3.4.2 Enhancing P. pastoris metabolic activities in the successive methanol induction phase
The major chemical reactions in heterogeneous protein by P. pastoris and relevant key enzymes involved have been widely reported previously.1,10 Functions of the key enzymes involved in methanol metabolism were as follows: AOX catalyzes the first chemical reaction in methanol metabolism (CH3OH+O2→HCHO+H2O2); FLD and FDH contribute to NADH and ATP regeneration ability in the formaldehyde dissimilation pathway and the role of this path was to supply a source of energy for heterogeneous proteins production. As shown in Fig.4II, when using the “high DO-low glycerol concentration” control strategy, specific activities of AOX, FLD and FDH were significantly repressed, leading to a limited methanol/oxygen consumption and hLYZ expression. As for the batch using the periodic DO control strategy, the specific activities of AOX, FLD and FDH were obviously enhanced. Accordingly, the average specific methanol consumption rate and OUR increased for about 40% with the periodic control strategy, which eventually led to an efficient hLYZ production.