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.