High-level expression of a truncated 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation.
1,3-1,4-beta-D-glucanase is an important endoglycosidase in the brewing and animal feed industries. To achieve high-level expression of recombinant glucanase in Pichia pastoris, we designed sequences encoding the alpha-factor signal peptide from Saccharomyces cerevisiae and the truncated 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes as a whole. The codons encoding the 52 amino acids of the signal peptide and 106 residues of the glucanase protein were optimized for expression in P. pastoris; 189 nucleotides were changed. The G + C content was adjusted to 48-49%, and AT-rich stretches were eliminated to avoid premature termination. The messenger ribonucleic acid secondary structure near the AUG start codon was also adjusted to ensure efficient translation; the resulting glucanase production was twofold higher compared with that achieved with gene structure optimization alone. We also propose a new fermentation strategy for the induction phase, in which 5/95% glycerol/methanol mixed feed was used in days 1-3 and 100% methanol was used on days 4-6. By comparison with methanol feed and glycerol/methanol-mixed feed alone, the yield of recombinant glucanase increased by 38.5 and 16.5%, respectively. The expressed optimized recombinant 1,3-1,4-beta-D-glucanase constituted approximately 90% of the total secreted protein, reaching up to 3 g l(-1) in the medium.
Huang
Huoqing
H
Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences, no. 12 Zhongguancun South Street, Beijing, 100081, People's Republic of China.
Yang
Peilong
P
Luo
Huiying
H
Tang
Huigui
H
Shao
Na
N
Yuan
Tiezheng
T
Wang
Yaru
Y
Bai
Yingguo
Y
Yao
Bin
B
D001482:Base Composition / Q000235:genetics; D001483:Base Sequence; D018533:Biomass; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D045848:Fibrobacter / Q000201:enzymology* / Q000235:genetics*; D005990:Glycerol / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics*; D000432:Methanol / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D013997:Time Factors
D003470:Culture Media; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006026:Glycoside Hydrolases; C028020:licheninase; D005990:Glycerol; D000432:Methanol