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Enhancing Intercellular Coordination: Rewiring Quorum Sensing Networks for Increased Protein Expression through Autonomous Induction
Abstract
While inducing agents are often used to redirect resources from growth and proliferation toward product outputs, they can be prohibitively expensive on the industrial scale. Previously, we developed an autonomously guided protein production system based on the rewiring of E. coli's native quorum sensing (QS) signal transduction cascade. Self-secreted autoinducer, AI-2, accumulated over time and actuated recombinant gene expression-its design, co-opting the collective nature of QS-mediated behavior. We recently demonstrated that desynchronization of autoinduced intercellular feedback leads to bimodality in QS activation. In this work, we developed a new QS-enabled system with enhanced feedback to reduce cell heterogeneity. This narrows the population distribution of protein expression, leading to significant per cell and overall increases in productivity. We believe directed engineering of cell populations and/or cell consortia will offer many such opportunities in future bioprocessing applications.
... 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. ...
... Growth rates were similar in all CT104 strains ( Figure S1). We note that distributed responses to AI-2 and other signaling or inducer molecules is common 10,13,18 and can be controlled. 10,19 For example, here, using the enhanced plasmids in the host strain CT104 resulted in an increase in fluorescence over use of the control plasmids at AI-2 concentrations of 4 μM or higher (Figure 3b). ...
... We note that distributed responses to AI-2 and other signaling or inducer molecules is common 10,13,18 and can be controlled. 10,19 For example, here, using the enhanced plasmids in the host strain CT104 resulted in an increase in fluorescence over use of the control plasmids at AI-2 concentrations of 4 μM or higher (Figure 3b). At the 4 μM AI-2 concentration, the fluorescent population was about twice as high in the cells with the enhanced feedback loops. ...
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 E. 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. This article is protected by copyright. All rights reserved.
... Synthetic biology, sometimes defined as the engineering of biology, has the potential to engineer genetic circuits to perform new functions for useful purposes in a systematic, predictable, robust, and efficient way [35,36]. In the last years, several synthetic circuits have been proposed with the ultimate goal of dealing with gene expression noise [20,22,[37][38][39][40]. ...
... This, in turn, contributes to protect from noise [28]. Thus, the idea of joining both intracellular negative feedback and extracellular feedback via quorum sensing is a natural one, that has been suggested in [38,39]. ...
Gene expression is a fundamental cellular process. Its stochastic fluctuations due to intrinsic and extrinsic sources, known generically as ‘gene expression noise’, trigger both beneficial and harmful consequences for the cell behavior.
Controlling gene expression noise is of interest in many applications in biotechnology, biomedicine and others. Yet, control of the mean expression level is an equally desirable goal. Here, we analyze a gene synthetic network designed to reduce gene expression noise while achieving a desired mean expression level. The circuit combines a negative feedback loop over the gene of interest, and a cell-to-cell communication mechanism based on quorum sensing. We analyze the ability of the circuit to reduce noise as a function of parameters that can be tuned in the wet-lab, and the role quorum sensing plays. Intrinsic noise is generated by the inherent stochasticity of biochemical reactions. On the other hand, extrinsic noise is due to variability in the cell environment and the amounts of cellular components that affect gene expression. We develop a realistic model of the gene synthetic circuit over the population of cells using mass action kinetics and the stochastic Chemical Langevin Equation to include intrinsic noise, with parameters drawn from a distribution to account for extrinsic noise. Stochastic simulations allow us to quantify the mean expression level and noise strength of all species under different scenarios, showing good agreement with system-wide available experimental data of protein abundance and noise in E. coli . Our in silico experiments reveal significant noise attenuation in gene expression through the interplay between quorum sensing and the negative feedback, allowing control of the mean expression and variance of the protein of interest. These in silico conclusions are validated by preliminary experimental results. This gene network could have important implications as a robust protein production system in industrial biotechnology.
Author Summary
Controlling gene expression level is of interest in many applications in biotechnology, biomedicine and others. Yet, the stochastic nature of biochemical reactions plays an important role in biological systems, and cannot be disregarded. Gene expression noise resulting from this stochasticity has been studied over the past years both in vivo , and in silico using mathematical models. Nowadays, synthetic biology approaches allow to design novel biological circuits, drawing on principles elucidated from biology and engineering, for the purpose of decoupled control of mean gene expression and its variance. We propose a gene synthetic circuit with these characteristics, using negative feedback and quorum sensing based cell-to-cell communication to induce population consensus. Our in silico analysis using stochastic simulations with a realistic model reveal significant noise attenuation in gene expression through the interplay between quorum sensing and the negative feedback, allowing control of the mean expression and variance of the protein of interest. Preliminary in vivo results fully agree with the computational ones.
... 8,9 We have previously shown that engineering the signal-responsive regulatory circuits can yield more homogeneous responses. 10 In the current work, we demonstrate that by partitioning the response into a tailored relay and amplification system, the overall outcome can consist of homogeneous, highly expressing reporter or "sentinel" strains. ...
