Abstract: Pichia pastoris has become one of the most popular eukaryotic expression systems for its strong promoter of alcohol oxidase Ⅰ (AOX1), PAOX1. However, the growth of the cell in methanol (short for MeOH) is obviously slower compared to glycerol. And glycerol can inhibit methanol metabolism. In order to clarify the mechanism how carbon sources affect yeast growth and metabolism, the effects of different carbon sources on Pichia pastoris were compared. All experimental results showed that the change of metabolic substrate and cell growth were involved in autophagy. When the carbon source was changed to methanol, the expression of Atg8 was up-regulated, while Atg30 was down-regulated. In methanol, higher level of macroautophagy resulted in slower cells growth, inhibited pexophagy and increased activity of AOX1. The change of autophagy in Glycerol transporter 1 knockout Pichia pastoris (short for P. pastoris ΔGT1) was compared with that in wild-type and found that the change of the expression level of Atg8 and Atg30 was smaller in P. pastoris ΔGT1, which conformed to the property that carbon source suppression was released in P. pastoris ΔGT1. It was further demonstrated that the autophagy was one of the main ways that carbon sources changed the cell growth and metabolism. This paper further analyzed the mechanism of methanol metabolism regulation of Pichia pastoris from the perspective of cell autophagy, and provided a new direction and theory to improve utilization of methanol and expression of heterogeneous proteins.

Key words: Pichia pastoris, growth and metabolism, pexophagy, macroautophagy, glycerol transporter