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Jie Rang - One of the best experts on this subject based on the ideXlab platform.

  • Identification of a TetR family regulator and a polyketide synthase gene cluster involved in growth development and butenyl-spinosyn biosynthesis of Saccharopolyspora Pogona
    Applied microbiology and biotechnology, 2021
    Co-Authors: Jie Rang, Jianli Tang, Jianming Chen, Zirong Zhu, Li Cao, Huang Weitao
    Abstract:

    Butenyl-spinosyn produced by Saccharopolyspora Pogona exhibits strong insecticidal activity and broad pesticidal spectrum. However, its synthetic level was low in the wild-type strain. At present, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently editing its genome to improve the butenyl-spinosyn yield. To accelerate the genetic modification of S. Pogona, we conducted comparative proteomics analysis to screen differentially expressed proteins related to butenyl-spinosyn biosynthesis. A TetR family regulatory protein was selected from the 289 differentially expressed proteins, and its encoding gene (SP_1288) was successfully deleted by CRISPR/Cas9 system. We further deleted a 32-kb polyketide synthase gene cluster (cluster 28) to reduce the competition for precursors. Phenotypic analysis revealed that the deletion of the SP_1288 and cluster 28 resulted in a 3.10-fold increase and a 35.4% decrease in the butenyl-spinosyn levels compared with the wild-type strain, respectively. The deletion of cluster 28 affected the cell growth, glucose consumption, mycelium morphology, and sporulation by controlling the expression of ptsH, ptsI, amfC, and other genes related to sporulation, whereas SP_1288 did not. These findings confirmed not only that the CRISPR/Cas9 system can be applied to the S. Pogona genome editing but also that SP_1288 and cluster 28 are closely related to the butenyl-spinosyn biosynthesis and growth development of S. Pogona. The strategy reported here will be useful to reveal the regulatory mechanism of butenyl-spinosyn and improve antibiotic production in other actinomycetes. KEY POINTS: • SP_1288 deletion can significantly promote the butenyl-spinosyn biosynthesis. • Cluster 28 deletion showed pleiotropic effects on S. Pogona. • SP_1288 and cluster 28 were deleted by CRISPR/Cas9 system in S. Pogona.

  • Deletion of a hybrid NRPS-T1PKS biosynthetic gene cluster via Latour gene knockout system in Saccharopolyspora Pogona and its effect on butenyl-spinosyn biosynthesis and growth development.
    Microbial biotechnology, 2020
    Co-Authors: Jie Rang, Li Cao, Ling Shuai, Yang Liu, Qianqian Wan, Yuewen Luo, Youming Zhang
    Abstract:

    Butenyl-spinosyn, a promising biopesticide produced by Saccharopolyspora Pogona, exhibits stronger insecticidal activity and a broader pesticidal spectrum. However, its titre in the wild-type S. Pogona strain is too low to meet the industrial production requirements. Deletion of non-target natural product biosynthetic gene clusters resident in the genome of S. Pogona could reduce the consumption of synthetic precursors, thereby promoting the biosynthesis of butenyl-spinosyn. However, it has always been a challenge for scientists to genetically engineer S. Pogona. In this study, the Latour gene knockout system (linear DNA fragment recombineering system) was established in S. Pogona. Using the Latour system, a hybrid NRPS-T1PKS cluster (~20 kb) which was responsible for phthoxazolin biosynthesis was efficiently deleted in S. Pogona. The resultant mutant S. Pogona-Δura4-Δc14 exhibited an extended logarithmic phase, increased biomass and a lower glucose consumption rate. Importantly, the production of butenyl-spinosyn in S. Pogona-Δura4-Δc14 was increased by 4.72-fold compared with that in the wild-type strain. qRT-PCR analysis revealed that phthoxazolin biosynthetic gene cluster deletion could promote the expression of the butenyl-spinosyn biosynthetic gene cluster. Furthermore, a TetR family transcriptional regulatory gene that could regulate the butenyl-spinosyn biosynthesis has been identified from the phthoxazolin biosynthetic gene cluster. Because dozens of natural product biosynthetic gene clusters exist in the genome of S. Pogona, the strategy reported here will be used to further promote the production of butenyl-spinosyn by deleting other secondary metabolite synthetic gene clusters.