... * S Supporting InformationThe Supporting Information is available free of charge on the ACS Publications website at DOI:10.1021/acssynbio.8b00146. Supplemental methods, tables of plasmids relevant to the study, figures to accompany the main body text, and subsequent discussion (PDF) Author Contributions R.M. provided concept design and conducted all experiments, analyzed data, and wrote the manuscript. ...
... Previously, we introduced the QS signaling responsive circuit into the engineered E. coli "reporter" cells which cannot produce native AI-2 but can sensitively respond to AI-2 and express the fluorescent marker, DsRed (Wu et al., 2013;Terrell et al., 2015;Servinsky et al., 2016;Zargar et al., 2016). Here, the engineered "reporter" cells and WT E. coli cells were collected using reverse charge coated MNPs from a relatively low cell density medium (turbidity of ~0.4) to focus the native QS signaling activity onto the reporter cells in a robust and defined manner ( Figure 1). ...
... The methodologies depicted here are simple and relatively fast when compared to selective plating and culturing (Chen et al., 2003). Additionally, we previously demonstrated that the reporter strains can also be viewed as production hosts in various bioprocess applications, such as the expression of recombinant proteins Zargar et al., 2016). The results shown here enable the induction of the recombinant strains without addition of exogenous inducers that may be costly or otherwise heterogeneously distributed among the producing cell populations. ...
Quorum sensing (QS) exists widely among bacteria, enabling a transition to multicellular behavior after bacterial populations reach a particular density. The coordination of multicellularity enables biotechnological application, dissolution of biofilms, coordination of virulence, etc. Here, a method to elicit and subsequently disperse multi‐cellular behavior among QS‐negative cells is developed using magnetic nanoparticle assembly. We fabricated magnetic nanoparticles (MNPs, ∼5 nm) that electrostatically collect wild‐type (WT) Escherichia coli BL21 cells and brings them into proximity of bioengineered E. coli (CT104 (W3110 lsrFG‐ luxS‐ pCT6+pET‐DsRed)) reporter cells that exhibit a QS response after receiving autoinducer‐2 (AI‐2). By shortening the distance between WT and reporter cells (e.g., increasing local available AI‐2 concentrations), the QS response signaling was amplified 4‐fold compared to that in native conditions without assembly. This study suggests potential applications in facilitating intercellular communication and modulating multicellular behaviors based on user‐specified designs. This article is protected by copyright. All rights reserved.
... 8,9 We have previously shown that engineering the signal-responsive regulatory circuits can yield more homogeneous responses. 10 In the current work, we demonstrate that by partitioning the response into a tailored relay and amplification system, the overall outcome can consist of homogeneous, highly expressing reporter or "sentinel" strains. ...
... * S Supporting InformationThe Supporting Information is available free of charge on the ACS Publications website at DOI:10.1021/acssynbio.8b00146. Supplemental methods, tables of plasmids relevant to the study, figures to accompany the main body text, and subsequent discussion (PDF) Author Contributions R.M. provided concept design and conducted all experiments, analyzed data, and wrote the manuscript. ...
... A recent discovery developed a new QS enabled system with enhanced feedback to reduce cell heterogeneity. This narrows the population distribution of protein expression, leading to significant per cell and overall increases in productivity (Zargar et al., 2016). ...
... Recent research shows the important role of quantum mechanics in photosynthetic proteins, vision, electron-and proton-tunneling, olfactory sensing, and magneto-reception. Erwin Schrödinger noted in his famous book BWhat is Life?^ (Schrödinger, 1944) that quantum mechanics accounts for the stability of living things and their cellular processes through our understanding via quantum mechanics of the stability of molecules, and the fact that quantum effects create, sometimes large, energy gaps between different states of chemical systems. ...
Quorum sensing is the efficient mode of communication in the bacterial world. After a lot of advancements in the classical theory of quorum sensing few basic questions of quorum sensing still remain unanswered. The sufficient progresses in quantum biology demands to explain these questions from the quantum perspective as non trivial quantum effects already have manifested in various biological processes like photosynthesis, magneto-reception etc. Therefore, it's the time to review the bacterial communication from the quantum view point. In this article we carefully accumulate the latest results and arguments to strengthen quantum biology through the addition of quorum sensing mechanism in the light of quantum mechanics.
... That said, QS-mediated autoinduction required a longer expression period, as our results showed low amounts of target protein at 2 h post-induction while at the same time the expression of IPTG-induced BL21 (DE3) was high. While we have demonstrated the benefits of QS-mediated expression for a cysteine-tagged protein, benefits for rewired QS circuitry have also appeared for non-Cys-tagged proteins including GFP (Tsao et al., 2010;Zargar et al., 2016), β-galactosidase (Tsao et al., 2010), chloramphenicol acetyltransferase (CAT) and an organophosphorus hydrolase (OPH) (Tsao et al., 2011), bacterial AI-2 regulators LsrK and LsrACDB . It may well be that this methodology is generally beneficial, including for proteins with designer tags. ...
