Comparison of constitutive and inducible β-fructofuranosidase production by recombinant Pichia pastoris in fed-batch culture using defined and semi-defined media

摘要

Short-chain fructooligosaccharides produced from sucrose by transfructosylation using β-fructofuranosidase (FFase), an industrially important enzyme, finds application in pre-biotics, sweeteners and confectionary products. Using recombinant Pichia pastoris , the influence of replacing the commonly-used Invitrogen ? medium with a semi-defined medium for FFase production under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) and alcohol oxidase (AOX) promoters was investigated. Replacing the trace metals (PTM 1 ) solution with yeast extract resulted in a 54.3% decrease in FFase volumetric activity under control of the AOX promoter, suggesting a distinct requirement for trace metals for recombinant protein synthesis during methanol induction, given that the biomass yield on methanol decreased by only 10%. The same medium adjustment had no effect on enzyme production under GAP promoter control, although AOX promoter control resulted in double the FFase volumetric activity compared to glycerol-fed cultures. Decreasing basal salts by half did not affect the cultures, but alleviated precipitation during sterilisation. Optimisation of the glycerol feed rate and dissolved oxygen tension in DO-stat fed-batch fermentations using the semi-defined medium resulted in 17% increase in volutmetric activity of FFase expressed under the GAP promoter. This study highlighted the influence of carbon source and trace metals on heterologous protein production by P. pastoris using constitutive and inducible promoters. Highlights ? Protein expression under AOX promoter was strongly influenced by trace salts. ? Glycerol-fed GAP strains may not require trace salts for protein production. ? The concentration of basal salts in the Invitrogen medium is over specified. ? Optimal glucose feed rate and DOT for the GAP strain were 40.3?g/h and 32.23%, respectively.