Engineered metabolic pathways constructed from enzymes heterologous to the production host often suffer from flux imbalances, as they typically lack the regulatory mechanisms characteristic of natural metabolism. In an attempt to increase the effective concentration of each component of a pathway of interest, we built synthetic protein scaffolds that spatially recruit metabolic enzymes in a designable manner. Scaffolds bearing interaction domains from metazoan signaling proteins specifically accrue pathway enzymes tagged with their cognate peptide ligands. The natural modularity of these domains enabled us to optimize the stoichiometry of three mevalonate biosynthetic enzymes recruited to a synthetic complex and thereby achieve 77-fold improvement in product titer with low enzyme expression and reduced metabolic load. One of the same scaffolds was used to triple the yield of glucaric acid, despite high titers (0.5 g/l) without the synthetic complex. These strategies should prove generalizeable to other metabolic pathways and programmable for fine-tuning pathway flux.

SignificanceDrought remains a critical obstacle to meeting the food demands of the coming century. Understanding the interplay between drought stress, plant development, and the plant microbiome is central to meeting this challenge. Here, we demonstrate that drought causes enrichment of a distinct set of microbes in roots, composed almost entirely of monoderms, which lack outer membranes and have thick cell walls. We demonstrate that under drought, roots increase the production of many metabolites, and that monoderms inhabiting the drought-treated rhizosphere exhibit increased activity of transporters connected with some of these same compounds. The discovery of this drought-induced enrichment and associated shifts in metabolite exchange between plant and microbe reveal a potential blueprint for manipulating plant microbiomes for improved crop fitness.

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