Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway
Abstract 4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major c...
Ausführliche Beschreibung
Autor*in: |
Shikui Song [verfasserIn] Jianxin He [verfasserIn] Meng Gao [verfasserIn] Yongqi Huang [verfasserIn] Xiyao Cheng [verfasserIn] Zhengding Su [verfasserIn] |
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E-Artikel |
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Sprache: |
Englisch |
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2023 |
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1,4-androstadiene-3,17-dione (ADD) 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) 3-ketosteroid-1,2-dehydrogenase (KstD) 3-ketosteroid-9α-hydroxylase (Ksh) |
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Übergeordnetes Werk: |
In: Microbial Cell Factories - BMC, 2003, 22(2023), 1, Seite 15 |
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Übergeordnetes Werk: |
volume:22 ; year:2023 ; number:1 ; pages:15 |
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DOI / URN: |
10.1186/s12934-022-02008-8 |
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Katalog-ID: |
DOAJ08096110X |
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520 | |a Abstract 4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2 Δhsd4A1 ) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2 Δhsd4A1 mutant, HGMS2 Δhsd4A1/Δkstd1 , enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2 Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2 Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2 Δhsd4A1/kstd2 and HGMS2 Δkstd1/Δhsd4A1/kshA1B1 , efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. | ||
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10.1186/s12934-022-02008-8 doi (DE-627)DOAJ08096110X (DE-599)DOAJ98287f4ccae24285822641b97da1bdb5 DE-627 ger DE-627 rakwb eng QR1-502 Shikui Song verfasserin aut Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract 4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2 Δhsd4A1 ) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2 Δhsd4A1 mutant, HGMS2 Δhsd4A1/Δkstd1 , enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2 Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2 Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2 Δhsd4A1/kstd2 and HGMS2 Δkstd1/Δhsd4A1/kshA1B1 , efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. 1,4-androstadiene-3,17-dione (ADD) 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) 3-ketosteroid-1,2-dehydrogenase (KstD) 3-ketosteroid-9α-hydroxylase (Ksh) 4-androstene-3,17-dione (4-AD) 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD) Microbiology Jianxin He verfasserin aut Meng Gao verfasserin aut Yongqi Huang verfasserin aut Xiyao Cheng verfasserin aut Zhengding Su verfasserin aut In Microbial Cell Factories BMC, 2003 22(2023), 1, Seite 15 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:22 year:2023 number:1 pages:15 https://doi.org/10.1186/s12934-022-02008-8 kostenfrei https://doaj.org/article/98287f4ccae24285822641b97da1bdb5 kostenfrei https://doi.org/10.1186/s12934-022-02008-8 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 22 2023 1 15 |
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10.1186/s12934-022-02008-8 doi (DE-627)DOAJ08096110X (DE-599)DOAJ98287f4ccae24285822641b97da1bdb5 DE-627 ger DE-627 rakwb eng QR1-502 Shikui Song verfasserin aut Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract 4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2 Δhsd4A1 ) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2 Δhsd4A1 mutant, HGMS2 Δhsd4A1/Δkstd1 , enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2 Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2 Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2 Δhsd4A1/kstd2 and HGMS2 Δkstd1/Δhsd4A1/kshA1B1 , efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. 1,4-androstadiene-3,17-dione (ADD) 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) 3-ketosteroid-1,2-dehydrogenase (KstD) 3-ketosteroid-9α-hydroxylase (Ksh) 4-androstene-3,17-dione (4-AD) 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD) Microbiology Jianxin He verfasserin aut Meng Gao verfasserin aut Yongqi Huang verfasserin aut Xiyao Cheng verfasserin aut Zhengding Su verfasserin aut In Microbial Cell Factories BMC, 2003 22(2023), 1, Seite 15 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:22 year:2023 number:1 pages:15 https://doi.org/10.1186/s12934-022-02008-8 kostenfrei https://doaj.org/article/98287f4ccae24285822641b97da1bdb5 kostenfrei https://doi.org/10.1186/s12934-022-02008-8 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 22 2023 1 15 |
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10.1186/s12934-022-02008-8 doi (DE-627)DOAJ08096110X (DE-599)DOAJ98287f4ccae24285822641b97da1bdb5 DE-627 ger DE-627 rakwb eng QR1-502 Shikui Song verfasserin aut Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract 4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2 Δhsd4A1 ) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2 Δhsd4A1 mutant, HGMS2 Δhsd4A1/Δkstd1 , enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2 Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2 Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2 Δhsd4A1/kstd2 and HGMS2 Δkstd1/Δhsd4A1/kshA1B1 , efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. 1,4-androstadiene-3,17-dione (ADD) 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) 3-ketosteroid-1,2-dehydrogenase (KstD) 3-ketosteroid-9α-hydroxylase (Ksh) 4-androstene-3,17-dione (4-AD) 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD) Microbiology Jianxin He verfasserin aut Meng Gao verfasserin aut Yongqi Huang verfasserin aut Xiyao Cheng verfasserin aut Zhengding Su verfasserin aut In Microbial Cell Factories BMC, 2003 22(2023), 1, Seite 15 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:22 year:2023 number:1 pages:15 https://doi.org/10.1186/s12934-022-02008-8 kostenfrei https://doaj.org/article/98287f4ccae24285822641b97da1bdb5 kostenfrei https://doi.org/10.1186/s12934-022-02008-8 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 22 2023 1 15 |
