Genomic Edition of Ashbya gossypii Using One-vector CRISPR/Cas9

The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of Ashbya gossypii. The filamentous fungus A. gossypii is currently used for the industrial production of riboflavin (vitamina B2). In addition, A. gossypii produces other high-value compounds such as folic acid, nucleosides and biolipids. A large molecular toolbox is available for the genomic manipulation of this fungus including gene targeting methods, rapid assembly of heterologous expression modules and, recently, a one-vector CRISPR/Cas9 editing system adapted for A. gossypii that allows marker-free engineering strategies to be implemented. The CRISPR/Cas9 system comprises an RNA guided DNA endonuclease (Cas9) and a guide RNA (gRNA), which is complementary to the genomic target region. The Cas9 nuclease requires a 5′-NGG-3′ trinucleotide, called protospacer adjacent motif (PAM), to generate a double-strand break (DSB) in the genomic target, which can be repaired with a synthetic mutagenic donor DNA (dDNA) by homologous recombination (HR), thus introducing a specific designed mutation. The CRISPR/Cas9 system adapted for A. gossypii largely facilitates the genomic edition of this industrial fungus

New Promoters for Metabolic Engineering of Ashbya gossypii

[EN]Ashbya gossypii is a filamentous fungus that is currently exploited for the industrial production of riboflavin. In addition, metabolically engineered strains of A. gossypii have also been described as valuable biocatalysts for the production of different metabolites such as folic acid, nucleosides, and biolipids. Hence, bioproduction in A. gossypii relies on the availability of well-performing gene expression systems both for endogenous and heterologous genes. In this regard, the identification of novel promoters, which are critical elements for gene expression, decisively helps to expand the A. gossypii molecular toolbox. In this work, we present an adaptation of the Dual Luciferase Reporter (DLR) Assay for promoter analysis in A. gossypii using integrative cassettes. We demonstrate the efficiency of the analysis through the identification of 10 new promoters with different features, including carbon source-regulatable abilities, that will highly improve the gene expression platforms used in A. gossypii. Three novel strong promoters (PCCW12, PSED1, and PTSA1) and seven medium/weak promoters (PHSP26, PAGL366C, PTMA10, PCWP1, PAFR038W, PPFS1, and PCDA2) are presented. The functionality of the promoters was further evaluated both for the overexpression and for the underexpression of the A. gossypii MSN2 gene, which induced significant changes in the sporulation ability of the mutant strains

Genomic profiling of fungal cell wall-interfering compounds: identification of a common gene signature

[Background]: The fungal cell wall forms a compact network whose integrity is essential for cell morphology and viability. Thus, fungal cells have evolved mechanisms to elicit adequate adaptive responses when cell wall integrity (CWI) is compromised. Functional genomic approaches provide a unique opportunity to globally characterize these adaptive mechanisms. To provide a global perspective on these CWI regulatory mechanisms, we developed chemical-genomic profiling of haploid mutant budding yeast cells to systematically identify in parallel those genes required to cope with stresses interfering the cell wall by different modes of action: β-1,3 glucanase and chitinase activities (zymolyase), inhibition of β-1,3 glucan synthase (caspofungin) and binding to chitin (Congo red). [Results]: Measurement of the relative fitness of the whole collection of 4786 haploid budding yeast knock-out mutants identified 222 mutants hypersensitive to caspofungin, 154 mutants hypersensitive to zymolyase, and 446 mutants hypersensitive to Congo red. Functional profiling uncovered both common and specific requirements to cope with different cell wall damages. We identified a cluster of 43 genes highly important for the integrity of the cell wall as the common >signature of cell wall maintenance (CWM)>. This cluster was enriched in genes related to vesicular trafficking and transport, cell wall remodeling and morphogenesis, transcription and chromatin remodeling, signal transduction and RNA metabolism. Although the CWI pathway is the main MAPK pathway regulating cell wall integrity, the collaboration with other signal transduction pathways like the HOG pathway and the invasive growth pathway is also required to cope with the cell wall damage depending on the nature of the stress. Finally, 25 mutant strains showed enhanced caspofungin resistance, including 13 that had not been previously identified. Only three of them, wsc1δ, elo2δ and elo3δ, showed a significant decrease in β-1,3-glucan synthase activity. [Conclusions]: This work provides a global perspective about the mechanisms involved in cell wall stress adaptive responses and the cellular functions required for cell wall integrity. The results may be useful to uncover new potential antifungal targets and develop efficient antifungal strategies by combination of two drugs, one targeting the cell wall and the other interfering with the adaptive mechanisms.This work was supported by grants BIO2010-22146, BIO2013-48136-P (MINECO, Spain) and S2010/BMD-2414 (Comunidad de Madrid) to J.A, and grant BIO2012-35372 (MINECO, Spain) to JCR.Peer Reviewe

