Megaprimer-Based PCR to Synthesize Fusion Genes for Cloning.

Megaprimer-based polymerase chain reaction (PCR) strategies allow the versatile and fast assembly and amplification of a myriad of tailor-made or random DNA sequences readily available for conventional or restriction-free (RF) cloning.In this chapter, we present a megaprimer-based PCR protocol that enables the expeditious construction of customized fusion genes ready for cloning into commercial expression plasmids. With the expanding use of protein tag technology in the most diverse application fields, this protocol remains a versatile and affordable solution for the synthesis and fusion of peptide tags/domains of interest.

Recombinant protein purification and immobilization strategies based on peptides with dual affinity to iron oxide and silica.

Iron oxide and silica-based materials have emerged as attractive protein purification and immobilization matrices. His6 has been reported as an effective affinity tag for both iron oxide and silica. Here, the silica-binding tags CotB1p and Car9 were shown to work as effectively as iron oxide-binding tags. Using EGFP as a model protein, commercially available bare iron oxide (BIONs) or silicon dioxide (BSiNs) nanoparticles as low-cost purification/immobilization matrices, and non-hazardous and mild binding and elution conditions, adsorption and desorption studies were performed with lysates from Escherichia coli-producing cells to compare the performance of these dual-affinity tags. Under the conditions tested, the His6 tag stood out as the best-performing tag, followed by CotB1p. Our findings concluded the promising combination of these tags, BIONs and BSiNs for one-step purification of recombinant proteins, and two-step purification and immobilization of recombinant proteins without intermediate buffer exchange. This proof of concept work set the ground for future evaluation of these purification and immobilization strategies using other proteins with different properties, which will be of interest to expand their utility and applicability.

Tag-mediated single-step purification and immobilization of recombinant proteins toward protein-engineered advanced materials.

Background: The potential applications of protein-engineered functional materials are so wide and exciting that the interest in these eco-friendly advanced materials will further expand in the future. Tag-mediated protein purification/immobilization technologies have emerged as green and cost-effective approaches for the fabrication of such materials. Strategies that combine the purification and immobilization of recombinant proteins/peptides onto/into natural, synthetic or hybrid materials in a single-step are arising and attracting increasing interest. Aim of Review: This review highlights the most significant advances of the last 5 years within the scope of tag-mediated protein purification/immobilization and elucidates their contributions for the development of efficient single-step purification and immobilization strategies. Recent progresses in the field of protein-engineered materials created using innovative protein-tag combinations and future opportunities created by these new technologies are also summarized and identified herein. Key Scientific Concepts of Review: Protein purification/immobilization tags present a remarkable ability to establish specific non-covalent/covalent interactions between solid materials and biological elements, which prompted the creation of tailor-made and advanced functional materials, and of next-generation hybrid materials. Affinity tags can bind to a wide range of materials (of synthetic, natural or hybrid nature), being most suitable for protein purification. Covalently binding tags are most suitable for long-term protein immobilization, but can only bind naturally to protein-based materials. Hybrid affinity-covalently binding tags have allowed efficient one-step purification and immobilization of proteins onto different materials, as well as the development of innovative protein-engineered materials. Self-aggregating tags have been particularly useful in combination with other tags for generating protein-engineered materials with self-assembling, flexible and/or responsive properties. While these tags have been mainly explored for independent protein purification, immobilization or functionalization purposes, efficient strategies that combine tag-mediated purification and immobilization/functionalization in a single-step will be essential to guarantee the sustainable manufacturing of advanced protein-engineered materials.

Microbial lipids from industrial wastes using xylose-utilizing Ashbya gossypii strains.

