Publications by authors named "Peter J Punt"

66 Publications

Deletion of the Pro-Protein Processing Protease Gene Results in a pH-Dependent Morphological Transition during Submerged Cultivations and Increases Cell Wall Chitin Content.

Microorganisms 2020 Dec 2;8(12). Epub 2020 Dec 2.

Institute of Biology Leiden, Microbial Sciences, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.

There is a growing interest in the use of post-fermentation mycelial waste to obtain cell wall chitin as an added-value product. In the pursuit to identify suitable production strains that can be used for post-fermentation cell wall harvesting, we turned to an strain in which the gene was deleted. Previous work has shown that the deletion of causes hyper-branching and thicker cell walls, traits that may be beneficial for the reduction in fermentation viscosity and lysis. Hyper-branching of was previously found to be pH-dependent on solid medium at pH 6.0, but was absent at pH 5.0. This phenotype was reported to be less pronounced during submerged growth. Here, we show a series of controlled batch cultivations at a pH range of 5, 5.5, and 6 to examine the pellet phenotype of in liquid medium. Morphological analysis showed that formed wild type-like pellets at pH 5.0, whereas the hyper-branching phenotype was found at pH 6.0. The transition of phenotypic plasticity was found in cultivations at pH 5.5, seen as an intermediate phenotype. Analyzing the cell walls of from these controlled pH-conditions showed an increase in chitin content compared to the wild type across all three pH values. Surprisingly, the increase in chitin content was found to be irrespective of the hyper-branching morphology. Evidence for alterations in cell wall make-up are corroborated by transcriptional analysis that showed a significant cell wall stress response in addition to the upregulation of genes encoding other unrelated cell wall biosynthetic genes.
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http://dx.doi.org/10.3390/microorganisms8121918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761569PMC
December 2020

Carbohydrate Binding Modules: Diversity of Domain Architecture in Amylases and Cellulases From Filamentous Microorganisms.

Front Bioeng Biotechnol 2020 31;8:871. Epub 2020 Jul 31.

Department of Microbial Biotechnology, Institute of Biology Leiden, Leiden, Netherlands.

Enzymatic degradation of abundant renewable polysaccharides such as cellulose and starch is a field that has the attention of both the industrial and scientific community. Most of the polysaccharide degrading enzymes are classified into several glycoside hydrolase families. They are often organized in a modular manner which includes a catalytic domain connected to one or more carbohydrate-binding modules. The carbohydrate-binding modules (CBM) have been shown to increase the proximity of the enzyme to its substrate, especially for insoluble substrates. Therefore, these modules are considered to enhance enzymatic hydrolysis. These properties have played an important role in many biotechnological applications with the aim to improve the efficiency of polysaccharide degradation. The domain organization of glycoside hydrolases (GHs) equipped with one or more CBM does vary within organisms. This review comprehensively highlights the presence of CBM as ancillary modules and explores the diversity of GHs carrying one or more of these modules that actively act either on cellulose or starch. Special emphasis is given to the cellulase and amylase distribution within the filamentous microorganisms from the genera of and that are well known to have a great capacity for secreting a wide range of these polysaccharide degrading enzyme. The potential of the CBM and other ancillary domains for the design of improved polysaccharide decomposing enzymes is discussed.
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http://dx.doi.org/10.3389/fbioe.2020.00871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410926PMC
July 2020

A seven-membered cell wall related transglycosylase gene family in is relevant for cell wall integrity in cell wall mutants with reduced α-glucan or galactomannan.

Cell Surf 2020 Dec 21;6:100039. Epub 2020 Mar 21.

Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, the Netherlands.

Chitin is an important fungal cell wall component that is cross-linked to β-glucan for structural integrity. Acquisition of chitin to glucan cross-links has previously been shown to be performed by transglycosylation enzymes in , called Congo Red hypersensitive (Crh) enzymes. Here, we characterized the impact of deleting all seven members of the gene family () in on cell wall integrity, cell wall composition and genome-wide gene expression. In this study, we show that the seven-fold knockout strain shows slightly compact growth on plates, but no increased sensitivity to cell wall perturbing compounds. Additionally, we found that the cell wall composition of this knockout strain was virtually identical to that of the wild type. In congruence with these data, genome-wide expression analysis revealed very limited changes in gene expression and no signs of activation of the cell wall integrity response pathway. However, deleting the entire gene family in cell wall mutants that are deficient in either galactofuranose or α-glucan, mainly α-1,3-glucan, resulted in a synthetic growth defect and an increased sensitivity towards Congo Red compared to the parental strains, respectively. Altogether, these results indicate that loss of the gene family in does not trigger the cell wall integrity response, but does play an important role in ensuring cell wall integrity in mutant strains with reduced galactofuranose or α-glucan.
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http://dx.doi.org/10.1016/j.tcsw.2020.100039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7389268PMC
December 2020

Interrogation of the cell wall integrity pathway in identifies a putative negative regulator of transcription involved in chitin deposition.

