XylB and XylC Inspiration and intention Build the Adaptors!

Based on the transcriptional factors bioinformatically mined from the database, we have built a large collection of well-characterized biosensors with low basal level and high induction ratio, including 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 that the detection profiles of these biosensors can be further expanded. For example, aromatics containing functional groups like hydroxyl and aldehyde groups often can not be readily detected by our biosensors, probably due to their poor performance in the host E.coli ^[1] or the unknown activation mechanism ^[2].

To broaden the detection profile of our toolkit, we set out to seek for possible solutions.

Previous studies that aimed to expand the detection profiles of biosensors were mainly protein engineering based on the sequence homology. For example, XylS protein with a R45T mutation could be activated by benzoate with substitutions at 2, 3 and 4 position of the aromatic ring, such as 4-methoxybenzoate (Fig. 1a) ^[3]. Victor de Lorenzo et al. generated a library of engineered transcription factors by shuffling homologous domains between DmpR and XylR. The product can sense not only xylene but also phenols (Fig. 1b) ^[4].

Therefore, new methods need to be developed for our goal. Because aromatic compounds can be inter-converted by the enzymes in the natural metabolic network, we reasoned that such enzymes may help to expand the detection profiles of existing biosensors. We analyzed the data of metabolic pathways provided by (KEGG: Kyoto Encyclopedia of Genes and Genomes), focusing on the potency of enzymes to that catalyze specific reaction to convert undetectable compounds into detectable compounds.

Excitingly, we found degradation enzymes can could serve our purpose well (Fig. 2). Analogous to the electronic adaptors that functionally connect two incompatible devices, these enzymes could be used as “Adaptors for biosensors circuits” to transform originally undetectable compounds into the aromatics that can be detected (Fig. 3).

To function properly as “Adaptors for biosensors”, the degradation enzymes need to be relatively biochemically 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 are expected to be detected by the downstream regulator proteins, XylS and NahR (Fig. 3).

The xyl gene cluster that specify the catabolism of toluene are organized in two units: the upper-operon xylUWCAMBN for the conversion of toluene into 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 the upper-operon ^[9-10], including xylB and xylC (Fig. 4).

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

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].

In our previous work on XylS, we found that benzaldehyde can indeed induce its response, but the induction ratio is apparent low comparing to its native inducer: 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 an originally undetectable compound benzyl alcohol.

After obtaining correct sequence of XylB and XylC via PCR from TOL plasmid pWW0 in Pseudomonas putida, we put XylC and XylB under the control of Pc promoters and expressed them on different plasmids (Fig. 5).

The performance of XylC Adaptor was tested in following protocol. Bacteria expressing XylC protein was cultured overnight in LB medium at 37 °C and then diluted 100 fold into Minimal M9 medium, growed for 12 hours at 30 °C to transform benzyladehydes into benzoates. The culture was then centrifuged, and supernatant was taken out to further culture XylS biosensor. Induction ratio was then measured by Flow Cytometry.

The Flow Cytometry test confirmed our hypothesis: the induction effect of 3-methyl-benzaldehyde and 3-chlorobenzaldehyde on XylS combined with adaptor XylC improved significantly comparing to biosensor XylS only (Fig. 6a). Furthermore, the dose-response curves for 3-methyl-benzaldehyde of adaptor-equipped XylS and unequipped XylC (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. Similarly, the detection limit of 3-chloro-benzaldehyde was decreased more than 1 order of magnitude (Fig. 6c). In addition, dose-response curves for 3-methyl-benzoate and 3-chlorobenzoate were also collected (Fig. 6d). The almost identical lines for 3-methyl-benzoate and 3-chloro-benzoate showed that adding Adaptor XylC does not significantly influence the original characteristics of the biosensor.

When further equipped with XylB, the biosensor was expected to obtain the ability to detect benzyl alcohol and its derivatives which cannot be detected directly by either biosensor XylS or XylS equipped with XylC.

Figure 1. Previous efforts to broaden the detection profiles of biosensors. (a)Mutagenesis: The XylS mutant (thr45), aromatics-responsive transcription regulator, showed much higher induction ratios than that of the wild-type. The inducers are not the natural for the wild-type XylS. (b) DNA shuffling: Shuffling N-terminal domains of the homologous regulators DmpR and XylR produced a transcriptional regulator that responds to both benzyl hydrocarbons and phenols.

Figure 2. The schematic diagram for the metabolic pathways concerning XylS and NahR. Aromatics boxed in green can be detected by our biosensor toolkit. Compounds boxed in the orange are those lacking accompanied aromatics-response proteins. All these compounds can be bridged by metabolite enzymes boxed in red. This inspired us to employ metabolic enzymes as the “Adaptors for biosensors” that transform originally undetectable upstream intermediates into detectable downstream aromatic compounds.

Figure 3. Schematic diagram for Adaptors in the context of metabolic pathways. Transcriptional factors (short for TF) XylS and NahR function to sense different aromatic 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 biosensor circuit and the Adaptor: XylB, XylC and XylS. A library of constitutive promoters was used to control XylB. XylC is expressed under constitutive promoter BBa_J23114. 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-methyl-benzaldehyde and 3-chloro-benzaldehyde 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-chloro-benzoate. The almost overlapping curves for the two compounds showed that the Adaptor XylC did not interfere the performance of XylS biosensor.