A dual-inducible control system for multistep biosynthetic pathways
Abstract Background The successful production of industrially relevant natural products hinges on two key factors: the cultivation of robust microbial chassis capable of synthesizing the desired compounds, and the availability of reliable genetic tools for expressing target genes. The development of...
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BMC
2024-11-01
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| Series: | Journal of Biological Engineering |
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| Online Access: | https://doi.org/10.1186/s13036-024-00462-z |
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| author | Andrés Felipe Carrillo Rincón Alexandra J. Cabral Andras Gyorgy Natalie G. Farny |
| author_facet | Andrés Felipe Carrillo Rincón Alexandra J. Cabral Andras Gyorgy Natalie G. Farny |
| author_sort | Andrés Felipe Carrillo Rincón |
| collection | DOAJ |
| description | Abstract Background The successful production of industrially relevant natural products hinges on two key factors: the cultivation of robust microbial chassis capable of synthesizing the desired compounds, and the availability of reliable genetic tools for expressing target genes. The development of versatile and portable genetic tools offers a streamlined pathway to efficiently produce a variety of compounds in well-established chassis organisms. The σ70 lac and tet expression systems – adaptations of the widely used lac and tet regulatory systems developed in our laboratory – have shown effective regulation and robust expression of recombinant proteins in various Gram-negative bacteria. Understanding the strengths and limitations of these regulatory systems in controlling recombinant protein production is essential for progress in this area. Results To assess their capacity for combinatorial control, both the σ70 lac and tet expression systems were combined into a single plasmid and assessed for their performance in producing fluorescent reporters as well as the terpenoids lycopene and β-carotene. We thoroughly characterized the induction range, potential for synergistic effects, and metabolic costs of our dual σ70 lac and tet expression system in the well-established microorganisms Escherichia coli, Pseudomonas putida, and Vibrio natriegens using combinations of fluorescent reporters. The dynamic range and basal transcriptional control of the σ70 expression systems were further improved through the incorporation of translational control mechanisms via toehold switches. This improvement was assessed using the highly sensitive luciferase reporter system. The improvement in control afforded by the integration of the toehold switches enabled the accumulation of a biosynthetic intermediate (lycopene) in the β-carotene synthesis pathway. Conclusion This study presents the development and remaining challenges of a set of versatile genetic tools that are portable across well-established gammaproteobacterial chassis and capable of controlling the expression of multigene biosynthetic pathways. The enhanced σ70 expression systems, combined with toehold switches, facilitate the biosynthesis and study of enzymes, recombinant proteins, and natural products, thus providing a valuable resource for producing a variety of compounds in microbial cell factories. Graphical Abstract |
| format | Article |
| id | doaj-art-eef8b05a445b4eedbb2e035e7d2526d0 |
| institution | Kabale University |
| issn | 1754-1611 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Biological Engineering |
| spelling | doaj-art-eef8b05a445b4eedbb2e035e7d2526d02024-11-24T12:29:33ZengBMCJournal of Biological Engineering1754-16112024-11-0118112110.1186/s13036-024-00462-zA dual-inducible control system for multistep biosynthetic pathwaysAndrés Felipe Carrillo Rincón0Alexandra J. Cabral1Andras Gyorgy2Natalie G. Farny3Department of Biology and Biotechnology, Worcester Polytechnic InstituteDepartment of Biology and Biotechnology, Worcester Polytechnic InstituteDivision of Engineering, New York University Abu DhabiDepartment of Biology and Biotechnology, Worcester Polytechnic InstituteAbstract Background The successful production of industrially relevant natural products hinges on two key factors: the cultivation of robust microbial chassis capable of synthesizing the desired compounds, and the availability of reliable genetic tools for expressing target genes. The development of versatile and portable genetic tools offers a streamlined pathway to efficiently produce a variety of compounds in well-established chassis organisms. The σ70 lac and tet expression systems – adaptations of the widely used lac and tet regulatory systems developed in our laboratory – have shown effective regulation and robust expression of recombinant proteins in various Gram-negative bacteria. Understanding the strengths and limitations of these regulatory systems in controlling recombinant protein production is essential for progress in this area. Results To assess their capacity for combinatorial control, both the σ70 lac and tet expression systems were combined into a single plasmid and assessed for their performance in producing fluorescent reporters as well as the terpenoids lycopene and β-carotene. We thoroughly characterized the induction range, potential for synergistic effects, and metabolic costs of our dual σ70 lac and tet expression system in the well-established microorganisms Escherichia coli, Pseudomonas putida, and Vibrio natriegens using combinations of fluorescent reporters. The dynamic range and basal transcriptional control of the σ70 expression systems were further improved through the incorporation of translational control mechanisms via toehold switches. This improvement was assessed using the highly sensitive luciferase reporter system. The improvement in control afforded by the integration of the toehold switches enabled the accumulation of a biosynthetic intermediate (lycopene) in the β-carotene synthesis pathway. Conclusion This study presents the development and remaining challenges of a set of versatile genetic tools that are portable across well-established gammaproteobacterial chassis and capable of controlling the expression of multigene biosynthetic pathways. The enhanced σ70 expression systems, combined with toehold switches, facilitate the biosynthesis and study of enzymes, recombinant proteins, and natural products, thus providing a valuable resource for producing a variety of compounds in microbial cell factories. Graphical Abstracthttps://doi.org/10.1186/s13036-024-00462-zInducible promotersDual expressionReporter systemLycopeneβ-caroteneLuciferase |
| spellingShingle | Andrés Felipe Carrillo Rincón Alexandra J. Cabral Andras Gyorgy Natalie G. Farny A dual-inducible control system for multistep biosynthetic pathways Journal of Biological Engineering Inducible promoters Dual expression Reporter system Lycopene β-carotene Luciferase |
| title | A dual-inducible control system for multistep biosynthetic pathways |
| title_full | A dual-inducible control system for multistep biosynthetic pathways |
| title_fullStr | A dual-inducible control system for multistep biosynthetic pathways |
| title_full_unstemmed | A dual-inducible control system for multistep biosynthetic pathways |
| title_short | A dual-inducible control system for multistep biosynthetic pathways |
| title_sort | dual inducible control system for multistep biosynthetic pathways |
| topic | Inducible promoters Dual expression Reporter system Lycopene β-carotene Luciferase |
| url | https://doi.org/10.1186/s13036-024-00462-z |
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