Effects of codon optimization and glycosylation on the high-level production of hydroxynitrile lyase from Chamberlinius hualienensis in Pichia pastoris

Abstract

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.

R‐hydroxynitrile lyase from the cyanogenic millipede, Chamberlinius hualienensis—A new entry to the carrier protein family Lipocalines

“…S1B and S1C). The recombinant ChuaHNL has been also confirmed that it was glycosylated at the same glycosylation sites (at position N109 and N123) in ChuaHNLs produced in Pichia pastoris expression system [27]. Although several Ser and Thr residues in the ChuaHNL structure are exposed to the solvent and there is only one predicted O ‐glycosylation site [5], the electron density of O ‐linked glycosylation site was not observed.…”

“…To prove the catalytic roles of the hydrophilic residues that interact with ( R )‐MAN as indicated by the results of the docking simulation, site‐directed mutagenesis was performed using the recombinant His‐ChuaHNL generated in P. pastoris by coexpression with protein disulfide bond isomerase (PpPDI) as described in our previous report [27]. The hydrophilic residues surrounding ( R )‐MAN in the docking model (R38, Y103, Y40, D56, and K117) were altered (Fig.…”

“…The site‐directed mutagenesis was performed using a Quick‐Change Lightning Site‐Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). The primer pairs are listed in Table S1 and pPICZαA‐His‐ori‐ChuaHNL vector [27] was used as template. The PCR products were treated with Dpn I (TaKaRa) at 37 ˚C for 1 h and then transformed to E. coli DH5α strain and selected on a low‐salt LB agar plate with Zeocin (25 µg·mL −1 ).…”

“…The mutant plasmids were isolated by a plasmid miniprep kit (Sigma‐Aldrich), and the amino acid exchanges were confirmed by DNA sequencing analysis. The constructed vector pPICZαA‐His‐ori‐ChuaHNL mutant was then linearized by digesting with Sac I and electroporated into P. pastoris PpPDI/GS115, according to our previous report [27].…”

“…Escherichia coli DH5a (Takara Bio, Otsu, Japan) was inoculated in Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) containing zeocin (25 µgÁmL À1 ) for plasmid propagation. P. pastoris cells harboring PpPDI/GS115 was used as host for the recombinant His-ChuaHNL expression as described previously [27]. The yeast strain was precultured in a buffered minimal glycerol (BMG) medium (1.34 % yeast nitrogen base without amino acids, 4 9 10 -5 % biotin, 100 mM potassium phosphate buffer (pH 7.0), and 1 % glycerol) and then the yeast cells were transferred to induction BMM medium (BMG in which 1 % glycerol was replaced by 1 % methanol).…”