  • Effects of lytS-L on the primary metabolism and butenyl-spinosyn biosynthesis in Saccharopolyspora Pogona
    Gene, 2020
    Co-Authors: Shengnan Peng, Jie Rang, Shuangqin Yuan, Jianli Tang, Zhudong Liu, Jianming Chen, Ziyuan Xia, Xuezhi Ding
    Abstract:

    Abstract The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora Pogona NRRL 30141 (S. Pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. Pogona and S. Pogona-ΔlytS-L: a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or

  • SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora Pogona.
    iScience, 2020
    Co-Authors: Jie Rang, Jianli Tang, Xuezhi Ding, Zhudong Liu, Youming Zhang, Jianming Chen, Ziyuan Xia, Liqiu Xia
    Abstract:

    Summary Butenyl-spinosyn produced by Saccharopolyspora Pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. Pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

  • deciphering the metabolic pathway difference between saccharopolyspora Pogona and saccharopolyspora spinosa by comparative proteomics and metabonomics
    Frontiers in Microbiology, 2020
    Co-Authors: Jie Rang, Ziquan Yu, Shengbiao Hu, Haocheng He, Shuangqin Yuan, Jianli Tang, Tahir Ali Khan, Yibo Hu, Weitao Huang, Xuezhi Ding
    Abstract:

    Butenyl-spinosyn, a secondary metabolite produced by Saccharopolyspora Pogona, exhibits strong insecticidal activity than spinosyn. However, the low synthesis capacity and unknown metabolic characteristics of butenyl-spinosyn in wild-type S. Pogona limit its broad application and metabolic engineering. Here, we showed that S. Pogona exhibited increased glucose consumption ability and growth rate compared with S. spinosa, but the production of butenyl-spinosyn was much lower than that of spinosyn. To further elucidate the metabolic mechanism of these different phenotypes, we performed a comparative proteomic and metabolomic study on S. Pogona and S. spinosa to identify the change in the abundance levels of proteins and metabolites. We found that the abundance of most proteins and metabolites associated with glucose transport, fatty acid metabolism, tricarboxylic acid cycle, amino acid metabolism, energy metabolism, purine and pyrimidine metabolism, and target product biosynthesis in S. Pogona was higher than that in S. spinosa. However, the overall abundance of proteins involved in butenyl-spinosyn biosynthesis was much lower than that of the high-abundance protein chaperonin GroEL, such as the enzymes related to rhamnose synthesis. We speculated that these protein and metabolite abundance changes may be directly responsible for the above phenotypic changes in S. Pogona and S. spinosa, especially affecting butenyl-spinosyn biosynthesis. Further studies revealed that the over-expression of the rhamnose synthetic genes and methionine adenosyltransferase gene could effectively improve the production of butenyl-spinosyn by 2.69-folds and 3.03-folds, respectively, confirming the reliability of this conjecture. This work presents the first comparative proteomics and metabolomics study of S. Pogona and S. spinosa, providing new insights into the novel links of phenotypic change and metabolic difference between two strains. The result will be valuable in designing strategies to promote the biosynthesis of butenyl-spinosyn by metabolic engineering.

Xuezhi Ding - One of the best experts on this subject based on the ideXlab platform.

  • Effects of lytS-L on the primary metabolism and butenyl-spinosyn biosynthesis in Saccharopolyspora Pogona
    Gene, 2020
    Co-Authors: Shengnan Peng, Jie Rang, Shuangqin Yuan, Jianli Tang, Zhudong Liu, Jianming Chen, Ziyuan Xia, Xuezhi Ding
    Abstract:

    Abstract The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora Pogona NRRL 30141 (S. Pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. Pogona and S. Pogona-ΔlytS-L: a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or

  • SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora Pogona.
    iScience, 2020
    Co-Authors: Jie Rang, Jianli Tang, Xuezhi Ding, Zhudong Liu, Youming Zhang, Jianming Chen, Ziyuan Xia, Liqiu Xia
    Abstract:

    Summary Butenyl-spinosyn produced by Saccharopolyspora Pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. Pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

  • deciphering the metabolic pathway difference between saccharopolyspora Pogona and saccharopolyspora spinosa by comparative proteomics and metabonomics
    Frontiers in Microbiology, 2020
    Co-Authors: Jie Rang, Ziquan Yu, Shengbiao Hu, Haocheng He, Shuangqin Yuan, Jianli Tang, Tahir Ali Khan, Yibo Hu, Weitao Huang, Xuezhi Ding
    Abstract:

    Butenyl-spinosyn, a secondary metabolite produced by Saccharopolyspora Pogona, exhibits strong insecticidal activity than spinosyn. However, the low synthesis capacity and unknown metabolic characteristics of butenyl-spinosyn in wild-type S. Pogona limit its broad application and metabolic engineering. Here, we showed that S. Pogona exhibited increased glucose consumption ability and growth rate compared with S. spinosa, but the production of butenyl-spinosyn was much lower than that of spinosyn. To further elucidate the metabolic mechanism of these different phenotypes, we performed a comparative proteomic and metabolomic study on S. Pogona and S. spinosa to identify the change in the abundance levels of proteins and metabolites. We found that the abundance of most proteins and metabolites associated with glucose transport, fatty acid metabolism, tricarboxylic acid cycle, amino acid metabolism, energy metabolism, purine and pyrimidine metabolism, and target product biosynthesis in S. Pogona was higher than that in S. spinosa. However, the overall abundance of proteins involved in butenyl-spinosyn biosynthesis was much lower than that of the high-abundance protein chaperonin GroEL, such as the enzymes related to rhamnose synthesis. We speculated that these protein and metabolite abundance changes may be directly responsible for the above phenotypic changes in S. Pogona and S. spinosa, especially affecting butenyl-spinosyn biosynthesis. Further studies revealed that the over-expression of the rhamnose synthetic genes and methionine adenosyltransferase gene could effectively improve the production of butenyl-spinosyn by 2.69-folds and 3.03-folds, respectively, confirming the reliability of this conjecture. This work presents the first comparative proteomics and metabolomics study of S. Pogona and S. spinosa, providing new insights into the novel links of phenotypic change and metabolic difference between two strains. The result will be valuable in designing strategies to promote the biosynthesis of butenyl-spinosyn by metabolic engineering.

  • Effect of the TetR family transcriptional regulator Sp1418 on the global metabolic network of Saccharopolyspora Pogona
    Microbial cell factories, 2020
    Co-Authors: Shuangqin Yuan, Jie Rang, Jianli Tang, Zhudong Liu, Jianming Chen, Ziyuan Xia, Xuezhi Ding
    Abstract:

    Saccharopolyspora Pogona is a prominent industrial strain due to its production of butenyl-spinosyn, a high-quality insecticide against a broad spectrum of insect pests. TetR family proteins are diverse in a tremendous number of microorganisms and some are been researched to have a key role in metabolic regulation. However, specific functions of TetR family proteins in S. Pogona are yet to characterize. In the present study, the overexpression of the tetR-like gene sp1418 in S. Pogona resulted in marked effects on vegetative growth, sporulation, butenyl-spinosyn biosynthesis, and oxidative stress. By using qRT-PCR analysis, mass spectrometry, enzyme activity detection, and sp1418 knockout verification, we showed that most of these effects could be attributed to the overexpression of Sp1418, which modulated enzymes related to the primary metabolism, oxidative stress and secondary metabolism, and thereby resulted in distinct growth characteristics and an unbalanced supply of precursor monomers for butenyl-spinosyn biosynthesis. This study revealed the function of Sp1418 and enhanced the understanding of the metabolic network in S. Pogona, and provided insights into the improvement of secondary metabolite production.

