Effect Of Mitophagy Related Gene Regulation On Antioxidant Activities Of Larger Yeast

Oxidative stress laid on yeast cells limits mitochondrial function,cell vitality and productivity in the serial beer brewing.Mitophagy is a process that cells selectively remove mitochondria via the autophagy pathway.It is crucial for preserving cellular homeostasis.The relationship between mitophagy and antioxidant activities of yeast cells is unclear.Previous research demonstrated that mitophagy significantly affected the antioxidant activities of brewer’s yeast.In this study,lager yeast Pilsner was used to study the impact of mitophagy-related genes’regulations on the antioxidant performance of yeast.Current work provides a new clue for investigating the antioxidant mechanism and strategies for enhancing the antioxidant performance of brewer’s yeast.The main findings are as follows:1.The mitophagy related genes were regulated in lager yeast Pilsner,and recombinant strains were successfully constructed.It was found that DNM1,ATG8,ATG11,ATG32 and MMM1 genes significantly affected on the antioxidant performance of yeast during beer brewing.2.Modification of mitophagy genes had a significant impact on the physiological performance of yeast under oxidative stress.Herein,overexpression of ATG8 and ATG11 genes significantly reduced the intracellular ROS contents of recombinant strains and helped to maintain high mitochondrial vitality.The ROS contents of P-O-ATG8 and P-O-ATG11 were52.05%and 22.57%of the initial state after 6 h stimulation of H₂O₂.On the other hand,strains with ATG11 and ATG32 overexpression showed higher ATP change rates than that of the original strain,which were 100.25%and 97.16%after 6 h stimulation of H₂O₂.Disruption of ATG11 and ATG32 caused the decrease of ATP,which were only 29.80%and 23.46%（6 h）of the initial state.3.Serial beer fermentation experiment was performed using the parental strain Pilsner and recombinant strains to evaluate the antioxidant activities of lager yeast during beer brewing.Strains with ATG8,ATG11 and ATG32 genes’disruption showed the imbalance of intracellular ROS homeostasis at the end of G5 fermentation.The ROS change rates of Pil-atg8Δ,Pil-atg11Δ,Pil-atg32Δwere 207.22%,320.91%and 529.80%（G5/G1）respectively,which were significantly higher than that of the original strain.On the contrary,strains with ATG8,ATG11and ATG32 genes overexpression could maintain intracellular ROS homeostasis during serial beer fermentation.The ROS change rates of P-O-ATG8,P-O-ATG11 and P-O-ATG32 were108.55%,141.77%and 99.08%（G5/G1）respectively.Similarly,Pil-atg8Δ,Pil-atg11Δand Pil-atg32Δdid not maintain the mitochondrial activity and intracellular energy metabolism during serial beer fermentation.4.Double disruption of ATG8 and ATG32 genes resulted in the severe imbalance of intracellular ROS homeostasis.The intracellular ROS content of Pil-atg8-32Δincreased to140.92%（6 h）of the initial state under oxidative stress;The intracellular ROS content of Pil-atg8-32Δsignificantly increased to 570.00%of that of G1 generation after G5 fermentation.Moreover,disruption of both ATG8 and ATG32 genes showed negative effects on mitochondrial activity and intracellular energy metabolism of yeast both under oxidative stress and during the serial beer fermentation.5.In order to expand the universality of the study,ATG8,ATG11 and ATG32 genes were modified in a model strain W303.The results were consistent with those obtained using lager yeast Pilsner as the host strain.