Gene X 2020 Dec 28;5:100028. Epub 2020 Jan 28.

Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, the Netherlands.

Post-fermentation fungal biomass waste provides a viable source for chitin. Cell wall chitin of filamentous fungi, and in particular its de-acetylated derivative chitosan, has a wide range of commercial applications. Although the cell wall of filamentous fungi comprises 10-30% chitin, these yields are too low for cost-effective production. Therefore, we aimed to identify the genes involved in increased chitin deposition by screening a collection of UV-derived cell wall mutants in . This screen revealed a mutant strain (RD15.4#55) that showed a 30-40% increase in cell wall chitin compared to the wild type. In addition to the cell wall chitin phenotype, this strain also exhibited sensitivity to SDS and produces an unknown yellow pigment. Genome sequencing combined with classical genetic linkage analysis identified two mutated genes on chromosome VII that were linked with the mutant phenotype. Single gene knockouts and subsequent complementation analysis revealed that an 8 bp deletion in NRRL3_09595 is solely responsible for the associated phenotypes of RD15.4#55. The mutated gene, which was named (), encodes an orthologue of Bypass of (), a negative regulator of transcription elongation. We propose that this conserved fungal protein is involved in preventing cell wall integrity signaling under non-inducing conditions, where loss of function results in constitutive activation of the cell wall stress response pathway, and consequently leads to increased chitin content in the mutant cell wall.
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http://dx.doi.org/10.1016/j.gene.2020.100028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285910PMC
December 2020

Profile Comparer Extended: phylogeny of lytic polysaccharide monooxygenase families using profile hidden Markov model alignments.

F1000Res 2019 31;8:1834. Epub 2019 Oct 31.

Department of Microbial Biotechnology and Health, Insitute of Biology Leiden, Leiden, 2333BE, The Netherlands.

Insight into the inter- and intra-family relationship of protein families is important, since it can aid understanding of substrate specificity evolution and assign putative functions to proteins with unknown function. To study both these inter- and intra-family relationships, the ability to build phylogenetic trees using the most sensitive sequence similarity search methods (e.g. profile hidden Markov model (pHMM)-pHMM alignments) is required. However, existing solutions require a very long calculation time to obtain the phylogenetic tree. Therefore, a faster protocol is required to make this approach efficient for research. To contribute to this goal, we extended the original Profile Comparer program (PRC) for the construction of large pHMM phylogenetic trees at speeds several orders of magnitude faster compared to pHMM-tree. As an example, PRC Extended (PRCx) was used to study the phylogeny of over 10,000 sequences of lytic polysaccharide monooxygenase (LPMO) from over seven families. Using the newly developed program we were able to reveal previously unknown homologs of LPMOs, namely the PFAM Egh16-like family. Moreover, we show that the substrate specificities have evolved independently several times within the LPMO superfamily. Furthermore, the LPMO phylogenetic tree, does not seem to follow taxonomy-based classification.
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http://dx.doi.org/10.12688/f1000research.21104.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950343PMC
June 2020

Rab GDP-dissociation inhibitor gdiA is an essential gene required for cell wall chitin deposition in Aspergillus niger.

Fungal Genet Biol 2020 03 27;136:103319. Epub 2019 Dec 27.

Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, the Netherlands. Electronic address:

The cell wall is a distinctive feature of filamentous fungi, providing them with structural integrity and protection from both biotic and abiotic factors. Unlike plant cell walls, fungi rely on structurally strong hydrophobic chitin core for mechanical strength together with alpha- and beta-glucans, galactomannans and glycoproteins. Cell wall stress conditions are known to alter the cell wall through the signaling cascade of the cell wall integrity (CWI) pathway and can result in increased cell wall chitin deposition. A previously isolated set of Aspergillus niger cell wall mutants was screened for increased cell wall chitin deposition. UV-mutant RD15.8#16 was found to contain approximately 60% more cell wall chitin than the wild type. In addition to the chitin phenotype, RD15.8#16 exhibits a compact colony morphology and increased sensitivity towards SDS. RD15.8#16 was subjected to classical genetic approach for identification of the underlying causative mutation, using co-segregation analysis and SNP genotyping. Genome sequencing of RD15.8#16 revealed eight SNPs in open reading frames (ORF) which were individually checked for co-segregation with the associated phenotypes, and showed the potential relevance of two genes located on chromosome IV. In situ re-creation of these ORF-located SNPs in a wild type background, using CRISPR/Cas9 genome editing, showed the importance Rab GTPase dissociation inhibitor A (gdiA) for the phenotypes of RD15.8#16. An alteration in the 5' donor splice site of gdiA reduced pre-mRNA splicing efficiency, causing aberrant cell wall assembly and increased chitin levels, whereas gene disruption attempts showed that a full gene deletion of gdiA is lethal.
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http://dx.doi.org/10.1016/j.fgb.2019.103319DOI Listing
March 2020

Identification of novel citramalate biosynthesis pathways in .

Fungal Biol Biotechnol 2019 19;6:19. Epub 2019 Nov 19.

Dutch DNA Biotech B.V., Padualaan 8, 3584 CH Utrecht, The Netherlands.

Background: The filamentous fungus is frequently used for industrial production of fermentative products such as enzymes, proteins and biochemicals. Notable examples of industrially produced fermentation products are glucoamylase and citric acid. Most notably, the industrial production of citric acid achieves high titers, yield and productivities, a feat that has prompted researchers to propose to serve as heterologous production host for the industrial production of itaconic acid (IA), a promising sustainable chemical building-block for the fabrication of various synthetic resins, coatings, and polymers. Heterologous production of IA in has resulted in unexpected levels of metabolic rewiring that has led us to the identification of IA biodegradation pathway in . In this study we have attempted to identify the final product of the IA biodegradation pathway and analyzed the effect of metabolic rewiring on the bioproduction of 9 industrially relevant organic acids.

Results: IA biodegradation manifests in diminishing titers of IA and the occurrence of an unidentified compound in the HPLC profile. Based on published results on the IA biodegradation pathway, we hypothesized that the final product of IA biodegradation in may be citramalic acid (CM). Based on detailed HPLC analysis, we concluded that the unidentified compound is indeed CM. Furthermore, by transcriptome analysis we explored the effect of metabolic rewiring on the production of 9 industrially relevant organic acids by transcriptome analysis of IA producing and WT strains. Interestingly, this analysis led to the identification of a previously unknown biosynthetic cluster that is proposed to be involved in the biosynthesis of CM. Upon overexpression of the putative citramalate synthase and a genomically clustered organic acid transporter, we have observed CM bioproduction by .

Conclusion: In this study, we have shown that the end product of IA biodegradation pathway in is CM. Knock-out of the IA biodegradation pathway results in the cessation of CM production. Furthermore, in this study we have identified a citramalate biosynthesis pathway, which upon overexpression drives citramalate bioproduction in .
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http://dx.doi.org/10.1186/s40694-019-0084-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6862759PMC
November 2019

Metabolic specialization in itaconic acid production: a tale of two fungi.

Curr Opin Biotechnol 2020 04 2;62:153-159. Epub 2019 Nov 2.

Dutch DNA Biotech BV Padualaan 8, 3584CH Utrecht, the Netherlands.

Some of the oldest and most established industrial biotechnology processes involve the fungal production of organic acids. In these fungi, the transport of metabolites between cellular compartments, and their secretion, is a major factor. In this review we exemplify the importance of both mitochondrial and plasma membrane transporters in the case of itaconic acid production in two very different fungal systems, Aspergillus and Ustilago. Homologous and heterologous overexpression of both types of transporters, and biochemical analysis of mitochondrial transporter function, show that these two fungi produce the same compound through very different pathways. The way these fungi respond to itaconate stress, especially at low pH, also differs, although this is still an open field which clearly needs additional research.
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http://dx.doi.org/10.1016/j.copbio.2019.09.014DOI Listing
April 2020

Disruption of a putative mitochondrial oxaloacetate shuttle protein in Aspergillus carbonarius results in secretion of malic acid at the expense of citric acid production.

BMC Biotechnol 2019 11 4;19(1):72. Epub 2019 Nov 4.

