XylB and XylC NahF Inspiration and intention Build the Adaptors!

Based on the transcriptional factors we mined bio-informatically from numerous papers, we have built fine-tuned and well-characterized biosensors with low basal signal, large induction ratio and wide detection range. For example, XylR for BTEX (short for benzene, toluene, ethyl-benzene and xylene), XylS for benzoates, NahR for salicylates, DmpR for phenols, HbpR for biphenyls and HcaR for phenylpropionate. However, we noticed the detection profile of these biosensors is not very satisfactory. For example, transcriptional factors for aromatics containing functional groups like hydroxyl and aldehyde groups are not readily detected by existing biosensors that may function in E.coli, either for their poor performance^[1] or their unclear activation mechanism^[2].

To broaden the detection profile of this toolkit, we set out to seek for methods to artificially expand inducer range for transcriptional factors.

Previous researches that focused on broadening the detection profiles of biosensors were mainly based on the putative structure of the transcriptional factor and sequence homology analysis. For example, XylS protein with a R45T mutation could be additionally activated by benzoate with substituents at 2, 3 and 4 position of the aromatic ring such as 4-Methoxybenzoate (Fig. 1a) ^[3]. Victor de Lorenzo generated a library of engineered transcription factors constructed by shuffling homologous domains between DmpR and XylR. The shuffled TF protein can sense not only xylene but also phenols (Fig. 1b)^[4]. These methodologies helped to enrich the inducers of individual transcription regulators. However, alcohols and aldehydes are still not incorporated into the detection profiles of existing biosensors.

Therefore, new methods need to be developed to broaden the detection profiles of biosensors.

Because aromatic compounds with diverse structures can be inter-converted by enzymes in the catabolic network (Fig. 2), we reasoned such enzymes may help to expand the detection profiles of existing biosensors. We surveyed through a wide range of metabolic pathways, keeping an eye on their potency to broaden the range of inducers. Excitingly, degradation enzymes along the pathway serve our purpose well. Analogous to electronic adaptors that connect two otherwise incompatible devices, these enzymes could be used as “Adaptors for biosensors” to transform originally undetectable compounds into ligands that can be detected, thus incorporating the undetectable upstream compounds into the detection profile of the existing downstream aromatics-responsive transcription regulators (Fig. 2)!

In order to function function properly as “Adaptors for biosensors”, degradation enzymes need to be relatively small and well-studied. XylB (benzaldehyde dehydrogenase) and XylC (benzyl alcohol dehydrogenase) from the toluene degradation pathway and NahF (salicylaldehyde dehydrogenase) from the naphthalene catabolic pathway originated in Pseudomonas putida meet these requirements (Fig. 3). Using them as Adaptors, various benzyl alcohols, benzaldehydes and salicylaldehydes should be detected by the downstream regulator proteins, namely XylS and NahR (Fig. 3).

The xyl genes of Pseudomonas putida TOL plasmid that specify catabolism of toluene are organized in two units: the upper-operon xylUWCAMBN for conversion of toluene to benzoate and the meta-operon xylXYZLTEGFJQKIH encoding enzymes for further degradation into TCA cycle intermediates ^[5]. The transcription factor XylR, which detects toluene, initiates transcription of genes in upper-operon ^[9-10], including xylB and xylC (Fig. 4).

XylB is benzyl alcohol dehydrogenase (BADH) that degrades benzyl alcohol to benzaldehyde ^[7]. Previous work showed the xylB gene can be expressed in E.coli, but enzyme activity of the protein is much lower compared to that when expressed in Pseudomonas putida. It is speculated that inefficient transcription of foreign gene in E.coli ^[7] and different regulation system altered the expression intensity of xylB gene as well as the enzyme's activity. So in order to optimize XylB's performance, it is significant to tune the expression level of XylB through selecting appropriate Pc promoters.

XylC is benzaldehyde dehydrogenase (BZDH) degrading benzaldehyde to benzoate in the upper operon ^[7]. Apart from benzaldehyde, XylC also displays activity towards methyl, nitro, methoxy, and chloro substituents of benzaldehyde ^[4]. This enzyme has been successfully expressed in E.coli and its catalysis ability in vitro has been verified. However, the substrates of XylC will inhibit enzyme activity when present in milli-molar concentrations ^[4].

According to our work concerning XylS, we have demonstrated that benzaldehyde is originally an inducer of this biosensor, but the induction ratio is apparent low comparing to benzoate. We speculated that the combination of XylS with enzyme XylC would increase the biosensor’s response to benzaldehyde, and further combination with XylB would enable XylS to detect originally undetectable compound benzyl alcohol.

After obtaining correct sequence of XylB and XylC via PCR from TOL plasmid pWW0 in Pseudomonas putida, we constructed recombinant plasmids expressing XylC and XylB respectively, in which the coding sequence locates downstream Pc promoter (Fig. 5).

