PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

Abstract

Background: Saccharomyces 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.

Findings: In 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.

Conclusions: The 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.

Figure 1

Efficiency of MNN5 gene amplification from wild type and modified strains. A) Percentage of colonies picked from Petri dishes that amplified MNN5. The experiment was carried out on 47 colonies for each strain. Dark grey: BY4742; light grey: YiMMOgène. Amplification was carried out using the DreamTaq or the Platinium Taq. B). PCR product visualisation, after amplification with the Platinium Taq, on a 1% agarose gel stained with SYBR safe. Left panel (1): BY4742, right panel (2): YiMMOgène, ML: Molecular ladder.

Figure 2

Lyticase treatment of the YiMMOgène colonies can improve PCR efficiency. A) OD₈₀₀ measurement during lyticase treatment, 2 independent assays. OD₈₀₀ was measured on an Ultrospec 2100pro (Amersham). B) Percentage of MNN5 amplification from 20 colonies picked on Petri dishes treated or untreated with lyticase for 2 h.

Figure 3

Efficiency of MNN5 gene amplification from liquid culture of modified strain. A) Percentage of MNN5 amplification from 47 colonies picked on Petri dishes and 47 colonies picked from liquid cultures in micro-plates. B) Growth curve in micro-plate (YPD medium), average of two colonies of YiMMOgène. The OD₆₀₀ was measured in a micro-plate reader. ♦: OD₆₀₀ after 6 hour culture (early exponential phase). ●: OD₆₀₀ after 17 hour culture (late exponential phase). ▄: OD₆₀₀ after 24 hour culture (early stationary phase). C) Percentage amplification of MNN5 from 47 YiMMOgène after different culture times in micro-plate. All amplifications have been carried out with the Platinium Taq.

Figure 4

MNN5 gene amplification efficiency on modified strain colonies picked from Petri dishes or micro-plate cultures. A) 3 independent assays carried out on 47 colonies for each condition. Dark grey: Petri dishes cultures, light grey: micro-plate cultures. B) Average of the 3 assays presented in A (standard deviation =22.07). C) PCR product visualised on a 1% agarose gel stained with SYBR safe. Left panel (1): Petri dishes, right panel (2): micro-plate cultures, ML: Molecular ladder. All amplifications were carried out using Platinium Taq.

Figure 5

MNN2 gene amplification efficiency on modified strain colonies picked from Petri dishes or micro-plate cultures. A) 3 independent assays carried out on 23 colonies for each condition. Dark grey: Petri dishes cultures, light grey: liquid cultures. B) Average of the 3 assays presented in A (standard deviation=19.55). C) PCR product visualized on a 1% agarose gel stained with SYBR safe. Left panel (1): Petri dishes, right panel (2): micro-plate cultures, ML: Molecular ladder. All amplifications have been carried out with the Platinium Taq.