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TorA-opdA

This is an improved part of opdA (BBa_K215090)

This part is a coding sequence (opdA) with a TorA signal peptide fused to its N-terminus for protein export to periplasmic space.

Biology

opdA encodes organophosphate hydrolase (OPH) which is a homodimeric organophosphate triesterase that requires metal ion as a cofactor to degrade a wide range of toxic organophosphates. OPH can hydrolyze various phosphorus-ester bonds including P-O, P-F, P-CN, and P-S bonds. Recombinant E. coli expressing OPH can degrade a variety of organophosphate, however, due to low protein solubility, OPH production is at low yields. Also, as a gram-negative bacteria, E. coli. Cell membrane can be a substrate diffusion barrier affecting whole cell biocatalytic efficiency.

TorA is an E. coli twin-arginine signal peptide bearing a consensus motif of SRRxFLK. The twin-arginine system a bacterial protein export pathway. Tat signal pepetides consist of three domains: a positively charged N-terminal domain, a hydrophobic domain, and a C-terminal domain. In E. coli, the Tat translocase consists of TatA, TatB, TatC proteins. TatBC is a signal peptide recognition complex, while TatA complex forms a channel for protein translocation across the cytoplasmic membrane. To express functional OPH molecules into the periplamic space, a twin-arginine signal peptide of E. coli trimethylamine N-oxide (TMAO) reductase (TorA), was added to the N-terminal of the opdA. TMAO reductase is a periplasmic enzyme that catalyzes reduction of TMAO to trimethylamine, and functions as a component of the anaerobic respiratory chain which provides energy for bacterial cell growth .

Design

In our project, to enable secretion of OPH (gene product of opdA) to the periplasm of E. coli for the development of live cell biocatalysts, the TorA signal peptide followed by four amino acid residues of the mature TorA protein is fused directly to the N-terminal of OPH domain. TorA signal peptide contains a twin-arginine motif of ‘SRRxFLA’, and a recognition site for type I signal peptidases (Figure 1).

Figure 1: diagram of TorA-opdA fusion design

TorA-opdA is bp in length. Figure 2 shows a colony PCR amplifying a section of TorA-opdA in pLEV1(408) vector.

                                       Figure 2: The agarose gel electrophoresis of TorA-opdA colony PCR product

Improvement:

This is an improved part of opdA (BBa_K215090), which has been codon optimized for E. coli chassis. By fusing a TorA signal peptide directly to the OPH domain, functional OPH has been exported to periplasmic space, and making the whole live cell ‘biocatalyst’. In nature, microorganisms generally evolve the ability of extracellular secretion of functional enzymes. Given that OPH is a degrading enzyme of pesticide, we believe that our improvement helps with using engineered bacteria for direct pesticide degradation in the field.

Reference

Alami, M.; Luke, I.; Deitermann, S.; Eisner, G.; Koch, H. G.; Brunner, J. & Muller, M. 2003. Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli. Mol. Cell, 12, 937–946. Kang, D. G., Lim, G-B. & Cha, H. J, 2005. Functional periplasmic secretion of organophosphorous hydrolase using the twin-arginine translocation pathway in Escherichia coli. Journal of Biotechnology, 118, 379-385. Lee, P. A.; Tullman-Ercek, D. & Georgiou, G. 2006. The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60, 373–395. Mulbry, W.W., Karns, J.S., 1989. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene andp rotein. J. Bacteriol. 171, 6740–6746.

Sequence and Features