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bioRxiv · 10.1101/2024.06.12.598700

A portable and dual-inducible control system for multistep biosynthetic pathways in Gram-negative bacteria

Rincon, A. F. C.·Cabral, A. J.·Gyorgy, A.·Farny, N. G.
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Abstract

BackgroundThe 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 {sigma}70 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. ResultsTo assess their capacity for combinatorial control, both the {sigma}70 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 {beta}-carotene. We thoroughly characterized the induction range, potential for synergistic effects, and metabolic costs of our dual {sigma}70 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 {sigma}70 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 {beta}-carotene synthesis pathway. ConclusionThis 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 {sigma}70 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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/598700v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@be801forg.highwire.dtl.DTLVardef@cda5deorg.highwire.dtl.DTLVardef@144b729org.highwire.dtl.DTLVardef@550011_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Rincon, A. F. C., Cabral, A. J., Gyorgy, A., Farny, N. G.. 2024-06-12. A portable and dual-inducible control system for multistep biosynthetic pathways in Gram-negative bacteria. https://doi.org/10.1101/2024.06.12.598700

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