Tatiana Quinta Aguiar

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 β-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 β-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.

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.

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 Saccharomycescerevisiae 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.

High-level expression of Aspergillus niger β-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 β‐galactosidase from Aspergillus niger was successfully expressed and secreted by A. gossypii from 2‐µm 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 β‐galactosidase: A. gossypii AgTEF and AgGPD promoters, and S. cerevisiae ScADH1 and ScPGK1 promoters. The native AgTEF promoter drove the highest expression levels of recombinant β‐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 β‐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 β‐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 β‐galactosidase by A. gossypii. Recombinant β‐galactosidase secreted by A. gossypii was extensively glycosylated, as are the native A. niger β‐galactosidase and recombinant β‐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].

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.

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.

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

BackgroundAshbya 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.ResultsAfter 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.ConclusionsThis 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.

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.

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.