Biofabrication utilizes biological materials and biological means, or mimics thereof, for assembly. When interfaced with microelectronics, electrobiofabricated assemblies enable exquisite sensing and reporting capabilities. We recently demonstrated that thiolated polyethylene glycol (PEG-SH) could be oxidatively assembled into a thin disulfide crosslinked hydrogel at an electrode surface; with sufficient oxidation, extra sulfenic acid groups are made available for covalent, disulfide coupling to sulfhydryl groups of proteins or peptides. We intentionally introduced a polycysteine tag (5xCys-tag) consisting of five consecutive cysteine residues at the C-terminus of a Streptococcal protein G to enable its covalent coupling to an electroassembled PEG-SH film. We found, however, that its expression and purification from E. coli was difficult, owing to the extra cysteine residues. We developed a redox-based autoinduction methodology that greatly enhanced the yield, especially in the soluble fraction of E. coli extracts. The redox component involved the deletion of oxyRS, a global regulator of the oxidative stress response and the autoinduction component integrated a quorum sensing (QS) switch that keys the secreted QS autoinducer-2 to induction. Interestingly, both methods helped when independently employed and further, when used in combination (i.e., autodinduced oxyRS mutant) the results were best—we found the highest total yield and highest yield in the soluble fraction. We hypothesize that the production host was less prone to severe metabolic perturbations that might reduce yield or drive sequestration of the -tagged protein into inclusion bodies. We expect this methodology will be useful for the expression of many such Cys-tagged proteins, ultimately enabling a diverse array of functionalized devices.
... Here, the hybrid tac promoter was used because it has a high background level of basal LsrK so that the prevailing AI-2 level is rapidly converted into viable genetic signal. 34,67,68 While not described thus far, our E. coli host strains were also engineered to achieve better control of protein expression levels. Notably, because our focus is on the homologous QS circuitry and the lsr promoter of E. coli, which is a component of native E. coli quorum sensing ( Figure 1A), our host cell has additional mutations (e.g., ∆ptsH) that ensure our host functions with advanced sensitivity to AI-2 and for a variety of environmental niches. ...
We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and by design, can be operated in three modes: a constitutive mode that enables consistent and high levels of expression; a tightly repressed mode in which there is very little background expression; and an inducible mode in which concentrations of two signals (arabinose and autoinducer-2) determine the net amplification of the gene(s)-of-interest. We demonstrate the utility of the circuit for the controlled expression of human granulocyte macrophage colony stimulating factor in an engineered probiotic E. coli. This dual-input genetic controller is the first homologous AI-2 QS circuit that has the ability to be operated in three different modes. We believe it has the potential for wide-ranging biotechnological applications due its versatile features.
... This, in turn, may contribute to protect from noise [33]. Thus, the idea of joining both intracellular negative feedback and extracellular feedback via quorum sensing is a natural one, that has been suggested in [1,37,42]. ...
... QS serves as an excellent platform for many technologies, particularly if one understands the regulatory reach of the genetic circuits. In the past two decades, the rewiring of native QS networks has enabled novel ways to engineer cell behavior, exemplified by such advances as programmed population controllers (36), synchronized genetic clocks (37), and population-based autonomous gene actuators (38,39). These studies have set the stage for the future development of a variety of innovative biotechnological applications, which are discussed in the following sections. ...
Quorum sensing (QS) is a molecular signaling modality that mediates molecular-based cell–cell communication. Prevalent in nature, QS networks provide bacteria with a method to gather information from the environment and make decisions based on the intel. With its ability to autonomously facilitate both inter- and intraspecies gene regulation, this process can be rewired to enable autonomously actuated, but molecularly programmed, genetic control. On the one hand, novel QS-based genetic circuits endow cells with smart functions that can be used in many fields of engineering, and on the other, repurposed QS circuitry promotes communication and aids in the development of synthetic microbial consortia. Furthermore, engineered QS systems can probe and intervene in interkingdom signaling between bacteria and their hosts. Lastly, QS is demonstrated to establish conversation with abiotic materials, especially by taking advantage of biological and even electronically induced assembly processes; such QS-incorporated biohybrid devices offer innovative ways to program cell behavior and biological function.
Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering, Volume 11 is June 8, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... For cell signaling-based induction, the native bacterial quorum-sensing system has been applied to establish autoinducible production by coupling gene expression to the concentration of secreted quorum signaling molecules (Gupta, Reizman, Reisch, & Prather, 2017;Kim et al., 2017). This type of system has also been shown to work when coupled to the T7 polymerase for further amplification of protein output (Zargar, Quan, & Bentley, 2016). While the expression is not always tightly regulated and the system might require tuning for each desired compound, further engineering could provide a system suitable for large-scale fermentation. ...
Inducible expression systems can be applied to control the expression of proteins or biochemical pathways in cell factories. However, several of the established systems require addition of expensive inducers, making them unfeasible for large scale production. Here, we establish a genome integrated trp-T7 expression system where tryptophan can be used to control the induction of a gene or a metabolic pathway. We show that the initiation of gene expression from low- and high-copy vectors can be tuned by varying the initial concentration of tryptophan or yeast extract, and that expression is tightly regulated and homogenous when compared to the commonly used lac-T7 system. Finally, we apply the trp-T7 expression system for production of L-serine, where we reach titers of 26 g/L in fed-batch fermentation. This article is protected by copyright. All rights reserved.