This work presents the exploitation of waste industrial by-products as raw materials for the production of microbial lipids in engineered strains of the filamentous fungus Ashbya gossypii. A lipogenic xylose-utilizing strain was used to apply a metabolic engineering approach aiming at relieving regulatory mechanisms to further increase the biosynthesis of lipids. Three genomic manipulations were applied: the overexpression of a feedback resistant form of the acetyl-CoA carboxylase enzyme; the expression of a truncated form of Mga2, a regulator of the main Δ9 desaturase gene; and the overexpression of an additional copy of DGA1 that codes for diacylglycerol acyltransferase. The performance of the engineered strain was evaluated in culture media containing mixed formulations of corn-cob hydrolysates, sugarcane molasses or crude glycerol. Our results demonstrate the efficiency of the engineered strains, which were able to accumulate about 40% of cell dry weight (CDW) in lipid content using organic industrial wastes as feedstocks.

Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactones.

BACKGROUND: Lactones are highly valuable cyclic esters of hydroxy fatty acids that find application as pure fragrances or as building blocks of speciality chemicals. While chemical synthesis often leads to undesired racemic mixtures, microbial production allows obtaining optically pure lactones. The production of a specific lactone by biotransformation depends on the supply of the corresponding hydroxy fatty acid, which has economic and industrial value similar to γ-lactones. Hence, the identification and exploration of microorganisms with the rare natural ability for de novo biosynthesis of lactones will contribute to the long-term sustainability of microbial production. In this study, the innate ability of Ashbya gossypii for de novo production of γ-lactones from glucose was evaluated and improved. RESULTS: Characterization of the volatile organic compounds produced by nine strains of this industrial filamentous fungus in glucose-based medium revealed the noteworthy presence of seven chemically different γ-lactones. To decipher and understand the de novo biosynthesis of γ-lactones from glucose, we developed metabolic engineering strategies focused on the fatty acid biosynthesis and the ß-oxidation pathways. Overexpression of AgDES589, encoding a desaturase for the conversion of oleic acid (C18:1) into linoleic acid (C18:2), and deletion of AgELO624, which encodes an elongase that catalyses the formation of C20:0 and C22:0 fatty acids, greatly increased the production of γ-lactones (up to 6.4-fold; (7.6 ± 0.8) × 103 µg/gCell Dry Weight). Further substitution of AgPOX1, encoding the exclusive acyl-CoA oxidase in A. gossypii, by a codon-optimized POX2 gene from Yarrowia lipolytica, which encodes a specific long chain acyl-CoA oxidase, fine-tuned the biosynthesis of γ-decalactone to a relative production of more than 99%. CONCLUSIONS: This study demonstrates the potential of A. gossypii as a model and future platform for de novo biosynthesis of γ-lactones. By means of metabolic engineering, key enzymatic steps involved in their production were elucidated. Moreover, the combinatorial metabolic engineering strategies developed resulted in improved de novo biosynthesis of γ-decalactone. In sum, these proof-of-concept data revealed yet unknown metabolic and genetic determinants important for the future exploration of the de novo production of γ-lactones as an alternative to biotransformation processes.

Light exposure during growth increases riboflavin production, reactive oxygen species accumulation and DNA damage in Ashbya gossypii riboflavin-overproducing strains.

The overproduction of riboflavin (vitamin B2) by Ashbya gossypii, one of the most distinctive traits of this filamentous hemiascomycete, has been proposed to act as an ecological defense mechanism, since it is triggered by environmental stress. The interaction of endogenous riboflavin with light generates reactive oxygen species (ROS) and induces oxidative DNA damage in mammalian cells, but exogenous riboflavin was shown to protect A. gossypii spores against ultraviolet light. Envisioning a better understanding of this biotechnologically relevant trait, here we investigated the putative genotoxic effects associated with the overproduction of riboflavin by A. gossypii. For assessing that we developed the Ashbya Comet Assay, which was able to reproducibly measure oxidative (H2O2/menadione-mediated) and non-oxidative (camptothecin-mediated) DNA damage in A. gossypii. Using this protocol, we determined that exposure to sunlight-mimicking light during growth significantly increased the DNA damage accumulation in riboflavin-overproducing cells, but not in non-overproducing ones. The exposure of overproducing cells to light induced the intracellular accumulation of ROS and increased the production of riboflavin 1.5-fold. These results show that riboflavin-overproducing strains are highly susceptible to photo-induced oxidative DNA damage and draw attention for the importance of controlling the exposure to light of biotechnological riboflavin production processes with A. gossypii.

