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). 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.

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 detection specificity of biosensor. ^[1]

MhpR is a 31,767 Da (281 amino acids) protein. It belongs to IclR family, which forms helix-turn-helix motif at N-terminal. MhpR behaves as an activator to initiate the expression of mhp cluster when contacts with its ligands, 3-hydroxyphenylpropionate (3-HPPA), 3-hydoxycinnamate (3-HCnA) and 3-(2, 3-dihydroxyphenyl) propionic acid (2,3-DHPPA). ^[2]

hca and mhp clusters are involved in the catabolism of PPA and CnA in E. coli (Fig.1). The enzymes encoded by hca cluster degrade PPA and CnA to 2,3-DHPPA and 2,3-DHCnA respectively, which serve as the substrates of the mhp cluster. The enzymes in mhp cluster function in the cleavage of aromatic ring.

Compared with the sole 2,3-DHPPA, the special induction effect of PPA and 2,3-DHPPA is obtained, although PPA don’t behave as ligand alone. Based on the result and the observation of different binding site of PPA with MhpR, it is deduced that PPA and 2,3-DHPPA have synergistic effect to the activation of MhpR expression ^[3]. (That is to say, PPA enhances the activation effect as a cooperator of 2,3-DHPPA instead of a ligand.) The same effect is observed in 3-HPPA along with PPA.

The synergistic effect seems to be explained by pre-activation mechanism. It is that 2,3-DHPPA is a product of PPA degradation by hca cluster, and it will accumulate before activating the expression of the downstream mhp cluster. 2,3-DHPPA has cytotoxicity to the bacteria. The pre-activation mechanism activates the downstream cluster at low ligand concentration so that bacteria consume it to prevent accumulation of toxicity. The mechanism reflects the precise control across several pathways in bacteria, and also contributes to the sensor application ^[3].

Based on the information, our team constructed the Ph/HcaR expression system. The coding sequence of HcaR was obtained from the genome of E. coli K12 via PCR. Constitutive Pc promoters are used to initiate the expression of hcaR on pSB4K5, and sfGFP, as a reporter gene, is under the control of Ph, the cognate promoter of HcaR.

We also created a Pc library to obtain the optical performance of this expression system which gets the best induction ratio. 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

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.