Tetracycline is a group of broad-spectrum antibiotic compounds with four hydrocarbon rings to form a stable structure which cannot be easily broken. Three major enzymes were identified from a large literature and a large amount of data. All of those enzymes belong to the reductase with strong reconversion, including lignin peroxidase, manganese peroxidase and laccase.

Lignin peroxidase belong to a glycosylated extracellular protein peroxidase secreted by fungi containing hematoxylin. The glycosylated heme protein of LiP is usually produced in a variety of complex forms, that is, it contains many isoenzymes.

Fig.1 The dimensional structure model of lignin peroxidase[3]

LiP can oxidate phenol type which are rich in electronic or non phenolic aromatic compounds, since the electron transport system can attack substrates. After that, it can take an electron from phenol or benzene rings of phenols to oxide subsrances into free radicals, which in turn to produce many different kinds of free radical chain reactions, which would lead to a series of pyrolysis reactions. [1].

Manganese peroxidase (MnP) is the most common lignin-modifying peroxidase produced by almost all wood-colonizing basidiomycetes causing white-rot and various soil-colonizing litter-decomposing fungi[2].

Fig.2 The dimensional structure model of Manganese peroxidase[3].

Laccases are copper-containing oxidase enzymes require oxygen as a second substrate for their enzymatic action. Laccase, Lip and Mnp comprise ligninolytic enzymes secreted by white rot fungi.

We have constructed those three enzymes with permutation combination method into a total seven gene circuits scheme in the below:

Fig.3 Degradation parts with Lip

Fig.4 Degradation parts with Mnp

Fig.5 Degradation parts with Laccase

Fig.6 Degradation parts with Lip and Mnp

Fig.7 Degradation parts with Lip and Laccase

Fig.8 Degradation parts with Mnp and Laccase

Fig.9 Degradation parts with Lip、Mnp and Laccase

In the process of experimental design, safety issues are always our concern. In order to avoid the problem of gene leakage of engineered bacteria, we introduced suicide genes, a lysis protein phiX174E, into the degradation chassis. The gene phiX174E is controlled by the lac promoter, and induced by IPTG.

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References:

1. Chopra, I.; Roberts, M. (1 June 2001). "Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance". Microbiology and Molecular Biology Reviews. 65 (2): 232–260. doi:10.1128/MMBR.65.2.232-260.2001

2. Hofrichter,M.(16 April 2002).”Review: lignin conversion by manganese peroxidase (MnP)”. Enzyme and Microbial Technology. Volume 30, Issue 4, Pages 454-466 doi.org/10.1016/S0141-0229(01)00528-2

3. Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R., Schwede, T. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46(W1), W296-W303 (2018).