Abstract
BackgroundSaccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies.FindingsIn order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications.ConclusionsThe developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications.
Highlights
Saccharomyces cerevisiae is extensively used in bio-industries
The developed protocol enables by-passing of many of the difficulties associated with Polymerase chain reaction (PCR) caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells
It has the potential to be extended to other yeast strains presenting cell wall structure modifications
Summary
Saccharomyces cerevisiae is extensively used in bio-industries. Humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies. With the progress in genetic manipulations, microorganisms’ engineering for research or industrial bio-compound production is very common. In 2009, Saccharomyces cerevisiae strains were engineered to obtain different flavonoids for use in high throughput screening of molecules in drugs discovery [10]. Saccharomyces cerevisiae is a efficient and attractive system for the bioproduction of therapeutic proteins. Large scale production allows high yields, secretion of post-translationally modified proteins and simplified downstream purification protocols. Productions of other human proteins are under active studies: α-amylase [11], HPV16IL [12], immunoglobulin G [13]
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