  • effect of fcl gene for butenyl spinosyn biosynthesis and growth of saccharopolyspora Pogona
    Chinese Journal of Biotechnology, 2019
    Co-Authors: Shengnan Peng, Jie Rang, Shuangqin Yuan, Weitao Huang, Xuezhi Ding, Yunjun Sun, Liqiu Xia
    Abstract:

    The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora Pogona was found through sequencing result of genome. The mutant S. Pogona-fcl and S. Pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora Pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. Pogona-Δfcl was 130% compared with that in S. Pogona, which reduced by 25% in S. Pogona-fcl. The results of determination of insecticidal activity showed that S. Pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. Pogona, while the S. Pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. Pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. Pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. Pogona-fcl and S. Pogona. Aboved all showed that deletion of fcl gene in S. Pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.

Jianli Tang - One of the best experts on this subject based on the ideXlab platform.

  • Identification of a TetR family regulator and a polyketide synthase gene cluster involved in growth development and butenyl-spinosyn biosynthesis of Saccharopolyspora Pogona
    Applied microbiology and biotechnology, 2021
    Co-Authors: Jie Rang, Jianli Tang, Jianming Chen, Zirong Zhu, Li Cao, Huang Weitao
    Abstract:

    Butenyl-spinosyn produced by Saccharopolyspora Pogona exhibits strong insecticidal activity and broad pesticidal spectrum. However, its synthetic level was low in the wild-type strain. At present, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently editing its genome to improve the butenyl-spinosyn yield. To accelerate the genetic modification of S. Pogona, we conducted comparative proteomics analysis to screen differentially expressed proteins related to butenyl-spinosyn biosynthesis. A TetR family regulatory protein was selected from the 289 differentially expressed proteins, and its encoding gene (SP_1288) was successfully deleted by CRISPR/Cas9 system. We further deleted a 32-kb polyketide synthase gene cluster (cluster 28) to reduce the competition for precursors. Phenotypic analysis revealed that the deletion of the SP_1288 and cluster 28 resulted in a 3.10-fold increase and a 35.4% decrease in the butenyl-spinosyn levels compared with the wild-type strain, respectively. The deletion of cluster 28 affected the cell growth, glucose consumption, mycelium morphology, and sporulation by controlling the expression of ptsH, ptsI, amfC, and other genes related to sporulation, whereas SP_1288 did not. These findings confirmed not only that the CRISPR/Cas9 system can be applied to the S. Pogona genome editing but also that SP_1288 and cluster 28 are closely related to the butenyl-spinosyn biosynthesis and growth development of S. Pogona. The strategy reported here will be useful to reveal the regulatory mechanism of butenyl-spinosyn and improve antibiotic production in other actinomycetes. KEY POINTS: • SP_1288 deletion can significantly promote the butenyl-spinosyn biosynthesis. • Cluster 28 deletion showed pleiotropic effects on S. Pogona. • SP_1288 and cluster 28 were deleted by CRISPR/Cas9 system in S. Pogona.

  • Effects of lytS-L on the primary metabolism and butenyl-spinosyn biosynthesis in Saccharopolyspora Pogona
    Gene, 2020
    Co-Authors: Shengnan Peng, Jie Rang, Shuangqin Yuan, Jianli Tang, Zhudong Liu, Jianming Chen, Ziyuan Xia, Xuezhi Ding
    Abstract:

    Abstract The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora Pogona NRRL 30141 (S. Pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. Pogona and S. Pogona-ΔlytS-L: a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or

  • SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora Pogona.
    iScience, 2020
    Co-Authors: Jie Rang, Jianli Tang, Xuezhi Ding, Zhudong Liu, Youming Zhang, Jianming Chen, Ziyuan Xia, Liqiu Xia
    Abstract:

    Summary Butenyl-spinosyn produced by Saccharopolyspora Pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. Pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

  • deciphering the metabolic pathway difference between saccharopolyspora Pogona and saccharopolyspora spinosa by comparative proteomics and metabonomics
    Frontiers in Microbiology, 2020
    Co-Authors: Jie Rang, Ziquan Yu, Shengbiao Hu, Haocheng He, Shuangqin Yuan, Jianli Tang, Tahir Ali Khan, Yibo Hu, Weitao Huang, Xuezhi Ding
    Abstract:

