Expanding protein sequence space beyond the bounds of evolution: Polyketide synthase-driven biosynthesis of designer amino acids

Engineering life beyond nature’s constraints would benefit greatly from an expanded set of amino acids beyond the 20 that are used to synthesize proteins in living organisms. This project will enable economical, large-scale production of proteins with non-standard amino acids for a variety of applications, including enzymes with improved stabilities or novel catalytic activities, biomaterials with enhanced properties, and pharmaceutical proteins.

The resources for producing these non-standard amino acids will be sugars derived from waste or from U.S. agriculture, which may lead to new markets for U.S. agricultural products. Postdoctoral fellows, undergraduate students and high school students will participate in this project, and will help construct educational and interactive public exhibits on sustainable manufacturing.

Engineering life beyond Nature’s constraints would benefit greatly from an expanded set of amino acids beyond the 20 that are used to synthesize proteins in living organisms. The goal of the proposed work is to develop a polyketide-based platform to produce many non-canonical amino acids (ncAAs) and demonstrate their incorporation into proteins expressed by Bacillus subtilis.

The investigators have chosen B. subtilis as the host for their system because of its GRAS (generally regarded as safe) status, its ability to express PKSs, its precedence and available tools for genetic code expansion, and its industrial use to produce recombinant proteins and enzymes. In the first aim, the investigators will develop an engineered polyketide synthase (PKS) system to produce threonine and complement the growth of an auxotrophic strain; they will use auxotrophy to evolve the PKS to be more productive.

In the second aim, multiple PKS loading modules, in conjunction with the previously employed PKS extension module from Aim 1, will be utilized to create a range of ncAAs. To broaden the spectrum of producible ncAAs, a full reduction loop will be introduced into the last extension module of the PKSs; the fully reducing PKSs will also enable production of two more natural amino acids, and their auxotrophies will be used to evolve the fully reducing PKS.

In the third aim, the investigators will introduce an orthogonal translation system into multiple ncAA producing B. subtilis strains and demonstrate the application of the platform to screen an amber codon mutant library of lipases for enhanced activity to depolymerize plastics. In the final aim, they will develop a containment system for the ncAA-producing B. subtilis and educate all members of the research team as well as the public about the safety, ethical, and security concerns around engineered biology.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.