The lysosomal hydrolase glucocerebrosidase catalyzes the penultimate step in the breakdown of membrane glycosphingolipids, An inherited deficiency in this enzyme leads to the onset of Gaucher disease, the most common lysosomal storage disorder. Exogenous sources of this protein are required for biochemical and biophysical investigations and enzyme replacement therapy of Gaucher disease. Heterologous expression of glucocerebrosidase has been successful in mammalian and insect cell lines and although its use in enzyme replacement therapy of Gaucher disease has proven efficacious. current production levels limit the availability of the enzyme. Initial attempts to express human glucocerebrosidase using the methylotrophie yeast Pichia pastoris had limited success, despite significant levels of transcription. Using fragments of the glucocerebrosidase cDNA fused to the luciferase cDNA as a translational read-through reporter, the impact of synonymous codon usage bias on protein expression in P. pastoris was examined. A table of preferred codons was determined for P. pastoris and the codon usage of a 186-bp fragment of the glucocerebrosidase gene was optimized to that of the P. pastoris preferred set. A second construct with altered G + C Content but no codon optimization was created for comparison. While the native glucocerebrosidase coding region limited luciferase activity to baseline levels, the codon optimized and G + C altered constructs increased luciferase activity 10.6- and 7.5-fold, respectively. Optimized G + C content, regardless of corresponding codon optimization, appears to he the major contributor to increased translational efficiency in this heterologous expression host. (C) 2002 Elsevier Science (USA). All rights reserved.