The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosa
Spinosad, a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa, has significant market potential. Despite its effectiveness, the regulatory mechanisms of spinosad biosynthesis remain unclear. Our investigation identified the crucial role of the LysR family transcriptional reg...
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KeAi Communications Co., Ltd.
2024-12-01
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| Series: | Synthetic and Systems Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X24000693 |
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| author | Xin Mu Ru Lei Shuqing Yan Zixin Deng Ran Liu Tiangang Liu |
| author_facet | Xin Mu Ru Lei Shuqing Yan Zixin Deng Ran Liu Tiangang Liu |
| author_sort | Xin Mu |
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| description | Spinosad, a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa, has significant market potential. Despite its effectiveness, the regulatory mechanisms of spinosad biosynthesis remain unclear. Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16, located upstream of spinosad biosynthetic genes, in spinosad biosynthesis. Through reverse transcription PCR (RT-PCR) and 5′-rapid amplification of cDNA ends (5′-Race), we unveiled that the spinosad biosynthetic gene cluster (BGC) contains six transcription units and seven promoters. Electrophoretic mobility shift assays (EMSAs) demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC, indicating its involvement in regulating spinosad biosynthesis. Notably, deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L, accompanied by decreased transcription levels of spinosad biosynthetic genes, confirming its positive regulatory function. Additionally, isothermal titration calorimetry (ITC) and EMSA confirmed that spinosyn A, the main product of the spinosad BGC, served as an effector of ORF-L16. Specifically, it decreased the binding affinity between ORF-L16 and spinosad BGC promoters, thus exerting negative feedback regulation on spinosad biosynthesis. This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S. spinosa. |
| format | Article |
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| institution | Kabale University |
| issn | 2405-805X |
| language | English |
| publishDate | 2024-12-01 |
| publisher | KeAi Communications Co., Ltd. |
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| series | Synthetic and Systems Biotechnology |
| spelling | doaj-art-fad55bbbb66b42e6b1aabee1a1c2c9f22024-12-15T06:15:58ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2024-12-0194609617The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosaXin Mu0Ru Lei1Shuqing Yan2Zixin Deng3Ran Liu4Tiangang Liu5Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430072, Hubei Province, ChinaKey Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430072, Hubei Province, ChinaKey Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430072, Hubei Province, ChinaKey Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430072, Hubei Province, China; State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, China; Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China; Corresponding author. State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, China.Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430072, Hubei Province, China; State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, China; Corresponding author. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430072, Hubei Province, China.Spinosad, a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa, has significant market potential. Despite its effectiveness, the regulatory mechanisms of spinosad biosynthesis remain unclear. Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16, located upstream of spinosad biosynthetic genes, in spinosad biosynthesis. Through reverse transcription PCR (RT-PCR) and 5′-rapid amplification of cDNA ends (5′-Race), we unveiled that the spinosad biosynthetic gene cluster (BGC) contains six transcription units and seven promoters. Electrophoretic mobility shift assays (EMSAs) demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC, indicating its involvement in regulating spinosad biosynthesis. Notably, deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L, accompanied by decreased transcription levels of spinosad biosynthetic genes, confirming its positive regulatory function. Additionally, isothermal titration calorimetry (ITC) and EMSA confirmed that spinosyn A, the main product of the spinosad BGC, served as an effector of ORF-L16. Specifically, it decreased the binding affinity between ORF-L16 and spinosad BGC promoters, thus exerting negative feedback regulation on spinosad biosynthesis. This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S. spinosa.http://www.sciencedirect.com/science/article/pii/S2405805X24000693SpinosadLysR family transcriptional regulatorSpinosyn ARegulationSaccharopolyspora spinosa |
| spellingShingle | Xin Mu Ru Lei Shuqing Yan Zixin Deng Ran Liu Tiangang Liu The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosa Synthetic and Systems Biotechnology Spinosad LysR family transcriptional regulator Spinosyn A Regulation Saccharopolyspora spinosa |
| title | The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosa |
| title_full | The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosa |
| title_fullStr | The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosa |
| title_full_unstemmed | The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosa |
| title_short | The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosa |
| title_sort | lysr family transcriptional regulator orf l16 regulates spinosad biosynthesis in saccharopolyspora spinosa |
| topic | Spinosad LysR family transcriptional regulator Spinosyn A Regulation Saccharopolyspora spinosa |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X24000693 |
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