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.

In fission yeast, knockout of the calcineurin gene resulted in hypersensitivity to Cl(-), and the overexpression of pmp1(+) encoding a dual-specificity phosphatase for Pmk1 mitogen-activated protein kinase (MAPK) or the knockout of the components of the Pmk1 pathway complemented the Cl(-) hypersensitivity of calcineurin deletion. Here, we showed that the overexpression of ptc1(+) and ptc3(+), both encoding type 2C protein phosphatase (PP2C), previously known to inactivate the Wis1-Spc1-Atf1 stress-activated MAPK signaling pathway, suppressed the Cl(-) hypersensitivity of calcineurin deletion. We also demonstrated that the mRNA levels of these two PP2Cs and pyp2(+), another negative regulator of Spc1, are dependent on Pmk1. Notably, the deletion of Atf1, but not that of Spc1, displayed hypersensitivity to the cell wall-damaging agents and also suppressed the Cl(-) hypersensitivity of calcineurin deletion, both of which are characteristic phenotypes shared by the mutation of the components of the Pmk1 MAPK pathway. Moreover, micafungin treatment induced Pmk1 hyperactivation that resulted in Atf1 hyperphosphorylation. Together, our results suggest that PP2C is involved in a negative feedback loop of the Pmk1 signaling, and results also demonstrate that Atf1 is a key component of the cell integrity signaling downstream of Pmk1 MAPK.

Calcineurin is an important mediator that connects the Ca 2؉ -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 Ca 2؉ 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, the prz1-null cells showed hypersensitivity to Ca 2؉ , consistent with a dramatic decrease in transcription of Pmc1 Ca 2؉ 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 Ca 2؉ hypersensitivity of prz1 deletion. In addition, mutations in the its2 ؉ /cps1 ؉ , its8 ؉ , and its10 ؉ /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.

SummaryAlthough 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 Ca 2+ -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 lkh1D, tup12D 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.

The function of two fission yeast genes, SPCC74.03c/ssp2 ؉ and SPAC23H4.02/ppk9 ؉ , encoding an Snf1-like protein kinase were investigated. Deletion of ssp2 ؉ caused a partial defect in glucose derepression of inv1 ؉ , fbp1 ؉ , and gld1 ؉ and in assimilation of sucrose and glycerol, while a mutation in ppk9 ؉ had no apparent effect. Scr1, a transcription factor involved in glucose repression, localized to the nucleus under glucose-rich conditions and to the cytoplasm during glucose starvation in wild-type cells. In contrast, in the ssp2⌬ mutant, Scr1 localized to the nucleus in cells grown in glucose-rich medium as well as in glucose-starved cells. Immunoblot analysis showed that Ssp2 is required for the phosphorylation of Scr1 upon glucose deprivation. Mutation of five putative Ssp2 recognition sites in Scr1 prevented glucose derepression of invertase in glucose-starved cells. These results indicate that Ssp2 regulates phosphorylation and subcellular localization of Scr1 in response to glucose.