Kaoru Takegawa

Engineering of protein secretion in yeast: strategies and impact on protein production

Yeasts combine the ease of genetic manipulation and fermentation of a microorganism with the capability to secrete and modify foreign proteins according to a general eukaryotic scheme. Their rapid growth, microbiological safety, and high-density fermentation in simplified medium have a high impact particularly in the large-scale industrial production of foreign proteins, where secretory expression is important for simplifying the downstream protein purification process. However, secretory expression of heterologous proteins in yeast is often subject to several bottlenecks that limit yield. Thus, many studies on yeast secretion systems have focused on the engineering of the fermentation process, vector systems, and host strains. Recently, strain engineering by genetic modification has been the most useful and effective method for overcoming the drawbacks in yeast secretion pathways. Such an approach is now being promoted strongly by current post-genomic technology and system biology tools. However, engineering of the yeast secretion system is complicated by the involvement of many cross-reacting factors. Tight interdependence of each of these factors makes genetic modification difficult. This indicates the necessity of developing a novel systematic modification strategy for genetic engineering of the yeast secretion system. This mini-review focuses on recent strategies and their advantages for systematic engineering of yeast strains for effective protein secretion.

A novel disaccharide substrate having 1,2-oxazoline moiety for detection of transglycosylating activity of endoglycosidases.

A disaccharide substrate of Manbeta1-4GlcNAc-oxazoline 2 was designed and synthesized as a novel probe for detection of the transglycosylating activity of endoglycosidases. A regio- and stereoselective transglycosylation reaction of 2 to GlcNAcbeta1-O-pNP or Dns-Asn(GlcNAc)-OH catalyzed by endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) and endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has been demonstrated for the first time, resulting in the core trisaccharide derivative Manbeta1-4GlcNAcbeta1-4GlcNAcbeta1-O-pNP 8 (or -(Dns)Asn-OH). Interestingly, the transglycosylation proceeds irreversibly; the resulting trisaccharide 8 was not hydrolyzed by Endo-M and Endo-A. Based on these results, a new mechanism including an oxazolinium ion intermediate has been proposed for the endoglycosidase-catalyzed hydrolysis or transglycosylation.

Critical Roles of Mucin 1 Glycosylation by Transactivated Polypeptide N-Acetylgalactosaminyltransferase 6 in Mammary Carcinogenesis

The structure of O-glycosylated proteins is altered in breast cancer cells, but the mechanisms of such an aberrant modification have been largely unknown. We here report critical roles of a novel druggable target, polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), which is upregulated in a great majority of breast cancers and encodes a glycosyltransferase responsible for initiating mucin-type O-glycosylation. Knockdown of GALNT6 by small interfering RNA significantly enhanced cell adhesion function and suppressed the growth of breast cancer cells. Western blot and immunostaining analyses indicated that wild-type GALNT6 protein could glycosylate and stabilize an oncoprotein mucin 1 (MUC1), which was upregulated with GALNT6 in breast cancer specimens. Furthermore, knockdown of GALNT6 or MUC1 led to similar morphologic changes of cancer cells accompanied by the increase of cell adhesion molecules beta-catenin and E-cadherin. Our findings implied that overexpression of GALNT6 might contribute to mammary carcinogenesis through aberrant glycosylation and stabilization of MUC1 and that screening of GALNT6 inhibitors would be valuable for the development of novel therapeutic modalities against breast cancer.

Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway

Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Δ, vps22Δ, and vps34Δ, were determined on account of their hGH secretion efficiency. The mutant vps10Δ was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe.

A simple and efficient procedure for transformation of Schizosaccharomyces pombe.

We describe a simple and efficient procedure for transformation of Schizosaccharomyces pombe. Sz. pombe colonies grown on minimal (SD) plates were directly removed and suspended in a 100 µl reaction mixture containing 70 µl PLATE solution (50% polyethylene glycol‐4000, 100 mM lithium acetate, 10 mM Tris–HCl, pH 4.9, and 1 mM EDTA), 10 µl plasmid DNA (1 µg), 10 µl carrier DNA (100 µg) and 10 µl sterile distilled water. After incubation at 30 °C for 1 h followed by heat shock treatment at 42 °C for 15 min, the reaction mixture was spread on a selection plate. The transformation efficiency obtained using the procedure was approximately 8000 transformants/µg DNA. The method is simple and time‐saving, making it especially useful for a large number of samples and when a high transformation efficiency is not required. Copyright

Multiple functions of ergosterol in the fission yeast Schizosaccharomyces pombe.

Sterols are a major class of membrane lipids in eukaryotes. In Schizosaccharomyces pombe, sterol 24-C-methyltransferase (Erg6p), C-8 sterol isomerase (Erg2p), C-5 sterol desaturase (Erg31p, Erg32p), C-22 sterol desaturase (Erg5p) and C-24 (28) sterol reductase (Sts1p/Erg4p) have been predicted, but not yet determined, to catalyse a sequence of reactions from zymosterol to ergosterol. Disruption mutants of these genes were unable to synthesize ergosterol, and most were tolerant to the polyene drugs amphotericin B and nystatin. Disruption of erg31(+) or erg32(+) did not cause ergosterol deficiency or tolerance to polyene drugs, indicating that the two C-5 sterol desaturases have overlapping functions. GFP-tagged DRM (detergent-resistant membrane)-associated protein Pma1p localized to the plasma membrane in ergDelta mutants. DRM fractionation revealed that the association between Pma1-GFP and DRM was weakened in erg6Delta but not in other erg mutants. Several GFP-tagged plasma membrane proteins were tested, and an amino acid permease homologue, SPBC359.03c, was found to mislocalize to intracellular punctate structures in the ergDelta mutants. These results indicate that these proteins are responsible for ergosterol biosynthesis in fission yeast, similar to the situation in Saccharomyces cerevisiae. Furthermore, in fission yeast, ergosterol is important for plasma membrane structure and function and for localization of plasma membrane proteins.

Schizosaccharomyces pombe minimum genome factory.

Various systems for the production of useful proteins have been developed using the fission yeast Schizosaccharomyces pombe as a host, and some are now being used commercially. It is necessary, however, to improve the system further for the production of low‐cost chemicals and commodities, so that the host becomes more economical and productive and can be widely used for the production of different molecules. We hypothesized that many S. pombe genes are not necessary under nutrient‐rich growth conditions; or rather, they serve only to waste energy when seen from the viewpoint of protein production, because their products are necessary only for adaptation to different environments. Thus we have tried to create S. pombe mutants that are dedicated to heterologous protein production by deleting as many non‐essential genes as possible. Putative essential genes were mapped using the genome information of S. pombe. The transcriptome of gene disruptants was analysed using microarrays and, using this system, a new promoter was identified. The method (called the Latour method) has been developed to delete efficiently a large region from the chromosome, resulting in the establishment of mutant strains lacking approx. 500 kb of genetic material. New experimental strains auxotrophic for six nutrients were established that were conveniently used for co‐expression of proteins using multiple plasmids. An efficient transformation method has also been developed that is useful for investigating heterologous protein production in a variety of strains. Incidentally, in heterologous protein production systems, products are often degraded, leading to a decline in production efficiency. Thus, to examine heterologous protein production, we created 52 S. pombe mutant strains in each of which a single protease gene was destroyed. We also successfully constructed strains in which multiple protease genes were disrupted. As a result, it was shown that the production of a model protein, human growth hormone, was increased in this strain. Furthermore, we obtained many strains that lacked genes related to glucose metabolism, intracellular transport or biosynthesis of sugar chains. The present minireview covers the results of functional analysis of these strains. By preparing strains in which large chromosomal regions have been deleted and then combining strains defective in various functional genes, the establishment of effective hosts will become possible.

Genome sequence of the white Koji mold aspergillus kawachii IFO 4308, used for brewing the Japanese distilled spirit shochu

ABSTRACT The filamentous fungus Aspergillus kawachii has traditionally been used for brewing the Japanese distilled spirit shochu. A. kawachii characteristically hyperproduces citric acid and a variety of polysaccharide glycoside hydrolases. Here the genome sequence of A. kawachii IFO 4308 was determined and annotated. Analysis of the sequence may provide insight into the properties of this fungus that make it superior for use in shochu production, leading to the further development of A. kawachii for industrial applications.

Loss-of-Function and Gain-of-Function Mutations in FAB1A/B Impair Endomembrane Homeostasis, Conferring Pleiotropic Developmental Abnormalities in Arabidopsis

In eukaryotic cells, PtdIns 3,5-kinase, Fab1/PIKfyve produces PtdIns (3,5) P2 from PtdIns 3-P, and functions in vacuole/lysosome homeostasis. Herein, we show that expression of Arabidopsis (Arabidopsis thaliana) FAB1A/B in fission yeast (Schizosaccharomyces pombe) fab1 knockout cells fully complements the vacuole morphology phenotype. Subcellular localizations of FAB1A and FAB1B fused with green fluorescent protein revealed that FAB1A/B-green fluorescent proteins localize to the endosomes in root epidermal cells of Arabidopsis. Furthermore, reduction in the expression levels of FAB1A/B by RNA interference impairs vacuolar acidification and endocytosis. These results indicate that Arabidopsis FAB1A/B functions as PtdIns 3,5-kinase in plants and in fission yeast. Conditional knockdown mutant shows various phenotypes including root growth inhibition, hyposensitivity to exogenous auxin, and disturbance of root gravitropism. These phenotypes are observed also in the overproducing mutants of FAB1A and FAB1B. The overproducing mutants reveal additional morphological phenotypes including dwarfism, male-gametophyte sterility, and abnormal floral organs. Taken together, this evidence indicates that imbalanced expression of FAB1A/B impairs endomembrane homeostasis including endocytosis, vacuole formation, and vacuolar acidification, which causes pleiotropic developmental phenotypes mostly related to the auxin signaling in Arabidopsis.

Production of heterologous proteins using the fission-yeast (Schizosaccharomyces pombe) expression system

The fission yeast Schizosaccharomyces pombe is a particularly useful model for studying the function and regulation of genes from higher eukaryotes. The genome of Sc. pombe has been sequenced, and DNA microarray, proteome and transcriptome analyses have been carried out. Among the well‐characterized yeast species, Sc. pombe is considered an attractive host for the production of heterologous proteins. Expression vectors for high‐level expression in Sc. pombe have been developed and many foreign proteins have been successfully expressed. However, further improvements in the protein‐expressing host systems are still required for the production of heterologous proteins involved in post‐translational modification, metabolism and intracellular trafficking. This minireview focuses on recent advances in heterologous protein production by use of engineered fission‐yeast strains.