Engineering Biosynthetic Pathways in Cell-Free Systems for Sustainability and Chemical Innovation

Abstract

This work presents the cell-free transcription-translation (TX-TL) system as a research and development platform for renewable synthesis and molecular discovery. TX-TL is easy to use and provides a biomolecular breadboard for the rapid prototyping and engineering of biosynthetic pathways. This work has validated the capabilities of the cell-free TX-TL system for simultaneous protein expression and chemical synthesis. Specifically, this work shows that TX-TL supports the conversion of intermediates from carbohydrate metabolism and amino acids into valuable compounds. Metabolic flux through cofactor dependent pathways confirms that active cofactor metabolism is occurring in TX-TL. This work has also demonstrated the industrial relevance of TX-TL through exploring design space of a biosynthetic pathway for improved product yield and expanding substrate scope of another biosynthetic pathway. Current methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. In this work, we have verified enzyme expression and enzyme activity and identified the conversion of 4-hydroxybutyrate to downstream metabolites as the pathway bottleneck. We demonstrate the reliability of using linear DNA in TX-TL as a tool for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Pathway constructs of varying pathway enzyme expression levels are tested in TX-TL and in vivo to identify correlations between the two systems, and we find that the production of BDO is correlated to the expression of enzyme ald in both systems. The use of TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Different pathway combinations are also tested in TX-TL for its application in pathway ranking. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development. TX-TL simulates a customizable cellular environment that can be controlled by manipulating pH, changing cellular components, or adding exogenous substrates. By adding linear DNA encoding individual enzymes of the violacein pathway and tryptophan analogs in TX-TL reactions, we have discovered new violacein analogs. TX-TL enables rapid production of natural product analogs with diverse substitution, which allows small-scale biosynthesis of potential drug candidates and offers a new platform for drug discovery. This work also presents TX-TL as a platform for protein engineering. Residues targeted for site-saturated mutagenesis were identified with protein-ligand docking. Linear DNAs of individual enzyme mutants were added into TX-TL reactions to screen for improved enzyme variant. Screening result indicates vioE mutant Y17H reduces byproduct formation and redirects metabolic flux towards target metabolites. Protein engineering for improved enzyme activity can further expand the substrate scope of a natural product pathway and result with more natural product analogs that can be applied for medical applications. This work demonstrates that the cell-free TX-TL system can become a valuable tool that complements the process of engineering biosynthesis in the whole cell in vivo system or the purified protein in vitro system. Future engineering and development of the TX-TL system can further expand the chemical space for biosynthesis.