Fusing enzymes to transcription activator LuxR for the rapid creation of metabolite sensors

Metabolite sensors have been applied for high-throughput screening for improved biosynthetic pathways, as well as for dynamic control of the metabolic networks. Obviously, however, current repertoire of natural sensors covers only a small fraction of the known metabolite. We have been developing the new robust workflow for the rapid creation of metabolite sensors where biosynthetic enzymes can be adopted as the sensory (recognizing) components. Most of the known metabolites act as the direct substrates of some enzymes, and they are recognized and converted by these enzymes in physiologically relevant concentrations. Thus, ever-increasing repertoire of available enzymes is a rich and reliable source of sensory units. We found that the transcription activator LuxR can be fused with various biosynthetic enzymes without losing its function. By adding moderately de-stabilizing mutations, typically by random mutagenesis of the resultant fusion proteins followed by screening a small number (~100) of variants, we could have quickly isolated variants that can activate LuxR-dependent promoter in response to the substrates of the enzymes fused to LuxR. In this presentation, we demonstrate various metabolites can be detected by this manner. Detailed analysis of the thus-obtained fusion proteins indicated that function of LuxR is dependent on the substrate binding-induced stabilization of the enzymes. The biosensors with this mode of action exhibited various unique features. For instance, we found that the sensitivity (EC50) and dynamic range of these sensors to the target metabolites can be flexibly altered by the concentration of homoserine lactones, the cognate ligand of LuxR, in the media. Also, this provides unique opportunity to indirectly visualizing the substrate-binding to the enzyme in high-throughput manner. Indeed, multi-round mutagenesis and screening of the fusion protein of isopentenyl diphosphate isomerase (IDI) with LuxR variant (IDI-LuxR) revealed that many of the mutations that improved sensory performance of IDI-LuxR also elevated the catalytic performance of IDI. Some of such mutations turned out to elevate IDI activity even without fusion partner LuxR. Altogether, by fusing to LuxR, random mutagenesis, and traditional reporter (fluorescence)-based screening, one can not only adopt a variety of biosynthetic enzymes as sensor components but also laboratory evolve their catalytic functions

Rapid and liquid-based selection of genetic switches using nucleoside kinase fused with aminoglycoside phosphotransferase.

The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON-) and negative (OFF-) selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3')-phosphotransferase (APH), we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling

Construction of a pathway to C50-ε-carotene.

Substrate tolerance of bacterial cyclases has been demonstrated in various contexts, but little is known about that of plant cyclases. Here, we tested two plant ε-cyclases to convert C50-lycopene, which we previously established by rounds of directed evolution. Unlike bacterial β-cyclases, two-end cyclase from lettuce exhibited complete specificity against this molecule, indicating that this enzyme has some mechanism that exerts size-specificity. Arabidopsis one-end cyclase At-y2 showed detectable activity to C50-lycopene. Interestingly, we found that it functions as a two-end cyclase in a C50 context. Based on this observation, a possible model for substrate discrimination of this enzyme is proposed

Dose-response of <i>lux</i> promoter variants to 3OC6-HSL activated by LuxR.

<p>Five individual <i>lux</i> promoter variants (#30, #08, #17, #01, and #05) were selected from 45-randomly picked ones from the pool shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120243#pone.0120243.s001" target="_blank">S1 Fig</a>. (C) (variants recovered from OFF-selected Library-2 that survived the ON selection (incubation with 50 μg/mL of Km for 4 h)) and quantitatively characterized using micro-titer plate measurement of sfGFP expression after 12 h growth in medium containing various concentrations of 3OC6-HSL (0–10⁵ nM). The bar heights show the average of 3 samples, and the error bars indicate the standard deviation.</p

<i>hsvtk</i>::<i>cat</i> fusion gene as a ON/OFF-selector.

<p>(A) The construct for the expression of hsvTK::CAT. The reading frames of hsvTK and CAT (excluding the start codon, 217 aa) were fused without a linker, resulting in <i>hsvtk</i>::<i>cat</i>. The fusion gene was placed under the T5 promoter (<i>p</i>_<i>T5</i>) with the translation initiation site (rbs score [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120243#pone.0120243.ref025" target="_blank">25</a>], 13843). (B) OFF-selection. <i>E</i>. <i>coli</i> MG1655 harboring either a plasmid expressing hsvTK::CAT or a plasmid expressing GFP^UV (negative control) were incubated with dP (0–1,000 nM), and the number of viable (colony forming) cells was measured after 3 h incubation. (C) ON-selection. <i>E</i>. <i>coli</i> MG1655 harboring either of the plasmids were treated with Cm (0–240 μg/mL), and the number of viable cells was measured after 3 h incubation. The bar heights show the average of 3 samples, and error bars indicate the standard deviation.</p

Selection of functional <i>lux</i> switches from <i>lux</i> box libraries.

<p>(A) Library design. Part (six nucleotides) of the <i>lux</i> box (LuxR-binding sites) was randomized by PCR mutagenesis, yielding Library-1 and -2. The fusion selector <i>hsvtk</i>::<i>aph</i>, together with <i>sfgfp</i>, was placed under the promoter library. LuxR was constitutively expressed from a different plasmid. (B) Selection procedure: first, approximately 10⁷ transformant cells were cultured for several hours in the presence of 1 μM dP (OFF-selection). Next, the cells were treated with 50–200 μg/mL Km (ON-selection) cultured for 1–6 hours in the presence of 1 μM 3OC6-HSL. The resultant cells were rinsed and re-grown in LB media. The cell pool was subjected to the flow cytometry in each step of this selection. (C) Flow cytometric analysis of the transformant pools before selection and after OFF/ON-selection (incubated in the absence/presence, respectively, of 1,000 nM 3OC6-HSL). Histograms in color indicate the fluorescent distribution in the presence of 3OC6-HSL, while those in gray indicate the fluorescent distribution in the absence of 3OC6-HSL.</p

Liquid-Based Iterative Recombineering Method Tolerant to Counter-Selection Escapes

<div><p>Selection-based recombineering is a flexible and proven technology to precisely modify bacterial genomes at single base resolution. It consists of two steps of homologous recombination followed by selection/counter-selection. However, the shortage of efficient counter-selectable markers limits the throughput of this method. Additionally, the emergence of ‘selection escapees’ can affect recombinant pools generated through this method, and they must be manually removed at each step of selection-based recombineering. Here, we report a series of efforts to improve the throughput and robustness of selection-based recombineering and to achieve seamless and automatable genome engineering. Using the nucleoside kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the non-natural nucleoside dP, a highly efficient, rapid, and liquid-based counter-selection system was established. By duplicating <i>hsvtk</i> gene, combined with careful control of the population size for the subsequent round, we effectively eliminated selection escapes, enabling seamless and multiple insertions/replacement of gene-size fragments in the chromosome. Four rounds of recombineering could thus be completed in 10 days, requiring only liquid handling and without any need for colony isolation or genotype confirmation. The simplicity and robustness of our method make it broadly accessible for multi-locus chromosomal modifications.</p></div