Functional genomics to study protein secretion stress in Aspergillus niger Silva Pinheiro Carvalho, N.D. Citation Silva Pinheiro Carvalho, N. D. (2011, June 7). Functional genomics to study protein secretion stress in Aspergillus niger. Retrieved from https://hdl.handle.net/1887/17685 Version: Corrected Publisher’s Version Licence agreement concerning inclusion of doctoral thesis in the License: Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/17685 Note: To cite this publication please use the final published version (if applicable). Summary Summary & Samenvatting & Supplementary material 122 Summary & Samenvatting & Supplementary material Summary Summary In its natural habitat, Aspergillus niger usually grows on complex plant biopolymers. To be able to consume these nutrients, the fungus secretes a wide range of enzymes like amylases, xylanases, pectinases, cellulases, proteases, to break down the polymers into smaller molecules (monosaccharides or amino acids), that can be then taken up by the cell. The high secretion capacity of A. niger has drawn a lot of research towards the production of homologous and heterologous proteins to be used in different branches of the food, medical and textile industries. While the production yields of homologous protein are often high, the production of heterologous proteins has revealed more problematic. Several studies suggest that these problems usually arise post-translationally, within different steps of the secretory pathway. A major bottleneck is the protein synthesis and subsequent folding of secretory proteins that takes place in the ER. If the flow of new proteins into this organelle is higher than the rate at which the proteins are folded and delivered to the next organelle in the pathway, proteins begin to accumulate in the ER lumen, posing a risk to the cell. To counteract this stress, the UPR pathway is triggered, which results in activating the HacA transcription factor responsible for the induced expression of foldases and chaperones that support protein folding. At the ER, proteins also encounter quality control checkpoints that validate their aptness to continue in the secretory pathway. Misfolded proteins are recognized and targeted to the ERAD pathway to become degraded by the proteasome. In this thesis we have focused our studies towards a better understanding of different processes involved in the protein secretion pathway that might act as bottlenecks for homologous and heterologous protein production. We have given particular attention to the molecular mechanisms of folding and quality control that take place in the ER, in order to be able to improve and develop new strategies for heterologous protein production by A. niger . In Chapter 1 a general outline is given of the secretory pathway of eukaryotes, with special attention to the UPR and ERAD pathways. Additionally, an overview of the commonly pointed bottlenecks that have been encountered on protein production, together with the current tools used by the modern science to broaden the knowledge on filamentous fungi. To generate gene knock-out mutants in filamentous fungi through homologous recombination is very time consuming which hampers the functional analysis of predicted genes from genome sequences. In Chapter 2 we present several methods that will boast functional genomics in A. niger . We describe the construction of a set of strains where the kusA gene has been deleted, resulting in recipient strains in which deletions of essential and non-essential genes can be easily achieved, as shown in the case of the ireA and hacA genes, respectively. To overcome the disadvantages of having a compromised NHEJ pathway, we also describe the construction of a transiently disrupted kusA strain. For complementation of ∆kusA mutants we present an approach based on autonomously replicating plasmids, in which the mutant phenotype can be maintained or lost by regulating (on/off) the selective pressure. Chapter 3 describes the cloning and functional characterization of the A. niger gmtA gene. Phylogenetic analysis showed that gmtA clusters 123 Summary Summary & Samenvatting & Supplementary material together with well characterized GDP-mannose transporters from other fungal species and YFP tagging of GmtA proved its localization at Golgi equivalents. The isolation and characterization of a secretion related small GTPase, srgC , is also described. The ∆srgC mutant phenotype showed reduced growth and the inability to form conidiospores at high temperatures and in addition, our microscopy results point to a role of SrgC in the organization, morphology and Golgi functions. Chapter 4 describes the effect of the deletion of five genes involved in ERAD under i) normal growth conditions, ii) conditions