Physiological characterization of a pyrimidine auxotroph exposes link between uracil phosphoribosyltransferase regulation and riboflavin production in Ashbya gossypii.

The blockage of the de novo pyrimidine biosynthetic pathway at the orotidine-5'-phosphate decarboxylase level was previously demonstrated to affect riboflavin production in the industrial producer fungus Ashbya gossypii. However, the molecular basis for the unusual sensitivity to uracil displayed by the pyrimidine auxotroph A. gossypii Agura3 was unknown. Here, uridine was shown to be the only intermediate of the pyrimidine salvage pathway able to fully restore this mutant's growth. Conversely, uracil, which is routinely used to rescue pyrimidine auxotrophs, had a dose-dependent growth-inhibitory effect. Uracil phosphoribosyltransferase (UPRT) is the pyrimidine salvage pathway enzyme responsible for converting uracil to uridine monophosphate in the presence of phosphoribosyl pyrophosphate (PRPP). Characterization of the A. gossypii UPRT, as produced and purified from Escherichia coli, revealed that uracil concentrations above 1 mM negatively affected its activity, thus explaining the hypersensitivity of the Agura3 mutant to uracil. Accordingly, overexpression of the AgUPRT encoding-gene in A. gossypii Agura3 led to similar growth on rich medium containing 5 mM uracil or uridine. Decreased UPRT activity ultimately favors the preservation of PRPP, which otherwise may be directed to other pathways. In A. gossypii, increased PRPP availability promotes overproduction of riboflavin. Thus, this UPRT modulation mechanism reveals a putative means of saving precursors essential for riboflavin overproduction by this fungus. A similar uracil-mediated regulation mechanism of the UPRT activity is reported only in two protozoan parasites, whose survival depends on the availability of PRPP. Physiological evidence here discussed indicate that it may be extended to other distantly related flavinogenic fungi.

Synthesis of Fusion Genes for Cloning by Megaprimer-Based PCR.

The polymerase chain reaction (PCR) is the technique of choice used to obtain DNA for cloning, because it rapidly provides high amounts of desired DNA fragments and allows the easy introduction of extremities adequate for enzyme restriction or homologous recombination, and of artificial, native, or modified sequence elements for specific applications. In this context, the use of megaprimer-based PCR strategies allows the versatile and fast assembly and amplification of tailor-made DNA sequences readily available for cloning.In this chapter, we describe the design and use of a megaprimer-based PCR protocol to construct customized fusion genes ready for cloning into commercial expression plasmids by restriction digestion and ligation.

New biotechnological applications for Ashbya gossypii: Challenges and perspectives.

The filamentous fungus Ashbya gossypii has long been considered a paradigm of the White Biotechnology in what concerns riboflavin production. Its industrial relevance led to the development of a significant molecular and in silico modeling toolbox for its manipulation. This, together with the increasing knowledge of its genome and metabolism has helped designing effective metabolic engineering strategies for optimizing riboflavin production, but also for developing new A. gossypii strains for novel biotechnological applications, such as production of recombinant proteins, single cell oils (SCOs), and flavour compounds. With the recent availability of its genome-scale metabolic model, the exploration of the full biotechnological potential of A. gossypii is now in the spotlight. Here, we will discuss some of the challenges that these emerging A. gossypii applications still need to overcome to become economically attractive and will present future perspectives for these and other possible biotechnological applications for A. gossypii.

Ashbya gossypii beyond industrial riboflavin production: A historical perspective and emerging biotechnological applications.

