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.