Constructing “quantized quorums” to guide emergent phenotypes through quorum quenching capsules

Abstract

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.

Modification and Assembly of a Versatile Lactonase for Bacterial Quorum Quenching

“…By attaching Sso Pox to the biopolymer chitosan, which, in turn, can be fabricated into several forms [ 27 , 32 ] and is used in drug delivery and wound healing applications [ 33 ], we may be able to provide new and innovative ways to deliver its lactonase and potentially, organophosphate hydrolase activity to various sites, including in humans. Here, lactonase activity is verified for the AI-1, N -(3-oxododecanoyl)- l -homoserine lactone (OdDHL), which stimulates inflammation in mammalian cells and promotes production of the toxin pyocyanin of P. aeruginosa [ 34 ].…”

Designer cells programming quorum-sensing interference with microbes

“…Once activated, the synthetic sensor cascade dynamically triggers the expression of AI-2 biosynthetic enzymes according to the levels of formylated peptides present, amplifies the input signal and provides strong and long-lasting enzymatic autoinducer release. Also, AI-2 is not expected to be toxic at the levels secreted by the designer cells, since AI-2 exposure is well tolerated by human cells 66 .…”

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

“…Previously, we developed inducible AI‐2 controller cells with enhanced ability to uptake and respond to AI‐2 by manipulating the AI‐2 quorum sensing network through overexpression of proteins responsible for uptake and phosphorylation of AI‐2, specifically LsrACDB and LsrK . We have also engineered cells that autonomously activate protein expression based on accumulation of AI‐2 and demonstrated that deleting genes responsible for degrading and altering AI‐2, lsrF and lsrG , results in activation of protein expression at lower AI‐2 levels .…”

“…7,8 Developing strains of Escherichia coli able to sense and respond to a range of AI-2 concentrations could be useful both for interrogating and manipulating native populations of bacteria and as tools for regulating communication in engineered ecosystems.Previously, we developed inducible AI-2 controller cells with enhanced ability to uptake and respond to AI-2 by manipulating the AI-2 quorum sensing network through overexpression of proteins responsible for uptake and phosphorylation of AI-2, specifically LsrACDB and LsrK. [9][10][11] We have also engineered cells that autonomously activate protein expression based on accumulation of AI-2 12 and demonstrated that deleting genes responsible for degrading and altering AI-2, lsrF and lsrG, results in activation of protein expression † These authors contributed equally to this work.…”