Hideki Tohda

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.

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.

Zinc Finger Protein Prz1 Regulates Ca2+ but Not Cl− Homeostasis in Fission Yeast IDENTIFICATION OF DISTINCT BRANCHES OF CALCINEURIN SIGNALING PATHWAY IN FISSION YEAST

Calcineurin is an important mediator that connects the Ca2+-dependent signaling to various cellular responses in a wide variety of cell types and organisms. In budding yeast, activated calcineurin exerts its function mainly by regulating the Crz1p/Tcn1 transcription factor. Here, we cloned the fission yeast prz1 + gene, which encodes a zinc finger transcription factor highly homologous to Crz1/Tcn1. Similar to the results in budding yeast, calcineurin dephosphorylated Prz1 and resulted in the trans-location of Prz1 from the cytoplasm to the nucleus. Prz1 expression was stimulated by high extracellular Ca2+ in a calcineurin-dependent fashion. However, unlike in budding yeast, the prz1-null cells did not show any phenotype similar to those previously reported in calcineurin deletion such as aberrant cell morphology, mating defect, or hypersensitivity to Cl−. Instead, theprz1-null cells showed hypersensitivity to Ca2+, consistent with a dramatic decrease in transcription of Pmc1 Ca2+ pump. Interestingly, overexpression of Prz1 did not suppress the Cl− hypersensitivity of calcineurin deletion, and overexpression of Pmp1 MAPK phosphatase suppressed the Cl− hypersensitivity of calcineurin deletion but not the Ca2+ hypersensitivity of prz1 deletion. In addition, mutations in theits2 + /cps1 + ,its8 + , andits10 + /cdc7 + genes that showed synthetic lethal genetic interaction with calcineurin deletion did not exhibit synthetic lethality with the prz1 deletion. Our results suggest that calcineurin activates at least two distinct signaling branches, i.e. the Prz1-dependent transcriptional regulation and an unknown mechanism, which functions antagonistically with the Pmk1 MAPK pathway.

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.

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.

Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe.

The creation of protease-deficient mutants to avoid product degradation is one of the current strategies employed to improve productivity and secretion efficiency of heterologous protein expression. We previously constructed a set of single protease-deficient mutants of the fission yeast Schizosaccharomyces pombe by respective disruption of 52 protease genes, and we succeeded in confirming useful disruptants (Idiris et al., Yeast 23:83–99, 2006). In the present study, we attempted multiple deletions of 13 protease genes, single deletions of which were previously confirmed as being beneficial for reducing extracellular product degradation. Using PCR-based gene replacement, a series of multiple deletion strains was constructed by multiple disruption of a maximum of seven protease genes. Effects of the resultant multiple deletion strains on heterologous expression were then measured by practical expression of a proteolytically sensitive model protein, the human growth hormone (hGH). Time profiles of hGH secretion from each resultant mutant demonstrated significantly enhanced hGH productivity with processing of the multiple protease deletions. The data clearly indicated that disruption of multiple protease genes in the fission yeast is an effective method for controlling proteolytic degradation of heterologous proteins particularly susceptible to proteases.

A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast

The technologies for chromosome modification developed to date are not satisfactorily universal, owing to the typical requirements for special enzymes and sequences. In the present report, we propose a new approach for chromosome modification in Schizosaccharomyces pombe that does not involve any special enzymes or sequences. This method, designated the ‘Latour system’, has wide applicability with extremely high efficiency, although both the basic principle and the operation are very simple. We demonstrate the ability of the Latour system to discriminate essential genes, with a long chromosomal area of 100 kb containing 33 genes deleted simultaneously and efficiently. Since no foreign sequences are retained after deletion using the Latour system, this system can be repeatedly applied at other sites. Provided that a negative selectable marker is available, the Latour system relies solely upon homologous recombination, which is highly conserved in living organisms. For this reason, it is expected that the system will be applicable to various yeasts.

Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products.

Abstract A cbh2 cDNA encoding Trichoderma reesei QM9414 cellobiohydrolase II, located on the expression vector whose copy number is controlled by the level of gentamicin, was successfully expressed under the control of a human cytomegalovirus promoter in the fission yeast, Schizosaccharomyces pombe. The 24-amino-acid leader peptide of the cbh2 gene was recognized by the yeast, enabling the efficient secretion of the heterologous cellobiohydrolase. The transformed S. pombe strain produced over 115 μg cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 μg/ml gentamicin. The molecular masses of the recombinant cellobiohydrolases, secreted as two molecular species, were estimated to be 70 kDa and 72 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation treatments revealed that the recombinant enzymes were overglycosylated and scarcely susceptible to α-mannosidase. The recombinant enzymes showed no carboxymethylcellulase activity, but showed similar characteristics to those of a native enzyme purified from T. reesei in their optimum pH and temperature, pH and temperature stabilities, and Vmax values toward phosphoric-acid-swollen cellulose as substrate, except that their Km values were about fourfold higher than that of the native enzyme.

Construction of a protease-deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease-sensitive heterologous proteins

One of the major problems hindering effective production and purification of heterologous proteins from the fission yeast Schizosaccharomyces pombe is proteolytic degradation of the recombinant gene products by host‐specific proteases. As an initial solution to this problem, we constructed a protease‐deficient disruptant set by respective disruption of 52 Sz. pombe protease genes. Functional screening of the resultant set was performed by observing secretory production of a proteolytically sensitive model protein, human growth hormone (hGH). The results indicated that some of the resultant disruptants were effective in reducing hGH degradation, as observed during the hGH expression procedure and mainly as a result of unknown serine‐ and/or cysteine‐type proteases in the culture medium. These findings also demonstrated that construction of a protease‐deficient strain set is not only useful for practical application in protein production, but also for functional screening, specification and modification of proteases in Sz. pombe, where further investigations of proteolytic processes and improvement through multiple gene manipulations are required. Copyright

Identification of a galactose-specific flocculin essential for non-sexual flocculation and filamentous growth in Schizosaccharomyces pombe

Although various mutant strains of the fission yeast Schizosaccharomyces pombe exhibit non‐sexual flocculation, little is known about the mechanistic basis for this phenomenon, nor have genes encoding the implicated flocculin been identified. In the budding yeast Saccharomyces cerevisiae, the transcription factor Flo8 controls expression of some of the genes involved in non‐sexual flocculation. We have found that overexpression of S. cerevisiae FLO8 induced non‐sexual flocculation in S. pombe. This non‐sexual flocculation was Ca2+‐dependent, and was inhibited by addition of galactose, but not by mannose, glucose or sucrose. In the FLO8‐overexpressing strain, a gene designated gsf2+ (galactose‐specific flocculation) was specifically induced. The gsf2+ gene was also highly expressed in lkh1Δ, tup12Δ and gsf1 mutants, all of which exhibited non‐sexual flocculation dependent on gsf2+. We show that the N‐terminal region of Gsf2 recognizes galactose in mediating cell–cell interaction. Disruption of gsf2+ also abolished the adhesion phenotype and invasive growth of the wild‐type strain cultured in low ammonium medium. The newly identified flocculin Gsf2 in fission yeast was not only required for non‐sexual flocculation but was also required for adhesion and filamentous growth through recognition of galactose residues on cell surface glycoconjugates.