Department of Chemistry and Bioscience, Section for Sustainable Biotechnology, Aalborg University, A.C. Meyers Vaenge 15, DK-2450, Copenhagen, SV, Denmark.

Background: In filamentous fungi, transport of organic acids across the mitochondrial membrane is facilitated by active transport via shuttle proteins. These transporters may transfer different organic acids across the membrane while taking others the opposite direction. In Aspergillus niger, accumulation of malate in the cytosol can trigger production of citric acid via the exchange of malate and citrate across the mitochondrial membrane. Several mitochondrial organic acid transporters were recently studied in A. niger showing their effects on organic acid production.

Results: In this work, we studied another citric acid producing fungus, Aspergillus carbonarius, and identified by genome-mining a putative mitochondrial transporter MtpA, which was not previously studied, that might be involved in production of citric acid. This gene named mtpA encoding a putative oxaloacetate transport protein was expressed constitutively in A. carbonarius based on transcription analysis. To study its role in organic acid production, we disrupted the gene and analyzed its effects on production of citric acid and other organic acids, such as malic acid. In total, 6 transformants with gene mtpA disrupted were obtained and they showed secretion of malic acid at the expense of citric acid production.

Conclusion: A putative oxaloacetate transporter gene which is potentially involved in organic acid production by A. carbonarius was identified and further investigated on its effects on production of citric acid and malic acid. The mtpA knockout strains obtained produced less citric acid and more malic acid than the wild type, in agreement with our original hypothesis. More extensive studies should be conducted in order to further reveal the mechanism of organic acid transport as mediated by the MtpA transporter.
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http://dx.doi.org/10.1186/s12896-019-0572-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829807PMC
November 2019

Metabolic engineering with ATP-citrate lyase and nitrogen source supplementation improves itaconic acid production in .

Biotechnol Biofuels 2019 30;12:233. Epub 2019 Sep 30.

Dutch DNA Biotech B.V., Padualaan 8, 3584 CH Utrecht, The Netherlands.

Background: Bio-based production of organic acids promises to be an attractive alternative for the chemicals industry to substitute petrochemicals as building-block chemicals. In recent years, itaconic acid (IA, methylenesuccinic acid) has been established as a sustainable building-block chemical for the manufacture of various products such as synthetic resins, coatings, and biofuels. The natural IA producer is currently used for industrial IA production; however, the filamentous fungus has been suggested to be a more suitable host for this purpose. In our previous report, we communicated the overexpression of a putative cytosolic citrate synthase in an strain carrying the full IA biosynthesis gene cluster from which resulted in the highest final titer reported for   (26.2 g/L IA). In this research, we have attempted to improve this pathway by increasing the cytosolic acetyl-CoA pool. Additionally, we have also performed fermentation optimization by varying the nitrogen source and concentration.

Results: To increase the cytosolic acetyl-CoA pool, we have overexpressed genes and that together encode for ATP-citrate lyase (ACL). Metabolic engineering of ACL resulted in improved IA production through an apparent increase in glycolytic flux. Strains that overexpress show an increased yield, titer and productivity in comparison with parental strain CitB#99. Furthermore, IA fermentation conditions were improved by nitrogen supplementation, which resulted in alkalization of the medium and thereby reducing IA-induced weak-acid stress. In turn, the alkalizing effect of nitrogen supplementation enabled an elongated idiophase and allowed final titers up to 42.7 g/L to be reached at a productivity of 0.18 g/L/h and yield of 0.26 g/g in 10-L bioreactors.

Conclusion: Ultimately, this study shows that metabolic engineering of ACL in our rewired IA biosynthesis pathway leads to improved IA production in due to an increase in glycolytic flux. Furthermore, IA fermentation conditions were improved by nitrogen supplementation that alleviates IA induced weak-acid stress and extends the idiophase.
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http://dx.doi.org/10.1186/s13068-019-1577-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767652PMC
September 2019

Efficient marker free CRISPR/Cas9 genome editing for functional analysis of gene families in filamentous fungi.

Fungal Biol Biotechnol 2019 21;6:13. Epub 2019 Sep 21.

1Department Molecular Microbiology and Biotechnology, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.

Background: CRISPR/Cas9 mediated genome editing has expedited the way of constructing multiple gene alterations in filamentous fungi, whereas traditional methods are time-consuming and can be of mutagenic nature. These developments allow the study of large gene families that contain putatively redundant genes, such as the seven-membered family of -genes encoding putative glucan-chitin crosslinking enzymes involved in cell wall biosynthesis.

