Influence of Long-Term Agar-Slant Preservation at 4 °C on the Recombinant Enzyme Activity of Engineered Yeast

Abstract

Strain preservation to maintain stable vitality and the recombinant enzyme activity plays a crucial role in industrial fermentation. A Pichia pastoris strain is routinely stored at −80 °C in a glycerol vial and activated on an antibiotic-containing YPD agar plate before being used for fermentation. Alternatively, the activated strain should be preserved in the agar slant at 2~4 °C (low-temperature storage) for a short period before use. To maximize the utilization of the low-temperature storage for fermentation, we evaluated this method by observing the capacity of both the vitality and the recombinant enzyme activity of the strain at different preservation durations. We found that engineered yeast could be preserved by low-temperature storage for at least 30 months without losing its vitality and biomass enzyme activity by the end of fermentation and could be directly used for the seed cultivation of fermentation, which is more time-saving than strain recovery from −80 °C in a glycerol vial. Moreover, the antibiotic added to the agar slant could be omitted if the heterologous gene was integrated into the host chromosome. Our approach may greatly elevate the production efficiency of the strain.