Transcription Factor-based Biosensors Research Articles

Transcription factor-based biosensors for detection of naturally occurring phenolic acids

Phenolic acids including hydroxybenzoic and hydroxycinnamic acids are secondary plant and fungal metabolites involved in many physiological processes offering health and dietary benefits. They are often utilised as precursors for production of value-added compounds. The limited availability of synthetic biology tools, such as whole-cell biosensors suitable for monitoring the dynamics of phenolic acids intracellularly and extracellularly, hinders the capabilities to develop high-throughput screens to study their metabolism and forward engineering. Here, by applying a multi-genome approach, we have identified phenolic acid-inducible gene expression systems composed of transcription factor-inducible promoter pairs responding to eleven different phenolic acids. Subsequently, they were used for the development of whole-cell biosensors based on model bacterial hosts, such as Escherichia coli, Cupriavidus necator and Pseudomonas putida. The dynamics and range of the biosensors were evaluated by establishing their response and sensitivity landscapes. The specificity and previously uncharacterised interactions between transcription factor and its effector(s) were identified by a screen of twenty major phenolic acids. To exemplify applicability, we utilise a protocatechuic acid-biosensor to identify enzymes with enhanced activity for conversion of p-hydroxybenzoate to protocatechuate. Transcription factor-based biosensors developed in this study will advance the analytics of phenolic acids and expedite research into their metabolism.

Development of a workflow for the selection, identification and optimization of lactic acid bacteria with high γ-aminobutyric acid production

Lactic acid bacteria produce γ-aminobutyric acid (GABA) as an acid stress response. GABA is a neurotransmitter that may improve sleep and resilience to mental stress. This study focused on the selection, identification and optimization of a bacterial strain with high GABA production, for development as a probiotic supplement. The scientific literature and an industry database were searched for probiotics and potential GABA producers. In silico screening was conducted to identify genes involved in GABA production. Subsequently, 17 candidates were screened for in vitro GABA production using thin layer chromatography, which identified three candidate probiotic strains Levilactobacillus brevis DSM 20054, Lactococcus lactis DS75843and Bifidobacterium adolescentis DSM 24849 as producing GABA. Two biosensors capable of detecting GABA were developed: 1. a transcription factor-based biosensor characterized by the interaction with the transcriptional regulator GabR was developed in Corynebacterium glutamicum; and 2. a growth factor-based biosensor was built in Escherichia coli, which used auxotrophic complementation by expressing 4-aminobutyrate transaminase (GABA-T) that transfers the GABA amino group to pyruvate, hereby forming alanine. Consequently, the feasibility of developing a workflow based on co-culture with producer strains and a biosensor was tested. The three GABA producers were identified and the biosensors were encapsulated in nanoliter reactors (NLRs) as alginate beads in defined gut-like conditions. The E. coli growth factor-based biosensor was able to detect changes in GABA concentrations in liquid culture and under gut-like conditions. L. brevis and L. lactis were successfully encapsulated in the NLRs and showed growth under miniaturized intestinal conditions.

MoBioS: Modular Platform Technology for High-Throughput Construction and Characterization of Tunable Transcriptional Biological Sensors.

All living organisms have evolved and fine-tuned specialized mechanisms to precisely monitor a vast array of different types of molecules. These natural mechanisms can be sourced by researchers to build Biological Sensors (BioS) by combining them with an easily measurable output, such as fluorescence. Because they are genetically encoded, BioS are cheap, fast, sustainable, portable, self-generating and highly sensitive and specific. Therefore, BioS hold the potential to become key enabling tools that stimulate innovation and scientific exploration in various disciplines. However, the main bottleneck in unlocking the full potential of BioS is the fact that there is no standardized, efficient and tunable platform available for the high-throughput construction and characterization of biosensors. Therefore, a modular, Golden Gate-based construction platform, called MoBioS, is introduced in this article. It allows for the fast and easy creation of transcription factor-based biosensor plasmids. As a proof of concept, its potential is demonstrated by creating eight different, functional and standardized biosensors that detect eight diverse molecules of industrial interest. In addition, the platform contains novel built-in features to facilitate fast and efficient biosensor engineering and response curve tuning.