of chemically induced ER stress and iii) by expressing an UPR-inducing heterologous protein (Glucoamylase-Gus). The results indicate that a functional ERAD pathway is not required for normal growth, but a defective ERAD pathway increases intracellular levels of the heterologous protein in study, indicating that this quality control pathway might impose a bottleneck on heterologous protein production by being (partially) responsible for their degradation. In Chapter 5, we have focused our study on i) the physiology of a genetically engineered A. niger strain that expresses only the activated form of the transcription factor HacA and ii) its transcriptome profile obtained using Affymetrix GeneChip analysis. GO enrichment analysis reflected a broader role of HacA in the cells, leading on one hand to the enrichment of secretory pathway processes (e.g.: folding and secretion) and an expansion of the endomembrane system ((phospho)lipid and inositol metabolism); on the other hand, a putative involvement of HacA on the negative regulation of extracellular enzymes expression (RESS mechanism) to counteract secretion stress. In the last Chapter ( Chapter 6 ) we summarize and discuss the main conclusions of this thesis and look ahead on how our studies on UPR and ERAD might help us to further improve heterologous protein production in A. niger 124 Summary & Samenvatting & Supplementary material Samenvatting Samenvatting Aspergillus niger groeit in zijn natuurlijke omgeving op complexe plantaardige biopolymeren. Om deze voedingsstoffen te kunnen consumeren, scheidt de schimmel een verscheidenheid aan enzymen uit, zoals amylases, xylanases, pectinases, cellulases en proteases, om de polymeren af te breken in kleinere moleculen (monosacchariden of aminozuren) die door de cel kunnen worden opgenomen. De hoge secretie capaciteit van A. niger heeft er voor gezorgd dat er veel onderzoek wordt gedaan naar de productie van homologe en heterologe eiwitten die kunnen worden gebruikt in verschillende takken van de medische-, textiel- en voedingsindustrie. De hoeveelheid geproduceerde homologe eiwitten is vaak groot, terwijl de opbrengst van heterologe eiwitten vaak problematisch laag is. Verscheidene studies wijzen erop dat deze problemen post-translationeel zijn en plaatsvinden tijdens verscheidene stappen in de secretie route. Een van de grootste problemen is de eiwitsynthese en de hierop volgende vouwing van secretie eiwitten in het endoplasmatisch reticulum (ER). Als de stroom van nieuwe eiwitten groter is dan het tempo waarin deze eiwitten kunnen worden gevouwen en afgeleverd bij het volgende organel in de route, vindt er eiwit accumulatie plaats, wat stress veroorzaakt in de cel. Om deze stress tegen te gaan wordt de Unfolded Protein Response (UPR) route geactiveerd, resulterend in activatie van de transcriptie factor HacA, welke verantwoordelijk is voor de geïnduceerde expressie van foldases en chaperones betrokken bij eiwit vouwing. In het ER worden eiwitten ook gecontroleerd of ze geschikt zijn om de secretieroute in te gaan. Niet goed gevouwen eiwitten worden herkend en via de ER Associated Degradation (ERAD) route en afgebroken in het proteasoom. In dit proefschrift hebben we onze studie gericht op het beter begrijpen van de verschillende processen die betrokken zijn bij eiwit secretie en mogelijk de oorzaak zijn van lage eiwitproductie. We hebben vooral onze aandacht gericht op het moleculaire mechanisme van eiwit vouwing en kwaliteitscontrole in het ER, om zo in staat te zijn nieuwe strategieën voor heterologe eiwitproductie in A. niger te ontwikkelen en te verbeteren. In Hoofdstuk 1 wordt een algemeen overzicht gegeven van de secretie route in eukaryoten, met speciale aandacht voor de UPR en ERAD routes. Bovendien worden de knelpunten besproken die zich voordoen bij eiwitproductie, samen met de huidige technieken die in de moderne wetenschap worden gebruikt om de kennis van filamenteuze schimmels te vergroten. Het verkrijgen van gen deletie mutanten door middel van homologe recombinatie is tijdrovend en hierdoor limiterend voor de functionele analyse van voorspelde genen in genoom sequenties. In Hoofdstuk 2 presenteren we verschillende methoden die de functionele genomics in A. niger kunnen verbeteren. We beschrijven de constructie van een aantal stammen waarin het kusA gen is gedeleteerd, resulterend in stammen waar deleties van essentiële en non-essentiële genen gemakkelijk kan worden verwezenlijkt, zoals met de ireA en hacA genen aangetoond is. Om de nadelen van een gecomprimeerde NHEJ route te overbruggen beschrijven we ook de constructie van een omkeerbare kusA deletie stam. Voor complementatie van ∆kusA mutanten stellen we een benadering voor die gebruik maakt van autoreplicerende plasmiden