The filamentous fungus Ashbya gossypii has been safely and successfully used for more than two decades in the commercial production of riboflavin (vitamin B2). Its industrial relevance combined with its high genetic similarity with Saccharomyces cerevisiae together promoted the accumulation of fundamental knowledge that has been efficiently converted into a significant molecular and in silico toolbox for its genetic engineering. This synergy has enabled a directed and sustained exploitation of A. gossypii as an industrial riboflavin producer. Although there is still room for optimizing riboflavin production, the recent years have seen an abundant advance in the exploration of A. gossypii for other biotechnological applications, such as the production of recombinant proteins, single cell oil and flavour compounds. Here, we will address the biotechnological potential of A. gossypii beyond riboflavin production by presenting (a) a physiological and metabolic perspective over this fungus; (b) the molecular toolbox available for its manipulation; and (c) commercial and emerging biotechnological applications for this industrially important fungus, together with the approaches adopted for its engineering.

Contribution of PRS3, RPB4 and ZWF1 to the resistance of industrial Saccharomyces cerevisiae CCUG53310 and PE-2 strains to lignocellulosic hydrolysate-derived inhibitors.

PRS3, RPB4 and ZWF1 were previously identified as key genes for yeast tolerance to lignocellulose-derived inhibitors. To better understand their contribution to yeast resistance to the multiple stresses occurring during lignocellulosic hydrolysate fermentations, we overexpressed these genes in two industrial Saccharomyces cerevisiae strains, CCUG53310 and PE-2, and evaluated their impact on the fermentation of Eucalyptus globulus wood and corn cob hydrolysates. PRS3 overexpression improved the fermentation rate (up to 32%) and productivity (up to 48%) in different hydrolysates. ZWF1 and RPB4 overexpression did not improve the fermentation performance, but their increased expression in the presence of acetic acid, furfural and hydroxymethylfurfural was found to contribute to yeast adaptation to these inhibitors. This study expands our understanding about the molecular mechanisms involved in industrial yeast tolerance to the stresses occurring during lignocellulosic bioethanol production and highlights the importance of selecting appropriate strain backgrounds/hydrolysates combinations when addressing further improvement of these processes.

Blockage of the pyrimidine biosynthetic pathway affects riboflavin production in Ashbya gossypii.

The Ashbya gossypii riboflavin biosynthetic pathway and its connection with the purine pathway have been well studied. However, the outcome of genetic alterations in the pyrimidine pathway on riboflavin production by A. gossypii had not yet been assessed. Here, we report that the blockage of the de novo pyrimidine biosynthetic pathway in the recently generated A. gossypii Agura3 uridine/uracil auxotrophic strain led to improved riboflavin production on standard agar-solidified complex medium. When extra uridine/uracil was supplied, the production of riboflavin by this auxotroph was repressed. High concentrations of uracil hampered this (and the parent) strain growth, whereas excess uridine favored the A. gossypii Agura3 growth. Considering that the riboflavin and the pyrimidine pathways share the same precursors and that riboflavin overproduction may be triggered by nutritional stress, we suggest that overproduction of riboflavin by the A. gossypii Agura3 may occur as an outcome of a nutritional stress response and/or of an increased availability in precursors for riboflavin biosynthesis, due to their reduced consumption by the pyrimidine pathway.

Investigation of protein secretion and secretion stress in Ashbya gossypii.