Results: Here, we present a CRISPR/Cas9 system for using a non-integrative plasmid, containing a selection marker, a Cas9 and a sgRNA expression cassette. Combined with selection marker free knockout repair DNA fragments, a set of the seven single knockout strains was obtained through homology directed repair (HDR) with an average efficiency of 90%. Cas9-sgRNA plasmids could effectively be cured by removing selection pressure, allowing the use of the same selection marker in successive transformations. Moreover, we show that either two or even three separate Cas9-sgRNA plasmids combined with marker-free knockout repair DNA fragments can be used in a single transformation to obtain double or triple knockouts with 89% and 38% efficiency, respectively. By employing this technique, a seven-membered -gene family knockout strain was acquired in a few rounds of transformation; three times faster than integrative selection marker () recycling transformations. An additional advantage of the use of marker-free gene editing is that negative effects of selection marker gene expression are evaded, as we observed in the case of disrupting virtually silent family members.

Conclusions: Our findings advocate the use of CRISPR/Cas9 to create multiple gene deletions in both a fast and reliable way, while simultaneously omitting possible locus-dependent-side-effects of poor auxotrophic marker expression.
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http://dx.doi.org/10.1186/s40694-019-0076-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754632PMC
September 2019

Correction to: Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions.

Appl Microbiol Biotechnol 2019 06;103(12):5063

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333, BE, Leiden, The Netherlands.

The correct title is: Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions.
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http://dx.doi.org/10.1007/s00253-019-09853-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828049PMC
June 2019

Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions [corrected].

Appl Microbiol Biotechnol 2019 05 8;103(10):4125-4136. Epub 2019 Apr 8.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

The AraR transcription factor of Aspergillus niger encodes a Zn(II)Cys transcription factor required for the induction of genes encoding arabinolytic enzymes. One of the target genes of AraR is abfA, encoding an arabinofuranosidase. The expression of abfA as well as other L-arabinose-induced genes in A. niger requires the presence of L-arabinose or its derivative L-arabitol as an inducer to activate AraR-dependant gene expression. In this study, mutants were isolated that express L-arabinose-induced genes independently of the presence of an inducer under derepressing conditions. To obtain these mutants, a reporter strain was constructed in a ΔcreA background containing the L-arabinose-responsive promoter (PabfA) fused to the acetamidase (amdS) gene. Spores of the ΔcreA PabfA-amdS reporter strain were UV-mutagenized and mutants were obtained by their ability to grow on acetamide without the presence of inducer. From a total of 164 mutants, 15 mutants were identified to contain transacting mutations resulting in high arabinofuranosidase activity in the medium after growth under non-inducing conditions. Sequencing of the araR gene of the 15 constitutive mutants revealed that 14 mutants carried a mutation in AraR. Some mutations were found more than once and in total nine different point mutations were identified in AraR. The AraR point mutation was reintroduced into a parental strain and confirmed that this point mutation leads to inducer-independent expression of AraR target genes. The inducer independent of L-arabinose-induced genes in the AraR mutant was found to be sensitive to carbon catabolite repression, indicating that the CreA-mediated carbon catabolite repression is dominant over the AraR mutant allele. These mutations in AraR provide new opportunities to improve arabinase production in industrial fungal strains.
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http://dx.doi.org/10.1007/s00253-019-09777-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486530PMC
May 2019

The interplay between transport and metabolism in fungal itaconic acid production.

Fungal Genet Biol 2019 04 28;125:45-52. Epub 2019 Jan 28.

iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich, Wilhelm-Johnen-Strasse, 52425 Jülich, Germany. Electronic address:

