Hypersecretory production of glucose oxidase in Pichia pastoris through combinatorial engineering of protein properties, synthesis, and secretion

Abstract Glucose oxidase (EC 1.1.3.4, GOD) is a widely used industrial enzyme. To construct a GOD‐hyperproducing Pichia pastoris strain, combinatorial strategies have been applied to improve GOD activity, synthesis, and secretion. First, wild‐type GOD was subjected to saturation mutagenesis to obtain an improved variant, MGOD1 (V20W/T30S), with 1.7‐fold higher k cat /K M . Subsequently, efficient signal peptides were screened, and the copy number of MGOD1 was optimized to generate a high‐producing strain, 8GM1, containing eight copies of AOX1 promoter‐ GAS1 signal peptide‐ MGOD1 expression cassette. Finally, the vesicle trafficking of 8GM1 was engineered to obtain the hyperproducing strain G1EeSe co‐expressing the trafficking components EES and SEC. 22 , and the EES gene (PAS_chr3_0685) was found to facilitate both protein secretion and production for the first time. Using these strategies, GOD secretion was enhanced 65.2‐fold. In the 5‐L bioreactor, conventional fed‐batch fermentation without any process optimization resulted in up to 7223.0 U/mL extracellular GOD activity (3.3‐fold higher than the highest level reported to date), with almost only GOD in the fermentation supernatant at a protein concentration of 30.7 g/L. Therefore, a GOD hyperproducing strain for industrial applications was developed, and this successful case can provide a valuable reference for the construction of high‐producing strains for other industrial enzymes.