Development of an unmarked gene deletion system for the filamentous fungi Aspergillus niger and Talaromyces versatilis.
ABSTRACT: In this article, we present a method to delete genes in filamentous fungi that allows recycling of the selection marker and is efficient in a nonhomologous end-joining (NHEJ)-proficient strain. We exemplify the approach by deletion of the gene encoding the transcriptional regulator XlnR in the fungus Aspergillus niger. To show the efficiency and advantages of the method, we deleted 8 other genes and constructed a double mutant in this species. Moreover, we showed that the same principle also functions in a different genus of filamentous fungus (Talaromyces versatilis, basionym Penicillium funiculosum). This technique will increase the versatility of the toolboxes for genome manipulation of model and industrially relevant fungi.
Project description:BACKGROUND:Research on filamentous fungi emphasized the remarkable redundancy in genes encoding hydrolytic enzymes, the similarities but also the large differences in their expression, especially through the role of the XlnR/XYR1 transcriptional activator. The purpose of this study was to evaluate the specificities of the industrial fungus Talaromyces versatilis, getting clues into the role of XlnR and the importance of glucose repression at the transcriptional level, to provide further levers for cocktail production. RESULTS:By studying a set of 62 redundant genes representative of several categories of enzymes, our results underlined the huge plasticity of transcriptional responses when changing nutritional status. As a general trend, the more heterogeneous the substrate, the more efficient to trigger activation. Genetic modifications of xlnR led to significant reorganisation of transcriptional patterns. Just a minimal set of genes actually fitted in a simplistic model of regulation by a transcriptional activator, and this under specific substrates. On the contrary, the diversity of xlnR+ versus ?xlnR responses illustrated the existence of complex and unpredicted patterns of co-regulated genes that were highly dependent on the culture condition, even between genes that encode members of a functional category of enzymes. They notably revealed a dual, substrate-dependant repressor-activator role of XlnR, with counter-intuitive transcripts regulations that targeted specific genes. About glucose, it appeared as a formal repressive sugar as we observed a massive repression of most genes upon glucose addition to the mycelium grown on wheat straw. However, we also noticed a positive role of this sugar on the basal expression of a few genes, (notably those encoding cellulases), showing again the strong dependence of these regulatory mechanisms upon promoter and nutritional contexts. CONCLUSIONS:The diversity of transcriptional patterns appeared to be the rule, while common and stable behaviour, both within gene families and with fungal literature, the exception. The setup of a new biotechnological process to reach optimized, if not customized expression patterns of enzymes, hence appeared tricky just relying on published data that can lead, in the best scenario, to approximate trends. We instead encourage preliminary experimental assays, carried out in the context of interest to reassess gene responses, as a mandatory step before thinking in (genetic) strategies for the improvement of enzyme production in fungi.
Project description:<h4>Background</h4>Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus.<h4>Results</h4>Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ?cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome.<h4>Conclusions</h4>In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.
Project description:Filamentous fungi are important producers of plant polysaccharide degrading enzymes that are used in many industrial applications. These enzymes are produced by the fungus to liberate monomeric sugars that are used as carbon source. Two of the main components of plant polysaccharides are L-arabinose and D-xylose, which are metabolized through the pentose catabolic pathway (PCP) in these fungi. In Aspergillus niger, the regulation of pentose release from polysaccharides and the PCP involves the transcriptional activators AraR and XlnR, which are also present in other Aspergilli such as Aspergillus nidulans. The comparative analysis revealed that the regulation of the PCP by AraR differs in A. nidulans and A. niger, whereas the regulation of the PCP by XlnR was similar in both species. This was demonstrated by the growth differences on L-arabinose between disruptant strains for araR and xlnR in A. nidulans and A. niger. In addition, the expression profiles of genes encoding L-arabinose reductase (larA), L-arabitol dehydrogenase (ladA) and xylitol dehydrogenase (xdhA) differed in these strains. This data suggests evolutionary changes in these two species that affect pentose utilisation. This study also implies that manipulating regulatory systems to improve the production of polysaccharide degrading enzymes, may give different results in different industrial fungi.
Project description:A critical step in the RT-qPCR workflow for studying gene expression is data normalization, one of the strategies being the use of reference genes. This study aimed to identify and validate a selection of reference genes for relative quantification in Talaromyces versatilis, a relevant industrial filamentous fungus. Beyond T. versatilis, this study also aimed to propose reference genes that are applicable more widely for RT-qPCR data normalization in filamentous fungi.A selection of stable, potential reference genes was carried out in silico from RNA-seq based transcriptomic data obtained from T. versatilis. A dozen functionally unrelated candidate genes were analysed by RT-qPCR assays over more than 30 relevant culture conditions. By using geNorm, we showed that most of these candidate genes had stable transcript levels in most of the conditions, from growth environments to conidial germination. The overall robustness of these genes was explored further by showing that any combination of 3 of them led to minimal normalization bias. To extend the relevance of the study beyond T. versatilis, we challenged their stability together with sixteen other classically used genes such as ?-tubulin or actin, in a representative sample of about 100 RNA-seq datasets. These datasets were obtained from 18 phylogenetically distant filamentous fungi exposed to prevalent experimental conditions. Although this wide analysis demonstrated that each of the chosen genes exhibited sporadic up- or down-regulation, their hierarchical clustering allowed the identification of a promising group of 6 genes, which presented weak expression changes and no tendency to up- or down-regulation over the whole set of conditions. This group included ubcB, sac7, fis1 and sarA genes, as well as TFC1 and UBC6 that were previously validated for their use in S. cerevisiae.We propose a set of 6 genes that can be used as reference genes in RT-qPCR data normalization in any field of fungal biology. However, we recommend that the uniform transcription of these genes is tested by systematic experimental validation and to use the geometric averaging of at least 3 of the best ones. This will minimize the bias in normalization and will support trustworthy biological conclusions.
