Team:WHU-China/Introduction

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Introduction

• 1 Dehalogenase
• 2 Cobalamin B12
• 3 Bacillus megaterium
• 4 2,4,6- trichlorophenol
• 5 Membrane bioreactor
• 6 Surface display system

Dehalogenase

    Reductive dehalogenases are enzymes which are characterized by the ability of dehalogenation with electron donor.

    They originated from organohalide respiring bacteria and contribute to the global halide cycle. These enzymes are usually membrane-associated, oxygen-sensitive and cobalamin (B12) dependent[1].

    Published in Nature in 2015, a paper succeeded in heterogeneously expressing and characterizing a novel reductive dehalogenase, RdhANP, soluble and oxygen-tolerant in vitro. As the authors have tested, RdhANP catalyzes the reduction of ortho-halogenated phenolic compounds using NADPH under aerobic conditions[2].

Cobalamin B12

    Cobalamin or vitamin B12, is an essential vitamin that is widely used in medical and food industries.

    B12 production in B. megaterium has served as a model system for biotechnology. Some usually used engineering strategies including overexpression and up- or down-regulation of the genes associated.

    In B. megaterium, B12 is synthesized via a branch of the modified tetrapyrrole pathway, with other branches leading to the production of heme and siroheme[3].

Bacillus megaterium

    First being described over 100 years ago, Bacillus megaterium has been increasingly gaining popularity in the field of biotechnology over the recent years. It has several remarkable advantages compared to two commonly used heterogeneous expression hosts, E. coli and B. subtilis.

    To begin with, unlike E. coli, B. megaterium is stained Gram's positive and doesn't contain endotoxins in its cell wall. Then, in the light protein expression, it has been reported that B. megaterium doesn't produce alkaline proteases such as those in B. subtilis. Protein stabilities can be remained in B. megaterium even up to 5h after induction[4].

    Having been found in many ecological niches, such as waste from petrochemical effluents and honey, B. megaterium is able to grow on a wide variety of carbon sources. Its degradation of persistent insecticides was also documented in 1987 and 1989, suggesting its potential application as detoxifying agent.

    What's more, industrial application of B. megaterium for vitamin B12 biosynthesis is an excellent example for development of a production strain using synthetic biological method[3][4]. There're also many other advantages of B. megaterium to make it an employable candidate for biological studies.

2,4,6- trichlorophenol

    2,4,6-trichlorophenol is a halogenated organic, phenol-structured and chlorinated in ortho- and para-position of the hydroxyl group.

    It has been used as fungicide, herbicide, insecticide, antiseptic and glue preservation.

    United States Environmental Protection Agency (EPA) has classified it as a Group B2, probable human carcinogen[5].

Membrane bioreactor

    Membrane bioreactor (MBR) is a membrane process combined with the activated sludge process. It has many advantages over conventional process and is now widely used in industrial and municipal wastewater treatment.

    In brief, the overall MBR process can be described as microbial fermentation to treat pollutants and microfiltration or ultrafiltration to prevent large particles and microorganisms.

    On that basis, hybrid membrane bioreactor (HMBR) was developed by introducing biofilm carriers into a conventional MBR and thus increased the total biomass. This not only improves the efficiency of biodegradation but may also contribute to the removal of small biological filaments and colloidal matter.

Surface display system[6]

    Surface display is a method to present target proteins on cell surfaces by membrane anchoring domain.

    Take BclA protein for example. It's derived from Bacillus anthracis (34F2) that covers the spores of B. anthracis. Although totally being 21 amino acid long, BclA is known to carry passenger proteins up to 120kDa.When expressed on the outer membrane of bacteria, its N-terminal domain faces intracellularly and C-terminal domain faces extracellularly, with internal repeating domain linking two terminals in between.

    If we coexpress cellulose binding domain (CBD) and BclA in our target host, Bacillus megaterium, it's proposed that B. megaterium should bind to cellulose membrane by CBD and carry out other designed biological functions.

References

[1] Jugder B-E, Ertan H, Bohl S, Lee M, Marquis CP and Manefield M (2016) Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation. Front. Microbiol. 7:249. doi: 10.3389/fmicb.2016.00249

[2] Karl AP Payne, Carolina P Quezada Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation Nature 2015 January 22; 517(7535): 513–516. doi:10.1038/nature13901

[3] Fang et al. Microb Cell Fact (2017) 16:15 DOI 10.1186/s12934-017-0631-y

[4] Mo Bi Tec. Bacillus megaterium Protein Expression System Product Information and Instructions October 2004

[5] U.S. Environmental Protection Agency. Integrated Risk Information System (IRIS) on 2,4,6-Trichlorophenol. National Center for Environmental Assessment, Office of Research and Development, Washington, DC. 1999.

[6]https://2016.igem.org/Team:Kyoto/Description#surface display