(290d) Cell-Free Protein Synthesis As a Tool for Prototyping Metabolic Pathways and Guiding Plant Genome Engineering

Metabolic engineering of organisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. In parallel, cell-free protein synthesis (CFPS) has matured into a powerful technique for characterizing metabolic enzymes and addressing challenges inherent in engineering life. In one example, CFPS enables in vitro prototyping of a pathway containing 9 heterologous steps from glucose to limonene with the goal of shortening design-build-test cycles for metabolic engineering. This effort measured the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. Leveraging the open nature of cell-free reactions, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production 25-fold from 23 to 610 mg/L. In a second example, CFPS has proved useful for expressing plant enzymes to guide genome engineering in plants. The wild tobacco relative Nicotiana benthamiana is commonly used for manufacturing proteins and reconstituting metabolic pathways which produce complex metabolites such as fragrances or medicines. However, small molecule compounds and their intermediate pathway metabolites produced in N. benthamiana are often over-glycosylated, oxidized/reduced, acylated or modified with glutathione by enzymes native to N. benthamiana. This unwanted activity has challenged efforts to reconstitute the 11-step pathway to strictosidine (an alkaloid precursor to the anti-cancer drug vinblastine) as well as many other pathways. To guide selection of endogenous candidate genes for knockout by Cas9-mediated mutagenesis, we have built a Golden Gate DNA assembly toolbox adapted for cell-free expression of plant proteins. Using a biofoundry-assisted workflow, we screened expression conditions for dozens of candidate glycosyltransferases and have subsequently measured glycosylation activity towards strictosidine pathway intermediates geraniol and cis-trans-nepetalactol. We anticipate that edited N. benthamiana lines will reduce unwanted metabolite derivatization and enable increased production of target molecules.