The Pichia pastoris formaldehyde dehydrogenase gene (FLD1) as a marker for selection of multicopy expression strains of P. pastoris

Abstract

The methylotrophic yeast Pichia pastoris is a popular host for the production of a variety of recombinant proteins . We describe the use of a novel selectable marker , the P. pastoris formaldehyde dehydrogenase gene ( FLD1 ) for DNA-mediated transformations of this yeast. The product of the FLD1 gene (Fld1p) is required for growth of P. pastoris on methanol as a carbon source and methylamine as a nitrogen source. In both these C 1 pathways, Fld1p oxidizes formaldehyde to formate, which is subsequently further oxidized by a second dehydrogenase to carbon dioxide. We show that the FLD1 gene can be used as a marker in transformations of a P. pastoris fld1 host by selection on plates containing methylamine. Furthermore, we demonstrate that populations of these transformants can be enriched for strains that receive multiple copies of an FLD1 -based vector by their increased resistance to formaldehyde. We provide the FLD1 selection system in a set of P. pastoris expression vectors that are composed almost entirely of P. pastoris DNA (except for the recombinant gene) and are devoid of antibiotic resistance genes or other sequences of bacterial origin. The vectors are useful for the selection of strains containing multiple copies of an expression vector and may be ideal for certain large-scale recombinant protein production processes where strains containing non- P. pastoris DNA sequences , particularly bacterial antibiotic resistance genes and replication origins , are considered a potential biological hazard to be avoided.