Besides enzymatic conversions, many eukaryotic metabolic pathways also involve transport proteins that shuttle molecules between subcellular compartments, or into the extracellular space. Fungal itaconate production involves two such transport steps, involving an itaconate transport protein (Itp), and a mitochondrial tricarboxylate transporter (Mtt). The filamentous ascomycete Aspergillus terreus and the unicellular basidiomycete Ustilago maydis both produce itaconate, but do so via very different molecular pathways, and under very different cultivation conditions. In contrast, the transport proteins of these two strains are assumed to have a similar function. This study aims to investigate the roles of both the extracellular and mitochondrial transporters from these two organisms by expressing them in the corresponding U. maydis knockouts and monitoring the extracellular product concentrations. Both transporters from A. terreus complemented their corresponding U. maydis knockouts in mediating itaconate production. Surprisingly, complementation with At_MfsA from A. terreus led to a partial switch from itaconate to (S)-2-hydroxyparaconate secretion. Apparently, the export protein from A. terreus has a higher affinity for (S)-2-hydroxyparaconate than for itaconate, even though this species is classically regarded as an itaconate producer. Complementation with At_MttA increased itaconate production by 2.3-fold compared to complementation with Um_Mtt1, indicating that the mitochondrial carrier from A. terreus supports a higher metabolic flux of itaconic acid precursors than its U. maydis counterpart. The biochemical implications of these differences are discussed in the context of the biotechnological application in U. maydis and A. terreus for the production of itaconate and (S)-2-hydroxyparaconate.
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http://dx.doi.org/10.1016/j.fgb.2019.01.011DOI Listing
April 2019

Itaconic acid degradation in : the role of unexpected bioconversion pathways.

Fungal Biol Biotechnol 2019 4;6. Epub 2019 Jan 4.

Dutch DNA Biotech B.V, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Background: Itaconic acid (IA), a C5-dicarboxylic acid, has previously been identified as one of the top twelve biochemicals that can be produced by biotechnological means. IA is naturally produced by , however, heterologous production in the related species has been proposed earlier. Remarkably, we observed that during high producing conditions and elevated titers . detoxifies the extracellular medium of IA. In order to determine the genes responsible for this decline in IA titers a transcriptome analysis was performed.

Results: Transcriptome analysis has led to the identification of two novel and previously unknown IA bioconversion pathways in . . One pathway is proposed to convert IA into pyruvate and acetyl-CoA through the action of itaconyl-CoA transferase (IctA), itaconyl-CoA hydratase (IchA) and citramalyl-CoA lyase, similar to the pathway identified in . . Another pathway putatively converts IA into 1-methyl itaconate through the action of trans-aconitate methyltransferase (TmtA). Upon deleting the key genes and we have observed increased IA production and titers and cessation of IA bioconversion. Surprisingly, deletion of lead to strong reduction of heterologous IA production.

Conclusion: Heterologous IA production in . induces the expression of IA bioconversion pathways. These pathways can be inhibited by deleting the key genes , and . Deletion of and resulted in increased IA production. Deletion of , however, resulted in almost complete cessation of IA production.
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http://dx.doi.org/10.1186/s40694-018-0062-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320622PMC
January 2019

The fungal composition of natural biofinishes on oil-treated wood.

Fungal Biol Biotechnol 2017 26;4. Epub 2017 Jan 26.

Applied and Industrial Mycology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands.

Background: Biofinished wood is considered to be a decorative and protective material for outdoor constructions, showing advantages compared to traditional treated wood in terms of sustainability and self-repair. Natural dark wood staining fungi are essential to biofinish formation on wood. Although all sorts of outdoor situated timber are subjected to fungal staining, the homogenous dark staining called biofinish has only been detected on specific vegetable oil-treated substrates. Revealing the fungal composition of various natural biofinishes on wood is a first step to understand and control biofinish formation for industrial application.

Results: A culture-based survey of fungi in natural biofinishes on oil-treated wood samples showed the common wood stain fungus and the recently described genus to be predominant constituents. A culture-independent approach, based on amplification of the internal transcribed spacer regions, cloning and Sanger sequencing, resulted in clone libraries of two types of biofinishes. was present in both biofinish types, but was only predominant in biofinishes on pine sapwood treated with raw linseed oil. Most cloned sequences of the other biofinish type (pine sapwood treated with olive oil) could not be identified. In addition, a more in-depth overview of the fungal composition of biofinishes was obtained with Illumina amplicon sequencing that targeted the internal transcribed spacer region 1. All investigated samples, that varied in wood species, (oil) treatments and exposure times, contained and this genus was predominant in the biofinishes on pine sapwood treated with raw linseed oil. was the predominant genus in most of the other biofinishes and present in all other samples. Surprisingly, , which was predominantly detected by the cultivation-based approach, could not be found with the Illumina sequencing approach, while was not detected in the culture-based approach.

Conclusions: Overall, the culture-based approach and two culture-independent methods that were used in this study revealed that natural biofinishes were composed of multiple fungal genera always containing the common wood staining mould . Besides , the use of other fungal genera for the production of biofinished wood has to be considered.
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http://dx.doi.org/10.1186/s40694-017-0030-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611603PMC
January 2017

whole-genome assembly of a wild type yeast isolate using nanopore sequencing.

