The feruloyl esterase B gene (faeB) is specifically induced by hydroxycinnamic acids (e.g. ferulic acid, caffeic acid and coumaric acid) but the transcriptional regulation network involved in faeB induction and ferulic acid metabolism has only been partially addressed. To identify transcription factors involved in ferulic acid metabolism we constructed and screened a transcription factor knockout library of 239 Aspergillus niger strains for mutants unable to utilize ferulic acid as a carbon source. The ΔfarA transcription factor mutant, already known to be involved in fatty acid metabolism, could not utilize ferulic acid and other hydroxycinnamic acids. In addition to screening the transcription factor mutant collection, a forward genetic screen was performed to isolate mutants unable to express faeB. For this screen a PfaeB-amdS and PfaeB-lux613 dual reporter strain was engineered. The rationale of the screen is that in this reporter strain ferulic acid induces amdS (acetamidase) expression via the faeB promoter resulting in lethality on fluoro-acetamide. Conidia of this reporter strain were UV-mutagenized and plated on fluoro-acetamide medium in the presence of ferulic acid. Mutants unable to induce faeB are expected to be fluoro-acetamide resistant and can be positively selected for. Using this screen, six fluoro-acetamide resistant mutants were obtained and phenotypically characterized. Three mutants had a phenotype identical to the farA mutant and sequencing the farA gene in these mutants indeed showed mutations in FarA which resulted in inability to growth on ferulic acid as well as on short and long chain fatty acids. The growth phenotype of the other three mutants was similar to the farA mutants in terms of the inability to grow on ferulic acid, but these mutants grew normally on short and long chain fatty acids. The genomes of these three mutants were sequenced and allelic mutations in one particular gene (NRRL3_09145) were found. The protein encoded by NRRL3_09145 shows similarity to the FarA and FarB transcription factors. However, whereas FarA and FarB contain both the Zn(II)2Cys6 domain and a fungal-specific transcription factor domain, the protein encoded by NRRL3_09145 (FarD) lacks the canonical Zn(II)2Cys6 domain and possesses only the fungal specific transcription factor domain.

Two-stage pretreatment conditions were optimized to convert corn fiber, separated from whole stillage in a corn dry grind ethanol plant, to fermentable sugars via hydrolysis. Liquid hot water pretreatment (25% solids) at 180 degrees C for 10 min, followed by three cycles of disk milling, provided maximum glucose, xylose, and arabinose yields of 88.5%, 41.0%, and 30.4% respectively after hydrolysis with Cellulase I. The glucose, xylose, and arabinose yields with Cellulase II at optimum conditions were 94.9%, 74.2%, and 66.3%, respectively. SSF of corn fiber using engineered yeast, with both Cellulase I and II, provided maximum ethanol concentrations of 2.13% and 2.73% (v/v). The protein content in the residual solid after fermentation was 47.95% and 52.05% for Cellulase I and II, respectively. This technology provides additional ethanol in a dry grind plant by converting corn fiber into ethanol and increases the protein content of DDGS, thereby improving the quality.

In this study, two acid catalysts, acetic acid (HAc) and sulfuric acid (H2SO4), were compared in thermal pretreatments of corn stover, in particular to assess the less understood fate of lignin. HAc-insoluble lignin, analyzed by pyrolysis GC-MS, showed decreasing levels (%) of C alpha-oxidized (from 3.7 +/- 0.2 to 1.8 +/- 0.1), propenyl (from 2.5 +/- 0.1 to 1.0 +/- 0.1), vinyl-G (from 34.5 +/- 1.8 to 28.4 +/- 0.9), vinyl-S (from 4.2 +/- 0.2 to 3.7 +/- 0.1) and methylated (from 4.9 +/- 0.04 to 2.8 +/- 0.1) lignin units at increasing HAc amounts. Concurrently, unsubstituted and vinyl-H units increased (from 7.5 +/- 0.5 to 11.3 +/- 0.2 and from 40.5 +/- 1.9 to 49.9 +/- 0.9, respectively). Similar trends were seen for residual lignin in H2SO4 catalyzed pretreatments, although the composition differed from that of residual HAc-lignin. In particular, H2SO4-lignin showed slightly lower values (%) for unsubstituted (9.9 +/- 0.2) and vinyl-H (45.7 +/- 4.1) units, while C alpha-oxidized (3.4 +/- 0.4), propenyl (1.9 +/- 0.1), vinyl-G (28.5 +/- 0.9), vinyl-S (4.4 +/- 0.6) and methylated (4.6 +/- 0.2) lignin units remained higher compared to HAc-catalysis at similar pH values. Xylan yields and corresponding enzymatic conversions of the solids were similar regardless the type of acid. Our findings show that HAc in pretreatments decreased lignin complexity, possibly due to cleavage reactions, although subsequent recondensation reactions increased solid lignin yields, more than H2SO4, while removal of xylan and enzymatic conversion of solids were equal.

Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large set, over 600 homologous and nearly 2,000 heterologous fungal genes, were overexpressed in Aspergillus niger using a standardized expression cassette and scored for high versus no production. Subsequently, sequence-based machine learning techniques were applied for identifying relevant DNA and protein sequence features. The amino-acid composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important: tyrosine and asparagine composition was found to have a positive correlation with high-level production, whereas for unsuccessful production, contributions were found for methionine and lysine composition. The predictor is available online at http://bioinformatics.tudelft.nl/hipsec. Subsequent work aims at validating these findings by protein engineering as a method for increasing expression levels per gene copy.

Material Supplemental http://genome.cshlp.org/content/suppl/2011/04/07/gr.112169.110.DC1.html References http://genome.cshlp.org/content/21/6/885.full.html#ref-list-1 This article cites 63 articles, 17 of which can be accessed free at: Open Access Freely available online through the Genome Research Open Access option. service Email alerting click here top right corner of the article or Receive free email alerts when new articles cite this article sign up in the box at the

The cell-factory Aspergillus niger is widely used for industrial enzyme production. To select potential proteins for large-scale production, we developed a sequence-based classifier that predicts if an over-expressed homologous protein will successfully be produced and secreted. A dataset of 638 proteins was used to train and validate a classifier, using a 10-fold cross-validation protocol. Using a linear discriminant classifier, an average accuracy of 0.85 was achieved. Feature selection results indicate what features are mostly defining for successful protein production, which could be an interesting lead to couple sequence characteristics to biological processes involved in protein production and secretion

The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing β-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.

Genetic recombination is an important tool in strain breeding in many organisms. We studied the possibilities of mitotic recombination in strain breeding of the asexual fungus Aspergillus niger. By identifying genes that complemented mapped auxotrophic mutations, the physical map was compared to the genetic map of chromosome III using the genome sequence. In a program to construct a chromosome III-specific marker strain by selecting mitotic crossing-over in diploids, a mitotic recombination hotspot was identified. Analysis of the mitotic recombination hotspot revealed some physical features, elevated basal transcription and a possible correlation with purine stretches.

Herman J Pel1, Johannes H de Winde1,2, David B Archer3, Paul S Dyer3, Gerald Hofmann4, Peter J Schaap5, Geoffrey Turner6, Ronald P de Vries7, Richard Albang8, Kaj Albermann8, Mikael R Andersen4, Jannick D Bendtsen9, Jacques A E Benen5, Marco van den Berg10, Stefaan Breestraat1, Mark X Caddick11, Roland Contreras12, Michael Cornell13, Pedro M Coutinho14, Etienne G J Danchin14, Alfons J M Debets15, Peter Dekker1, Piet W M van Dijck1, Alard van Dijk1, Lubbert Dijkhuizen16,17, Arnold J M Driessen17, Christophe d’Enfert18, Steven Geysens12, Coenie Goosen16,17, Gert S P Groot1, Piet W J de Groot19, Thomas Guillemette20, Bernard Henrissat14, Marga Herweijer1, Johannes P T W van den Hombergh1, Cees A M J J van den Hondel21, Rene T J M van der Heijden22, Rachel M van der Kaaij16,17, Frans M Klis19, Harrie J Kools5, Christian P Kubicek23, Patricia A van Kuyk21, Jürgen Lauber24, Xin Lu25, Marc J E C van der Maarel16, Rogier Meulenberg1, Hildegard Menke1, Martin A Mortimer11, Jens Nielsen4, Stephen G Oliver13, Maurien Olsthoorn1, Karoly Pal15,26, Noël N M E van Peij1, Arthur F J Ram21, Ursula Rinas25, Johannes A Roubos1, Cees M J Sagt1, Monika Schmoll23, Jibin Sun25, David Ussery27, Janos Varga26,28, Wouter Vervecken12, Peter J J van de Vondervoort21, Holger Wedler24, Han A B Wösten7, An-Ping Zeng25, Albert J J van Ooyen1, Jaap Visser29 & Hein Stam1

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.