Engineering Tunable, Low Latency Spatial Computation with Dual Input Quorum Sensing Promoters.

Quorum sensing signals have evolved for population-level signaling in bacterial communities and are versatile tools for engineering cell-cell signaling in synthetic biology projects. Here, we characterize the spatial diffusion of a palette of quorum sensing signals and find that their diffusion in agar can be predicted from their molecular weight with a simple power law. We also engineer novel dual- and multi-input promoters that respond to quorum-sensing diffusive signals for use in engineered genetic systems. We engineer a promoter scaffold that can be adapted for activation and repression by multiple diffusers simultaneously. Lastly, we combine the knowledge on diffusion dynamics with the novel genetic components to build a new generation of spatial, stripe-forming systems with a simplified design, improved robustness, tuneability, and response time.

Genome-scale target identification in Escherichia coli for high-titer production of free fatty acids.

To construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL--aidB+-ryfAM--gadAH-, producing 30.0 g L-1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.