Robust gene circuit construction requires use of promoters exhibiting low crosstalk. Orthogonal promoters have been engineered utilizing an assortment of natural and synthetic transcription factors, but design of large orthogonal promoter-repressor sets is complicated, labor-intensive, and often results in unanticipated crosstalk. The specificity and ease of targeting the RNA-guided DNA-binding protein dCas9 to any 20 bp user-defined DNA sequence makes it a promising candidate for orthogonal promoter regulation. Here, we rapidly construct orthogonal variants of the classic T7-lac promoter using site-directed mutagenesis, generating a panel of inducible hybrid promoters regulated by both LacI and dCas9. Remarkably, orthogonality is mediated by only two to three nucleotide mismatches in a narrow window of the RNA:DNA hybrid, neighboring the protospacer adjacent motif. We demonstrate that, contrary to many reports, one PAM-proximal mismatch is insufficient to abolish dCas9-mediated repression, and we show for the first time that mismatch tolerance is a function of target copy number. Finally, these promoters were incorporated into the branched violacein biosynthetic pathway as dCas9-dependent switches capable of throttling and selectively redirecting carbon flux in Escherichia coli We anticipate this strategy is relevant for any promoter and will be adopted for many applications at the interface of synthetic biology and metabolic engineering.

Fermentation-based chemical production strategies provide a feasible route for the rapid, safe, and sustainable production of a wide variety of important chemical products, ranging from fuels to pharmaceuticals. These strategies have yet to find wide industrial utilization due to their inability to economically compete with traditional extraction and chemical production methods. Here, we engineer for the first time the complex microbial biosynthesis of an anthocyanin plant natural product, starting from sugar. This was accomplished through the development of a synthetic, 4-strain Escherichia coli polyculture collectively expressing 15 exogenous or modified pathway enzymes from diverse plants and other microbes. This synthetic consortium-based approach enables the functional expression and connection of lengthy pathways while effectively managing the accompanying metabolic burden. The de novo production of specific anthocyanin molecules, such as calistephin, has been an elusive metabolic engineering target for over a decade. The utilization of our polyculture strategy affords milligram-per-liter production titers. This study also lays the groundwork for significant advances in strain and process design toward the development of cost-competitive biochemical production hosts through nontraditional methodologies.IMPORTANCE To efficiently express active extensive recombinant pathways with high flux in microbial hosts requires careful balance and allocation of metabolic resources such as ATP, reducing equivalents, and malonyl coenzyme A (malonyl-CoA), as well as various other pathway-dependent cofactors and precursors. To address this issue, we report the design, characterization, and implementation of the first synthetic 4-strain polyculture. Division of the overexpression of 15 enzymes and transcription factors over 4 independent strain modules allowed for the division of metabolic burden and for independent strain optimization for module-specific metabolite needs. This study represents the most complex synthetic consortia constructed to date for metabolic engineering applications and provides a new paradigm in metabolic engineering for the reconstitution of extensive metabolic pathways in nonnative hosts.