1 INTRODUCTION
Heterologous protein production by methanotrophic yeast P. pastoris is basically divided into two discrete and successive phases: (1) a cell growth phase to achieve high cell-density cultivation using glycerol as the sole carbon source; (2) a successive methanol induction phase to initiate the targeted recombinant protein production by feeding methanol.1-3 In general, achievement of high cell-density cultivation at the end of cell growth phase (beginning of the methanol induction phase) is acknowledged as the precondition for efficient heterologous protein production in the successive methanol induction phase. Therefore, different online and offline glycerol feeding control strategies (DO-stat feeding, constant feeding or exponential feeding etc.) were carried out to achieve a higher cell concentration at the end of cell growth phase.4,5Among these control strategies, the online DO-stat glycerol feeding strategy, namely the environment of “high DO-low glycerol concentration” (DO 20-80%, glycerol concentration 0-1 g L-1), have been considered as the most simple and effective method to achieve high cell-density cultivation within short time.5 However, it was well known that severe reactive oxygen species (ROS) accumulation occurred when cells were exposed to the oxygen-enriched (high DO) environments for long time.6,7 In this case, the structural and functional cell components were obviously damaged, leading to the loss of cell growth ability or even cell death. As a result, in human lysozyme (hLYZ) production by the aerobic P. pastoris , severe ROS accumulation must be managed when using the traditional DO-stat glycerol feeding strategy.
In adaptive laboratory evolution, the increased toxicity due to environmental stresses could be effectively ameliorated by implementing a recovery treatment after environmental stimulation.8In this study, a similar control strategy, namely periodic DO control strategy, was developed to repress the severe ROS accumulation under the oxygen-enriched environments by alternatively switching the cultivation environment from “high DO-low glycerol concentration” (DO-stat control mode) to “low DO-high glycerol concentration” for 4 cycles., aiming to achieve a higher cell concentration at the end of cell growth phase and further enhance hLYZ production in the successive methanol induction phase.