Implication of the adaptive laboratory evolution method to improve recombinant Pichia pastoris productivity

INTRODUCTION: Adaptive laboratory evolution (ALE) is a common methods in biological studies for improving growth and production of different cells in a long term duration and under the specified conditions. OBJECTIVE: The aim of present study was to investigate the adaptive laboratory evolution of Pichia Pastoris yeast cells, cultured in medium containing 1% methanol, in order to enhance the expression of recombinant human vascular endothelial growth factor (VEGF) protein.MATERIALS AND METHODS: The X33 strain of yeast Pichia pastoris containing human VEGF gene under the control of alcohol oxidase 1 (AOX1) promoter, was used. The generation time of yeast cells in a complex (YPM) and defined (FM22) media, and with methanol as the carbon source, was calculated. Four repeat for each medium were cultured for 490 and 230 generations in complex and defined media, containing 1% methanol, respectively. The efficiency of final cells for production of recombinant VEGF was measured using ELISA.RESULTS: The generation time for Pichia pastoris cells in YPM and FM22 was 3.4 and 7.3 h/generation respectively. In compared with the prototype, the obtained clones after 490 generation in YPM showed no significant differences in growth and VEGF production. While, the production of recombinant VEGF increased 1.3 fold in one of the clones grown in the FM22 medium.

کلیدواژه ها:

Adaptive laboratory evolution ، Pichia pastoris ، VEGF ، generation time