High-level expression of an Aspergillus niger endo-beta-1,4-glucanase in Pichia pastoris through gene codon optimization and synthesis.

Abstract

To improve the expression efficiency of recombinant endo-beta-1,4-glucanase in P. pastoris, the endo-beta-1,4-glucanase (egI) gene from Aspergillus niger was synthesized using optimized codons. Fourteen pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egI gene was generated by two-step PCR-based DNA synthesis. In the synthesized endo-beta-1,4-glucanase gene syn-egI, 193 nucleotides were changed, and the G+C content was decreased from 54% to 44.2%. The syn-egI gene was inserted into pPIC9K and transformed into P. pastoris GS115 by electroporation. The enzyme activity of recombinant P. pastoris stain 2-7# reached 20.3 U/ml with 1% barley beta-glucan and 3.3 U/ml with 1% carboxymethylcellulose (CMC) as substrate in shake flasks versus 1270.3 U/ml and 220.7 U/ml for the same substrates in 50 l fermentors. The molecular mass of the recombinant protein was approximately 40 kDa as determined by SDS-PAGE analysis, the optimal temperature for recombinant enzyme activity was 70 degrees C and the optimal pH was 5.0 when CMC was used as the substrate.