High-level expression of l-glutamate oxidase in Pichia pastoris using multi-copy expression strains and high cell density cultivation.
l-glutamate oxidase (GLOD), encoded by the gox gene, catalyses the transformation of l-glutamic acid into α-ketoglutaric acid (α-KG). In the present study, Pichia pastoris was used for heterologous production of GLOD following optimization of the gox coding sequence for expression in the yeast host. A series of constructs based on the pHBM905BDM plasmid were engineered and transformed into P. pastoris to increase the gox copy number. The results indicated that GLOD protein levels and enzyme activity increased with increasing gox copy number. Strain PGLOD4, which contained four copies of the target gene, was chosen for subsequent fermentation experiments, and a fermentation strategy involving two exponential feeding phases was developed. During the preinduction phase, glycerol was fed exponentially at μG= 0.15/h. When the cell density reached 300 g/l, methanol was fed exponentially at μM= 0.03/h to induce GLOD production. After 84 h of cultivation, the final cell density and total enzyme activity reached 420 g/L and 247.8 U/mL, respectively. The recombinant enzyme displayed an optimum temperature of 40 °C, which was higher than recombinant enzyme expressed in E. coli. This is important because increasing the temperature could accelerate enzymatic transformation of l-glutamic acid to α-KG. Experiments also demonstrated superior thermo-stability for the enzyme produced in yeast, which further enhances its potential for industrial applications.
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province 430062, People's Republic of China.
Wang
W
YaPing
National Biopesticide Engineering Research Center, Hubei Academy of Agricultural, Wuhan, Hubei Province 430064, People's Republic of China.
Rao
R
Ben
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province 430062, People's Republic of China.
Yan
Y
Hong
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province 430062, People's Republic of China.
Han
H
Rui
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province 430062, People's Republic of China.
Li
L
Li
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province 430062, People's Republic of China.
Liao
L
Ping'an
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province 430062, People's Republic of China. Electronic address: malixing98@163.com.
Ma
M
Lixin
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018628:Gene Dosage; D015870:Gene Expression; D010843:Pichia / Q000235:genetics / Q000254:growth & development*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C023061:L-glutamate oxidase
Copy number; Exponential feeding; Fermentation; Pichia pastoris; l-glutamate oxidase