Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria

Abstract 4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Xin Li [verfasserIn] Tian Chen [verfasserIn] Fei Peng [verfasserIn] Shikui Song [verfasserIn] Jingpeng Yu [verfasserIn] Douanla Njimeli Sidoine [verfasserIn] Xiyao Cheng [verfasserIn] Yongqi Huang [verfasserIn] Yijun He [verfasserIn] Zhengding Su [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2021

Schlagwörter:	1,4-Androstadiene-3,17-dione (ADD) 21-Hydroxy-20-methylpregn-4-en-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)

Übergeordnetes Werk:	In: Microbial Cell Factories - BMC, 2003, 20(2021), 1, Seite 15
Übergeordnetes Werk:	volume:20 ; year:2021 ; number:1 ; pages:15

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.1186/s12934-021-01653-9

Katalog-ID:	DOAJ002946157

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520			\|a Abstract 4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.
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allfields	10.1186/s12934-021-01653-9 doi (DE-627)DOAJ002946157 (DE-599)DOAJ1ae1bd6405d64294b85a2ec11c50a548 DE-627 ger DE-627 rakwb eng QR1-502 Xin Li verfasserin aut Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract 4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. 1,4-Androstadiene-3,17-dione (ADD) 21-Hydroxy-20-methylpregn-4-en-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 Tian Chen verfasserin aut Fei Peng verfasserin aut Shikui Song verfasserin aut Jingpeng Yu verfasserin aut Douanla Njimeli Sidoine verfasserin aut Xiyao Cheng verfasserin aut Yongqi Huang verfasserin aut Yijun He verfasserin aut Zhengding Su verfasserin aut In Microbial Cell Factories BMC, 2003 20(2021), 1, Seite 15 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:20 year:2021 number:1 pages:15 https://doi.org/10.1186/s12934-021-01653-9 kostenfrei https://doaj.org/article/1ae1bd6405d64294b85a2ec11c50a548 kostenfrei https://doi.org/10.1186/s12934-021-01653-9 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA 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 20 2021 1 15
spelling	10.1186/s12934-021-01653-9 doi (DE-627)DOAJ002946157 (DE-599)DOAJ1ae1bd6405d64294b85a2ec11c50a548 DE-627 ger DE-627 rakwb eng QR1-502 Xin Li verfasserin aut Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract 4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. 1,4-Androstadiene-3,17-dione (ADD) 21-Hydroxy-20-methylpregn-4-en-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 Tian Chen verfasserin aut Fei Peng verfasserin aut Shikui Song verfasserin aut Jingpeng Yu verfasserin aut Douanla Njimeli Sidoine verfasserin aut Xiyao Cheng verfasserin aut Yongqi Huang verfasserin aut Yijun He verfasserin aut Zhengding Su verfasserin aut In Microbial Cell Factories BMC, 2003 20(2021), 1, Seite 15 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:20 year:2021 number:1 pages:15 https://doi.org/10.1186/s12934-021-01653-9 kostenfrei https://doaj.org/article/1ae1bd6405d64294b85a2ec11c50a548 kostenfrei https://doi.org/10.1186/s12934-021-01653-9 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA 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 20 2021 1 15
allfields_unstemmed	10.1186/s12934-021-01653-9 doi (DE-627)DOAJ002946157 (DE-599)DOAJ1ae1bd6405d64294b85a2ec11c50a548 DE-627 ger DE-627 rakwb eng QR1-502 Xin Li verfasserin aut Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract 4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. 1,4-Androstadiene-3,17-dione (ADD) 21-Hydroxy-20-methylpregn-4-en-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 Tian Chen verfasserin aut Fei Peng verfasserin aut Shikui Song verfasserin aut Jingpeng Yu verfasserin aut Douanla Njimeli Sidoine verfasserin aut Xiyao Cheng verfasserin aut Yongqi Huang verfasserin aut Yijun He verfasserin aut Zhengding Su verfasserin aut In Microbial Cell Factories BMC, 2003 20(2021), 1, Seite 15 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:20 year:2021 number:1 pages:15 https://doi.org/10.1186/s12934-021-01653-9 kostenfrei https://doaj.org/article/1ae1bd6405d64294b85a2ec11c50a548 kostenfrei https://doi.org/10.1186/s12934-021-01653-9 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA 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 20 2021 1 15
