Part:BBa_K3829012
P-ss-PETase-V5tag-Anchor protein 4609-T
The composite Part is used to express PETase. SS secretes PETase out of the cell, while anchor protein anchor 4609 anchor it on the cell surface. This part is based onBBa_K3829010, replacing GFP with PETase.
Characterization
Anchor proteins 4609 was screened by model prediction and yeGFP characterization. We then used it for the following experiment. On the basis of BBa_K3829010, we replaced PETase with yeGFP to obtain the composite parts BBa_K3829012.
Fig.1 The structure of the gene circuit.
The overall enzyme activity of PETase was measured. Since PETase could also catalyze substrates into p-nitrophenol, a crude test was carried out to show the enzyme activity at different temperatures. The results showed that PET-4609 and 5105 performed better than the wild type ATCC20336 and cytPET (Figure 2).
Fig.2 Determination of enzyme activity of PETase.
It was acknowledged that PETase was able to degrade PET into TPA and MHET. According to the HPLC detection results, the product content of the degraded PET powder of each strain were plotted (Figure 3). It was demonstrated that target products were not detected in the group of ATCC 20336. Compared with the control strain, degradation products were obviously presented in the strain PET-4609 and PET-5105, indicating PETase was displayed on the surface of Candida tropicalis with high enzyme activity. However, several products were detected in the strain cytPET which should not be detected theoretically. It was speculated that a part of PETase was released due to the lysis of cells.
In conclusion, these results demonstrated that the surface display system was indeed able to degrade PET, which was consistent with the previous results.
Fig.3 Hydrolysate content of PET powder. Content of PET powder: 10 mg, thallus: OD=5, reaction system: 1 mL, reaction time: 18 h.
References
1.Eisenhaber, Birgit, et al. "A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe." Journal of molecular biology 337.2 (2004): 243-253.
2.Möller, Steffen, Michael DR Croning, and Rolf Apweiler. "Evaluation of methods for the prediction of membrane spanning regions." Bioinformatics 17.7 (2001): 646-653.
3.Smith MR, Khera E, Wen F. “Engineering Novel and Improved Biocatalysts by Cell Surface Display.” Ind Eng Chem Res, volume 53, issue 16, 29 April 2015, pp. 4021-4032.
4.Tanaka T, Yamada R, Ogino C, Kondo A. “Recent Developments in Yeast Cell Surface Display toward Extended Applications in Biotechnology.” Appl Microbiol Biotechnol, volume 75, issue 3, August 2012, pp. 577-591.
5.Andreu C, Del Olmo ML. “Yeast Arming Systems: pros and cons of different protein anchors and other elements required for display.” Appl Microbiol Biotechnol, volume 102, issue 6, Mar 2018, pp. 2543-2561.
Sequence and Features| None |