Transcription factor-based biosensors: a molecular-guided approach for natural product engineering

Abstract

Natural products and their derivatives offer a rich source of chemical and biological diversity; however, traditional engineering of their biosynthetic pathways to improve yields and access to unnatural derivatives requires a precise understanding of their enzymatic processes. High-throughput screening platforms based on allosteric transcription-factor based biosensors can be leveraged to overcome the screening bottleneck to enable searching through large libraries of pathway/strain variants. Herein, the development and application of engineered allosteric transcription factor-based biosensors is described that enable optimization of precursor availability, product titers, and downstream product tailoring for advancing the natural product bioeconomy. We discuss recent successes for tailoring biosensor design, including computationally-based approaches, and present our future outlook with the integration of cell-free technologies and de novo protein design for rapidly generating biosensor tools.

Figure 1.

Coupling genetically-encoded aTF biosensor platforms to natural product biosynthesis and engineering. Natural product biosynthetic logic is frequently divided into precursor generation, scaffold assembly, and tailoring, thereby providing several entry points for detection by aTFs. A variety of biosynthetic precursors (outlined in blue) and mature natural products (outlined in red), have been detected by aTF-based biosensors.

Figure 2.

Engineering and optimizing transcription factor-based biosensors. (a) Genetic circuit of a biosensor platform whereby the aTF regulates the expression of downstream genes by binding to its cognitive operator sequence. In the presence of an inducer, the system will derepress, allowing for the expression of a reporter signal. The corresponding dose response curve describing the response as a function of effector concentration is used to evaluate the biosensor transfer functions, as defined by the Hill equation. (b) aTF biosensor platforms can be tailored for a preferred function, including (i) sensitivity, (ii) dynamic range, (iii) effector specificity, (iv) control over transcription and translation via promoter and RBS engineering, (v) tailored substrate selectivity and/or promiscuity via protein engineering.

Figure 3.

Basic mechanism of (a) polyketide biosynthesis catalyzed by modular Type I PKSs and (b) the mevalonate pathway for hemiterpene production.