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Applied and Environmental Microbiology, September 2003, p. 5238-5242, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5238-5242.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Enhanced Production of {alpha}-Galactosyl Epitopes by Metabolically Engineered Pichia pastoris

Jun Shao,1 Takahisa Hayashi,2 and Peng George Wang1*

Department of Chemistry, Wayne State University, Detroit, Michigan 48202,1 Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan2

Received 18 March 2003/ Accepted 9 June 2003

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 (Gal{alpha}1,3Galß1,4Glc) 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.

* Corresponding author. Mailing address: Department of Chemistry, Wayne State University, Detroit, MI 48202. Phone: (313) 993-6759. Fax: (313) 577-9241. E-mail: pwang{at}chem.wayne.edu.

Applied and Environmental Microbiology, September 2003, p. 5238-5242, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5238-5242.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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