... More endeavors have been made [56] to increase protein yield in this autonomous system through a different approach. With the same intention in mind [49,50], a new study showed that reduced heterogeneity between independent cells could be achieved by inserting an enhanced feedback loop to the E. coli's native AI-2 QS system. ...
... We constructed a simple synthetic H 2 O 2 -responsive promoter system and used the system to guide bacterial swimming. Complementary studies [6,17,45,46] demonstrate that additional regulatory constructs based on engineered plasmid controllers and/or engineered host cells enable tight or "tuned" control of gene expression based on signal molecule concentration. That is, the current system restores swimming motility while only minimally altering native circuitry. ...
Synthetic biologists construct innovative genetic/biological systems to treat environmental, energy, and health problems. Many systems employ rewired cells for non-native product synthesis, while a few have employed the rewired cells as ‘smart’ devices with programmable function. Building on the latter, we developed a genetic construct to control and direct bacterial motility towards hydrogen peroxide, one of the body’s immune response signaling molecules. A motivation for this work is the creation of cells that can target and autonomously treat disease, the latter signaled by hydrogen peroxide release. Bacteria naturally move towards a variety of molecular cues (e.g., nutrients) in the process of chemotaxis. In this work, we engineered bacteria to recognize and move towards hydrogen peroxide, a non-native chemoattractant and potential toxin. Our system exploits oxyRS, the native oxidative stress regulon of E. coli. We first demonstrated H2O2-mediated upregulation motility regulator, CheZ. Using transwell assays, we showed a two-fold increase in net motility towards H2O2. Then, using a 2D cell tracking system, we quantified bacterial motility descriptors including velocity, % running (of tumble/run motions), and a dynamic net directionality towards the molecular cue. In CheZ mutants, we found that increased H2O2 concentration (0–200 μM) and induction time resulted in increased running speeds, ultimately reaching the native E. coli wild-type speed of ~22 μm/s with a ~45–65% ratio of running to tumbling. Finally, using a microfluidic device with stable H2O2 gradients, we characterized responses and the potential for “programmed” directionality towards H2O2 in quiescent fluids. Overall, the synthetic biology framework and tracking analysis in this work will provide a framework for investigating controlled motility of E. coli and other ‘smart’ probiotics for signal-directed treatment.
Quorum sensing, a bacterial process for coordinating community behavior, has inspired scientists to engineer cell‐cell communication for diverse applications. Fundamental knowledge of the molecular underpinnings of quorum sensing systems enabled engineers to rewire quorum sensing circuits in order to alter quorum sensing processes, program control of bacterial populations, and engineer cell‐cell communication. Further, scientific advancements from diverse engineering disciplines have contributed to the design of devices enabling new modes of manipulating or communicating with biological cells. This perspective reviews early and current developments in engineering cell‐cell communication and its applications. Influence of the quorum sensing field on the authors, both engineers, is briefly discussed.
In nature, quorum sensing is one of the mechanism bacterial populations use to communicate with their own species or across species to coordinate behaviours. For the last 20 years, synthetic biologists have recognised the remarkable properties of quorum sensing to build genetic circuits responsive to population density. This has led to progress in designing dynamic, coordinated and sometimes multi-cellular systems for bio-production in metabolic engineering and for increased spatial and temporal complexity in synthetic biology. In this review, we highlight recent works focused on using quorum sensing to engineer cell-cell behaviour.
Bacteria exist as communities in diverse multispecies environments. Quorum sensing, a process for cell–cell communication, allows individual bacteria to glean information about their surroundings and coordinate activities with their neighbors. Recent studies indicate the importance of quorum sensing in microbiomes, but many questions remain regarding how quorum sensing may influence the composition and function of these communities. Synthetic biology, a field where scientists seek to design biological systems with predictable behavior, may provide tools to probe and manipulate quorum sensing behavior in natural consortia. In parallel, quorum sensing processes can be used as a tool in synthetic biology to construct synthetic cocultures with desired behavior. Here, we review recent synthetic biology strategies for manipulating quorum sensing processes in microbial consortia.
Stochastic fluctuations in gene expression trigger both beneficial and harmful consequences for cell behavior. Therefore, achieving a desired mean protein expression level while minimizing noise is of interest in many applications, including robust protein production systems in industrial biotechnology. Here we consider a synthetic gene circuit combining intracellular negative feedback and cell-to-cell communication based on quorum sensing. Accounting for both intrinsic and extrinsic noise, stochastic simulations allow us to analyze the capability of the circuit to reduce noise strength as a function of its parameters. We obtain mean expression level and noise strength for all species under different scenarios showing good agreement with system-wide available experimental data of protein abundance and noise in \emph{E. coli}. Our \emph{in silico} experiments, validated by preliminary \textit{in vivo} results, reveal significant noise attenuation in gene expression through the interplay between quorum sensing and negative feedback, and highlight the differential role they play in regard to intrinsic and extrinsic noise.