The performance of XylC Adaptor was tested in following protocol. Bacteria carrying XylC enzyme was overnight-cultured in LB containing kanamycin at 37℃ and then diluted 100 fold into Minimal M9 medium added kanamycin, growing for 12 hours at 30℃ to transform benzyladehydes into benzoates. After the Minimal M9 medium was centrifuged, supernatant medium was taken out and added ampicillin to further culture XylS biosensor. Induction ratio was then obtained following test protocol 1.

The flow cytometry test confirmed our hypothesis: the induction effect of 3-methyl-benzaldehyde and 3-chlorobenzaldehyde towards XylS biosensor equipped with Adaptor XylC ameliorates evidently comparing to biosensor XylS only (Fig. 6a). Furthermore, the dose-response curves of XylS equipped with or without Adaptor XylC for 3-methyl-benzaldehyde (Fig. 6b) showed that the Adaptor XylC could reduce the detection limit (the concentration of inducer at which an output three times the basal single is generated) of 3-methyl-benzaldehyde from 100 μM to 1 μM. Likewise, the detection limit of 3-Chloro-benzaldehyde was also decreased more than 1 order (Fig. 6c). In addition, dose-response curves for the corresponding acids 3-methyl-benzoate and 3-chlorobenzoate were also collected (Fig. 6d). The almost coincident lines for 3-Methyl-benzoate and 3-chloro-benzoate showed that adding Adaptor XylC does not largely influence the original characteristics of the biosensor.

Further equipped with XylB, the biosensor might obtain the ability to detect benzyl alcohol which cannot be detected directly by neither biosensor XylS nor XylS equipped with XylC. The experiments are undergoing.

NahF is a 50.8 KDa protein functioning as salicylaldehyde dehydrogenase to transform salicylaldehyde into salicylic acid (salicylate) using NAD+ (Fig. 7). It is encoded in the naphthalene degradation plasmid from Pseudomonas putida, in which the bacterial oxidation of naphthalene has been extensively investigated. Plasmid pDTG1, NAH7 and pND6-1 identified in different P. putida strains all act to degrade naphthalene and share high identity in amino acid sequences ^[11].

NahF from plasmid NAH7 is mostly widely studied. It has a wide range of substrates, including salicylaldehyde, 5-chloro-salicylaldehyde, 3-nitro-benzaldehyde, 2-methoxy-benzaldehyde etc. and can be activated to 140.3% enzyme activity in the presence of Fe2+ ^[12]. The wide scope of substrates makes it a commendable candidate to be an Adaptor since many aldehydes can be transformed to the corresponding acids that are detected by NahR biosensor (for salicylates) or biosensor XylS (for benzoates).

NahF has been expressed in E.coli and its ability to catalyze the reaction in vitro and in vivo has been proved ^[13-14]. However, the reaction efficiency of E.coli was only about 3% of that of P. putida possibly due to the difference of expression regulation in these two bacteria ^[13]. Therefore, it is necessary to fine-tune the expression level of NahF in E.coli. We built a library of constitutive promoters for tuning the expression of NahF, and NahR biosensor was used to detect the possible salicylates transformed from salicylaldehydes (Fig. 8).

The performance of NahF Adaptor was tested in the protocol similar to that of XylC. Bacteria carrying NahF enzyme was overnight-cultured in LB containing chloromycetin at 37℃ and then diluted 100 fold into Minimal M9 medium added chloromycetin, growing for 12 hours at 30℃ to transform salicylaldehyde into salicylate. After the Minimal M9 medium was centrifuged, supernatant medium was taken out to further culture NahR biosensor. Induction ratio was then obtained following test protocol 1.

The comparison of induction ratio (calculated as previously mentioned) of NahR biosensor equipped with or without the Adaptor NahF showed that NahF highly improved the induction performance of biosensor NahR elicited by salicylaldehyde and 5-chloro-salicylaldehyde (Fig. 9a). Dose response curves of biosensor NahR equipped with Adaptor NahF exposed to salicylaldehydes of different concentration were also obtained (Fig. 9b). Comparing it with the dose-response curve of biosensor NahR for salicylaldehydes in the same test protocol (Fig. 9b,c), we demonstrated that Adaptor NahF also functioned to lower the detection limit (the concentration of inducer at which an output three times the basal single is generated) of salicylaldehyde from 300 μM to 1 μM (Fig. 9b) and 5-chloro-salicylaldehyde from 300 μM to 3 μM (Fig. 9c). Dose response curves of biosensor NahR for corresponding acids processed by Adaptor NahF were obtained as well, showing the orthogonality of biosensor NahR and biosensor NahR equipped with Adaptor NahF (Fig. 9d).

