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

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 σ ⁷⁰ lac and tet expression systems have shown effective regulation and robust expression of recombinant proteins across various Gram-negative bacteria. To leverage their advantages, here both 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. This rapid approach enabled the straightforward transformation of the well-established microorganisms Escherichia coli , Pseudomonas putida , and Vibrio natriegens into efficient microbial cell factories. The dynamic range and the basal expression levels of the σ ⁷⁰ expression systems were further enhanced through the incorporation of translational control mechanisms via toehold switches. This improvement was assessed using the highly sensitive luciferase reporter system. This study presents the development and remaining challenges of a versatile genetic tool that is portable across well-established bacterial chassis and capable of controlling the expression of multiple genes, thus facilitating the biosynthesis and study of natural products.

Graphical Abstract

Highlights

• A dual-inducible duet-expression system is described for Gram-negative organisms.

• Production of lycopene and β-carotene is demonstrated in E. coli, P. putida, and V. natriegens .

• Incorporation of a toehold switch effectively reduces leaky expression of target genes in the uninduced state.

• The σ ⁷⁰ duet lac/tet expression system is a versatile tool for multi-gene biosynthesis across different bacterial chassis.