Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastoris.

Genetic manipulation of lipid biosynthetic enzymes allows modification of cellular membranes. We made use of this strategy and constructed mutants in phospholipid metabolism of Pichia pastoris, which is widely used in biotechnology for expression of heterologous proteins. Here we describe identification of two P. pastoris phosphatidylserine decarboxylases (PSDs) encoded by genes homologous to PSD1 and PSD2 from Saccharomyces cerevisiae. Using P. pastoris psd1Delta and psd2Delta mutants we investigated the contribution of the respective gene products to phosphatidylethanolamine synthesis, membrane composition and cell growth. Deletion of PSD1 caused loss of PSD activity in mitochondria, a severe growth defect on minimal media and depletion of cellular and mitochondrial phosphatidylethanolamine levels. This defect could not be compensated by Psd2p, but by supplementation with ethanolamine, which is the substrate for the cytidine diphosphate (CDP)-ethanolamine pathway, the third route of phosphatidylethanolamine synthesis in yeast. Fatty acid analysis showed selectivity of both Psd1p and Psd2p in vivo for the synthesis of unsaturated phosphatidylethanolamine species. Phosphatidylethanolamine species containing palmitic acid (16:0), however, were preferentially assembled into mitochondria. In summary, this study provides first insight into membrane manipulation of P. pastoris, which may serve as a useful method to modify cell biological properties of this microorganism for biotechnological purposes.

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

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.

Posttransformational vector amplification in the yeast Pichia pastoris.

Generating a high yield of recombinant protein is a major goal when expressing a foreign gene in any expression system. In the methylotrophic yeast Pichia pastoris, a common means of achieving this end is to select for transformants containing multiple integrated copies of an expression vector by plating them on high levels of a selectable marker drug followed by screening for rare colonies with multiple copies. We describe a more convenient method to select for such clones. Using Zeocin-resistance-based vectors, we demonstrate that strains transformed with only one or a few vector copies can, long after transformation, be subjected to further selection at high levels of drug. This resulted in the frequent selection of clones containing increased copy numbers of the vector. This posttransformational vector amplification (PTVA) process resulted in strains containing multiple head-to-tail copies of the entire vector integrated at a single locus in the genome. Of our PTVA selected clones, 40% showed a three- to fivefold increase in vector copy number. So-called 'jackpot' clones with >10 copies of the expression vector represented 5-6% of selected clones and had a proportional increase in recombinant protein.