The core of our project was to develop a whole-cell biocatalyst by displaying PETase and MHETase on the surface of yeast cells (Candida tropicalis). With careful consideration and effective improvement, we finally succeeded in achieving the purpose of our project step by step. We screened a promoter (BBa_K3829001: P-GAPDH), a terminator (BBa_K3829000: T-GAPDH), and a reporter (BBa_K3829002: yeGFP), which could effectively screen anchor proteins.
Part 1 Construction of surface display system
1.1 Background
Surface display technology can expose peptides or proteins on the cell surface, which is a powerful tool used in many fields such as medicine. It has been reported that the enzymes anchored on the cell surface have better activity and stability compared with free enzymes. Furthermore, the decrease of enzyme activity is not obvious after the completion of enzymatic recycling. The surface display system can also eliminate the step of enzyme purification, making industrial applications more feasible. Recently, some researchers have displayed PETase enzyme on the surface of pichia pastoris and its activity is 36 times as much as that of free enzyme, proving that PETase enzyme is suitable for functional display on yeast.
At present, cell surface display technology mainly focuses on some model strains and some specific anchor proteins. To expand the application value of the technology, non-model strain display platforms and more efficient anchor proteins should be developed.
1.2 Research
Chassis microorganism
Candida tropicalis is capable of absorbing alkanes and fatty acids as carbon sources and surviving in environments containing acids and phenolic substances. Compared with other surface display systems, the surface display technology on Candida tropicalis has not been fully developed. Therefore, it is worthy to establish the system on Candida tropicalis. In addition, Candida tropicalis is maturely used in the production of long-chain diacid, which lays a good foundation for its industrial applications.
Anchor protein
GPI-anchored proteins have three main characteristics: (1) N-terminal contains secretion signal peptide sequence (2) Mature protein has no transmembrane domain (3) C-terminal contains GPI signal peptide. Based on these characteristics, bioinformatics analysis was carried out to predict related anchoring proteins.
1.3 Imagine
We can build a surface display system for Candida tropicalis. BioBricks are promoters, reporters, and terminators. First, we need to ensure that reporter GFP can express fluorescence. Then GFP is fused with GPI-anchored proteins to reflect protein expression through fluorescence.
1.4 Design
According to the reported characteristics of GPI-anchored proteins, anchor protein candidates were screened from the Candida tropicalis genome using bioinformatics methods. Then, the yeast green fluorescent protein (yeGFP) was fused with the potential GPI-anchored protein for preliminary identification. Subsequently, the subcellular location was determined by immunofluorescence and Western bolt. Finally, GPI-anchored proteins that could be used to display proteins were screened and flow cytometry was used to evaluate the display efficiency of the screened GPI-anchored proteins.
Screening of anchored proteins
yeGFP (yeast enhanced green fluorescent protein) was detected to obtain the bacterial strain that displayed fluorescence on the outer side of the cell periplasm. Then immunofluorescence and Western Bolt were used to further screen the strains located in the cell wall.
1.5 Build
In order to determine that the protein can be successfully expressed, we have to select a promoter that can efficiently initiate transcription of downstream genes in Candida tropicalis. Therefore, so the promoter of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was chosen and the corresponding terminator was selected. To facilitate the display of protein expression, we chose GFP as the reporter. In addition, we have performed codon optimization (BBa_K3829002) for the existing part-reporter GFP (BBa_K3402050) to improve the expression efficiency in Candida tropicalis.
Fig.1 Composition of the basic Bio-bricks.
1.6 Test
The bacteria were collected, resuspend in buffer, and then observe with a confocal microscope. The results were shown in the figure 2. Compared with the negative control, that is, the wild strain (URA3-KO), the fluorescence of yeGFP (BBa_K3829002) in the positive control can be expressed.
Fig.2 Representative images of yeGFP expression.
1.7 Learn
It has been proved that yeGFP (BBa_K3829002) can be expressed intracellularly. On this basis, we fused a signal peptide (ss) to the N-terminus of yeGFP, so that downstream genes can be guided through the cell membrane to the periplasm.
Fig.3 Composition of the gene circuit after adding signal peptide (SS).
1.8 Improve
The ability of the new gene circuit to display the protein on the cell surface is also affected by different anchor proteins. Therefore, 129 anchor proteins were measured and merely 25 anchor proteins were found to work (Figure 4).