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.
Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms.
Heterologous pathways used in metabolic engineering may produce intermediates toxic to the cell. Dynamic control of pathway enzymes could prevent the accumulation of these metabolites, but such a strategy requires sensors, which are largely unknown, that can detect and respond to the metabolite. Here we applied whole-genome transcript arrays to identify promoters that respond to the accumulation of toxic intermediates, and then used these promoters to control accumulation of the intermediate and improve the final titers of a desired product. We apply this approach to regulate farnesyl pyrophosphate (FPP) production in the isoprenoid biosynthetic pathway in Escherichia coli. This strategy improved production of amorphadiene, the final product, by twofold over that from inducible or constitutive promoters, eliminated the need for expensive inducers, reduced acetate accumulation and improved growth. We extended this approach to another toxic intermediate to demonstrate the broad utility of identifying novel sensor-regulator systems for dynamic regulation.
Recent reports have shown that bacterial cell–cell communication or quorum sensing is quite prevalent in pathogenic Escherichia coli, especially at high cell density; however, the role of quorum sensing in nonpathogenic E. coli is less clear and, in particular, there is no information regarding the role of quorum sensing in overexpression of plasmid-encoded genes. In this work, it was found that the activity of a quorum signaling molecule, autoinducer-2 (AI-2), decreased significantly following induction of several plasmid-encoded genes in both low and high-cell-density cultures of E. coli. Furthermore, we show that AI-2 signaling level was linearly related to the accumulation level of each protein product and that, in general, the highest rates of recombinant protein accumulation resulted in the greatest attenuation of AI-2 signaling. Importantly, our findings demonstrate for the first time that recombinant E. coli communicate the stress or burden of overexpressing heterologous genes through the quorum-based AI-2 signaling pathway. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 439–450, 2001.
We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
Different species of bacteria were tested for production of extracellular autoinducer-like activities that could stimulate the expression of the luminescence genes in Vibrio harveyi. Several species of bacteria, including the pathogens Vibrio cholerae and Vibrio parahaemolyticus, were found to produce such activities. Possible physiological roles for the two V. harveyi detection-response systems and their joint regulation are discussed.
Gene expression from plasmids containing the araBAD promoter can be regulated by the concentration of arabinose in the growth medium. Guzman et al. [Guzman, L.-M., Belin, D., Carson, M. J. & Beckwith, J. (1995) J. Bacteriol. 177, 4121-4130] showed that expression of a cloned gene could be modulated over several orders of magnitude in cultures grown in the presence of subsaturating concentrations of arabinose. We constructed plasmids expressing a fast-folding mutant Aequorea victoria green fluorescent protein from the araBAD promoter to examine the distribution of expressed gene products in individual cells at intermediate induction levels. Microscopic examination of cells grown at low arabinose concentrations shows mixtures of brightly fluorescent and dark cells, suggesting that intermediate expression levels in cultures reflect a population average of induced and uninduced cells. The kinetics of green fluorescent protein induction suggest that this reflects an "autocatalytic" induction mechanism due to accumulation of the inducer by active transport. This mechanism, which is analogous to the induction of the lac operon at subsaturating inducer concentrations in lacY+ cells, was described 40 years ago by Novick and Weiner [Novick, A. & Weiner, M. (1957) Proc. Natl. Acad. Sci. USA 43, 553-566].
AI-2 is a quorum-sensing signaling molecule proposed to be involved in interspecies communication. In Escherichia coli and Salmonella enterica serovar Typhimurium, extracellular AI-2 accumulates in exponential phase, but the amount decreases drastically upon entry
into stationary phase. In S. enterica serovar Typhimurium, the reduction in activity is due to import and processing of AI-2 by the Lsr transporter. We show that
the Lsr transporter is functional in E. coli, and screening for mutants defective in AI-2 internalization revealed lsrK and glpD. Unlike the wild type, lsrK and glpD mutants do not activate transcription of the lsr operon in response to AI-2. lsrK encodes the AI-2 kinase, and the lsrK mutant fails to activate lsr expression because it cannot produce phospho-AI-2, which is the lsr operon inducer. glpD encodes the glycerol-3-phosphate (G3P) dehydrogenase, which is involved in glycerol and G3P metabolism. G3P accumulates in
the glpD mutant and represses lsr transcription by preventing cyclic AMP (cAMP)-catabolite activator protein (CAP)-dependent activation. Dihydroxyacetone phosphate
(DHAP) also accumulates in the glpD mutant, and DHAP represses lsr transcription by a cAMP-CAP-independent mechanism involving LsrR, the lsr operon repressor. The requirement for cAMP-CAP in lsr activation explains why AI-2 persists in culture fluids of bacteria grown in media containing sugars that cause catabolite
repression. These findings show that, depending on the prevailing growth conditions, the amount of time that the AI-2 signal
is present and, in turn, the time that a given community of bacteria remains exposed to this signal can vary greatly.
