Effects of codon optimization and glycosylation on the high-level production of hydroxynitrile lyase from Chamberlinius hualienensis in Pichia pastoris
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A hydroxynitrile lyase (HNL) from the millipede Chamberlinius hualienensis has high potential for industrial use in the synthesis of cyanohydrins. However, obtaining sufficient amounts of millipedes is difficult, and the production of the Chamberlinius hualienensis HNL (ChuaHNL) in E. coli has not been very successful. Therefore, we investigated the conditions required for high-yield heterologous production of this enzyme using Pichia pastoris. When we employed P. pastoris to express His-ChuaHNL, the yield was very low (22.6 ± 3.8 U/L culture). Hence, we investigated the effects of ChuaHNL codon optimization and the co-production of two protein disulfide isomerases (PDIs) [from P. pastoris (PpPDI) and C. hualienensis (ChuaPDI1, ChuaPDI2)] on His-ChuaHNL production. The productivity of His-ChuaHNL was increased approximately 140 times per unit culture to 3170 ± 144.7 U/L by the co-expression of codon-optimized ChuaHNL and PpPDI. Moreover, we revealed that the N-glycosylation on ChuaHNL had a large effect on the stability, enzyme secretion, and catalytic properties of ChuaHNL in P. pastoris. This study demonstrates an economical and efficient approach for the production of HNL, and the data show that glycosylation has a large effect on the enzyme properties and the P. pastoris expression system.
KeywordsHydroxynitrile lyase Chamberlinius hualienensis Protein disulfide isomerase Pichia pastoris Codon optimization Deglycosylation
We thank Dr. Yuko Ishida for analyzing the RNA sequence of C. hualienensis. This work was supported by ERATO (Exploratory Research for Advanced Technology Program), Asano Active Enzyme Molecule Project of Japan Science and Technology Agency (Grant No. JPMJER1102). This research was also supported in part by a grant-in-aid for Scientific Research (S) from The Japan Society for Promotion of Sciences (Grant No. 17H06169) to Y. Asano. The financial support given to A. Nuylert from Ministry of Education, Culture, Sport, Science and Technology (MEXT) of Japan is deeply appreciated.
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