Band-pass filters, which selectively transmit signals within a defined range of input magnitudes, are fundamental components of signal-processing systems. In cellular gene circuits, band-pass behavior has likewise been pursued as a mean to implement complex signal-processing functions. However, previously reported genetic band-pass circuits have typically relied not only on a large number of regulatory components but also on transcriptional cascades involving multiple transcription factors, resulting in long DNA sequences and increased circuit complexity. Here, we first propose a band-pass gene circuit that operates without transcriptional cascades. By co-expressing two variants of the transcription factor BetI that exhibit opposite input-response behaviors-one acting as an inducer-dependent activator and the other as an inducer-dependent repressor-band-pass filtering is achieved solely through differential tuning of their inducer sensitivities. This minimal architecture enables gene expression only within a specific range of intracellular choline concentrations. Furthermore, we demonstrate that this cascade-free band-pass circuit can be exploited to generate spatial expression patterns in Escherichia coli populations in response to a choline diffusion gradient, illustrating its utility for pattern formation in multicellular contexts.

The fusion of different proteins can result in the linkage-dependent emergence of molecular switches. We inserted chloramphenicol acetyltransferase (CAT) from Escherichia coli into a loop of a d-fucose-responsive mutant of transcription factor AraC, using linker libraries with various lengths. We found that on-switches tend to emerge when two proteins are fused with linkers long enough to fill the gap of the distance of residues to be connected, while fusing with short or zero linkers results in the frequent emergence of off-switches. Both types of switches rapidly evolved their switching efficiency upon mutations, establishing the d-fucose-on and -off regulations of CAT activity without disrupting the d-fucose-inducible logic of AraC function. To our surprise, both one-input/two-output split gates thus obtained could be easily interconverted upon mutations. Through mutations, fusion proteins rapidly establish and evolve mutual regulatory relationships with unrelated partner proteins, enabling diverse functional outcomes. Furthermore, random mutagenesis can alter the behavior of these emergent regulatory relationships, such as interconverting the activation or deactivation of the partner protein upon ligand binding, sometimes at a surprisingly high frequency.

Arsenite [As(III)] is a toxic substance widely present on Earth, and the development of low-cost and simple microbial-based As(III) sensors has been attracting attention. Recently, we discovered that the protein LuxR, which contains multiple cysteine residues with high affinity for As(III), forms an insoluble structure upon binding to As(III) and exhibits OFF-switching properties as a quorum sensing transcriptional activator. Based on this property, the LuxR sensor operates on a new principle distinct from conventional whole-cell As(III) sensors; however, its sensitivity remains a challenge. In this study, we aimed to improve the sensitivity of the whole-cell OFF-type As(III) sensor by increasing the frequency of intracellular interactions between the sensor protein and As(III). We utilized the super-repressor properties of ArsR, a transcriptional repressor of the As(III)-metabolizing ars operon, achieved by replacing C34 in its As(III)-binding domain with Y. By linking ArsRC34Y with the OFF-type As(III) sensor protein LuxR, we constructed a single plasmid to create a portable ArsRC34Y-LuxR sensor protein. By suppressing the expression of ArsB, an As(III) efflux transporter encoded in the ars operon, using ArsRC34Y, we successfully enhanced the sensitivity of the OFF-type As(III) response.

Methyl methacrylate (MMA), the primary raw material of acrylic resin, is an important polymeric material due to its increasing demand and ease of recycling. The most promising biosynthetic route for MMA involves the condensation of methanol with methacrylyl-CoA (MAA-CoA), an intermediate in the valine degradation pathway. The toxicity of MAA-CoA, poor stability and low activity of the heterologous pathway enzymes make this biosynthetic pathway less feasible. For enabling the evolutionary engineering of this pathway and its components (enzymes), we constructed a biosensor system in which the cellular level of key intermediate MAA-CoA can be evaluated in a high-throughput manner. With the aid of this MAA-CoA sensory system, we could establish the functional pathway from isobutyric acid to MAA-CoA. The sensor described in this paper should be valuable tool in the design-build-test-learn cycle for optimizing and breeding this MMA pathway.

The development of a low-cost and user-friendly sensor using microorganisms to monitor the presence of As(III) on earth has garnered significant attention. In conventional research on microbial As(III) sensors, the focus has been on transcription factor ArsR, which plays a role in As(III) metabolism. However, we recently discovered that LuxR, a quorum-sensing control factor in Vibrio fischeri that contains multiple cysteine residues, acted as an As(III) sensor despite having no role in As(III) metabolism. This finding suggested that any protein could be an As(III) sensor if cysteine residues were incorporated. In this study, we aimed to confer As(III) responsiveness to BetI, a transcriptional repressor of the TetR family involved in osmotic regulation of the choline response, unrelated to As(III) metabolism. Based on the BetI structure constructed using molecular dynamics calculations, we generated a series of mutants in which each of the three amino acids not critical for function was substituted with cysteine. Subsequent examination of their response to As(III) revealed that the cysteine-substituted mutant, incorporating all three substitutions, demonstrated As(III) responsiveness. This was evidenced by the fluorescence intensity of the downstream reporter superfolder green fluorescent protein expression regulated by the operator region. Intriguingly, the BetI cysteine mutant maintained its binding responsiveness to the natural ligand choline. We successfully engineered an OR logic gate capable of responding to two orthogonal ligands using a single protein.

Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couples gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as PT5 system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.

SAM (S-adenosylmethionine) is an important metabolite that operates as a major donor of methyl groups and is a controller of various physiological processes. Its availability is also believed to be a major bottleneck in the biological production of numerous high-value metabolites. Here, we constructed SAM-sensing systems using MetJ, an SAM-dependent transcriptional regulator, as a core component. SAM is a corepressor of MetJ, which suppresses the MetJ promoter with an increasing cellular concentration of SAM (SAM-OFF sensor). The application of transcriptional interference and evolutionary tuning effectively inverted its response, yielding a SAM-ON sensor (signal increases with increasing SAM concentration). By linking two genes encoding fluorescent protein reporters in such a way that their transcription events interfere with each other's and by placing one of them under the control of MetJ, we could increase the effective signal-to-noise ratio of the SAM sensor while decreasing the batch-to-batch deviation in signal output, likely by canceling out the growth-associated fluctuation in translational resources. By taking the ratio of SAM-ON/SAM-OFF signals and by resetting the default pool size of SAM, we could rapidly identify SAM synthetase (MetK) mutants with increased cellular activity from a random library. The strategy described herein should be widely applicable for identifying activity mutants, which would be otherwise overlooked because of the strong homeostasis of metabolic networks.

S-adenosylmethionine (SAM) is an important biomolecule that mainly acts as a methyl donor and plays many roles in a variety of biological functions. SAM is also required for the biosynthesis of valuable methylated compounds, but its supply is a bottleneck for these biosynthetic pathways. To overcome this bottleneck and to reconfigure SAM homeostasis, a high-throughput sensing system for changes in intracellular SAM availability is required. We constructed a plasmid that can detect the factors that can alter SAM availability using minimal components. It does so by placing a fluorescent protein under a promoter controlled by endogenous MetJ, a transcription factor that represses its own regulons upon binding with SAM. Next, to validate SAM-responsive behavior, we systematically reconstructed 10 synthetic promoters with different positions and with different number of metbox sites. We found that a position between the -35 box and the -10 box was the most effective for repression and that this setup was suitable for detecting the genetic or environmental factors that can deplete and recover the intracellular SAM availability. Overall, the response patterns of the synthetic MetJ-regulated promoters characterized in this study may be useful for the development of better SAM biosensing systems.

Binding-induced folding 1–4 (BIF) is a promising mechanism that can be used to rapidly convert binders into sensors/regulators without allosteric design. Here we showed that allosteric regulatory proteins AraC can acquire BIF mechanism without compromising their inherent allosteric mechanisms, with high frequency upon mutations. This opened an opportunity to compare the evolutionary capacity of the allosteric and non-allosteric modes of a specific sensory protein. We found that AraC evolved novel sensory function far more rapidly in BIF mode than in allosteric mode. This newly acquired (non-allosteric) sensory function is distinguishable both in its response logic and in sensitivity from original (allosteric) one, and they can be operated simultaneously, independently, and cooperatively, allowing the construction of complex regulatory networks behaviours such as a selective NIMPLY/OR converter and width-tuneable band-pass filter. Together with its high frequency of emergence, BIF can be an overlooked evolutionary driver of the invention of novel biosensors and complex regulatory networks in nature and laboratory.

Whole-cell sensors for arsenite detection have been developed exclusively based on the natural arsenite (As(III)) sensory protein ArsR for arsenic metabolism. This study reports that the quorum-sensing LuxR/Plux system from Vibrio fischeri, which is completely unrelated to arsenic metabolism, responds to As(III) in a dose-dependent manner. Due to as many as 9 cysteine residues, which has a high binding affinity with As(III), LuxR underwent As(III)-induced insoluble form, thereby reducing its effective cellular concentration. Accordingly, the expression level of green fluorescent protein under the control of Plux gradually decreased with increasing As(III) concentration in the medium. This is a novel As(III)-detection system that has never been proposed before, with a unique ON-to-OFF transfer function.

We constructed a methodology for thermostabilizing a G-protein coupled receptor (GPCR) in the inactive state whose wild-type (WT) structure is unknown solely by multiple amino-acid mutations without the ligand binding. It is a combination of our recently developed theory based on statistical thermodynamics and site-directed saturation mutagenesis, a method often employed in evolutionary molecular engineering. First, the WT structure is predicted using the homology modeling. Second, a key residue is determined by our statistical-thermodynamics theory using suitably modeled mutant structures. Many of 19 different single mutations for the key residue are expected to produce significantly higher stabilization. Third, we undertake to mutate not only the key residue but also a few more residues whose side chains are close to the side chain of the key residue. The whole mutational space is then efficiently explored by introducing site-directed saturation mutations, and a gene (mutant) library is constructed using the small-intelligent and fully automatic single-tube recombination methods. Each mutant is expressed in Escherichia coli cells, and highly stabilized mutants are sorted out using a fluorescence-screening technique. The methodology was illustrated for the serotonin 2A receptor, 5-HT 2A R, for stabilizing its inactive state. We could identify a double mutant whose apparent midpoint temperature of thermal denaturation is higher than that of a thermostabilized double mutant previously reported by ~8.9°C and that of the WT by over 15°C. Moreover, it exhibits higher binding affinity for spiperone, an antagonist which was previously proved to stabilize 5-HT 2A R in the inactive state.

