| Content Provider | Springer Nature : BioMed Central |
|---|---|
| Author | Passolunghi, Simone Riboldi, Luca Dato, Laura Porro, Danilo Branduardi, Paola |
| Abstract | Background Zygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a β-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS 1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS 1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion. Results The GAS 1 homologue of Z. bailii was cloned by PCR, and when expressed in a S. cerevisiae GAS 1 null mutant was able to restore the parental phenotype. The respective Z. bailii Δgas 1 deleted strain was obtained by targeted deletion of both alleles of the ZbGAS 1 gene with deletion cassettes having flanking regions of ~400 bp. The morphological and physiological characterization of the Z. bailii null mutant resulted very similar to that of the corresponding S. cerevisiae mutant. As for S. cerevisiae, in the Z. bailii Δgas 1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the Candida rugosa lipase CRL1 and of the Yarrowia lipolytica protease XPR2, while for human IL-1β secretion disruption had no relevant effect. Conclusions The data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that Z. bailii is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as already widely reported in literature, our data confirmed that an \"always working\" solution to the problems related to recombinant protein production can be hardly, if never, found; instead, manipulations have to be finely tuned for each specific product and/or combination of host cell and product. |
| Related Links | https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/1475-2859-9-7.pdf |
| Ending Page | 11 |
| Page Count | 11 |
| Starting Page | 1 |
| File Format | HTM / HTML |
| ISSN | 14752859 |
| DOI | 10.1186/1475-2859-9-7 |
| Journal | Microbial Cell Factories |
| Issue Number | 1 |
| Volume Number | 9 |
| Language | English |
| Publisher | BioMed Central |
| Publisher Date | 2010-01-26 |
| Access Restriction | Open |
| Subject Keyword | Applied Microbiology Biotechnology Microbiology Microbial Genetics and Genomics Enzymology Genetic Engineering Lipase Wild Type Strain Glucan Shake Flask Heterologous Protein |
| Content Type | Text |
| Resource Type | Article |
| Subject | Applied Microbiology and Biotechnology Bioengineering Biotechnology |
| Journal Impact Factor | 4.3/2023 |
| 5-Year Journal Impact Factor | 5.5/2023 |
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