TFBMiner: A User-Friendly Command Line Tool for the Rapid Mining of Transcription Factor-Based Biosensors

Transcription factors responsive to small molecules are essential elements in synthetic biology designs. They are often used as genetically encoded biosensors with applications ranging from the detection of environmental contaminants and biomarkers to microbial strain engineering. Despite our efforts to expand the space of compounds that can be detected using biosensors, the identification and characterization of transcription factors and their corresponding inducer molecules remain labor- and time-intensive tasks. Here, we introduce TFBMiner, a new data mining and analysis pipeline that enables the automated and rapid identification of putative metabolite-responsive transcription factor-based biosensors (TFBs). This user-friendly command line tool harnesses a heuristic rule-based model of gene organization to identify both gene clusters involved in the catabolism of user-defined molecules and their associated transcriptional regulators. Ultimately, biosensors are scored based on how well they fit the model, providing wet-lab scientists with a ranked list of candidates that can be experimentally tested. We validated the pipeline using a set of molecules for which TFBs have been reported previously, including sensors responding to sugars, amino acids, and aromatic compounds, among others. We further demonstrated the utility of TFBMiner by identifying a biosensor for S-mandelic acid, an aromatic compound for which a responsive transcription factor had not been found previously. Using a combinatorial library of mandelate-producing microbial strains, the newly identified biosensor was able to distinguish between low- and high-producing strain candidates. This work will aid in the unraveling of metabolite-responsive microbial gene regulatory networks and expand the synthetic biology toolbox to allow for the construction of more sophisticated self-regulating biosynthetic pathways.

Transcription factor-based biosensors for screening and dynamic regulation.

Advances in synthetic biology and genetic engineering are bringing into the spotlight a wide range of bio-based applications that demand better sensing and control of biological behaviours. Transcription factor (TF)-based biosensors are promising tools that can be used to detect several types of chemical compounds and elicit a response according to the desired application. However, the wider use of this type of device is still hindered by several challenges, which can be addressed by increasing the current metabolite-activated transcription factor knowledge base, developing better methods to identify new transcription factors, and improving the overall workflow for the design of novel biosensor circuits. These improvements are particularly important in the bioproduction field, where researchers need better biosensor-based approaches for screening production-strains and precise dynamic regulation strategies. In this work, we summarize what is currently known about transcription factor-based biosensors, discuss recent experimental and computational approaches targeted at their modification and improvement, and suggest possible future research directions based on two applications: bioproduction screening and dynamic regulation of genetic circuits.

Engineering an AI-based forward-reverse platform for the design of cross-ribosome binding sites of a transcription factor biosensor

A cross-ribosome binding site (cRBS) adjusts the dynamic range of transcription factor-based biosensors (TFBs) by controlling protein expression and folding. The rational design of a cRBS with desired TFB dynamic range remains an important issue in TFB forward and reverse engineering. Here, we report a novel artificial intelligence (AI)-based forward-reverse engineering platform for TFB dynamic range prediction and de novo cRBS design with selected TFB dynamic ranges. The platform demonstrated superior in processing unbalanced minority-class datasets and was guided by sequence characteristics from trained cRBSs. The platform identified correlations between cRBSs and dynamic ranges to mimic bidirectional design between these factors based on Wasserstein generative adversarial network (GAN) with a gradient penalty (GP) (WGAN-GP) and balancing GAN with GP (BAGAN-GP). For forward and reverse engineering, the predictive accuracy was up to 98% and 82%, respectively. Collectively, we generated an AI-based method for the rational design of TFBs with desired dynamic ranges.

Detection of Bile Acids in Complex Matrices Using a Transcription Factor-Based Biosensor.

Bile acids play an important role in digestion and human health, are found throughout the gastrointestinal tract, and are excreted in feces. Therefore, bile acids are promising biomarkers for monitoring health and detecting fecal contamination in water sources. Here, we engineered a bile acid sensor by expressing the transcription factor BreR, a TetR-like repressor from Vibrio cholorae, in Escherichia coli. The sensor was further optimized by screening a promoter library. To further characterize the BreR sensor and increase its utility, we moved expression to a cell-free expression (CFE) system, resulting in an approximately 3 orders of magnitude increase in deoxycholic acid sensitivity. We next optimized this sensor to detect bile acids in fecal water, wastewater, and serum and transferred the CFE sensor to a paper-based assay to enhance fieldability.

A Synthetic Biosensor for Detecting Putrescine in Beef Samples.