    Butenyl-spinosyn, a secondary metabolite produced by Saccharopolyspora Pogona, exhibits strong insecticidal activity than spinosyn. However, the low synthesis capacity and unknown metabolic characteristics of butenyl-spinosyn in wild-type S. Pogona limit its broad application and metabolic engineering. Here, we showed that S. Pogona exhibited increased glucose consumption ability and growth rate compared with S. spinosa, but the production of butenyl-spinosyn was much lower than that of spinosyn. To further elucidate the metabolic mechanism of these different phenotypes, we performed a comparative proteomic and metabolomic study on S. Pogona and S. spinosa to identify the change in the abundance levels of proteins and metabolites. We found that the abundance of most proteins and metabolites associated with glucose transport, fatty acid metabolism, tricarboxylic acid cycle, amino acid metabolism, energy metabolism, purine and pyrimidine metabolism, and target product biosynthesis in S. Pogona was higher than that in S. spinosa. However, the overall abundance of proteins involved in butenyl-spinosyn biosynthesis was much lower than that of the high-abundance protein chaperonin GroEL, such as the enzymes related to rhamnose synthesis. We speculated that these protein and metabolite abundance changes may be directly responsible for the above phenotypic changes in S. Pogona and S. spinosa, especially affecting butenyl-spinosyn biosynthesis. Further studies revealed that the over-expression of the rhamnose synthetic genes and methionine adenosyltransferase gene could effectively improve the production of butenyl-spinosyn by 2.69-folds and 3.03-folds, respectively, confirming the reliability of this conjecture. This work presents the first comparative proteomics and metabolomics study of S. Pogona and S. spinosa, providing new insights into the novel links of phenotypic change and metabolic difference between two strains. The result will be valuable in designing strategies to promote the biosynthesis of butenyl-spinosyn by metabolic engineering.

  • Effect of the TetR family transcriptional regulator Sp1418 on the global metabolic network of Saccharopolyspora Pogona
    Microbial cell factories, 2020
    Co-Authors: Shuangqin Yuan, Jie Rang, Jianli Tang, Zhudong Liu, Jianming Chen, Ziyuan Xia, Xuezhi Ding
    Abstract:

    Saccharopolyspora Pogona is a prominent industrial strain due to its production of butenyl-spinosyn, a high-quality insecticide against a broad spectrum of insect pests. TetR family proteins are diverse in a tremendous number of microorganisms and some are been researched to have a key role in metabolic regulation. However, specific functions of TetR family proteins in S. Pogona are yet to characterize. In the present study, the overexpression of the tetR-like gene sp1418 in S. Pogona resulted in marked effects on vegetative growth, sporulation, butenyl-spinosyn biosynthesis, and oxidative stress. By using qRT-PCR analysis, mass spectrometry, enzyme activity detection, and sp1418 knockout verification, we showed that most of these effects could be attributed to the overexpression of Sp1418, which modulated enzymes related to the primary metabolism, oxidative stress and secondary metabolism, and thereby resulted in distinct growth characteristics and an unbalanced supply of precursor monomers for butenyl-spinosyn biosynthesis. This study revealed the function of Sp1418 and enhanced the understanding of the metabolic network in S. Pogona, and provided insights into the improvement of secondary metabolite production.

Shuangqin Yuan - One of the best experts on this subject based on the ideXlab platform.

  • Effects of lytS-L on the primary metabolism and butenyl-spinosyn biosynthesis in Saccharopolyspora Pogona
    Gene, 2020
    Co-Authors: Shengnan Peng, Jie Rang, Shuangqin Yuan, Jianli Tang, Zhudong Liu, Jianming Chen, Ziyuan Xia, Xuezhi Ding
    Abstract:

    Abstract The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora Pogona NRRL 30141 (S. Pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. Pogona and S. Pogona-ΔlytS-L: a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or

