Team:BJEA China/Engineering



Our project idea focuses on one of the most common plastics, polyethylene terephthalate (PET). We inspired by the mutant LCC (mLCC) that has published on nature recently which can hydrolyze 90% of PET in plastic bottles into its monomers - ethylene glycol and terephthalic acid in just 10 hours. Although the recycling of plastic bottles plays an important role in environmental protection, but the fact is with only 9% of PET material being recycled. Based on the fact, we decide to enhance the activity of mLCC by proceeding two approaches, which are constructing a fusion protein of mLCC and hydrophobins and using the technique of Bacillus subtilis surface display.

1. Create a fusion protein by jointing mLCC with hydrophobins.

Why use Hydrophobin?

Hydrophobin is an amphiphilic protein. Therefore, by linking mLCC and hydrophobin to form a fusion protein, the adsorption of mLCC on PET can be increased.
We chose 3 types of hydrophobins. The first is BslA[1], which is a hydrophobin derived from bacteria. The other two are mHGFI and mHFBI. HFBI[2] and HGFI[3] are hydrophobins derived from fungi, HGFI is a class I hydrophobin, and HFBI is a class II hydrophobin. In 2015, the team Tianjin mutated these two hydrophobins, and the resulting mHGFI (inJanus-m, BBa_K1582002) and mHFBI (sJanus-m, BBa_K1582003) can be expressed in bacteria. This greatly shortens the production time and reduces the difficulty of protein expression. Therefore, we chose to use these two parts submitted by the Tianjin team.
We decided to express the fusion protein in E. coli, because E. coli is a mature, low-cost model organism. We are not sure which kind of hydrophobin can succeed, so we tried 3 kinds of hydrophobins for fusion.
We choose pET28a as the expression vector, and its his-tag can be used for protein purification (using affinity chromatography). We designed 4 constructs, one mLCC(BBa_K3759000), three constructs of mLCC fused with BslA(BBa_K3759001), mHGFI or mHFBI with a GS linker [4].

(Figure 1. Plasmid design for jointing mLCC with hydrophobins)

How did we test it?


Because the pET28a vector has His-tag, therefore we used affinity chromatography to purify the supernatant obtained after bacteria clastogenesis using nickel. The result of our proteogels after elution with 200 mm imidazole and 300 mm imidazole is shown below. We successfully purified mLCC and mLCC-linker-BslA (BBa_K3759019) . At the same time, we can also see that the two proteins, mLCC-mHGFI, mLCC-mHFBI, are expressed, but the protein expression level is very low.


Figure.2. (a). M: marker;
Lane 1, 2: mLCC-linker-BslA 43kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 3, 4: mLCC 28kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 5, 6: mLCC-linker-mHFBI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 7, 8: mLCC-linker-mHGFI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;

After the successful purify of the enzymes, we had to test the activity of those. We used PET film (GoodFellow) as a substrate for the mLCC and its fusion protein with hydrophobin. After the incubation time, we tested the product TPA by testing 240nm UV value with nanodrop. The samples were incubated for 18 hours at 70°C.


Figure 3.
(a). The adsorption value of mLCC, mLCC-Linker-BslA of UV240 under the condition of pH8, 70 celsius degree, reaction time of 18h.

2. Construct Cell surface display of mLCC in Bacillus subtilis

Why use Bacillus subtilis?

mLCC is a thermostable enzyme, and its optimum temperature is 70°C. Therefore, we need to find a chassis organism that is also thermally stable to use as a whole-cell biocatalyst to degrade PET at 70 degrees. Bacillus subtilis as a chassis, it also has the advantages of a short reproduction cycle and a mature expression system.
Anchor protein is a surface-exposed coat component that has the purpose of displaying protein at the spore surface on the bacteria cells, function as an anchoring motif, support the attachment for the fused protein and chassis. Similarly, we are not sure which anchor protein will be successful, so we finally selected the four most studied anchor proteins by looking up references, namely CotB, CotC, CotG and CotX [5].
Therefore, our goal is to display mLCC on the surface of Bacillus subtilis to construct a whole-cell biocatalyst.
We choose pHT43 as the expression vector, which is a shuttle plasmid, and added flag-tag at the C-terminal. This can provide conditions for the use of fluorescence to detect the target protein after it is displayed on the cell surface. We designed 4 constructs of CotB, CotC, CotG and CotX fused with mLCC with a GS linker [6].

(Figure 4. Plasmid design for Cell surface display of mLCC in Bacillus subtilis)


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[2] J Vereman, Thysens T , Derdelinckx G , et al. Extraction and spray drying of Class Ⅱ hydrophobin HFBI produced by Trichoderma reesei[J]. Process Biochemistry, 2019, 77(FEB.):159-163.
[3] Song D , Wang X , Gao Z , et al. Expression, Purification and Characterization of Hydrophobin HGFI from Grifola Frondosa in Saccharomyces Cerevisiae[J]. Acta Scientiarum Naturalium Universitatis Nankaiensis, 2018.
[4] Malin G , Janne L , Stuart D , et al. Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris.[J]. Protein Eng, 2001(9):711-715.
[5] Xiao-Xia Y U , Tian J , Ning-Feng W U . Research Progress on Bacillus subtilis Spore Display of Recombinant Proteins[J]. Journal of Agricultural Science and Technology, 2013.
[6] Futatsumori-Sugai M , Abe R , Watanabe M , et al. Utilization of Arg-elution method for FLAG-tag based chromatography[J]. Protein Expression & Purification, 2009, 67(2):148-155.