Protein engineering for natural product biosynthesis and synthetic biology applications.

As protein engineering grows more salient, many strategies have emerged to alter protein structure and function, with the goal of redesigning and optimizing natural product biosynthesis. Computational tools, including machine learning and molecular dynamics simulations, have enabled the rational mutagenesis of key catalytic residues for enhanced or altered biocatalysis. Semi-rational, directed evolution and microenvironment engineering strategies have optimized catalysis for native substrates and increased enzyme promiscuity beyond the scope of traditional rational approaches. These advances are made possible using novel high-throughput screens, including designer protein-based biosensors with engineered ligand specificity. Herein, we detail the most recent of these advances, focusing on polyketides, non-ribosomal peptides and isoprenoids, including their native biosynthetic logic to provide clarity for future applications of these technologies for natural product synthetic biology.

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

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.