Fig.4 Representative images of 25 screened anchor proteins.
Then immunofluorescence was performed to verify that if the protein was successfully displayed outside the cell membrane. The results suggested that nine of the 25 anchor proteins worked well. The results of 4609 were shown in figure 5.
Fig.5 Representative images of 4609 (further screened anchor protein).
Part 2 PET Degradation
2.1 Research
In 2016, Japanese scientist Yoshida discovered the bacterium Ideonella sakaiensis 201-F6 from the sludge of a bottle recycling plant in Osaka., was able to decompose PET plastic. The bacterium could completely degrade the low-crystallinity PET film in 6 weeks under the reaction conditions of 30℃, which was currently known to be the best bacteria for PET degradation. PETase and MHETase were found to be the key enzymes. PETase was able to degrade polyethylene terephthalate (PET) into bis-hydroxyethyl terephthalate (BHET), mono-(2-hydroxyethyl) terephthalic acid (MHET) and terephthalic acid (TPA). MHETase was able to further degrade MHET into TPA and ethylene glycol (EG).
Fig.6 Enzymes and their products involved in PET degradation
2.2 Imagine
PETase and MHETase can be fused with GPI-anchored proteins with high surface display efficiency respectively. Afterwards, enzyme activity of the targeted proteins can be determined. Subsequently, the efficiency of the surface display system should be optimized, and the PET film can be used as the substrate to evaluate the ability of the dual-enzyme display system to degrade plastics in whole cells. Finally, Candida tropicalis should be further modified to convert the hydrolyzed substrates TPA and EG into high value-added products, such as protocatechuic acid (PCA) and glycolic acid (GLA) etc.
2.3 Design
We obtained the candidate anchor proteins through modeling analysis. The analysis steps were as follows. First, analyze the 10221 amino acid sequences of the genome (Candida tropicalis ATCC 20336). Then combined with the database, the sequences without secretion signal peptide and without GPI anchor signal were eliminated. Furthermore, proteins without transmembrane structure would be removed. Then, the sequences were further analyzed to obtain the targeted proteins. The anchor protein screening system was further designed to verify their functions. Finally, yeGFP would be replaced with PETase and MHETase.
2.4 Build
GPI-anchored proteins 5105 and 4609 were selected as the target proteins for surface display system. Then, the strains respectively displaying PETase and MHETase were constructed. The schematic diagram of gene expression boxes was shown in figure 7.
Fig.7 The composition of the genetic circuit after replacing yeGFP with PETase and MHETase
2.5 Test
After the targeted gene was successfully integrated into Candida tropicalis genome, we verified the gene function. Due to the advantages of the surface display system, we only need to add the same amount of cell suspension for measurement of enzyme activity instead of enzyme extraction. First, preliminary measurement of enzyme activity was conducted. The coefficient of determination was 0.9997, indicating that the standard curve was reliable and could be used to calculate the activity.
Fig.8 standard curve of p-Nitrophenol.
We determined the enzyme activity of the wild strain ATCC20336 (Negative control), which knocked out the URA3 gene, the strain expressing PETase intracellularly (cytPET), the strain displaying PETase 4609 (PET-4609), and the strain showing PETase 5105. (PET-5105). The catalytic activity of 4-Nitrophenyl acetate (CAS:830-03-5) at 30°C and 37°C was measured respectively. The results were shown in figure 9. Notes-The catalytic product was p-Nitrophenol, and the amount of p-Nitrophenol generated was calculated according to the standard curve.
Fig.9 Determination of enzyme activity at different temperatures.
2.6 Learn and Improve
In the experiment, the PET film used was 0.175mm which was too thick. It took time finish the degradation cycle and the effect was not detectable. Therefore, we started to try PET powder or thinner plastic film (0.0025mm) in our experiment. Finally, it was found that PET powder had a better effect.
References
Tanasupawat, Somboon; Takehana, Toshihiko; Yoshida, Shosuke; Hiraga, Kazumi; Oda, Kohei. ”Ideonella sakaiensis sp. nov., isolated from a microbial consortium that degrades poly(ethylene terephthalate).” International Journal of Systematic and Evolutionary Microbiology, August 2016, volume 66, issue 8, pp. 2813-2818, ISSN 1466-5034. PMID 27045688. doi:10.1099/ijsem.0.001058.
