Enhanced production of alpha-galactosyl epitopes by metabolically engineered Pichia pastoris.
A metabolically engineered Pichia pastoris strain was constructed that harbored three heterologous enzymes: an S11E mutated sucrose synthase from Vigna radiata, a truncated UDP-glucose C4 epimerase from Saccharomyces cerevisiae, and a truncated bovine alpha-1,3-galactosyltransferase. Each gene has its own methanol-inducible alcohol oxidase 1 promoter and transcription terminator on the chromosomal DNA of P. pastoris strain GS115. The proteins were coexpressed intracellularly under the induction of methanol. After permeabilization, the whole P. pastoris cells were used to synthesize alpha-galactosyl (alpha-Gal) trisaccharide (Galalpha1,3Galbeta1,4Glc) with in situ regeneration of UDP-galactose. Up to 28 mM alpha-Gal was accumulated in a 200-ml reaction. The Pichia system described here is simple and flexible. This work demonstrates that recombinant P. pastoris is an excellent alternative to Escherichia coli transformants in large-scale synthesis of oligosaccharides.
Shao
Jun
J
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Hayashi
Takahisa
T
Wang
Peng George
PG
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers; D000939:Epitopes / Q000378:metabolism*; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics; D005697:Galactosides / Q000378:metabolism*; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014534:UDPglucose 4-Epimerase / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D000939:Epitopes; D005697:Galactosides; D011994:Recombinant Proteins; D005700:Galactosyltransferases; D005964:Glucosyltransferases; C022699:sucrose synthase; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase; D014534:UDPglucose 4-Epimerase