Unknown,Transcriptomics,Genomics,Proteomics

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Functional genomics study of Aspergillus niger biofilms for the identification and characterization of genes of lignocellulolytic enzymes of industrial interest

ABSTRACT: Aspergillus niger produces a variety of lignocellulolytic enzymes (cellulases, hemicellulases, among others) and is regarded as cell factory for the production of heterologous proteins. Therefore, there is a growing interest in the study of its genes and the understanding of the cellular mechanisms in order to expand its applications. On the other hand, we have shown that enzyme production by A. niger is higher when grown forming biofilms than when grown conventionally in submerged systems. The objective of this study was to perform a global transcriptomic analysis and an expression analysis of both lignocellulases and biofilm regulatory genes as compared to A. niger in submerged culture. DNA microarray assays were performed to investigate the global gene expression which yielded information on the expression of more than 90% of A. niger genes. To further this comparison, the two culture systems were supplemented with different carbon sources (glucose, lactose, xylose and maltose) to establish a differential gene expression under different culture conditions. Also, to validate the differential expression qPCR was performed for quantitative comparison of the transcriptional level of genes in both culture systems. Organism : Aspergillus niger, Agilent Aspergillus niger Gene expression 4x44k Array AMADID: 032510 Grant Information: Grant NÂº 072-FINCyT-PIN2008 from the National Program of Science and Technology of Peru Contributor: Institut Pasteur de Montevideo, Uruguay

ORGANISM(S): Aspergillus niger

SUBMITTER: Genotypic technology

PROVIDER: E-GEOD-85945 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This work presents an exploration of submerged differentiation of the ubiquitous saprophyte and industrially important fungus, Aspergillus niger, in response to a limited availability of a sole carbon and energy source, maltose. In aspergilli and other mold fungi, asexual reproduction through formation of elaborate conidiogenic structures normally requires an aerial interface. This requirement is bypassed in submerged culture in response to severe nutrient limitation. Continuous cultures with cell retention (retentostat cultures) were applied to generate a fundamental physiological state, where the specific growth rate approaches zero, as the density of the cell population adapts to the supply of the limiting energy source. Temporal differentiation of mycelium structure and commitment to asexual reproduction were major phenomena, apparent on biochemical, morphological, physiological, and transcriptomic level. The severe substrate limitation had a rapid negative impact on cytoplasmic processes, and promoted endo- and exogenous nutrient mobilization, and hyphal compartmentalisation. The first conidiogenic structures appeared after one day with little additional differentiation until Day 4 to 6, where a transition to full commitment to reproductive growth took place. Submerged conidiation in A. niger involved transcriptional regulation of homologs of the regulatory pathway, centered around the Bristle gene (brlA), and structural genes previously described in other aspergilli. Comparison of transcriptomes, revealed a number of co-regulated gene clusters, which appear to encode secondary metabolite biosynthetic potential. We discuss the concept of maintenance energy in the context of differentiation, a possible physiological trigger for sporulation and the special physiological adaptations of the starved mycelium. We also present a simple and efficient method for in situ retention of filamentous organisms. The dataset consists of 9 Affymetrix arrays derived from defined growth conditions of lab-scale bioreactor cultures (5L). Total RNA was extracted from biomass harvested at three different growth phases: exponential growth phase, 2 and 8 days of retentostat cultivation. For each of the phases, the data is derived from three biological replicates.

Project description:Background: Saprobic fungi are the predominant industrial sources of Carbohydrate Active enZymes (CAZymes) used for the saccharification of lignocellulose during the production of second generation biofuels. The production of more effective enzyme cocktails is a key objective for efficient biofuel production. To achieve this objective, it is crucial to understand the response of fungi to lignocellulose substrates. Our previous study used RNA-seq to identify the genes induced in Aspergillus niger in response to wheat straw, a biofuel feedstock, and showed that the range of genes induced was greater than previously seen with simple inducers [GSE33852]. Results: In this work we used RNA-seq to identify the genes induced in A. niger in response to short rotation coppice willow and compared this with the response to wheat straw from our previous study, at the same time-point. The response to willow showed a large increase in expression of genes encoding CAZymes. Genes encoding the major activities required to saccharify lignocellulose were induced on willow such as endoglucanases, cellobiohydrolases and xylanases. The transcriptome response to willow had many similarities with the response to straw with some significant differences in the expression levels of individual genes which are discussed in relation to differences in substrate composition or other factors. Differences in transcript levels include higher levels on wheat straw from genes encoding enzymes classified as members of GH62 (an arabinofuranosidase) and CE1 (a feruloyl esterase) CAZy families whereas two genes encoding endoglucanases classified as members of the GH5 family had higher transcript levels when exposed to willow. There were changes in the cocktail of enzymes secreted by A. niger when cultured with willow or straw. Assays for particular enzymes as well as saccharification assays were used to compare the enzyme activities of the cocktails. Wheat straw induced an enzyme cocktail that saccharified wheat straw to a greater extent than willow. Genes not encoding CAZymes were also induced on willow such as hydrophobins as well as genes of unknown function. Several genes were identified as promising targets for future study. Conclusions: By comparing this first study of the global transcriptional response of a fungus to willow with the response to straw, we have shown that the inducing lignocellulosic substrate has a marked effect upon the range of transcripts and enzymes expressed by A. niger. The use by industry of complex substrates such as wheat straw or willow could benefit efficient biofuel production. Six samples in total consisting of duplicate shake flask Aspergillus niger cultures from three conditions: glucose 48 h, willow 24 h, willow 24 h + glucose 5 h

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Functional genomics study of Aspergillus niger biofilms for the identification and characterization of genes of lignocellulolytic enzymes of industrial interest

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