Aspergillus nidulans como modelo para manipulação de genes envolvidos no processo de unfolded protein response

Orientadores: André Ricardo de Lima Damásio, Fábio Márcio SquinaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaAgência de fomento: CAPESResumo: Em eucariotos, o unfolded protein response (UPR) regula positivamente genes responsáveis por restaurar a homeostase no retículo endoplasmático (RE) durante o acúmulo de proteínas enoveladas incorretamente. A homeostase é restaurada devido à ativação de genes relacionados à via secretória, como aqueles que codificam chaperonas e foldases, o que aumenta, por sua vez, a capacidade de enovelamento de proteínas pelo RE. Alguns sistemas de produção de proteínas heterólogas têm sido desenvolvidos com a super-expressão individual de chaperonas e foldases nas células. Entretanto, a taxa de sucesso com a aplicação dessa estratégia é baixa. Estudos têm mostrado que a manipulação de genes que respondem ao UPR em linhagens fúngicas podem levar ao aumento na produção de proteínas de interesse. Neste trabalho, inicialmente estudamos e identificamos o perfil de proteínas que são recrutadas para expressar e produzir proteínas heterólogas em A. nidulans por espectrometria de massas. Posteriormente, identificamos nas cepas de A. nidulans os genes que respondem ao tratamento com ditiotreitol e tunicamicina, drogas que induzem o UPR. Finalmente, selecionamos 12 genes associados à via de secreção em A. nidulans, os quais foram deletados em cepas recombinantes de A. nidulans, uma que secreta xilanase homóloga (xlnE, 5B3 strain) e outra xilanase heteróloga (tpet_0854, 854 strain). A deleção de uma ciclofilina e de uma chaperona molecular Hsp40 resultou no aumento de 1,25 e 1,70 vezes na secreção de xlnE, respectivamente. Da mesma forma, a deleção de uma tiorredoxina e uma manosiltransferase também aumentou, ainda que em níveis mais baixos, a secreção de xlnE. Os resultados ainda mostraram que a secreção de proteínas totais diminuiu nessas cepas delatadas. Considerando os resultados, essa abordagem demonstrou aumento expressivo na produção de enzimas-alvo, sugerindo que a manosiltransferase, a chaperona Hsp40, a ciclofilina e a tiorredoxina codificadas pelos genes deletados desempenham um papel importante na regulação da produção de proteína em A. nidulans. Entretanto, ainda não entendemos o mecanismo envolvido no aumento da secreção de xlnE após as deleções. Sugerimos que a maior produção de enzimas nas cepas deletadas possa estar relacionada à ativação do UPR e também a um "afrouxamento" no rigor do enovelamento de proteínas pela célula, resultando em um controle de qualidade mais brando e maior secreção de proteínas para o meio extracelularAbstract: In eukaryotes, the unfolded protein response (UPR) positively regulates genes responsible for restoring homeostasis in the endoplasmic reticulum (ER) during accumulation of misfolded proteins. The homeostasis is restored due to the activation of genes related to proteins secretion such as those encoding for chaperones and foldases, which, in turn, increase protein folding capacity by RE. In fact, some systems for heterologous protein production have been developed by the individual overexpression of chaperones and foldases in the hosting cells. However, the success rate of this strategy usually is quite low. Studies on the manipulation of genes that respond to UPR in fungal strains are interesting aiming a higher production of proteins. In this work, we initially identified the profile of proteins that are recruited to express and produce heterologous proteins in A. nidulans by mass spectrometry. Subsequently, we proceeded the identification of genes that respond to UPR-activating chemicals such as dithiothreitol and tunicamycin. Finally, we selected 12 genes with a predicted function in the A. nidulans secretion pathway. These 12 genes were deleted in an A. nidulans recombinant strain producing an homologous xylanase (xlnE, 5B3 strain) and another recombinant strain producing an heterologous xylanase (tpet_0854, 854 strain). The deletion of cyclophilin and a molecular chaperone Hsp40 resulted in an increase around 1.25 and 1.70-fold in the xlnE activity, respectively. Similarly, the deletion of thioredoxin and glycosyl phosphatidyl inositol-mannosyltransferase also increased the xlnE secretion even at lower levels. The results also showed a decreased production of total proteins production in these deleted strains. Thus, our results suggest that proteins such as glycosyl phosphatidyl inositol-mannosyltransferase, chaperone Hsp40, cyclophilin and thioredoxin play an important role in the regulation of proteins production by A. nidulans. However, we still do not understand the mechanism involved in increased secretion of 5B3 after the deletions. We suggest that the increased production of enzymes in the deleted strains is related to the activation of the UPR and a "less stringent" protein folding by the cell, resulting in a mild quality control and higher secretion of proteins into the extracellular mediumDoutoradoBioquímicaDoutora em Biologia Funcional e Molecular2014/15403-6FAPES

Analysis of a conserved cellulase transcriptional regulator reveals inducer-independent production of cellulolytic enzymes in Neurospora crassa.

Cellulose is recalcitrant to deconstruction to glucose for use in fermentation strategies for biofuels and chemicals derived from lignocellulose. In Neurospora crassa, the transcriptional regulator, CLR-2, is required for cellulolytic gene expression and cellulose deconstruction. To assess conservation and divergence of cellulase gene regulation between fungi from different ecological niches, we compared clr-2 function with its ortholog (clrB) in the distantly related species, Aspergillus nidulans. Transcriptional profiles induced by exposure to crystalline cellulose were similar in both species. Approximately 50% of the cellulose-responsive genes showed strict dependence on functional clr-2/clrB, with a subset of 28 genes encoding plant biomass degrading enzymes that were conserved between N. crassa and A. nidulans. Importantly, misexpression of clr-2 under noninducing conditions was sufficient to drive cellulase gene expression, secretion, and activity in N. crassa, to a level comparable to wild type exposed to Avicel. However, misexpression of clrB in A. nidulans was not sufficient to drive cellulase gene expression under noninducing conditions, although an increase in cellulase activity was observed under crystalline cellulose conditions. Manipulation of clr-2 orthologs among filamentous fungi may enable regulated cellulosic enzyme production in a wide array of culture conditions and host strains, potentially reducing costs associated with enzyme production for plant cell wall deconstruction. However, this functionality may require additional engineering in some species

Metabolically Engineering Aspergillus nidulans for Client Protein Production

The filamentous fungi, A. nidulans, can produce nearly 100 grams per liter of industrially relevant proteins under optimal conditions. However, many of these proteins are degraded or produced alongside other proteins, which drastically reduce their efficacy in a cellulose fermentation reaction.The aim of this work is to redesign the regulatory genetic circuitry of Aspergillus nidulans to efficiently produce client proteins. We have successfully reengineered the cellulase regulatory network to produce cellulases in the presence of the C5-sugar xylose. By replacing expensive substrates with a cheap by-product carbon source we reduce enzyme production costs and lower operational costs by eliminating the need for off-site enzyme production, purification, concentration, transport and dilution.We also propose a novel mechanism, utilizing RNA interference, to combinatorially silence genes, which degrade or contaminate client proteins. Using dual promoters, we will flank a sequence containing 30 or 40bp complementary sequences for multiple client genes. This will induce double stranded RNA production, in turn loading these individual complementary sequences into the Argonaute complex, silencing the messenger RNA for each target gene.We have also utilized LC-MS/MS to examine changes in the proteome of our silenced strains. We have seen marked decreases in our target gene sequences as well as the induction of new proteins, acting as a compensation mechanism for the fungus.Our silenced strains, when transformed to produce client proteins, have also had a marked change in the amount of protein produced, as well as how long it lasts in the media during production. We have continued this work by silencing genes responsible for unwanted amylolytic activity in client protein production

How a fungus shapes biotechnology: 100 years of Aspergillus niger research

In 1917, a food chemist named James Currie made a promising discovery: any strain of the filamentous mould Aspergillus niger would produce high concentrations of citric acid when grown in sugar medium. This tricarboxylic acid, which we now know is an intermediate of the Krebs cycle, had previously been extracted from citrus fruits for applications in food and beverage production. Two years after Currie’s discovery, industrial-level production using A. niger began, the biochemical fermentation industry started to flourish, and industrial biotechnology was born. A century later, citric acid production using this mould is a multi-billion dollar industry, with A. niger additionally producing a diverse range of proteins, enzymes and secondary metabolites. In this review, we assess main developments in the field of A. niger biology over the last 100 years and highlight scientific breakthroughs and discoveries which were influential for both basic and applied fungal research in and outside the A. niger community. We give special focus to two developments of the last decade: systems biology and genome editing. We also summarize the current international A. niger research community, and end by speculating on the future of fundamental research on this fascinating fungus and its exploitation in industrial biotechnology.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität Berli

Comparative analysis of the heterologous protein secretion in Aspergillus nidulans

Orientadores: André Ricardo de Lima Damásio, Gabriela Felix PersinotiDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Como forma de diminuir o uso de energias de origem fóssil e aumentar a utilização de energias renováveis, há um grande incentivo mundial recente no desenvolvimento de biorrefinarias, o que envolve processos de conversão integrada de biomassa vegetal e equipamentos para produção de energia, combustíveis e produtos químicos renováveis. Nesta perspectiva o etanol de segunda geração obtido a partir da conversão de biomassa vegetal lignocelulósica em açúcares fermentáveis tem sido foco de muitas pesquisas atualmente a fim de viabilizar seu uso. No entanto, devido à natureza recalcitrante da biomassa lignocelulósica, o custo de processamento corrente na produção de biocombustíveis se torna um importante gargalo a ser superado. Uma das formas de superar este problema é melhorar a produção de enzimas necessárias na etapa de hidrólise enzimática da biomassa vegetal, um processo ainda hoje considerado muito caro. Os fungos filamentosos se destacam na produção e secreção de enzimas lignocelulolíticas de forma homóloga e heteróloga, entretanto pouco se sabe sobre a regulação dos mecanismos de secreção de proteínas nestes microrganismos. Pensando nisso, a utilização do fungo filamentoso modelo Aspergillus nidulans se torna um importante recurso de estudos por ser geneticamente bem caracterizado. Nosso objetivo neste trabalho foi realizar a análise comparativa dos mecanismos de secreção de diferentes proteínas heterólogas em A. nidulans a partir de dados de transcriptoma gerados pela técnica de RNA-seq (Next Generation RNA sequencing). Para monitorar o processo de secreção, adotamos 3 genes alvo: alfa-arabinofuranosidase GH51 (AbfA) e beta-glucosidase GH3 (BglC) de A. fumigatus, e mananase GH5 termofílica (1542) de Thermotoga petrophila. Estes genes foram transformados em A. nidulans, sendo que para AbfA atingiu-se altos níveis de secreção, diferente de BglC e 1542, onde os níveis de secreção foram mínimos. Com isso foram definidos perfis transcriptômicos dos genes relacionados a secreção de proteínas em A. nidulans, representando a resposta da super expressão de três genes heterólogos em diferentes tempos de indução. Em geral houveram processos biológicos super-regulados relacionados principalmente ao metabolismo, proteínas com função ligante, transporte e defesa celulares. Além disso, foram identificados 17 genes diferencialmente expressos envolvidos diretamente com a via de secreção de A. nidulans, representando importantes alvos para manipulação genética deste organismo. Este foi o primeiro estudo que descreve a resposta transcriptômica da secreção de proteínas heterólogas em A. nidulans, gerando uma nova perspectiva ao comparar três diferentes proteínas heterólogas com níveis crescentes de secreçãoAbstract: As a way to reduce the use of fossil fuels and increase the use of renewable energies, there is a major recent worldwide incentive in the development of biorefineries, which involves integrated conversion processes of plant biomass and equipment for the production of energy, fuels and renewable chemicals. In this perspective, the second generation of ethanol obtained from the conversion of lignocellulosic biomass to fermentable sugars has been the focus of many currently researches in order to make its use feasible. However, due to the recalcitrant nature of lignocellulosic biomass, the current processing cost in the production of biofuels becomes an important bottleneck to be overcome. One of the ways to overcome this problem is to improve the production of enzymes required in the enzymatic hydrolysis of plant biomass, a process still considered very expensive nowadays. The filamentous fungi stand out in the production and secretion of lignocellulolytic enzymes in a homologous and heterologous way, however little is known about the regulation of the protein secretion mechanisms in these microorganisms. Considering this, the use of the filamentous fungus model Aspergillus nidulans becomes an important resource of studies to be well genetically characterized. Our objective in this work was to perform a comparative analysis of the mechanisms of secretion of different heterologous proteins in A. nidulans through transcriptome data analysis using RNA-seq technique (Next Generation RNA sequencing). To monitor the secretion process, we adopted 3 target genes: alfa-arabinofuranosidase GH51 (AbfA) and beta-glucosidase GH3 (BglC) from A. fumigatus, and thermophilic mannanase GH5 (1542) from Thermotoga petrophila. These genes were transformed into A. nidulans, and for AbfA high secretion levels were reached, different from BglC and 1542, where secretion levels were minimal. Thereby, transcriptomic profiles of the genes related to protein secretion were defined in A. nidulans, representing the super expression response of three heterologous genes at different induction times. In general there were overrepresented biological processes related mainly to metabolism, protein with binding function, cellular transport and defense. In addition, 17 differentially expressed genes directly involved with the A. nidulans secretion pathway were identified, representing important targets for the genetic manipulation of this organism. This was the first study to describe the transcriptomic response of heterologous protein secretion in A. nidulans, generating a new perspective when comparing three different heterologous proteins with increasing levels of secretionMestradoFármacos, Medicamentos e Insumos para SaúdeMestre em Ciências2014/23051-2FAPES

Shotgun proteomics of Aspergillus niger microsomes upon D-xylose induction

Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which-many of them hypothetical proteins-were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelle

Practical guidance for the implementation of the CRISPR genome editing tool in filamentous fungi

Background: Within the last years, numerous reports described successful application of the CRISPR nucleases Cas9 and Cpf1 for genome editing in filamentous fungi. However, still a lot of efforts are invested to develop and improve protocols for the fungus and genes of interest with respect to applicability, scalability and targeting efficiencies. These efforts are often hampered by the fact that—although many different protocols are available— one have systematically analysed and compared different CRISPR nucleases and different application procedures thereof for the efficiency of single- and multiplex-targeting approaches in the same fungus. Results: We present here data for successful genome editing in the cell factory Thermothelomyces thermophilus, formerly known as Myceliophthora thermophila, using the three different nucleases SpCas9, FnCpf1, AsCpf1 guided to four different gene targets of our interest. These included a polyketide synthase (pks4.2), an alkaline protease (alp1), a SNARE protein (snc1) and a potential transcription factor (ptf1). For all four genes, guide RNAs were developed which enabled successful single-targeting and multiplex-targeting. CRISPR nucleases were either delivered to T. thermophilus on plasmids or preassembled with in vitro transcribed gRNA to form ribonucleoproteins (RNPs). We also evaluated the efficiency of single oligonucleotides for site-directed mutagenesis. Finally, we were able to scale down the transformation protocol to microtiter plate format which generated high numbers of positive transformants and will thus pave the way for future high-throughput investigations. Conclusion: We provide here the first comprehensive analysis and evaluation of different CRISPR approaches for a filamentous fungus. All approaches followed enabled successful genome editing in T. thermophilus; however, with different success rates. In addition, we show that the success rate depends on the respective nuclease and on the targeted gene locus. We finally present a practical guidance for experimental considerations aiming to guide the reader for successful implementation of CRISPR technology for other fungi.TU Berlin, Open-Access-Mittel - 201