Bacterial autoinducer 2 (AI-2) is proposed to be an interspecies mediator of cell-cell communication that enables cells to
operate at the multicellular level. Many environmental stimuli have been shown to affect the extracellular AI-2 levels, carbon
sources being among the most important. In this report, we show that both AI-2 synthesis and uptake in Escherichia coli are subject to catabolite repression through the cyclic AMP (cAMP)-CRP complex, which directly stimulates transcription of
the lsr (for “luxS regulated”) operon and indirectly represses luxS expression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of the lsr promoter and works with the LsrR repressor to regulate AI-2 uptake. The functions of the lsr operon and its regulators, LsrR and LsrK, previously reported in Salmonella enterica serovar Typhimurium, are confirmed here for E. coli. The elucidation of cAMP-CRP involvement in E. coli autoinduction impacts many areas, including the growth of E. coli in fermentation processes.
Many reports have elucidated the mechanisms and consequences of bacterial quorum sensing (QS), a molecular communication system by which bacterial cells enumerate their cell density and organize collective behavior. In few cases, however, the numbers of bacteria exhibiting this collective behavior have been reported, either as a number concentration or a fraction of the whole. Not all cells in the population, for example, take on the collective phenotype. Thus, the specific attribution of the postulated benefit can remain obscure. This is partly due to our inability to independently assemble a defined quorum, for natural and most artificial systems the quorum itself is a consequence of the biological context (niche and signaling mechanisms). Here, we describe the intentional assembly of quantized quorums. These are made possible by independently engineering the autoinducer signal transduction cascade of Escherichia coli (E. coli) and the sensitivity of detector cells so that upon encountering a particular autoinducer level, a discretized sub-population of cells emerges with the desired phenotype. In our case, the emergent cells all express an equivalent amount of marker protein, DsRed, as an indicator of a specific QS-mediated activity. The process is robust, as detector cells are engineered to target both large and small quorums. The process takes about 6 h, irrespective of quorum level. We demonstrate sensitive detection of autoinducer-2 (AI-2) as an application stemming from quantized quorums. We then demonstrate sub-population partitioning in that AI-2-secreting cells can 'call' groups neighboring cells that 'travel' and establish a QS-mediated phenotype upon reaching the new locale.The ISME Journal advance online publication, 5 June 2015; doi:10.1038/ismej.2015.89.
Coordination between cell populations via prevailing metabolic cues has been noted as a promising approach to connect synthetic devices and drive phenotypic or product outcomes. However, there has been little progress in developing 'controller cells' to modulate metabolic cues and guide these systems. In this work, we developed 'controller cells' that manipulate the molecular connection between cells by modulating the bacterial signal molecule, autoinducer-2, that is secreted as a quorum sensing (QS) signal by many bacterial species. Specifically, we have engineered E. coli to overexpress components responsible for autoinducer uptake (lsrACDB), phosphorylation (lsrK), and degradation (lsrFG), thereby attenuating cell-cell communication among populations. Further, we developed a simple mathematical model that recapitulates experimental data and characterizes the dynamic balance among the various uptake mechanisms. This study revealed two controller "knobs" that serve to increase AI-2 uptake: overexpression of the AI-2 transporter, LsrACDB, which controls removal of extracellular AI-2, and overexpression of the AI-2 kinase, LsrK, which increases the net uptake rate by limiting secretion of AI-2 back into the extracellular environment. We find that the overexpression of lsrACDBFG results in an extraordinarily high AI-2 uptake rate that is capable of completely silencing QS-mediated gene expression among wild-type cells. We demonstrate utility by modulating naturally occurring processes of chemotaxis and biofilm formation. We envision that 'controller cells' that modulate bacterial behavior by manipulating molecular communication, will find use in a variety of applications, particularly those employing natural or synthetic bacterial consortia.
Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
Chemicals production by engineered microorganisms often requires induction of target gene expression at an appropriate cell density to reduce conflict with cell growth. The lux system in Vibrio fischeri is a well-characterized model for cell density-dependent regulation of gene expression termed quorum sensing (QS). However, there are currently no reports for application of the lux system to microbial chemical production. Here, we constructed a synthetic lux system as a tunable cell density sensor-regulator using a synthetic lux promoter and a positive feedback loop in Escherichia coli. In this system, self-induction of a target gene expression is driven by QS-signal, and its threshold cell density can be changed depending on the concentration of a chemical inducer. We demonstrate auto-redirection of metabolic flux from central metabolic pathways toward a synthetic isopropanol pathway at a desired cell density resulting in a significant increase in isopropanol production.
Copyright © 2015. Published by Elsevier Inc.
Metabolic engineering strategies have enabled improvements in yield and titer for a variety of valuable small molecules produced naturally in microorganisms, as well as those produced via heterologous pathways. Typically, the approaches have been focused on up- and downregulation of genes to redistribute steady-state pathway fluxes, but more recently a number of groups have developed strategies for dynamic regulation, which allows rebalancing of fluxes according to changing conditions in the cell or the fermentation medium. This review highlights some of the recently published work related to dynamic metabolic engineering strategies and explores how advances in high-throughput screening and synthetic biology can support development of new dynamic systems. Dynamic gene expression profiles allow trade-offs between growth and production to be better managed and can help avoid build-up of undesired intermediates. The implementation is more complex relative to static control, but advances in screening techniques and DNA synthesis will continue to drive innovation in this field.
Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
1,4-Butanediol (BD) is an important chemical that is widely used in industry with an annual demand of one million metric tons. Here we report a modular development of engineered bacteria for successful BD bio-production. Using a systems engineering concept, we partitioned our development into two parts, namely BD biosynthesis and production control. The former was implemented through a de novo pathway that functions as an enzymatic reactor, while the latter was accomplished via synthetic circuits serving as genetic controllers. To facilitate development, the carbon utilizations were also partitioned into two routes. D-xylose was exclusively designated for BD production with other carbon sources utilized for cellular growth. Additionally, a quorum-sensing mechanism was exploited for production control, and the resulting strain was capable of autonomous production of BD. This study represents an example of the synergy between synthetic biology and metabolic engineering, affirming the need for deeper integration of the two fields.
Copyright © 2015. Published by Elsevier Inc.
Significance
One important synthetic chemistry reaction endowed by nature is the decarboxylative carbon condensation reaction using malonyl-CoA as carbon donor. Previous metabolic engineering efforts centered on the malonyl-CoA–dependent pathway have resulted in the production of many value-added compounds. Here we mimicked the native biological systems and used a dynamic regulatory network to optimize production titers and yield. The naturally existing transcriptional regulator FapR was rewired to dynamically control gene expressions involved in the supply and consumption of malonyl-CoA. Applying this metabolic control allowed the engineered cell to dynamically regulate pathway expression and compensated the metabolic activity of critical enzymes. The synthetic malonyl-CoA switch engineered in this study opens up new venues for dynamic pathway optimization and efficient production of malonyl-CoA–derived compounds.
Metabolic engineering emerged 20 years ago as the discipline occupied with the directed modification of metabolic pathways for the microbial synthesis of various products. As such, it deals with the engineering (design, construction, and optimization) of native as well as non-natural routes of product synthesis, aided in this task by the availability of synthetic DNA, the core enabling technology of synthetic biology. The two fields, however, only partially overlap in their interest in pathway engineering. While fabrication of biobricks, synthetic cells, genetic circuits, and nonlinear cell dynamics, along with pathway engineering, have occupied researchers in the field of synthetic biology, the sum total of these areas does not constitute a coherent definition of synthetic biology with a distinct intellectual foundation and well-defined areas of application. This paper reviews the origins of the two fields and advances two distinct paradigms for each of them: that of unit operations for metabolic engineering and electronic circuits for synthetic biology. In this context, metabolic engineering is about engineering cell factories for the biological manufacturing of chemical and pharmaceutical products, whereas the main focus of synthetic biology is fundamental biological research facilitated by the use of synthetic DNA and genetic circuits.
Microbial oil production by heterotrophic organisms is a promising path for the cost-effective production of biofuels from renewable resources provided high conversion yields can be achieved. To this end, we have engineered the oleaginous yeast Yarrowia lipolytica. We first established an expression platform for high expression using an intron-containing translation elongation factor-1α (TEF) promoter and showed that this expression system is capable of increasing gene expression 17-fold over the intronless TEF promoter. We then used this platform for the overexpression of diacylglycerol acyltransferase (DGA1), the final step of the triglyceride (TAG) synthesis pathway, which yielded a 4-fold increase in lipid production over control, to a lipid content of 33.8% of dry cell weight (DCW). We also show that the overexpression of acetyl-CoA carboxylase (ACC1), the first committed step of fatty acid synthesis, increased lipid content 2-fold over control, or 17.9% lipid content. Next we combined the two genes in a tandem gene construct for the simultaneous coexpression of ACC1 and DGA1, which further increased lipid content to 41.4%, demonstrating synergistic effects of ACC1+DGA1 coexpression. The lipid production characteristics of the ACC1+DGA1 transformant were explored in a 2-L bioreactor fermentation, achieving 61.7% lipid content after 120h. The overall yield and productivity were 0.195g/g and 0.143g/L/h, respectively, while the maximum yield and productivity were 0.270g/g and 0.253g/L/h during the lipid accumulation phase of the fermentation. This work demonstrates the excellent capacity for lipid production by the oleaginous yeast Y. lipolytica and the effects of metabolic engineering of two important steps of the lipid synthesis pathway, which acts to divert flux towards the lipid synthesis and creates driving force for TAG synthesis.
With increasing price volatility and growing awareness of the lack of sustainability of traditional chemical synthesis, microbial chemical production has been tapped as a promising renewable alternative for the generation of diverse, stereospecific compounds. Nonetheless, many attempts to generate them are not yet economically viable. Due to the zero-sum nature of microbial resources, traditional strategies of pathway optimization are attaining minimal returns. This result is in part a consequence of the gross changes in host physiology resulting from such efforts and underscores the need for more precise and subtle forms of gene modulation. In this review, we describe alternative strategies and emerging paradigms to address this problem and highlight potential solutions from the emerging field of synthetic biology.
