Engineering Self-Remodeling Protein Complexes for Synthetic Biology

This project will establish a synthetic biology platform to build enzyme complexes that rapidly sense-and-respond to their environment, with applications towards building programmable biomolecular systems for bioprocessing, bioproduction, and novel biomaterial development. Biological systems frequently assemble multiple proteins with complementary functions into complexes to accelerate catalysis. Predictable control over which proteins assemble, when they assemble, and how they assemble would prove a major advance in our ability to engineer biology.

Here, we take inspiration from the multi-enzyme complexes (cellulosomes) made by anaerobic fungi to degrade plant matter in the guts of herbivores (Figure A). Fungal cellulosomes are an ideal template for self-remodeling protein complexes because their modular, highly engineerable components scaffold many proteins of varied structure and function. Cellulosomes assemble via non-covalent interactions between enzyme-fused dockerin domains and cohesin domains repeated on a central scaffoldin protein, but fungal cellulosome parts have never been explored for engineering because the sequence and structure of cohesin remain unknown. We developed a toolkit for building synthetic fungal cellulosomes made from polypeptides containing two dockerin domains (a “double dockerin”) and a non-native cohesin substitute called a nanobody, which binds with nanomolar affinity (Figure B). By leveraging atomistic modeling and experimental high-throughput screening, we engineered a library of double dockerin-nanobody pairs with pH-sensitive binding behaviors, enabling synthesis of protein complexes that alter their assembly state in response to pH changes (Figure C).