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-micron 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 approximately 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.Project AshByofactory (grant PTDC/EBB-EBI/101985/2008 - FCOMP-01-0124-FEDER-009701), MIT-Portugal Program (PhD grant SFRH/BD/39112/2007 to Tatiana Q. Aguiar) and grant SFRH/BDP/63831/2009 to Carla Oliveir

Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in Ashbya gossypii

Additional files: Additional file 1: List of primers used in this study Additional file 2: Alignments of PRS proteins. Underlined in red are the residues involved in ribose-5-phosphate binding; underlined in blue are the residues involved in pyrophosphate binding. Red circles indicate residues that have been described to participate in catalysis in the B. subtilis, E. coli and human PRPP synthetasesBackground Phosphoribosyl pyrophosphate (PRPP) is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism. PRPP is directly involved in the de novo and salvage biosynthesis of GTP, which is the immediate precursor of riboflavin. The industrial production of this vitamin using the fungus Ashbya gossypii is an important biotechnological process that is strongly influenced by substrate availability. Results Here we describe the characterization and manipulation of two genes of A. gossypii encoding PRPP synthetase (AGR371C and AGL080C). We show that the AGR371C and AGL080C gene products participate in PRPP synthesis and exhibit inhibition by ADP. We also observed a major contribution of AGL080C to total PRPP synthetase activity, which was confirmed by an evident growth defect of the Δagl080c strain. Moreover, we report the overexpression of wild-type and mutant deregulated isoforms of Agr371cp and Agl080cp that significantly enhanced the production of riboflavin in the engineered A. gossypii strains. Conclusion It is shown that alterations in PRPP synthetase activity have pleiotropic effects on the fungal growth pattern and that an increase in PRPP synthetase enzymatic activity can be used to enhance riboflavin production in A. gossypii.This work was supported in part by BASF AG and grant AGL2005-07245-C03-03 from the Ministerio de Educación y Ciencia, Spain.Peer reviewe

Raman spectroscopy adds complementary detail to the high-resolution X-ray crystal structure of photosynthetic PsbP from Spinacia oleracea

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally “dry,” has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.Support by the Academy of Sciences of the Czech Republic (RVO: 61388971, AVOZ60870520), the Ministry of Education of the Czech Republic (MSM6007665808, ME09062, COST LD11011), the Czech Science Foundation (grant 203/08/0114 to RE), and the Grant Agency of the Academy of Sciences of the Czech Republic (number IAA608170901 to DK and AD, number KJB101120805 to VK and KH) is gratefully acknowledged. JLR and JBA thank the Spanish Ministerio de Ciencia e Innovación (reference numbers BIO2008-00194 and BF2007-68107-C02-02/BMC).Peer reviewe

The protein factor-arrest 11 (Far11) is essential for the toxicity of human caspase-10 in yeast and participates in the regulation of autophagy and the DNA damage signaling

The heterologous expression of human caspase-10 in Saccharomyces cerevisiae induces a lethal phenotype, which includes some hallmarks of apoptosis and autophagy, alterations in the intra-S checkpoint, and cell death. To determine the cellular processes and pathways that are responsible of the caspase-10-induced cell death we have designed a loss-of-function screening system to identify genes that are essential for the lethal phenotype. We observed that the ER-Golgi-localized family of proteins Far, MAPK signaling, the autophagy machinery, and several kinases and phosphatases are essential for caspase-10 toxicity. We also found that the expression of caspase-10 elicits a simultaneous activation of the MAP kinases Fus3, Kss1, and Slt2. Furthermore, the protein Far11, which is a target of MAP kinases, is essential for the dephosphorylation of Atg13 and, consequently, for the induction of autophagy. In addition, Far11 participates in the regulation of the DNA damage response through the dephosphorylation of Rad53. Finally, we have also demonstrated that Far11 is able to physically interact with the phosphatases Pph21, Pph22, and Pph3. Overall, our results indicate that the expression of human caspase-10 in S. cerevisiae activates an intracellular death signal that depends on the Far protein complex and that Far11 may function as a regulator subunit of phosphatases in different processes, thus representing a mechanistic link between them. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported in part by Junta de Castilla y León Grants SA008B08 (to A. J.) and GR147 (to J. L. R.) and Ministerio de Ciencia y Innovación Grant BIO2008-00194.Peer Reviewe