  • deciphering the metabolic pathway difference between saccharopolyspora Pogona and saccharopolyspora spinosa by comparative proteomics and metabonomics
    Frontiers in Microbiology, 2020
    Co-Authors: Jie Rang, Ziquan Yu, Shengbiao Hu, Haocheng He, Shuangqin Yuan, Jianli Tang, Tahir Ali Khan, Yibo Hu, Weitao Huang, Xuezhi Ding
    Abstract:

    Butenyl-spinosyn, a secondary metabolite produced by Saccharopolyspora Pogona, exhibits strong insecticidal activity than spinosyn. However, the low synthesis capacity and unknown metabolic characteristics of butenyl-spinosyn in wild-type S. Pogona limit its broad application and metabolic engineering. Here, we showed that S. Pogona exhibited increased glucose consumption ability and growth rate compared with S. spinosa, but the production of butenyl-spinosyn was much lower than that of spinosyn. To further elucidate the metabolic mechanism of these different phenotypes, we performed a comparative proteomic and metabolomic study on S. Pogona and S. spinosa to identify the change in the abundance levels of proteins and metabolites. We found that the abundance of most proteins and metabolites associated with glucose transport, fatty acid metabolism, tricarboxylic acid cycle, amino acid metabolism, energy metabolism, purine and pyrimidine metabolism, and target product biosynthesis in S. Pogona was higher than that in S. spinosa. However, the overall abundance of proteins involved in butenyl-spinosyn biosynthesis was much lower than that of the high-abundance protein chaperonin GroEL, such as the enzymes related to rhamnose synthesis. We speculated that these protein and metabolite abundance changes may be directly responsible for the above phenotypic changes in S. Pogona and S. spinosa, especially affecting butenyl-spinosyn biosynthesis. Further studies revealed that the over-expression of the rhamnose synthetic genes and methionine adenosyltransferase gene could effectively improve the production of butenyl-spinosyn by 2.69-folds and 3.03-folds, respectively, confirming the reliability of this conjecture. This work presents the first comparative proteomics and metabolomics study of S. Pogona and S. spinosa, providing new insights into the novel links of phenotypic change and metabolic difference between two strains. The result will be valuable in designing strategies to promote the biosynthesis of butenyl-spinosyn by metabolic engineering.

  • Effect of the TetR family transcriptional regulator Sp1418 on the global metabolic network of Saccharopolyspora Pogona
    Microbial cell factories, 2020
    Co-Authors: Shuangqin Yuan, Jie Rang, Jianli Tang, Zhudong Liu, Jianming Chen, Ziyuan Xia, Xuezhi Ding
    Abstract:

    Saccharopolyspora Pogona is a prominent industrial strain due to its production of butenyl-spinosyn, a high-quality insecticide against a broad spectrum of insect pests. TetR family proteins are diverse in a tremendous number of microorganisms and some are been researched to have a key role in metabolic regulation. However, specific functions of TetR family proteins in S. Pogona are yet to characterize. In the present study, the overexpression of the tetR-like gene sp1418 in S. Pogona resulted in marked effects on vegetative growth, sporulation, butenyl-spinosyn biosynthesis, and oxidative stress. By using qRT-PCR analysis, mass spectrometry, enzyme activity detection, and sp1418 knockout verification, we showed that most of these effects could be attributed to the overexpression of Sp1418, which modulated enzymes related to the primary metabolism, oxidative stress and secondary metabolism, and thereby resulted in distinct growth characteristics and an unbalanced supply of precursor monomers for butenyl-spinosyn biosynthesis. This study revealed the function of Sp1418 and enhanced the understanding of the metabolic network in S. Pogona, and provided insights into the improvement of secondary metabolite production.

  • effect of fcl gene for butenyl spinosyn biosynthesis and growth of saccharopolyspora Pogona
    Chinese Journal of Biotechnology, 2019
    Co-Authors: Shengnan Peng, Jie Rang, Shuangqin Yuan, Weitao Huang, Xuezhi Ding, Yunjun Sun, Liqiu Xia
    Abstract:

    The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora Pogona was found through sequencing result of genome. The mutant S. Pogona-fcl and S. Pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora Pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. Pogona-Δfcl was 130% compared with that in S. Pogona, which reduced by 25% in S. Pogona-fcl. The results of determination of insecticidal activity showed that S. Pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. Pogona, while the S. Pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. Pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. Pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. Pogona-fcl and S. Pogona. Aboved all showed that deletion of fcl gene in S. Pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.

