGMS6 Δadhc/Δglpk in an aqueous phase fermentation medium and determined that fermentation media consisting of 10% glycerol and 0.5% HP-β-CD provided the optimal conversion efficiency. In pilot-scale fermentation experiments, this mutant was highly efficient at converting phytosterols, achieving a remarkable conversion rate of up to 83.8% (mol/mol%). This bioconversion level was on par with that of HGMS6 and HGMS6 Δadhc/Δglpk when operating in vegetable oil- or HP-β-CD-containing fermentation media. This study provides a promising strategy for enhancing the efficiency of mycobacterial phytosterol conversion. Key points • The GLPK and ADHC of M. neoaurum were confirmed and examined. • The enzymology of ADHC and GLPK was characterized in vitro. • An adhc/glpk-knockout mutant efficiently converts phytosterols in fermentation media containing 10% (w/v) glycerol and 0.5% (w/v) HP-β-CD. |
| format | Article |
| id | doaj-art-323bc56b5f084370bfd54a31cac0832d |
| institution | Kabale University |
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| language | English |
| publishDate | 2025-07-01 |
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| series | Applied Microbiology and Biotechnology |
| spelling | doaj-art-323bc56b5f084370bfd54a31cac0832d2025-08-20T04:01:52ZengSpringerApplied Microbiology and Biotechnology1432-06142025-07-01109111510.1007/s00253-024-13360-7Knockout of glycerol metabolic pathways enables efficient mycolicibacterial phytosterol conversion using glycerol as cosovlentShikui Song0Ye Wang1Jinpeng Yu2Qianqian Ma3Xiyao Cheng4Baoguo Zhang5Zhengding Su6Xinjiang Key Laboratory of Special Species Conservation and Regulatory Biology, College of Life Sciences, Xinjiang Normal UniversityInstitute of Materia Medica, School of Pharmaceutical Sciences, Xinjiang UniversityProtein Engineering and Biopharmaceutical Sciences Laboratory, Hubei University of TechnologyInstitute of Materia Medica, School of Pharmaceutical Sciences, Xinjiang UniversitySchool of Light Industry and Food Engineering, Guangxi UniversityLab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of SciencesInstitute of Materia Medica, School of Pharmaceutical Sciences, Xinjiang UniversityAbstract Mycobacterial phytosterol conversion naturally occurs within a vegetable oil environment, a process that has been widely adopted in water‒oil two-phase fermentation to produce active pharmaceutical intermediates of steroids. The use of hydroxypropyl-β-CD (HP-β-CD) as a potential replacement for vegetable oils has been explored. However, both approaches encounter challenges, particularly the need for a high concentration of either vegetable oil or HP-β-CD in the fermentation medium, which significantly affects the efficiency of mycobacterial phytosterol conversion as well as the recovery of the resulting products. In this study, we demonstrated that the knockout of glycerol metabolic pathways enables the utilization of glycerol as cosovlent for efficient phytosterol conversion in Mycolicibacterium neoaurum HGMS6, an industrial strain known for its production of 4-androstene-3,17-dione (4-AD). Our bioinformatic analysis revealed two parallel glycerol metabolic pathways in the HGMS6 strain. These pathways are independently initiated by glycerol kinase (GLPK) and glycerol dehydrogenase (ADHC). To investigate their roles, we conducted gene knockout experiments in which both the glycerol kinase gene (glpk) and the glycerol dehydrogenase gene (adhc) were targeted in HGMS6, resulting in the creation of a glycerol metabolism-deficient mutant, denoted HGMS6 Δadhc/Δglpk . This double mutant exhibited complete inactivation of mycobacterial glycerol metabolism without any adverse effects on mycobacterial growth when cultured in conventional fermentation medium containing glucose. We then evaluated HGMS6 Δadhc/Δglpk in an aqueous phase fermentation medium and determined that fermentation media consisting of 10% glycerol and 0.5% HP-β-CD provided the optimal conversion efficiency. In pilot-scale fermentation experiments, this mutant was highly efficient at converting phytosterols, achieving a remarkable conversion rate of up to 83.8% (mol/mol%). This bioconversion level was on par with that of HGMS6 and HGMS6 Δadhc/Δglpk when operating in vegetable oil- or HP-β-CD-containing fermentation media. This study provides a promising strategy for enhancing the efficiency of mycobacterial phytosterol conversion. Key points • The GLPK and ADHC of M. neoaurum were confirmed and examined. • The enzymology of ADHC and GLPK was characterized in vitro. • An adhc/glpk-knockout mutant efficiently converts phytosterols in fermentation media containing 10% (w/v) glycerol and 0.5% (w/v) HP-β-CD.https://doi.org/10.1007/s00253-024-13360-7Mycolicibacterium sp. strainGlycerol metabolismPhytosterol conversionGlycerol dehydrogenaseGlycerol kinase |
| spellingShingle | Shikui Song Ye Wang Jinpeng Yu Qianqian Ma Xiyao Cheng Baoguo Zhang Zhengding Su Knockout of glycerol metabolic pathways enables efficient mycolicibacterial phytosterol conversion using glycerol as cosovlent Applied Microbiology and Biotechnology Mycolicibacterium sp. strain Glycerol metabolism Phytosterol conversion Glycerol dehydrogenase Glycerol kinase |
| title | Knockout of glycerol metabolic pathways enables efficient mycolicibacterial phytosterol conversion using glycerol as cosovlent |
| title_full | Knockout of glycerol metabolic pathways enables efficient mycolicibacterial phytosterol conversion using glycerol as cosovlent |
| title_fullStr | Knockout of glycerol metabolic pathways enables efficient mycolicibacterial phytosterol conversion using glycerol as cosovlent |
| title_full_unstemmed | Knockout of glycerol metabolic pathways enables efficient mycolicibacterial phytosterol conversion using glycerol as cosovlent |
| title_short | Knockout of glycerol metabolic pathways enables efficient mycolicibacterial phytosterol conversion using glycerol as cosovlent |
| title_sort | knockout of glycerol metabolic pathways enables efficient mycolicibacterial phytosterol conversion using glycerol as cosovlent |
| topic | Mycolicibacterium sp. strain Glycerol metabolism Phytosterol conversion Glycerol dehydrogenase Glycerol kinase |
| url | https://doi.org/10.1007/s00253-024-13360-7 |
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