allfieldsGer	10.1186/s12934-021-01653-9 doi (DE-627)DOAJ002946157 (DE-599)DOAJ1ae1bd6405d64294b85a2ec11c50a548 DE-627 ger DE-627 rakwb eng QR1-502 Xin Li verfasserin aut Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract 4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. 1,4-Androstadiene-3,17-dione (ADD) 21-Hydroxy-20-methylpregn-4-en-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 Tian Chen verfasserin aut Fei Peng verfasserin aut Shikui Song verfasserin aut Jingpeng Yu verfasserin aut Douanla Njimeli Sidoine verfasserin aut Xiyao Cheng verfasserin aut Yongqi Huang verfasserin aut Yijun He verfasserin aut Zhengding Su verfasserin aut In Microbial Cell Factories BMC, 2003 20(2021), 1, Seite 15 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:20 year:2021 number:1 pages:15 https://doi.org/10.1186/s12934-021-01653-9 kostenfrei https://doaj.org/article/1ae1bd6405d64294b85a2ec11c50a548 kostenfrei https://doi.org/10.1186/s12934-021-01653-9 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA 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 20 2021 1 15
allfieldsSound	10.1186/s12934-021-01653-9 doi (DE-627)DOAJ002946157 (DE-599)DOAJ1ae1bd6405d64294b85a2ec11c50a548 DE-627 ger DE-627 rakwb eng QR1-502 Xin Li verfasserin aut Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract 4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds. 1,4-Androstadiene-3,17-dione (ADD) 21-Hydroxy-20-methylpregn-4-en-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 Tian Chen verfasserin aut Fei Peng verfasserin aut Shikui Song verfasserin aut Jingpeng Yu verfasserin aut Douanla Njimeli Sidoine verfasserin aut Xiyao Cheng verfasserin aut Yongqi Huang verfasserin aut Yijun He verfasserin aut Zhengding Su verfasserin aut In Microbial Cell Factories BMC, 2003 20(2021), 1, Seite 15 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:20 year:2021 number:1 pages:15 https://doi.org/10.1186/s12934-021-01653-9 kostenfrei https://doaj.org/article/1ae1bd6405d64294b85a2ec11c50a548 kostenfrei https://doi.org/10.1186/s12934-021-01653-9 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA 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 20 2021 1 15
language	English
source	In Microbial Cell Factories 20(2021), 1, Seite 15 volume:20 year:2021 number:1 pages:15
sourceStr	In Microbial Cell Factories 20(2021), 1, Seite 15 volume:20 year:2021 number:1 pages:15
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	1,4-Androstadiene-3,17-dione (ADD) 21-Hydroxy-20-methylpregn-4-en-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
isfreeaccess_bool	true
container_title	Microbial Cell Factories
authorswithroles_txt_mv	Xin Li @@aut@@ Tian Chen @@aut@@ Fei Peng @@aut@@ Shikui Song @@aut@@ Jingpeng Yu @@aut@@ Douanla Njimeli Sidoine @@aut@@ Xiyao Cheng @@aut@@ Yongqi Huang @@aut@@ Yijun He @@aut@@ Zhengding Su @@aut@@
publishDateDaySort_date	2021-01-01T00:00:00Z
hierarchy_top_id	355987651
id	DOAJ002946157
language_de	englisch
fullrecord	<?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01000caa a22002652 4500</leader><controlfield tag="001">DOAJ002946157</controlfield><controlfield tag="003">DE-627</controlfield><controlfield tag="005">20230503151345.0</controlfield><controlfield tag="007">cr uuu---uuuuu</controlfield><controlfield tag="008">230225s2021 xx \|\|\|\|\|o 00\| \|\|eng c</controlfield><datafield tag="024" ind1="7" ind2=" "><subfield code="a">10.1186/s12934-021-01653-9</subfield><subfield code="2">doi</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-627)DOAJ002946157</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-599)DOAJ1ae1bd6405d64294b85a2ec11c50a548</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-627</subfield><subfield code="b">ger</subfield><subfield code="c">DE-627</subfield><subfield code="e">rakwb</subfield></datafield><datafield tag="041" ind1=" " ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="050" ind1=" " ind2="0"><subfield code="a">QR1-502</subfield></datafield><datafield tag="100" ind1="0" ind2=" "><subfield code="a">Xin Li</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2021</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Abstract 4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and 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">21-Hydroxy-20-methylpregn-4-en-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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callnumber-first	Q - Science
author	Xin Li
spellingShingle	Xin Li misc QR1-502 misc 1,4-Androstadiene-3,17-dione (ADD) misc 21-Hydroxy-20-methylpregn-4-en-3-one (BA) misc 3-Ketosteroid-1,2-dehydrogenase (KstD) misc 3-Ketosteroid-9α-hydroxylase (Ksh) misc 4-Androstene-3,17-dione (4-AD) misc 9α-Hydroxyl-4-androstene-3,17-dione (9OH-AD) misc Microbiology Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria
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topic_title	QR1-502 Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria 1,4-Androstadiene-3,17-dione (ADD) 21-Hydroxy-20-methylpregn-4-en-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)
topic	misc QR1-502 misc 1,4-Androstadiene-3,17-dione (ADD) misc 21-Hydroxy-20-methylpregn-4-en-3-one (BA) misc 3-Ketosteroid-1,2-dehydrogenase (KstD) misc 3-Ketosteroid-9α-hydroxylase (Ksh) misc 4-Androstene-3,17-dione (4-AD) misc 9α-Hydroxyl-4-androstene-3,17-dione (9OH-AD) misc Microbiology
topic_unstemmed	misc QR1-502 misc 1,4-Androstadiene-3,17-dione (ADD) misc 21-Hydroxy-20-methylpregn-4-en-3-one (BA) misc 3-Ketosteroid-1,2-dehydrogenase (KstD) misc 3-Ketosteroid-9α-hydroxylase (Ksh) misc 4-Androstene-3,17-dione (4-AD) misc 9α-Hydroxyl-4-androstene-3,17-dione (9OH-AD) misc Microbiology
topic_browse	misc QR1-502 misc 1,4-Androstadiene-3,17-dione (ADD) misc 21-Hydroxy-20-methylpregn-4-en-3-one (BA) misc 3-Ketosteroid-1,2-dehydrogenase (KstD) misc 3-Ketosteroid-9α-hydroxylase (Ksh) misc 4-Androstene-3,17-dione (4-AD) misc 9α-Hydroxyl-4-androstene-3,17-dione (9OH-AD) misc Microbiology
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title	Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria
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title_full	Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria
author_sort	Xin Li
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author_browse	Xin Li Tian Chen Fei Peng Shikui Song Jingpeng Yu Douanla Njimeli Sidoine Xiyao Cheng Yongqi Huang Yijun He Zhengding Su
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title_sort	efficient conversion of phytosterols into 4-androstene-3,17-dione and its c1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered mycobacteria
callnumber	QR1-502
title_auth	Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria
abstract	Abstract 4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and 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), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and 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), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.
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title_short	Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria
url	https://doi.org/10.1186/s12934-021-01653-9 https://doaj.org/article/1ae1bd6405d64294b85a2ec11c50a548 https://doaj.org/toc/1475-2859
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up_date	2024-07-03T15:02:43.415Z
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fullrecord_marcxml	<?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01000caa a22002652 4500</leader><controlfield tag="001">DOAJ002946157</controlfield><controlfield tag="003">DE-627</controlfield><controlfield tag="005">20230503151345.0</controlfield><controlfield tag="007">cr uuu---uuuuu</controlfield><controlfield tag="008">230225s2021 xx \|\|\|\|\|o 00\| \|\|eng c</controlfield><datafield tag="024" ind1="7" ind2=" "><subfield code="a">10.1186/s12934-021-01653-9</subfield><subfield code="2">doi</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-627)DOAJ002946157</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-599)DOAJ1ae1bd6405d64294b85a2ec11c50a548</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-627</subfield><subfield code="b">ger</subfield><subfield code="c">DE-627</subfield><subfield code="e">rakwb</subfield></datafield><datafield tag="041" ind1=" " ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="050" ind1=" " ind2="0"><subfield code="a">QR1-502</subfield></datafield><datafield tag="100" ind1="0" ind2=" "><subfield code="a">Xin Li</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2021</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Abstract 4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2 Δkstd211 + ΔkshB122 , showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2 kstd2 + Δkstd211+ΔkshB122 and HGMS2 kshA51 + Δkstd211+ΔkshA226 , by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and 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">21-Hydroxy-20-methylpregn-4-en-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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Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria

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