In recent years, advances in bioengineering and synthetic biology techniques have been used to create carotenoid diversity in the laboratory. In this chapter, we describe the step-by-step method to perform directed evolution of carotenoid biosynthetic enzymes. We first explain how to establish an efficient Escherichia coli colony-based screening, including a detailed description of plasmid DNA construction design as well as tips and tricks to handle and manipulate cells to produce stable colonies. As an example for the directed evolution experiment, we engineer a bacterial phytoene desaturase CrtI to obtain a C50-phytoene desaturase, which catalyzes formation of a non-natural long-chain carotenoid. The method described in this chapter can be applied to many carotenoid biosynthetic enzymes, whose numbers have been rapidly expanding with recent advances in genomics. The use of directed evolution for carotenoid enzymes will contribute not only to the discovery of novel carotenoids but also to a deeper understanding of the creation and evolution of carotenoid biosynthetic pathways in nature.

Over 800 known carotenoids are synthesized from phytoene or 4,4′-diapophytoene (dehydrosqualene) characterized by three conjugated double bonds. In this paper, we report that carotenoid desaturase CrtN from Staphylococcus aureus and Methylomonas can accept oxidosqualene, which is the precursor for plant- or animal-type triterpenoids, yielding the yellow carotenoid pigments with 8, 9, or 10 conjugated double bonds. The resulting pathway is the second nonnatural route for carotenoid pigments and the first pathway for carotenoid pigments not biosynthesized via (diapo)phytoene.

One of the most prominent features of genetically encoded biosensors (GEBs) is their evolvability-the ability to invent new sensory functions using mutations. Among the GEBs, the transcription factor-based biosensors (TF-biosensors) is the focus of this review. We also discuss how this class of sensors can be highly evolvable and how we can exploit it. With an established platform for directed evolution, researchers can create, or evolve, new TF-biosensors. Directed evolution experiments have revealed the TF-biosensors' evolvability, which is based partially on their characteristic physicochemical properties.

A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae . Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system’s utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.

Stringency (low leak) is one of the most important specifications required for genetic circuits and induction systems, but it is challenging to evolve without sacrificing the maximum output level. This problem also comes from the absence of truly tunable negative selection methods. This paper reports that stringently switching variants can sometimes emerge with surprising frequency upon mutations. We randomly mutated the previously generated leaky variants of LuxR, the quorum-sensing transcription activator from Vibrio fischeri, to restore the stringency. We found as much as 10-20% of the entire population exhibited significantly improved signal-to-noise ratios compared with their parents. This indicated that these mutants arose by the loss of folding capability by accumulating destabilizing mutations, not by introducing rare adaptive mutations, thereby becoming AHL-dependent folders. Only four rounds of mutagenesis and ON-state selection resulted in the domination of the entire population by the improved variants with low leak, without direct selection pressure for stringency. With this surprising frequency, conversion into the "ligand-addicted folders" should be one of the prevailing modes of evolving stringency both in the laboratory and in nature, and the workflow described here provides a rapid and versatile method of improving the signal-to-noise ratio of -various genetic switches.

Genetic switches provide core components of gene expression induction systems, genetic circuits, and metabolite sensors, including those for high-throughput screening and selection of enzyme functions. However, it is rare that natural transcription factors meet the required specifications for each application. Fortunately, the directed evolution of transcription switches is a straightforward process, given that the two states of genetic switches (on and off) are both selectable, using a wide range of positive and negative selection tools developed in the field of molecular genetics. On/off-state selections based on bactericidal mechanism allow greatly accelerate the entire process. The key to success is finding the selection conditions that minimize false-positive and false-negative clones as with any directed evolution experiment. We introduce a reliable and automatable directed evolution platform to rapidly evolve genetic switches with desired specifications in this chapter. Highlighting the importance and ease of screening for selection conditions, we demonstrate how selection conditions influence the resultant population of the selected pools.

Heterologous production of a useful carotenoid astaxanthin was achieved in a cyanobacterium Synechocystis sp. PCC 6803 with the aid of marine bacterial genes. Astaxanthin and its intermediates emerged at high levels, whereas β-carotene and zeaxanthin disappeared in the strain. Total carotenoid accumulation was nearly two fold compared with wild type. The astaxanthin-producing strain was capable of only growing heterotrophically, which was likely due to the absence of β-carotene. Further enhanced accumulation was pursued by gene overexpression for possible rate-limiting steps in the biosynthesis pathway.