Metabolic engineering has the potential to produce from simple, readily available, inexpensive starting materials a large number of chemicals that are currently derived from nonrenewable resources or limited natural resources. Microbial production of natural products has been achieved by transferring product-specific enzymes or entire metabolic pathways from rare or genetically intractable organisms to those that can be readily engineered, and production of unnatural specialty chemicals, bulk chemicals, and fuels has been enabled by combining enzymes or pathways from different hosts into a single microorganism and by engineering enzymes to have new function. Whereas existing production routes use well-known, safe, industrial microorganisms, future production schemes may include designer cells that are tailor-made for the desired chemical and production process. In any future, metabolic engineering will soon rival and potentially eclipse synthetic organic chemistry.
Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or "program" entire populations of cells. We re-engineered the native QS regulon so that individual cell signals (autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and beta-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions.
Metabolic engineering has achieved encouraging success in producing foreign metabolites in a variety of hosts. However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to metabolic imbalance and suboptimal productivity. Here we have demonstrated the second stage of metabolic engineering effort by designing and engineering a regulatory circuit to control gene expression in response to intracellular metabolic states. Specifically, we recruited and altered one of the global regulatory systems in Escherichia coli, the Ntr regulon, to control the engineered lycopene biosynthesis pathway. The artificially engineered regulon, stimulated by excess glycolytic flux through sensing of an intracellular metabolite, acetyl phosphate, controls the expression of two key enzymes in lycopene synthesis in response to flux dynamics. This intracellular control loop significantly enhanced lycopene production while reducing the negative impact caused by metabolic imbalance. Although we demonstrated this strategy for metabolite production, it can be extended into other fields where gene expression must be closely controlled by intracellular physiology, such as gene therapy.
Genetically identical cells and organisms exhibit remarkable diversity even when they have identical histories of environmental exposure. Noise, or variation, in the process of gene expression may contribute to this phenotypic variability. Recent studies suggest that this noise has multiple sources, including the stochastic or inherently random nature of the biochemical reactions of gene expression. In this review, we summarize noise terminology and comment on recent investigations into the sources, consequences, and control of noise in gene expression.
A more complete understanding of the causes and effects of cell-cell variability in gene expression is needed to elucidate whether the resulting phenotypes are disadvantageous or confer some adaptive advantage. Here we show that increased variability in gene expression, affected by the sequence of the TATA box, can be beneficial after an acute change in environmental conditions. We rationally introduce mutations within the TATA region of an engineered Saccharomyces cerevisiae GAL1 promoter and measure promoter responses that can be characterized as being either highly variable and rapid or steady and slow. We computationally illustrate how a stable transcription scaffold can result in "bursts" of gene expression, enabling rapid individual cell responses in the transient and increased cell-cell variability at steady state. We experimentally verify computational predictions that the rapid response and increased cell-cell variability enabled by TATA-containing promoters confer a clear benefit in the face of an acute environmental stress.
A segregated model of multicopy plasmid propagation has been formulated which incorporates plasmid replication and partition functions, as well as the effect of plasmid presence on host growth rate. Growth of plasmid-free cells in selective medium is explicitly analyzed. The model parameters can be determined from experimentally measurable quantities. Propagation of a recombinant multicopy plasmid in the yeast Saccharomyces cerevisiae is analyzed using this model.
Experimental elucidation of the metabolic load placed on bacteria by the expression of foreign protein is presented. The host/vector system is Escherichia coli RR1/pBR329 (ampr, camr, and letr). Plasmid content results, which indicate that the plasmid copy number monotonically increases with decreasing growth rate, are consistent with the literature on ColE1-like plasmids. More significantly, we have experimentally quantified the reduction in growth rate brought about by the expression of chloramphenicol-acetyl-transferase (CAT) and β-lactamase. Results indicate a nearly linear decrease in growth rate with increasing foreign protein content. Also, the change in growth rate due to foreign protein expression depends on the growth rate of the cells. The observed linear relationship is media independent and, to our knowledge, previously undocumented. Furthermore, the induction of CAT, mediated by the presence of chloramphenicol, is shown to occur only at low growth rates, which further increases the metabolic load.
Results are vdelineated with the aid of a structured kinetic model representing the metabolism of recombinant E. coli. In this article, several previous hypotheses and model predictions are justified and validated. This work provides an important step in the development of comprehensive, methabolically-structured, kinetic models capable of prediciting optimal conditions for maximizing product yield.
Several researchers have demonstrated that the presence of a heterologous protein in recombinant Escherichia coli elicits a response similar to the heat-shock response, which includes enhanced protease expression. The present work detects, quantifies, and characterizes intracellular protease activity in E. coli that are "shocked" by the induction of a recombinant protein, CAT, which is an endogenous protein in some E. coli strains. A novel, sodium dodecyl sulfate gelatin poly-acrylamide gel electrophoresis (SDS-GPAGE) method is used to detect, quantify, and characterize the presence of these proteases. A hypothesis is proposed which links the amplified protease activity to a temporary depletion of specific amino acid pools, and a stringent-like stress response.