Efficient production of heterologous proteins with yeasts and other eukaryotic hosts is often hampered by inefficient secretion of the product. Limitation of protein secretion has been attributed to a low folding rate, and a rational solution is the overexpression of proteins supporting folding, like protein disulfide isomerase (Pdi), or the unfolded protein response transcription factor Hac1. Assuming that other protein factors which are not directly involved in protein folding may also support secretion of heterologous proteins, we set out to analyze the differential transcriptome of a Pichia pastoris strain overexpressing human trypsinogen versus that of a nonexpressing strain. Five hundred twenty-four genes were identified to be significantly regulated. Excluding those genes with totally divergent functions (like, e.g., core metabolism), we reduced this number to 13 genes which were upregulated in the expression strain having potential function in the secretion machinery and in stress regulation. The respective Saccharomyces cerevisiae homologs of these genes, including the previously characterized secretion helpers PDI1, ERO1, SSO2, KAR2/BiP, and HAC1 as positive controls, were cloned and overexpressed in a P. pastoris strain expressing a human antibody Fab fragment. All genes except one showed a positive effect on Fab fragment secretion, as did the controls. Six out of these novel secretion helper factors, more precisely Bfr2 and Bmh2 (involved in protein transport), the chaperones Ssa4 and Sse1, the vacuolar ATPase subunit Cup5, and Kin2 (a protein kinase connected to exocytosis), proved their benefits for practical application in laboratory-scale production processes by increasing both specific production rates and the volumetric productivity of an antibody fragment up to 2.5-fold in fed-batch fermentations of P. pastoris.Successful secretion of proteins has been accomplished with a variety of fungal hosts, including the yeasts Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, and Kluyveromyces lactis and filamentous fungi like Aspergillus awamori and Trichoderma reesei. While the secretion of some proteins is readily achieved at high rates, many other proteins are secreted only at comparatively low levels (20,24,25). Improvement of the secretion of a recombinant protein was first attempted by random mutagenesis (1, 16). The major disadvantage of this method is usually that any positive result cannot be transferred to other strains.It has been shown in several cases that the secretion process can be enhanced by cooverexpression of proteins which support the folding and processing of other proteins (recently reviewed in references 12 and 5). Some of these supporting factors, like protein disulfide isomerase (Pdi), have catalytic activity on the proteins, and others act by binding to the proteins and preventing them from aggregation (chaperones, e.g., BiP) or by stimulating the release of the protein to the cell exterior at a later step in the secretory pathway (Sso proteins). Another approa...
