Saccharomyces cerevisiae’s culture conditions for heterologous expression: A detailed investigation using reporter gene eGFP and Escherichia coli’s heat-labile toxin B subunit

Abstract

The Saccharomyces cerevisiae recombinant expression host 2805 has been employed extensively for various applications, ranging from recombinant enzyme production, oral vaccine development, and viral antigen production. For all of its previous applications, recombinant yeast cells have been cultured under a conventional three-stage protocol: after being revived and grown on selective agar plates, (i) cells are seeded in 5 mL Uracil deficient medium (–Ura medium) for two days; (ii) cells are diluted at a 1:8 ratio in the complete medium YEPD and cultured for 16 hours; (iii) flask culture in YEPD for 1 to 5 days at 30 °C. Despite its routine use, the rationale and performance of this culture regime have not been systematically examined. In this study, using a synthetic, yeast codon optimised gene encoding the B subunit of Escherichia coli heat-labile toxin (LTB) fused to the reporter enhanced Green Fluorescent Protein (eGFP), we employed flow cytometry to monitor the expression dynamics in the recombinant 2805 yeast strains across all cultural stages. The results show that LTB-eGFP expression peaked on day 1 of the first seeding in the –Ura medium, dropped on day 2, then increased again after being transferred to YEPD, and then declined steadily in flask culture from day 1 to day 5. Across conditions, the percentage of fluorescent positive cells was approximately 45–60%. These results indicate that the re-optimisation of the culture protocol is necessary in order to maximise the expression efficiency.