  • AfsR is an important regulatory factor for growth and butenyl-spinosyn biosynthesis of Saccharopolyspora Pogona
    Annals of Microbiology, 2019
    Co-Authors: Liang Gong, Jie Rang, Shuangqin Yuan, Jianli Tang, Zhudong Liu, Shengnan Peng, Xuezhi Ding
    Abstract:

    To generate a AfsR-like (AfsR-L) overexpression strain Saccharopolyspora Pogona-AfsR-L and investigate its effects on the morphology and metabolism of S. Pogona. Firstly, we generated the overexpression vector pOJ260-PermE-afsR-L via overlap extension PCR. Then, the recombination strain S. Pogona-AfsR-L was constructed via conjugal transfer. To monitor the growth and morphology, mycelia and sporulation were observed. The distinctive proteins and butenyl-spinosyn biosynthesis were investigated by SDS-PAGE, HPLC, and mass spectrometry. And the transcriptional level of afsR-L and other relative functional genes in S. Pogona-AfsR-L was analyzed by qRT-PCR. Western blot verified the increased amount of AfsR-L protein in the overexpression strain. Growth curve and mycelia observation showed that afsR-L overexpression make the stationary phase of S. Pogona-AfsR-L longer than that of wild S. Pogona by approximate 3 days. Moreover, S. Pogona-AfsR-L exhibited a more obvious white phenotype on the solid medium, which means afsR-L overexpression affects the sporulation ability of S. Pogona. HPLC analysis revealed that the peak area of the butenyl-spinosyn yield of S. Pogona-AfsR-L was 293.6, while that of S. Pogona was 250.9. SDS-PAGE analysis showed that the two strains had different whole protein expression profiles, and the distinctive proteins were further identified by LC-MS/MS identification, which showed the possible control mechanism of afsR-L gene in S. Pogona. We concluded that AfsR could directly or indirectly positively regulate the biosynthesis of butenyl-spinosyn and affect the growth features of S. Pogona. We envisioned that this result can be expanded to other Streptomyces for strain improvement.

Zhudong Liu - One of the best experts on this subject based on the ideXlab platform.

  • Effects of lytS-L on the primary metabolism and butenyl-spinosyn biosynthesis in Saccharopolyspora Pogona
    Gene, 2020
    Co-Authors: Shengnan Peng, Jie Rang, Shuangqin Yuan, Jianli Tang, Zhudong Liu, Jianming Chen, Ziyuan Xia, Xuezhi Ding
    Abstract:

    Abstract The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora Pogona NRRL 30141 (S. Pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. Pogona and S. Pogona-ΔlytS-L: a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or

  • SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora Pogona.
    iScience, 2020
    Co-Authors: Jie Rang, Jianli Tang, Xuezhi Ding, Zhudong Liu, Youming Zhang, Jianming Chen, Ziyuan Xia, Liqiu Xia
    Abstract:

    Summary Butenyl-spinosyn produced by Saccharopolyspora Pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. Pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

  • Effect of the TetR family transcriptional regulator Sp1418 on the global metabolic network of Saccharopolyspora Pogona
    Microbial cell factories, 2020
    Co-Authors: Shuangqin Yuan, Jie Rang, Jianli Tang, Zhudong Liu, Jianming Chen, Ziyuan Xia, Xuezhi Ding
    Abstract:

    Saccharopolyspora Pogona is a prominent industrial strain due to its production of butenyl-spinosyn, a high-quality insecticide against a broad spectrum of insect pests. TetR family proteins are diverse in a tremendous number of microorganisms and some are been researched to have a key role in metabolic regulation. However, specific functions of TetR family proteins in S. Pogona are yet to characterize. In the present study, the overexpression of the tetR-like gene sp1418 in S. Pogona resulted in marked effects on vegetative growth, sporulation, butenyl-spinosyn biosynthesis, and oxidative stress. By using qRT-PCR analysis, mass spectrometry, enzyme activity detection, and sp1418 knockout verification, we showed that most of these effects could be attributed to the overexpression of Sp1418, which modulated enzymes related to the primary metabolism, oxidative stress and secondary metabolism, and thereby resulted in distinct growth characteristics and an unbalanced supply of precursor monomers for butenyl-spinosyn biosynthesis. This study revealed the function of Sp1418 and enhanced the understanding of the metabolic network in S. Pogona, and provided insights into the improvement of secondary metabolite production.

