HIGH-LEVEL PRODUCTION OF SPINACH GLYCOLATE OXIDASE IN THE METHYLOTROPHIC YEAST PICHIA-PASTORIS - ENGINEERING A BIOCATALYST

Glycolate oxidase (GO) is a flavo-enzyme that catalyzes the oxidation of glycolate, and is useful for the biocatalytic production of glyoxyl ate. We have produced high levels of spinach GO in the methylotrophic yeast Pichia pastoris (Pp), by chromosomal integration of multiple cop ies of an expression cassette containing the GO coding sequence under control of the methanol-inducible alcohol oxidase I promoter. Under fe rmentation conditions, greater than 250 units of GO per gram of cells (wet weight) was obtained, corresponding to roughly 20-30% of soluble cell protein. This recombinant Pp strain was used as a whole-cell bioc atalyst for conversion of glycolic acid to glyoxylic acid.