Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

Large parts of the endoplasmic reticulum of the yeast, Saccharomyces cerevisiae, are located close to intracellular organelles, i.e. mitochondria and the plasma membrane, as shown by fluorescence and electron microscopy. Here we report the isolation and characterization of the subfraction of the endoplasmic reticulum that is closely associated with the plasma membrane. This plasma membrane associated membrane (PAM) is characterized by its high capacity to synthesize phosphatidylserine and phosphatidylinositol. As such, PAM is reminiscent of MAM, a mitochondria associated membrane fraction of the yeast [Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F. & Daum, G. (1995) Biochim.Biophys. Acta 1234, 214±220], although the specific activity of phosphatidylserine synthase and phosphatidylinositol synthase in PAM exceeds several-fold the activity in MAM and also in the bulk endoplasmic reticulum. In addition, several enzymes involved in ergosterol biosynthesis, namely squalene synthase (Erg9p), squalene epoxidase (Erg1p) and sterolD 24 -methyltransferase (Erg6p), are highly enriched in PAM. A possible role of PAM in the supply of lipids to the plasma membrane is discussed.

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Delta, erg6Delta, and erg2Deltaerg6Delta) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergDelta mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergDelta mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.

Sterols and sphingolipids are limited to eukaryotic cells, and their interaction has been proposed to favor formation of lipid microdomains. Although there is abundant biophysical evidence demonstrating their interaction in simple systems, convincing evidence is lacking to show that they function together in cells. Using lipid analysis by mass spectrometry and a genetic approach on mutants in sterol metabolism, we show that cells adjust their membrane composition in response to mutant sterol structures preferentially by changing their sphingolipid composition. Systematic combination of mutations in sterol biosynthesis with mutants in sphingolipid hydroxylation and head group turnover give a large number of synthetic and suppression phenotypes. Our unbiased approach provides compelling evidence that sterols and sphingolipids function together in cells. We were not able to correlate any cellular phenotype we measured with plasma membrane fluidity as measured using fluorescence anisotropy. This questions whether the increase in liquid order phases that can be induced by sterol-sphingolipid interactions plays an important role in cells. Our data revealing that cells have a mechanism to sense the quality of their membrane sterol composition has led us to suggest that proteins might recognize sterol-sphingolipid complexes and to hypothesize the coevolution of sterols and sphingolipids.

Hydratases provide access to secondary and tertiary alcohols by regio- and/or stereospecifically adding water to carbon-carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure-based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two-electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio- and stereoselective hydration.

Sterols are essential factors for endocytosis in animals and yeast. To investigate the sterol structural requirements for yeast endocytosis, we created a variety of erg⌬ mutants, each accumulating a distinct set of sterols different from ergosterol. Mutant erg2⌬erg6⌬ and erg3⌬erg6⌬ cells exhibit a strong internalization defect of the ␣-factor receptor (Ste2p). Specific sterol structures are necessary for pheromone-dependent receptor hyperphosphorylation, a prerequisite for internalization. The lack of phosphorylation is not due to a defect in Ste2p localization or in ligandreceptor interaction. Contrary to most known endocytic factors, sterols seem to function in internalization independently of actin. Furthermore, sterol structures are required at a postinternalization step of endocytosis. erg⌬ cells were able to take up the membrane marker FM4-64, but exhibited defects in FM4-64 movement through endosomal compartments to the vacuole. Therefore, there are at least two roles for sterols in endocytosis. Based on sterol analysis, the sterol structural requirements for these two processes were different, suggesting that sterols may have distinct functions at different places in the endocytic pathway. Interestingly, sterol structures unable to support endocytosis allowed transport of the glycosylphosphatidylinositol-anchored protein Gas1p from the endoplasmic reticulum to Golgi compartment.

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Glycosylphosphatidylinositol (GPI), covalently attached to many eukaryotic proteins, not only acts as a membrane anchor but is also thought to be a sorting signal for GPI-anchored proteins that are associated with sphingolipid and sterol-enriched domains. GPI anchors contain a core structure conserved among all species. The core structure is synthesized in two topologically distinct stages on the leaflets of the endoplasmic reticulum (ER). Early GPI intermediates are assembled on the cytoplasmic side of the ER and then are flipped into the ER lumen where a complete GPI precursor is synthesized and transferred to protein. The flipping process is predicted to be mediated by a protein referred as flippase; however, its existence has not been proven. Here we show that yeast Arv1p is an important protein required for the delivery of an early GPI intermediate, GlcN-acylPI, to the first mannosyltransferase of GPI synthesis in the ER lumen. We also provide evidence that ARV1 deletion and mutations in other proteins involved in GPI anchor synthesis affect inositol phosphorylceramide synthesis as well as the intracellular distribution and amounts of sterols, suggesting a role of GPI anchor synthesis in lipid flow from the ER.