  • AfsR is an important regulatory factor for growth and butenyl-spinosyn biosynthesis of Saccharopolyspora Pogona
    Annals of Microbiology, 2019
    Co-Authors: Liang Gong, Jie Rang, Shuangqin Yuan, Jianli Tang, Zhudong Liu, Shengnan Peng, Xuezhi Ding
    Abstract:

    To generate a AfsR-like (AfsR-L) overexpression strain Saccharopolyspora Pogona-AfsR-L and investigate its effects on the morphology and metabolism of S. Pogona. Firstly, we generated the overexpression vector pOJ260-PermE-afsR-L via overlap extension PCR. Then, the recombination strain S. Pogona-AfsR-L was constructed via conjugal transfer. To monitor the growth and morphology, mycelia and sporulation were observed. The distinctive proteins and butenyl-spinosyn biosynthesis were investigated by SDS-PAGE, HPLC, and mass spectrometry. And the transcriptional level of afsR-L and other relative functional genes in S. Pogona-AfsR-L was analyzed by qRT-PCR. Western blot verified the increased amount of AfsR-L protein in the overexpression strain. Growth curve and mycelia observation showed that afsR-L overexpression make the stationary phase of S. Pogona-AfsR-L longer than that of wild S. Pogona by approximate 3 days. Moreover, S. Pogona-AfsR-L exhibited a more obvious white phenotype on the solid medium, which means afsR-L overexpression affects the sporulation ability of S. Pogona. HPLC analysis revealed that the peak area of the butenyl-spinosyn yield of S. Pogona-AfsR-L was 293.6, while that of S. Pogona was 250.9. SDS-PAGE analysis showed that the two strains had different whole protein expression profiles, and the distinctive proteins were further identified by LC-MS/MS identification, which showed the possible control mechanism of afsR-L gene in S. Pogona. We concluded that AfsR could directly or indirectly positively regulate the biosynthesis of butenyl-spinosyn and affect the growth features of S. Pogona. We envisioned that this result can be expanded to other Streptomyces for strain improvement.

  • Impact on strain growth and butenyl-spinosyn biosynthesis by overexpression of polynucleotide phosphorylase gene in Saccharopolyspora Pogona
    Applied microbiology and biotechnology, 2018
    Co-Authors: Jie Rang, Jianli Tang, Zhudong Liu, Jie Xiao
    Abstract:

    Polynucleotide phosphorylase is a highly conserved protein found in bacteria and fungi that can regulate the transcription of related enzymes involved in amino acid metabolism, organic acid metabolism, and cell biosynthesis. We studied the effect of polynucleotide phosphorylase on Saccharopolyspora Pogona (S. Pogona) growth and the synthesis of secondary metabolites. First, we generated the overexpression vector pOJ260-PermE-pnp via overlap extension PCR. The vector pOJ260-PermE-pnp was then introduced into S. Pogona by conjugal transfer, thereby generating the recombination strain S. Pogona-Pnp. Results showed that engineering strains possessed higher biomass than those of the wild-type strains. Moreover, the ability of these strains to produce spores on solid medium was stronger than that of the wild-type strains. HPLC results revealed that the butenyl-spinosyn yield in S. Pogona-Pnp increased by 1.92-fold compared with that of S. Pogona alone. These findings revealed that overexpression of polynucleotide phosphorylase effectively promoted butenyl-spinosyn biosynthesis in S. Pogona. This result may be extended to other Streptomyces for strain improvement.