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Team:Hong Kong CUHK/pah
From 2013.igem.org
PAH Degradation
T7-RBS-QsrR (Device BBa_K1092001)
Quinone sensing and response repressor (QsrR) is a novel kind of thiol-stress-sensing regulator YodB family transcriptional regulator found in the notorious pathogen Staphylococcus aureus. [2] It specifically binds to a palindromic DNA sequence, blocking RNA polymerase and thus repressing transcription. [2] However, with the presence of quinone, it reshapes to bind with quinone molecules and leaves the target DNA, which allows the transcription process to start. [2] Since laccase is used at first to degrade PAHs into quinones for the proposed pathway, the quinone-like compounds produced as intermediates can function as signaling molecules that induce the expression of other enzymes responsible for subsequent degradations. This method can save energy for the cell that expresses those PAH-degrading enzymes. The protein sequence of QsrR is readily available at GenBank, with the reference code 4HQE_A for side chain A, and 4HQE_B for side chain B.
QsrR Binding region:
5’ – GTATAN{5}TATAC – 3’ [2]
The redox-sensing regulator YodB family utilizes disulfide bond formation between cystine residuals for the transformation of repressor protein. [1][2] The presence of these repressors in some pathogen bacteria may relate to bacterial defense of quinone-like immune molecules. [2]
References:
[1] CHI, B.K., ALBRECHT, D., GRONAU, K., BECHER, D., HECKER, M. and ANTELMANN, H., 2010. The redox-sensing regulator YodB senses quinones and diamide via a thiol-disulfide switch in Bacillus subtilis. Proteomics, 10(17), pp. 3155-3164.
[2] JI, Q., ZHANG, L., JONES, M.B., SUN, F., DENG, X., LIANG, H., CHO, H., BRUGAROLAS, P., GAO, Y.N., PETERSON, S.N., LAN, L., BAE, T. and HE, C., 2013. Molecular mechanism of quinone signaling mediated through S-quinonization of a YodB family repressor QsrR. Proceedings of the National Academy of Sciences, 110(13), pp. 5010-5015.
T7-RBS-Laccase (Device BBa_K1092005)
The laccase (cotA) found in Bacillus sp. HR03 is a bacterial laccase that have the potential ability to degrade a variety of aromatic compounds. [3] Previous study showed that it can be successfully expressed in Escherichia coli strain BL-21(DE3), and was correctly folded. [4] Moreover, the complete gene sequence of this laccase was readily recorded in GenBank, with the reference code of FJ663050.1. It is thus used as one of the two major enzymes for PAH degradation.
Properties of the laccase:
Optimal temperature: ~70°C;
Optimal pH: ~7. [3]
Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) are a type of oxidative enzymes that show the ability of oxidizing a variety of compounds ranging from phenolic compounds to polycyclic aromatic hydrocarbons (PAHs). [1][2][3] They can be found both in bacteria and fungi, especially white-rot fungi that have the ability to degrade lignin, an important structural component of plants. [1][5] The experiment of Hadibarata et al. suggested that laccase could play a very significant role in benzo[a]pyrene degradation, a reaction that hardly occurs. [1] And the previous research done by Hadibarata et al. and Zeng et al. suggested that laccase degraded PAHs by adding oxygen atoms into the ring, forming quinone-like intermediates. [1][6] After such transformation, hydrophilic quinones can be further converted into other metabolites by other enzymes.
References:
[1] HADIBARATA, T. and KRISTANTI, R.A., 2012. Identification of metabolites from benzo[a]pyrene oxidation by ligninolytic enzymes of Polyporus sp. S133. Journal of environmental management, 111(0), pp. 115-119.
[2] HADIBARATA, T., TEH, Z., RUBIYATNO, ZUBIR, M., KHUDHAIR, A., YUSOFF, A., SALIM, M. and HIDAYAT, T., 2013. Identification of naphthalene metabolism by white rot fungus Pleurotus eryngii. Bioprocess and Biosystems Engineering, 36(10), pp. 1455-1461.
[3] MOHAMMADIAN, M., FATHI-ROUDSARI, M., MOLLANIA, N., BADOEI-DALFARD, A. and KHAJEH, K., 2010. Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization. Journal of industrial microbiology & biotechnology, 37(8), pp. 863-869.
[4] MOLLANIA, N., KHAJEH, K., RANJBAR, B., RASHNO, F., AKBARI, N. and FATHI-ROUDSARI, M., 2013. An efficient in vitro refolding of recombinant bacterial laccase in Escherichia coli. Enzyme and microbial technology, 52(6–7), pp. 325-330.
[5] SHARMA, P., GOEL, R. and CAPALASH, N., 2007. Bacterial laccases. World Journal of Microbiology and Biotechnology, 23(6), pp. 823-832.
[6] ZENG, J., LIN, X., ZHANG, J., ZHU, H., CHEN, H. and WONG, M., 2013. Successive transformation of benzo[a]pyrene by laccase of Trametes versicolor and pyrene-degrading Mycobacterium strains. Applied Microbiology and Biotechnology, 97(7), pp. 3183-3194.
FL3-Lgt-FL3 (Protein Domain BBa_K1092103)
Transmembrane protein Lgt with two flexible linkers FL3 This is a biobrick we adapted from SJTU 2012 team, BBa_K771102. Prolipoprotein diacylglyceryl transferase (Lgt) is an inner membrane protein of E.coli. Lgt serves as transmembrane domain in our project. To link upstream and downstream domains and enzymes with Lgt, we add a flexible linker FL3 on both sides of Lgt.
