Codon optimization of Bacillus licheniformis beta-1,3-1,4-glucanase gene and its expression in Pichia pastoris.
Beta-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive industry. To improve expression efficiency of recombinant beta-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding beta-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously increased from 43.6 to 45.5%. At shaking flask level, beta-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml(-1) with barley beta-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration is approximately 250 mg l(-1). The beta-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml(-1) with barley beta-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed. Compared to the nonoptimized control, expression level of the optimized beta-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal acidity and temperature of this recombinant enzyme were pH 6.0 and 45 degrees C, respectively.
Teng
Da
D
Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
Fan
Ying
Y
Yang
Ya-Lin
YL
Tian
Zi-Gang
ZG
Luo
Jin
J
Wang
Jian-Hua
JH
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics*; D008969:Molecular Sequence Data; D010843:Pichia; D017422:Sequence Analysis, DNA
D003062:Codon; D006026:Glycoside Hydrolases; C028020:licheninase