We present a new application for the well known expression system Pichia pastoris. Up to now P. pastoris has been used extensively and successfully for the expression of heterologous recombinant proteins. With the availability of the P. pastoris genome sequence a huge field of possibilities has arisen. Combining the knowledge about this excellent protein and enzyme producer with the information about the metabolic pathways derived from the genome sequence we have been able to take the first steps towards a whole cell biocatalyst P. pastoris system. For testing this new strategy we chose an already known pathway for the application in white biotech: the riboflavin synthesis pathway.

To overcome the obstacle of the tight regulation of the genes within the riboflavin pathway on the transcription level the native promoter regions were replaced by the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP). These promoter replacements had to be done stepwise following the pathway from the precursors GTP and ribulose-5-phosphate to the final product riboflavin. Already the first deregulation turned P. pastoris to a so called flavinogenic yeast. The more the pathway got deregulated the more riboflavin accumulated in the cells and also in the culture supernatants. Based on these results, developments are ongoing to establish a P. pastoris system for whole cell biocatalysis.