Project description:There is an urgent requirement for second-generation bio-based industries for economical yet efficient enzymatic cocktail to convert diverse cellulosic biomass into fermentable sugars. In our previous study, secretome of Penicillium funiculosum NCIM1228 showed high commercial potential by exhibiting high biomass hydrolyzing efficiency. To develop NCIM1228 further as an industrial workhorse, one of the major genetic interventions needed is global deregulation of cellulolytic genes to achieve higher enzyme production. Mig1 orthologs found in all yeast and filamentous fungi are transcriptional regulators that maintain carbon homeostasis by negatively regulating genes of secondary carbon source utilization. Their disruption has long been known to be beneficial for increasing the production of secreted enzymes for alternate carbon source utilization.Upon detailed genotypic and phenotypic analysis, we observed that NCIM1228 harbors a truncated yet functional allele of homolog of a well-known catabolite repressor, Mig1. Alleviation of carbon repression in NCIM1228 was attained by replacing functional Mig1134 allele with null allele Mig188. P. funiculosum having Mig188 null allele showed better growth characteristics and 1.75-fold better glucose utilization than parent strain. We also showed that visibly small colony size, one of the major characteristics of CCR disruptant strains in filamentous fungi, was not due to retarded growth, but altered hyphal morphology. CCR-disrupted strain PfMig188 showed profuse branching pattern in terminal hyphae resulting in small and compact colonies with compromised filamentous proliferation. We further observed that basal level expression of two major classes of cellulases, namely, cellobiohydrolase and endoglucanase, was regulated by Mig1134 in NCIM1228, whereas other two major classes, namely, xylanases and β-glucosidase, were only marginally regulated. Finally, CCR disruption in P. funiculosum NCIM1228 led to prolonged cellulase induction in production medium resulting in twofold increased cellulase activity than the parent strain with maximum secreted protein titer being > 14 g/l.CCR-disrupted P. funiculosum showed better growth, enhanced carbon source utilization, profuse branching pattern in terminal hyphae, and higher cellulase activity than parent strain. Our findings are particularly important in shedding light on important functions performed by Mig1 in addition to its role as negative regulator of alternate carbon source utilization in filamentous fungi.
Project description:Aspergilli are commonly found in soil and on decaying plant material. D-xylose and L-arabinose are highly abundant components of plant biomass. They are released from polysaccharides by fungi using a set of extracellular enzymes and subsequently converted intracellularly through the pentose catabolic pathway (PCP).In this study, the L-arabinose responsive transcriptional activator (AraR) is identified in Aspergillus niger and was shown to control the L-arabinose catabolic pathway as well as expression of genes encoding extracellular L-arabinose releasing enzymes. AraR interacts with the D-xylose-responsive transcriptional activator XlnR in the regulation of the pentose catabolic pathway, but not with respect to release of L-arabinose and D-xylose.AraR was only identified in the Eurotiales, more specifically in the family Trichocomaceae and appears to have originated from a gene duplication event (from XlnR) after this order or family split from the other filamentous ascomycetes. XlnR is present in all filamentous ascomycetes with the exception of members of the Onygenales. Since the Onygenales and Eurotiales are both part of the subclass Eurotiomycetidae, this indicates that strong adaptation of the regulation of pentose utilisation has occurred at this evolutionary node. In Eurotiales a unique two-component regulatory system for pentose release and metabolism has evolved, while the regulatory system was lost in the Onygenales. The observed evolutionary changes (in Eurotiomycetidae) mainly affect the regulatory system as in contrast, homologues for most genes of the L-arabinose/D-xylose catabolic pathway are present in all the filamentous fungi, irrespective of the presence of XlnR and/or AraR.
Project description:The expression of genes encoding enzymes involved in xylan degradation and two endoglucanases involved in cellulose degradation was studied at the mRNA level in the filamentous fungus Aspergillus niger. A strain with a loss-of-function mutation in the xlnR gene encoding the transcriptional activator XlnR and a strain with multiple copies of this gene were investigated in order to define which genes are controlled by XlnR. The data presented in this paper show that the transcriptional activator XlnR regulates the transcription of the xlnB, xlnC, and xlnD genes encoding the main xylanolytic enzymes (endoxylanases B and C and beta-xylosidase, respectively). Also, the transcription of the genes encoding the accessory enzymes involved in xylan degradation, including alpha-glucuronidase A, acetylxylan esterase A, arabinoxylan arabinofuranohydrolase A, and feruloyl esterase A, was found to be controlled by XlnR. In addition, XlnR also activates transcription of two endoglucanase-encoding genes, eglA and eglB, indicating that transcriptional regulation by XlnR goes beyond the genes encoding xylanolytic enzymes and includes regulation of two endoglucanase-encoding genes.
Project description:Fungi are ubiquitous and are often confronted with the need to secure utilisable carbon from their external growth milieu through the use of extracellular proteins to scavenge for carbon from a vast array of complex polymeric carbon sources. This attribute is conserved across evolution in fungi. To understand how filamentous fungi extracellular proteins are modulated in response to the presence polymeric carbons in the environment, we have typed the array of the main extracellular proteins involved and their dynamics using a known hypercellulolytic fungus – Penicillium funiculosum (NCIM 1228), through multiplexed quantitative proteomics
Project description:Under the rules for the naming of fungi with pleomorphic life-cycles adopted in July 2011, the nomenclaturally correct name for the fungus causing the current ash dieback in Europe is determined to be Hymenoscyphus fraxineus, with the basionym Chalara fraxinea, and Hymenoscyphus pseudoalbidus as a taxonomic synonym of H. fraxineus.
Project description:Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular ?-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a "seesaw model" in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors.