High-level expression of an Aspergillus niger endo-beta-1,4-glucanase in Pichia pastoris through gene codon optimization and synthesis.
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.
Shumiao
Zhao
Z
State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China.
Huang
Jun
J
Zhang
Changyi
C
Deng
Ling
L
Hu
Nan
N
Liang
Yunxiang
Y
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D003062:Codon; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D005285:Fermentation; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D014170:Transformation, Genetic; D047071:beta-Glucans / Q000378:metabolism
D003062:Codon; D011994:Recombinant Proteins; D047071:beta-Glucans; C064197:beta-1,6-glucan; D004247:DNA; D002480:Cellulase; C019826:carboxymethylcellulase