Abstract
BackgroundThermotolerant yeast has outstanding potential in industrial applications. Komagataella phaffii (Pichia pastoris) is a common cell factory for industrial production of heterologous proteins.ResultsHerein, we obtained a thermotolerant K. phaffii mutant G14 by mutagenesis and adaptive evolution. G14 exhibited oxidative and thermal stress cross-tolerance and high heterologous protein production efficiency. The reactive oxygen species (ROS) level and lipid peroxidation in G14 were reduced compared to the parent. Oxidative stress response (OSR) and heat shock response (HSR) are two major responses to thermal stress, but the activation of them was different in G14 and its parent. Compared with the parent, G14 acquired the better performance owing to its stronger OSR. Peroxisomes, as the main cellular site for cellular ROS generation and detoxification, had larger volume in G14 than the parent. And, the peroxisomal catalase activity and expression level in G14 was also higher than that of the parent. Excitingly, the gene knockdown of CAT encoding peroxisomal catalase by dCas9 severely reduced the oxidative and thermal stress cross-tolerance of G14. These results suggested that the augmented OSR was responsible for the oxidative and thermal stress cross-tolerance of G14. Nevertheless, OSR was not strong enough to protect the parent from thermal stress, even when HSR was initiated. Therefore, the parent cannot recover, thereby inducing the autophagy pathway and resulting in severe cell death.ConclusionsOur findings indicate the importance of peroxisome and the significance of redox balance in thermotolerance of yeasts.
Highlights
IntroductionKomagataella phaffii (Pichia pastoris) is a common cell factory for industrial production of heterologous proteins
Thermotolerant yeast has outstanding potential in industrial applications
Yeasts with 5–10 °C higher growth temperature than their parents are labelled as thermotolerant yeasts [28]
Summary
Komagataella phaffii (Pichia pastoris) is a common cell factory for industrial production of heterologous proteins. Komagataella phaffii (formally known as Pichia pastoris) is a widely used platform for heterologous protein productions and has become a potential choice for metabolic engineering as well [1]. Cross-talk of protective mechanisms occurs in yeasts when responding to various environmental stresses. The functional role of transcription factors (TFs) in cells is clear, at least 11 stressresponsive TFs change simultaneously under oxidative or thermal stress in S. cerevisiae [9]. Thermal stress simultaneously induces HSR and OSR under an aerobic environment [11, 12]. HSR and OSR have been substantially explored, their ordinated regulation mechanism is poorly understood. The cross-talk of protective mechanism still needs to be further explored
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