BACKGROUND: Ashbya gossypii is a filamentous Saccharomycete used for the industrial production of riboflavin that has been recently explored as a host system for recombinant protein production. To gain insight into the protein secretory pathway of this biotechnologically relevant fungus, we undertook genome-wide analyses to explore its secretome and its transcriptional responses to protein secretion stress. RESULTS: A computational pipeline was used to predict the inventory of proteins putatively secreted by A. gossypii via the general secretory pathway. The proteins actually secreted by this fungus into the supernatants of submerged cultures in minimal and rich medium were mapped by two-dimensional gel electrophoresis, revealing that most of the A. gossypii secreted proteins have an isoelectric point between 4 and 6, and a molecular mass above 25 kDa. These analyses together indicated that 1-4% of A. gossypii proteins are likely to be secreted, of which less than 33% are putative hydrolases. Furthermore, transcriptomic analyses carried out in A. gossypii cells under recombinant protein secretion conditions and dithiothreitol-induced secretion stress unexpectedly revealed that a conventional unfolded protein response (UPR) was not activated in any of the conditions, as the expression levels of several well-known UPR target genes (e.g. IRE1, KAR2, HAC1 and PDI1 homologs) remained unaffected. However, several other genes involved in protein unfolding, endoplasmatic reticulum-associated degradation, proteolysis, vesicle trafficking, vacuolar protein sorting, secretion and mRNA degradation were up-regulated by dithiothreitol-induced secretion stress. Conversely, the transcription of several genes encoding secretory proteins, such as components of the glycosylation pathway, was severely repressed by dithiothreitol CONCLUSIONS: This study provides the first insights into the secretion stress response of A. gossypii, as well as a basic understanding of its protein secretion potential, which is more similar to that of yeast than to that of other filamentous fungi. Contrary to what has been widely described for yeast and fungi, a conventional UPR was not observed in A. gossypii, but alternative protein quality control mechanisms enabled it to cope with secretion stress. These data will help provide strategies for improving heterologous protein secretion in A. gossypii.

Genome-wide metabolic re-annotation of Ashbya gossypii: new insights into its metabolism through a comparative analysis with Saccharomyces cerevisiae and Kluyveromyces lactis.

BACKGROUND: Ashbya gossypii is an industrially relevant microorganism traditionally used for riboflavin production. Despite the high gene homology and gene order conservation comparatively with Saccharomyces cerevisiae, it presents a lower level of genomic complexity. Its type of growth, placing it among filamentous fungi, questions how close it really is from the budding yeast, namely in terms of metabolism, therefore raising the need for an extensive and thorough study of its entire metabolism. This work reports the first manual enzymatic genome-wide re-annotation of A. gossypii as well as the first annotation of membrane transport proteins. RESULTS: After applying a developed enzymatic re-annotation pipeline, 847 genes were assigned with metabolic functions. Comparatively to KEGG's annotation, these data corrected the function for 14% of the common genes and increased the information for 52 genes, either completing existing partial EC numbers or adding new ones. Furthermore, 22 unreported enzymatic functions were found, corresponding to a significant increase in the knowledge of the metabolism of this organism. The information retrieved from the metabolic re-annotation and transport annotation was used for a comprehensive analysis of A. gossypii's metabolism in comparison to the one of S. cerevisiae (post-WGD - whole genome duplication) and Kluyveromyces lactis (pre-WGD), suggesting some relevant differences in several parts of their metabolism, with the majority being found for the metabolism of purines, pyrimidines, nitrogen and lipids. A considerable number of enzymes were found exclusively in A. gossypii comparatively with K. lactis (90) and S. cerevisiae (13). In a similar way, 176 and 123 enzymatic functions were absent on A. gossypii comparatively to K. lactis and S. cerevisiae, respectively, confirming some of the well-known phenotypes of this organism. CONCLUSIONS: This high quality metabolic re-annotation, together with the first membrane transporters annotation and the metabolic comparative analysis, represents a new important tool for the study and better understanding of A. gossypii's metabolism.

High-level expression of Aspergillus niger b-galactosidase in Ashbya gossypii.

