Constructing "quantized quorums" to guide emergent phenotypes through quorum quenching capsules.

Microbial cells have for many years been engineered to facilitate efficient production of biologics, chemicals, and other compounds. As the "metabolic" burden of synthetic genetic components can impair cell performance, microbial consortia are being developed to piece together specialized subpopulations that collectively produce desired products. Their use, however, has been limited by the inability to control their composition and function. One approach to leverage advantages of the division of labor within consortia is to link microbial subpopulations together through quorum sensing (QS) molecules. Previously, we directed the assembly of "quantized quorums," microbial subpopulations that are parsed through QS activation, by the exogenous addition of QS signal molecules to QS synthase mutants. In this work, we develop a more facile and general platform for creating "quantized quorums." Moreover, the methodology is not restricted to QS-mutant populations. We constructed quorum quenching capsules that partition QS-mediated phenotypes into discrete subpopulations. This compartmentalization guides QS subpopulations in a dose-dependent manner, parsing cell populations into activated or deactivated groups. The capsular "devices" consist of polyelectrolyte alginate-chitosan beads that encapsulate high-efficiency (HE) "controller cells" that, in turn, provide rapid uptake of the QS signal molecule AI-2 from culture fluids. In this methodology, instead of adding AI-2 to parse QS-mutants into subpopulations, we engineered cells to encapsulate them into compartments, and they serve to deplete AI-2 from wild-type populations. These encapsulated bacteria therefore, provide orthogonal control of population composition while allowing only minimal interaction with the product-producing cell population or consortia. We envision that compartmentalized control of QS should have applications in both metabolic engineering and human disease. Biotechnol. Bioeng. 2017;114: 407-415. © 2016 Wiley Periodicals, Inc.

Engineering Escherichia coli for enhanced sensitivity to the autoinducer-2 quorum sensing signal.

The autoinducer-2 (AI-2) quorum sensing system is involved in a range of population-based bacterial behaviors and has been engineered for cell-cell communication in synthetic biology systems. Investigation into the cellular mechanisms of AI-2 processing has determined that overexpression of uptake genes increases AI-2 uptake rate, and genomic deletions of degradation genes lowers the AI-2 level required for activation of reporter genes. Here, we combine these two strategies to engineer an Escherichia coli strain with enhanced ability to detect and respond to AI-2. In an E. coli strain that does not produce AI-2, we monitored AI-2 uptake and reporter protein expression in a strain that overproduced the AI-2 uptake or phosphorylation units LsrACDB or LsrK, a strain with the deletion of AI-2 degradation units LsrF and LsrG, and an "enhanced" strain with both overproduction of AI-2 uptake and deletion of AI-2 degradation elements. By adding up to 40 µM AI-2 to growing cell cultures, we determine that this "enhanced" AI-2 sensitive strain both uptakes AI-2 more rapidly and responds with increased reporter protein expression than the others. This work expands the toolbox for manipulating AI-2 quorum sensing processes both in native environments and for synthetic biology applications.