Biogenic amines (BAs) are toxicological risks present in many food products. Putrescine is the most common foodborne BA and is frequently used as a quality control marker. Currently, there is a lack of regulation concerning safe putrescine limits in food as well as outdated food handling practices leading to unnecessary putrescine intake. Conventional methods used to evaluate BAs in food are generally time-consuming and resource-heavy with few options for on-site analysis. In response to this challenge, we have developed a transcription factor-based biosensor for the quantification of putrescine in beef samples. In this work, we use a naturally occurring putrescine responsive repressor-operator pair (PuuR-puuO) native to Escherichia coli. Moreover, we demonstrate the use of the cell-free putrescine biosensor on a paper-based device that enables rapid low-cost detection of putrescine in beef samples stored at different temperatures. The results presented demonstrate the potential role of using paper-based biosensors for on-site testing, particularly as an index for determining meat product stability and quality.

Surveying the Genetic Design Space for Transcription Factor-Based Metabolite Biosensors: Synthetic Gamma-Aminobutyric Acid and Propionate Biosensors in E. coli Nissle 1917.

Engineered probiotic bacteria have been proposed as a next-generation strategy for noninvasively detecting biomarkers in the gastrointestinal tract and interrogating the gut-brain axis. A major challenge impeding the implementation of this strategy has been the difficulty to engineer the necessary whole-cell biosensors. Creation of transcription factor-based biosensors in a clinically-relevant strain often requires significant tuning of the genetic parts and gene expression to achieve the dynamic range and sensitivity required. Here, we propose an approach to efficiently engineer transcription-factor based metabolite biosensors that uses a design prototyping construct to quickly assay the gene expression design space and identify an optimal genetic design. We demonstrate this approach using the probiotic bacterium Escherichia coli Nissle 1917 (EcN) and two neuroactive gut metabolites: the neurotransmitter gamma-aminobutyric acid (GABA) and the short-chain fatty acid propionate. The EcN propionate sensor, utilizing the PrpR transcriptional activator from E. coli, has a large 59-fold dynamic range and >500-fold increased sensitivity that matches biologically-relevant concentrations. Our EcN GABA biosensor uses the GabR transcriptional repressor from Bacillus subtilis and a synthetic GabR-regulated promoter created in this study. This work reports the first known synthetic microbial whole-cell biosensor for GABA, which has an observed 138-fold activation in EcN at biologically-relevant concentrations. Using this rapid design prototyping approach, we engineer highly functional biosensors for specified in vivo metabolite concentrations that achieve a large dynamic range and high output promoter activity upon activation. This strategy may be broadly useful for accelerating the engineering of metabolite biosensors for living diagnostics and therapeutics.

Transcription Factor-Based Biosensors for Detecting Pathogens

Microorganisms are omnipresent and inseparable from our life. Many of them are beneficial to humans, while some are not. Importantly, foods and beverages are susceptible to microbial contamination, with their toxins causing illnesses and even death in some cases. Therefore, monitoring and detecting harmful microorganisms are critical to ensuring human health and safety. For several decades, many methods have been developed to detect and monitor microorganisms and their toxicants. Conventionally, nucleic acid analysis and antibody-based analysis were used to detect pathogens. Additionally, diverse chromatographic methods were employed to detect toxins based on their chemical and structural properties. However, conventional techniques have several disadvantages concerning analysis time, sensitivity, and expense. With the advances in biotechnology, new approaches to detect pathogens and toxins have been reported to compensate for the disadvantages of conventional analysis from different research fields, including electrochemistry, nanotechnology, and molecular biology. Among them, we focused on the recent studies of transcription factor (TF)-based biosensors to detect microorganisms and discuss their perspectives and applications. Additionally, the other biosensors for detecting microorganisms reported in recent studies were also introduced in this review.

Assessment of fluorescent protein candidates for multi-color flow cytometry analysis of Saccharomyces cerevisiae

Transcription factor-based biosensors represent promising tools in the construction and evaluation of efficient cell factories for the sustainable production of fuels, chemicals and pharmaceuticals. They can notably be designed to follow the production of a target compound or to monitor key cellular properties, such as stress or starvation. In most cases, the biosensors are built with fluorescent protein (FP) genes as reporter genes because of the direct correlation between promoter activity and fluorescence level that can be measured using, for instance, flow cytometry or fluorometry. The expansion of available FPs offers the possibility of using several FPs - and biosensors – in parallel in one host, with simultaneous detection using multicolor flow cytometry. However, the technique is currently limited by the unavailability of combinations of FP whose genes can be successfully expressed in the host and whose fluorescence can be efficiently distinguished from each other.In the present study, the broad collection of available FPs was explored and four different FPs were successfully expressed in the yeast Saccharomyces cerevisiae: yEGFP, mEGFP, CyOFP1opt and mBeRFPopt. After studying their fluorescence signals, population heterogeneity and possible interactions, we recommend two original combinations of FPs for bi-color flow cytometry: mEGFP together with either CyOFP1opt or mBeRFPopt, as well as the combination of all three FPs mEGFP, CyOFP1opt and mBeRFPopt for tri-color flow cytometry. These combinations will allow to perform different types of bi-color or possibly tri-color flow cytometry and FACS experiments with yeast, such as phenotype evaluation, screening or sorting, by single-laser excitation with a standard 488 nm blue laser.

Engineering Transcription Factor BmoR Mutants for Constructing Multifunctional Alcohol Biosensors.

Native transcription factor-based biosensors (TFBs) have the potential for the in situ detection of value-added chemicals or byproducts. However, their industrial application is limited by their ligand promiscuity, low sensitivity, and narrow detection range. Alcohols exhibit similar structures, and no reported TFB can distinguish a specific alcohol from its analogues. Here, we engineered an alcohol-regulated transcription factor, BmoR, and obtained various mutants with remarkable properties. For example, the generated signal-molecule-specific BmoRs could distinguish the constitutional isomers n-butanol and isobutanol, with insensitivity up to an ethanol concentration of 800 mM (36.9 g/L). Linear detection of 0-60 mM of a specific higher alcohol could be achieved in the presence of up to 500 mM (23.0 g/L) ethanol as background noise. Furthermore, we obtained two mutants with raised outputs and over 107-fold higher sensitivity and one mutant with an increased upper detection limit (14.8 g/L n-butanol or isobutanol). Using BmoR as an example, this study systematically explored the ultimate detection limit of a TFB toward its small-molecule ligands, paving the way for in situ detection in biofuel and wine industries.

Directed Evolution of Transcription Factor-Based Biosensors for Altered Effector Specificity.

Transcription factor-based biosensors are important tools in Synthetic Biology for the sensing of industrially valuable molecules and clinically important metabolites, therefore presenting applications in the bioremediation, industrial biotechnology, and biomedical fields. The directed evolution of allosteric transcription factors (aTFs) with the aim of altering effector specificity has the potential for the development of new biosensors to detect natural and nonnatural molecules, expanding the scope of available aTF-based biosensors. In this chapter, we delineate a general method for the directed evolution of aTFs. The theory of library design is discussed, along with the detailed methodology for an improved transformation of combined libraries, and the experimental search space by counterselection using fluorescence-activated cell sorting (FACS) is presented.

Biosensor-based isolation of amino acid-producing Vibrio natriegens strains

The marine bacterium Vibrio natriegens has recently been demonstrated to be a promising new host for molecular biology and next generation bioprocesses. V. natriegens is a Gram-negative, non-pathogenic slight-halophilic bacterium, with a high nutrient versatility and a reported doubling time of under 10 min. However, V. natriegens is not an established model organism yet, and further research is required to promote its transformation into a microbial workhorse.In this work, the potential of V. natriegens as an amino acid producer was investigated. First, the transcription factor-based biosensor LysG, from Corynebacterium glutamicum, was adapted for expression in V. natriegens to facilitate the detection of positively charged amino acids. A set of different biosensor variants were constructed and characterized, using the expression of a fluorescent protein as sensor output. After random mutagenesis, one of the LysG-based sensors was used to screen for amino acid producer strains. Here, fluorescence-activated cell sorting enabled the selective sorting of highly fluorescent cells, i.e. potential producer cells. Using this approach, individual L-lysine, L-arginine and L-histidine producers could be obtained producing up to 1 mM of the effector amino acid, extracellularly. Genome sequencing of the producer strains provided insight into the amino acid production metabolism of V. natriegens.This work demonstrates the successful expression and application of transcription factor-based biosensors in V. natriegens and provides insight into the underlying physiology, forming a solid basis for further development of this promising microbe.

Biosensor-based growth-coupling and spatial separation as an evolution strategy to improve small molecule production of Corynebacterium glutamicum

Evolutionary engineering is a powerful method to improve the performance of microbial cell factories, but can typically not be applied to enhance the production of chemicals due to the lack of an appropriate selection regime. We report here on a new strategy based on transcription factor-based biosensors, which directly couple production to growth. The growth of Corynebacterium glutamicum was coupled to the intracellular concentration of branched-chain amino acids, by integrating a synthetic circuit based on the Lrp biosensor upstream of two growth-regulating genes, pfkA and hisD. Modelling and experimental data highlight spatial separation as key strategy to limit the selection of ‘cheater’ strains that escaped the evolutionary pressure. This approach facilitated the isolation of strains featuring specific causal mutations enhancing amino acid production. We envision that this strategy can be applied with the plethora of known biosensors in various microbes, unlocking evolution as a feasible strategy to improve production of chemicals.

Development of an Haa1-based biosensor for acetic acid sensing in Saccharomyces cerevisiae

ABSTRACTAcetic acid is one of the main inhibitors of lignocellulosic hydrolysates and acetic acid tolerance is crucial for the development of robust cell factories for conversion of biomass. As a precursor of acetyl-coenzyme A, it also plays an important role in central carbon metabolism. Thus, monitoring acetic acid levels is a crucial aspect when cultivating yeast. Transcription factor-based biosensors represent useful tools to follow metabolite concentrations. Here, we present the development of an acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1 that upon binding to acetic acid relocates to the nucleus. In the biosensor, a synthetic transcription factor consisting of Haa1 and BM3R1 from Bacillus megaterium was used to control expression of a reporter gene under a promoter containing BM3R1 binding sites. The biosensor did not drive expression under a promoter containing Haa1 binding sites and responded to acetic acid over a linear range spanning from 10 to 60 mM. To validate its applicability, the biosensor was integrated into acetic acid-producing strains. A direct correlation between biosensor output and acetic acid production was detected. The developed biosensor enables high-throughput screening of strains producing acetic acid and could also be used to investigate acetic acid-tolerant strain libraries.

Effective use of biosensors for high-throughput library screening for metabolite production.

The development of fast and affordable microbial production from recombinant pathways is a challenging endeavor, with targeted improvements difficult to predict due to the complex nature of living systems. To address the limitations in biosynthetic pathways, much work has been done to generate large libraries of various genetic parts (promoters, RBSs, enzymes, etc.) to discover library members that bring about significantly improved levels of metabolite production. To evaluate these large libraries, high throughput approaches are necessary, such as those that rely on biosensors. There are various modes of operation to apply biosensors to library screens that are available at different scales of throughput. The effectiveness of each biosensor-based method is dependent on the pathway or strain to which it is applied, and all approaches have strengths and weaknesses to be carefully considered for any high throughput library screen. In this review, we discuss the various approaches used in biosensor screening for improved metabolite production, focusing on transcription factor-based biosensors.

Transcription-Factor-based Biosensor Engineering for Applications in Synthetic Biology.

Transcription-factor-based biosensors (TFBs) are often used for metabolite detection, adaptive evolution, and metabolic flux control. However, designing TFBs with superior performance for applications in synthetic biology remains challenging. Specifically, natural TFBs often do not meet real-time detection requirements owing to their slow response times and inappropriate dynamic ranges, detection ranges, sensitivity, and selectivity. Furthermore, designing and optimizing complex dynamic regulation networks is time-consuming and labor-intensive. This Review highlights TFB-based applications and recent engineering strategies ranging from traditional trial-and-error approaches to novel computer-model-based rational design approaches. The limitations of the applications and these engineering strategies are additionally reviewed.

Progress in transcription factor-based metabolite biosensors

Transcription factor-based biosensors (TFBs) play an essential role in metabolic engineering and synthetic biology. TFBs sense the metabolite concentration signals and convert them into specific signal output. They hold high sensitivity, strong specificity, brief analysis speed, and are widely used in response to target metabolites. Here we reviewe the principles of TFBs, the application examples, and challenges faced in recent years in microbial cells, including detecting target metabolite concentrations, high-throughput screening, adaptive laboratory evolutionary selection, and dynamic control. Simultaneously, to overcome the challenges in the application, we also focus on reviewing the performance tuning strategies of TFBs, mainly including traditional and computer-aided tuning strategies. We also discuss the opportunities and challenges that TFBs may face in practical applications, and propose the future research trend.

Transcription Factor-Based Biosensor for Dynamic Control in Yeast for Natural Product Synthesis.

The synthesis of natural products in yeast has gained remarkable achievements with intensive metabolic engineering efforts. In particular, transcription factor (TF)-based biosensors for dynamic control of gene circuits could facilitate strain evaluation, high-throughput screening (HTS), and adaptive laboratory evolution (ALE) for natural product synthesis. In this review, we summarized recent developments of several TF-based biosensors for core intermediates in natural product synthesis through three important pathways, i.e., fatty acid synthesis pathway, shikimate pathway, and methylerythritol-4-phosphate (MEP)/mevalonate (MVA) pathway. Moreover, we have shown how these biosensors are implemented in synthetic circuits for dynamic control of natural product synthesis and also discussed the design/evaluation principles for improved biosensor performance.