Effect Of Different Carbon Source On Cell Metabolism And Autophagy In Pichia Pastoris

Based on an effective AOX1(alcohol oxidase 1)promoter,which induced by methanol,Pichia pastoris is developed one of the most commonly used eukaryotic expression systems.However,the growth of the cell in methanol(short for Me OH)is obviously slower than in other carbon resource like glycerol.And glycerol can inhibit methanol metabolism.This paper aims to analyzed the mechanism of methanol metabolism regulation of P.pastoris and optimize the metabolism of methanol in Pichia pastoris.There was also a strong link between autophagy and cell growth,so we want to know how carbon resource influence autophagy.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 methanol autophagy associated gene and protein was increased,but the expression of pexophagy associated gene and protein was decreased.In methanol,the level of macroautophagy was higher,which resulted that cells grew slowly;the pexophagy was inhibited,which increased the activity of AOX1.Previous study found that knockout of Glycerol transporter 1(GT1)in P.Pastoris can make its AOX1 express under glycerol.We compared the change of autophagy of P.Pastoris with GT1 knockout(short for ?GT1)and wild-type.It was found that the change of the autophagy and pexophagy caused by different carbon resource was smaller in ?GT1,which was conform to the property of carbon source suppression was released in ?GT1.It was further demonstrated that the autophagy was one of the main way that carbon sources changed the cell growth and metabolism.This paper provided a new direction and theoretical basis to improve utilization of methanol and expression of heterogeneous proteins from the perspective of cell autophagy.On that basis,we constructed mutants in which pexophagy is depressed through Atg30 knock-out,Pex1 over-expression or Pex6 over-expression and it was observed that the activity of AOX1 in mutants increased,especially the mutant with three gene changed.Its AOX1 activity increased 1.8 times more than AOX1 activity in wild-type,after grew in methanol medium 24 hours.And its AOX1 activity increased 2.5 times more than AOX1 activity in wild-type,with GT1 knockout.Our study of the effects of different carbon sources on the growth,metabolism and autophagy of Pichia pastoris,is expected to have a broad application prospect in heterologous proteins industrial production.We developed a mutant strain with high expression of PAOX1,which effectively increased the production of exogenous protein and optimized the yeast expression system.