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 versatile and portable genetic tools offers a streamlined pathway to efficiently produce a variety of compounds in well-established chassis organisms. The σ⁷⁰lac 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 σ⁷⁰lac 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 σ⁷⁰lac 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.