In summary, Adaptors XylC and NahF functioned not only to optimize the response to several originally detectable compounds, but also expand detection profile in a new way besides modification on coding sequence such as mutative selection or DNA shuffling. The advantages of the new concept that using Adaptor to expand detection profile lie in that adding enzymes does not influence the original characteristics of the biosensors they adapt to, and this methodology does not require labor-intensive mutant screening work.

Figure 1. Previous researches about broadening the detection profile of biosensors and their improvements. (a) Point mutation: The mutant thr45 XylS aromatics-response transcription regulator showed much higher induction ratios than that of the Wild-type (WT) XylR. (b) DNA shuffling: Shuffled N-terminal domains of the homologous regulators DmpR and XylR generated a transcriptional regulator that responds to both benzyl hydrocarbons and phenols.

Figure 2. Typical metabolic pathway from prokaryotes. Aromatics inside the green box could already been detected respectively by the aromatics-response transcription regulator written beside their structure formula. Compounds marked in the orange boxes are those lacking corresponding aromatics-response proteins. All these compounds are integrated by metabolite enzymes (marked in red box located near the arrows that connect two intermediates), which inspired us to employ metabolic enzymes as the “Adaptors for biosensors” that transform originally undetectable upstream intermediates into the detectable downstream ligands of regulator proteins, making the upstream intermediates detectable.

Figure 3. Schematic diagram for Adaptors based on metabolic pathways. Transcriptional factors (short for TF) XylS and NahR function to sense different acids. With Adaptors like XylC and NahF transforming aldehydes to acids, aldehydes can be detected. Likewise, with Adaptor XylB transforming alcohols to aldehydes, alcohols can also be detected.

Figure 4. Xylene degradation metabolite operon in TOL plasmid and the catabolic pathway from benzyl alcohol to benzoic acid. (a) Relationship of gene cluster and regulatory protein in the TOL plasmid: XylR controls Pu promoter and Ps2 promoter. Ps1 is a weak constitutive promoter, which controls the level of XylS. When xylene or its homologous compounds exist, the upper pathway and another promoter called XylS was activated. (b) Biochemical reaction catalyzed by enzyme XylB and XylC: Benzyl alcohol is transformed into benzaldehyde by XylB and then benzaldehyde is converted to benzoic acid via the catalysis of XylC.

Figure 5. Schematic diagrams for the plasmid circuits used as Adaptor: XylB, XylC and the Sensor: XylS. A library of constitutive promoters is being constructed for XylB. XylC is expressed under constitutive promoter BBa_J23114. These parts are used as Adaptors. XylS biosensor circuit is shown in the bottom. Promoters are presented in orange, RBS in light green, coding sequence in dark cyan, and terminators in dark red.

Figure 6. Data plots that demonstrate the performance of Apator XylC.(a) Fluorescence intensity of XylS and XylS equipped with Adaptor XylC elicited by 3-MeBAD and 3-ClBAD at the concentration of 1 mM. XylS with Adaptor XylC showed higher fluorescence intensity and induction ratio than XylS. (b) Dose-response curves of XylS and XylS equipped with XylC for 3-Methyl-benzaldehyde. Use of Adaptor XylC significantly reduced the detection limit by 100 folds. (c) Dose-response curves of XylS and XylS equipped with XylC for 3-Chloro-benzaldehyde. (d) Dose-response curves of XylS and XylS equipped with XylC for 3-Methyl-benzoate and 3-chlorobenzoate. The coincident curves for the two compounds showed that the Adaptor XylC did not interfere the performance of XylS biosensor.

Figure 7. Biochemical reaction catalyzed by enzyme NahF: Salicylaldehyde is transformed into salicylic acid (salicylate) accompanied by the reduction of NAD+ to NADH.

Figure 8. Schematic diagrams for the plasmid circuits used as Adaptor: NahF and the Sensor: NahR. A constitutive promoter library for the expression of NahF was constructed to obtain the most appropriate expression level of NahF enzyme in E.coli. The number of the Standard Biological constitutive promoter Parts used in this study and its initiation strength is listed in the left portion of the figure. Promoters are presented in orange, RBS in light green, coding sequence in dark cyan, and terminators in dark red.

Figure 9. Data plots that demonstrate the performance of Apator NahF. (a) Induction ratio of biosensor NahR and NahR equipped with Adaptor NahF elicited by SaD at the concentration of 1 mM and 5-ClSaD at the concentration of 0.1 mM. Biosensor NahR with Adaptor NahF showed higher induction ratio than NahR. (b) Dose-response curves of Biosensor NahR and NahR equipped with NahF for salicylaldehyde. Use of Adaptor NahF significantly reduced the detection limit by more than 100 folds. (c) Dose-response curves of Biosensor NahR and NahR equipped with NahF for 5-Chloro-salicylaldehyde. (d) Dose-response curves of Biosensor NahR and NahR equipped with NahF for salicylate and 5-chloro-salicylate. The coincident curves for the two compounds showed that the Adaptor NahF did not interfere the performance of NahR biosensor.

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