Ashbya gossypii has been recently considered as a host for the expression of recombinant proteins. The production levels achieved thus far were similar to those obtained with Saccharomyces cerevisiae for the same proteins. Here, the b-galactosidase from Aspergillus niger was successfully expressed and secreted by A. gossypii from 2-mm plasmids carrying the native signal sequence at higher levels than those secreted by S. cerevisiae laboratorial strains. Four different constitutive promoters were used to regulate the expression of bgalactosidase: A. gossypii AgTEF and AgGPD promoters, and S. cerevisiae ScADH1 and ScPGK1 promoters. The native AgTEF promoter drove the highest expression levels of recombinant b-galactosidase in A. gossypii, leading to 2- and 8-fold higher extracellular activity than the AgGPD promoter and the heterologous promoters, respectively. In similar production conditions, the levels of active b-galactosidase secreted by A. gossypii were up to 37 times higher than those secreted by recombinant S. cerevisiae and 2.5 times higher than those previously reported for the b-galactosidase-high producing S. cerevisiae NCYC869-A3/pVK1.1. The substitution of glucose by glycerol in the production medium led to a 1.5-fold increase in the secretion of active b-galactosidase by A. gossypii. Recombinant b-galactosidase secreted by A. gossypii was extensively glycosylated, as are the native A. niger b-galactosidase and recombinant b-galactosidase produced by yeast. These results highlight the potential of A. gossypii as a recombinant protein producer and open new perspectives to further optimize recombinant protein secretion in this fungus.

Cre-loxP-based system for removal and reuse of selection markers in Ashbya gossypii targeted engineering.

The filamentous ascomycete Ashbya gossypii is amenable to genetic manipulation and is an excellent model system for studying eukaryotic cell biology. However, the number of selection markers in current use for both targeted gene integration and disruption in this fungus are very limited. Therefore, the Cre-loxP recombination system was adapted for use in A. gossypii and its effectiveness in recycling marker genes was demonstrated by constructing both single and double deleted Agura3 and Agade1 auxotrophic strains free of exogenous markers. In spite of its wide use in other organisms, including other Ascomycete fungi, this is the first report describing Cre-loxP-based methodology for A. gossypii, opening new perspectives for targeted engineering of this fungus with several promising biotechnological applications [corrected].

Molecular and functional characterization of an invertase secreted by Ashbya gossypii.

The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity toward sucrose was approximately 10 times higher than toward raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-µm-plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii.

Characterization of the Ashbya gossypii secreted N-glycome and genomic insights into its N-glycosylation pathway.

The riboflavin producer Ashbya gossypii is a filamentous hemiascomycete, closely related to the yeast Saccharomyces cerevisiae, that has been used as a model organism to study fungal developmental biology. It has also been explored as a host for the expression of recombinant proteins. However, although N-glycosylation plays important roles in protein secretion, morphogenesis, and the development of multicellular organisms, the N-glycan structures synthesised by A. gossypii had not been elucidated. In this study, we report the first characterization of A. gossypii N-glycans and provide valuable insights into their biosynthetic pathway. By combined matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry profiling and nuclear magnetic resonance (NMR) spectroscopy we determined that the A. gossypii secreted N-glycome is characterized by high-mannose type structures in the range Man4-18GlcNAc2, mostly containing neutral core-type N-glycans with 8-10 mannoses. Cultivation in defined minimal media induced the production of acidic mannosylphosphorylated N-glycans, generally more elongated than the neutral N-glycans. Truncated neutral N-glycan structures similar to those found in other filamentous fungi (Man4-7GlcNAc2) were detected, suggesting the possible existence of trimming activity in A. gossypii. Homologs for all of the S. cerevisiae genes known to be involved in the endoplasmatic reticulum and Golgi N-glycan processing were found in the A. gossypii genome. However, processing of N-glycans by A. gossypii differs considerably from that by S. cerevisiae, allowing much shorter N-glycans. Genes for two putative N-glycan processing enzymes were identified, that did not have homologs in S. cerevisiae.

Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii.

To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native ß-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities.   A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an ß-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth, similarly to what has been reported for GAS1-defficient yeast. Thus, secretion could not be tested in these mutants.