HcaR is a 32,838 Da (296 amino acids) protein regulator mined from Escherichia coli. The gene cluster regulated by HcaR is hca operon (for 3-phenylpropionic acid and cinnamic acid), encoding enzymes that degrade PPA and CnA to 2, 3-DHPPA and 2, 3-DHCnA, respectively (Fig. 1a).

HcaR belongs to LysR family. Its N-terminal domain functions in DNA binding via a helix-turn-helix motif, while the C-terminal domain functions in multimerization. As an activator, HcaR activates the expression of hca cluster in the presence of aromatic effectors.

The cognate promoter of HcaR, ph, is quite regular: it is σ⁷⁰-dependent and functions via contacting the α-unit of RNAP. The presence of aromatic effectors will cause the HcaR to dimerize and to bind to sequence-specific DNA operator in the ph promoter (Fig. 1b).

According to these properties of HcaR, we could design an HcaR biosensor that is supposed to detect 3-phenylpropionic acid, cinnamic acid and their derivatives. It aromatics-sensing profile is quite narrow, supposed to be 3-phenylpropionic acid (PPA) and cinnamic acid (CnA) only, thus to guarantee the detection specificity of the biosensor.

Based on the design frame of biosensors we've discussed in the Biosensor Introduction section, we constructed a HcaR biosensor using Ph/HcaR pair obtained from the genome of E. coli strain K12. The constitutive promoter (Pc) to control the expression of HcaR is BBa_J23106 and the RBS preceding sfGFP is BBa_B0034.

This primary design, however, did not work. Therefore, we used a library of combinations of Pc promoters and RBS sequences to fine-tune the performance of HcaR biosensor. The library consists of a series of Pc promoters with different expression intensity, including BBa_J23113, J23109, J23114 and J23106. Primary test following protocol 1 showed that HcaR performed best under the control of BBa_J23106. Then the best performed expression system is subjected to the On-Off test about 78 aromatics according to protocol 1. Results showed that HcaR worked as a specific sensor to PPA (Fig.2).

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Figure 1. The degradation pathway of PPA and CnA. The enzymes that catalyze each step of the pathway are also indicated; they are encoded by the hca gene cluster. PPA and CnA will finally be degraded into 2,3-DHPPA and 2,3-DHCnA, respectively.

Figure 2. Results of On-Off test about biosensor HcaR. HcaR specifically responds to PPA (1000 μM) with the induction ratio over 10, which is consistent with the paper's result.

Figure 3. RBS and Pc constitutive promoter library for HcaR biosensor. X-axis stands for different construction of biosensor HcaR. Y-axis denotes induction ratios. The HcaR biosensor with J23106 is a strong constitutive promoter, and B0031 is a weak RBS, and this construction performed well, which showed the induction ratio higher than 25 folds.

Figure 4. Dose-response curve of HcaR biosensor responding to PPA. X-axis stands for concentration gradient of inducers at 10µM, 30µM, 100µM, 300µM and 1000µM. Y-axis denotes induction ratios.

Reference:
[1] Díaz, E., Ferrández, A., & García, J. L. (1998). Characterization of the hca Cluster Encoding the Dioxygenolytic Pathway for Initial Catabolism of 3-Phenylpropionic Acid in Escherichia coliK-12. Journal of bacteriology, 180(11), 2915-2923.
[2] Ferrández, A., García, J. L., & Díaz, E. (1997). Genetic characterization and expression in heterologous hosts of the 3-(3-hydroxyphenyl) propionate catabolic pathway of Escherichia coli K-12. Journal of bacteriology, 179(8), 2573-2581.
[3] Manso, I., Torres, B., Andreu, J. M., Menéndez, M., Rivas, G., Alfonso, C., ... & Galán, B. (2009). 3-Hydroxyphenylpropionate and phenylpropionate are synergistic activators of the MhpR transcriptional regulator from Escherichia coli. Journal of Biological Chemistry, 284(32), 21218-21228.