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10.1186/s12934-022-02008-8 doi (DE-627)DOAJ08096110X (DE-599)DOAJ98287f4ccae24285822641b97da1bdb5 DE-627 ger DE-627 rakwb eng QR1-502 Shikui Song verfasserin aut Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract 4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2 Δhsd4A1 ) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2 Δhsd4A1 mutant, HGMS2 Δhsd4A1/Δkstd1 , enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2 Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2 Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2 Δhsd4A1/kstd2 and HGMS2 Δkstd1/Δhsd4A1/kshA1B1 , efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. 1,4-androstadiene-3,17-dione (ADD) 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) 3-ketosteroid-1,2-dehydrogenase (KstD) 3-ketosteroid-9α-hydroxylase (Ksh) 4-androstene-3,17-dione (4-AD) 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD) Microbiology Jianxin He verfasserin aut Meng Gao verfasserin aut Yongqi Huang verfasserin aut Xiyao Cheng verfasserin aut Zhengding Su verfasserin aut In Microbial Cell Factories BMC, 2003 22(2023), 1, Seite 15 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:22 year:2023 number:1 pages:15 https://doi.org/10.1186/s12934-022-02008-8 kostenfrei https://doaj.org/article/98287f4ccae24285822641b97da1bdb5 kostenfrei https://doi.org/10.1186/s12934-022-02008-8 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 22 2023 1 15 |
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10.1186/s12934-022-02008-8 doi (DE-627)DOAJ08096110X (DE-599)DOAJ98287f4ccae24285822641b97da1bdb5 DE-627 ger DE-627 rakwb eng QR1-502 Shikui Song verfasserin aut Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract 4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2 Δhsd4A1 ) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2 Δhsd4A1 mutant, HGMS2 Δhsd4A1/Δkstd1 , enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2 Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2 Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2 Δhsd4A1/kstd2 and HGMS2 Δkstd1/Δhsd4A1/kshA1B1 , efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. 1,4-androstadiene-3,17-dione (ADD) 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) 3-ketosteroid-1,2-dehydrogenase (KstD) 3-ketosteroid-9α-hydroxylase (Ksh) 4-androstene-3,17-dione (4-AD) 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD) Microbiology Jianxin He verfasserin aut Meng Gao verfasserin aut Yongqi Huang verfasserin aut Xiyao Cheng verfasserin aut Zhengding Su verfasserin aut In Microbial Cell Factories BMC, 2003 22(2023), 1, Seite 15 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:22 year:2023 number:1 pages:15 https://doi.org/10.1186/s12934-022-02008-8 kostenfrei https://doaj.org/article/98287f4ccae24285822641b97da1bdb5 kostenfrei https://doi.org/10.1186/s12934-022-02008-8 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 22 2023 1 15 |
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1,4-androstadiene-3,17-dione (ADD) 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) 3-ketosteroid-1,2-dehydrogenase (KstD) 3-ketosteroid-9α-hydroxylase (Ksh) 4-androstene-3,17-dione (4-AD) 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD) Microbiology |
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In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2 Δhsd4A1 ) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2 Δhsd4A1 mutant, HGMS2 Δhsd4A1/Δkstd1 , enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2 Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2 Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2 Δhsd4A1/kstd2 and HGMS2 Δkstd1/Δhsd4A1/kshA1B1 , efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. 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QR1-502 Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway 1,4-androstadiene-3,17-dione (ADD) 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) 3-ketosteroid-1,2-dehydrogenase (KstD) 3-ketosteroid-9α-hydroxylase (Ksh) 4-androstene-3,17-dione (4-AD) 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD) |
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Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway |
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Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway |
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Abstract 4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2 Δhsd4A1 ) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2 Δhsd4A1 mutant, HGMS2 Δhsd4A1/Δkstd1 , enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2 Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2 Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2 Δhsd4A1/kstd2 and HGMS2 Δkstd1/Δhsd4A1/kshA1B1 , efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. |
abstractGer |
Abstract 4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2 Δhsd4A1 ) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2 Δhsd4A1 mutant, HGMS2 Δhsd4A1/Δkstd1 , enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2 Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2 Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2 Δhsd4A1/kstd2 and HGMS2 Δkstd1/Δhsd4A1/kshA1B1 , efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. |
abstract_unstemmed |
Abstract 4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2 Δhsd4A1 ) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2 Δhsd4A1 mutant, HGMS2 Δhsd4A1/Δkstd1 , enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2 Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2 Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2 Δhsd4A1/kstd2 and HGMS2 Δkstd1/Δhsd4A1/kshA1B1 , efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. |
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Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway |
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The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2 Δhsd4A1 ) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2 Δhsd4A1 mutant, HGMS2 Δhsd4A1/Δkstd1 , enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2 Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2 Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2 Δhsd4A1/kstd2 and HGMS2 Δkstd1/Δhsd4A1/kshA1B1 , efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">1,4-androstadiene-3,17-dione (ADD)</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA)</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">3-ketosteroid-1,2-dehydrogenase (KstD)</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">3-ketosteroid-9α-hydroxylase (Ksh)</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">4-androstene-3,17-dione (4-AD)</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">9α-hydroxyl-4-androstene-3,17-dione (9OH-AD)</subfield></datafield><datafield tag="653" ind1=" " 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