F1000Res 2017;6:618. Epub 2017 May 3.

Institute of Biology, Leiden University, Leiden, 2300 RA, Netherlands.

The introduction of the MinION sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. Nanopore sequence data offers great potential for assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. In this study, we used this new technology to sequence and assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named ( ) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the nanopore sequencing protocol, showing that nanopore-derived assemblies provide the highest contiguity. The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published species. In conclusion, MinION-mediated long read sequencing can be used for high quality assembly of new eukaryotic microbial genomes.
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http://dx.doi.org/10.12688/f1000research.11146.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6081980PMC
May 2017

The discovery of novel LPMO families with a new Hidden Markov model.

BMC Res Notes 2017 Feb 21;10(1):105. Epub 2017 Feb 21.

Molecular Microbiology and Health, Institute of Biology Leiden, Leiden University, Leiden, The Netherlands.

Background: Renewable biopolymers, such as cellulose, starch and chitin are highly resistance to enzymatic degradation. Therefore, there is a need to upgrade current degradation processes by including novel enzymes. Lytic polysaccharide mono-oxygenases (LPMOs) can disrupt recalcitrant biopolymers, thereby enhancing hydrolysis by conventional enzymes. However, novel LPMO families are difficult to identify using existing methods. Therefore, we developed a novel profile Hidden Markov model (HMM) and used it to mine genomes of ascomycetous fungi for novel LPMOs.

Results: We constructed a structural alignment and verified that the alignment was correct. In the alignment we identified several known conserved features, such as the histidine brace and the N/Q/E-X-F/Y motif and previously unidentified conserved proline and glycine residues. These residues are distal from the active site, suggesting a role in structure rather than activity. The multiple protein alignment was subsequently used to build a profile Hidden Markov model. This model was initially tested on manually curated datasets and proved to be both sensitive (no false negatives) and specific (no false positives). In some of the genomes analyzed we identified a yet unknown LPMO family. This new family is mostly confined to the phyla of Ascomycota and Basidiomycota and the class of Oomycota. Genomic clustering indicated that at least some members might be involved in the degradation of β-glucans, while transcriptomic data suggested that others are possibly involved in the degradation of pectin.

Conclusions: The newly developed profile hidden Markov Model was successfully used to mine fungal genomes for a novel family of LPMOs. However, the model is not limited to bacterial and fungal genomes. This is illustrated by the fact that the model was also able to identify another new LPMO family in Drosophila melanogaster. Furthermore, the Hidden Markov model was used to verify the more distant blast hits from the new fungal family of LPMOs, which belong to the Bivalves, Stony corals and Sea anemones. So this Hidden Markov model (Additional file 3) will help the broader scientific community in identifying other yet unknown LPMOs.
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http://dx.doi.org/10.1186/s13104-017-2429-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320794PMC
February 2017

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

Authors:
Ronald P de Vries Robert Riley Ad Wiebenga Guillermo Aguilar-Osorio Sotiris Amillis Cristiane Akemi Uchima Gregor Anderluh Mojtaba Asadollahi Marion Askin Kerrie Barry Evy Battaglia Özgür Bayram Tiziano Benocci Susanna A Braus-Stromeyer Camila Caldana David Cánovas Gustavo C Cerqueira Fusheng Chen Wanping Chen Cindy Choi Alicia Clum Renato Augusto Corrêa Dos Santos André Ricardo de Lima Damásio George Diallinas Tamás Emri Erzsébet Fekete Michel Flipphi Susanne Freyberg Antonia Gallo Christos Gournas Rob Habgood Matthieu Hainaut María Laura Harispe Bernard Henrissat Kristiina S Hildén Ryan Hope Abeer Hossain Eugenia Karabika Levente Karaffa Zsolt Karányi Nada Kraševec Alan Kuo Harald Kusch Kurt LaButti Ellen L Lagendijk Alla Lapidus Anthony Levasseur Erika Lindquist Anna Lipzen Antonio F Logrieco Andrew MacCabe Miia R Mäkelä Iran Malavazi Petter Melin Vera Meyer Natalia Mielnichuk Márton Miskei Ákos P Molnár Giuseppina Mulé Chew Yee Ngan Margarita Orejas Erzsébet Orosz Jean Paul Ouedraogo Karin M Overkamp Hee-Soo Park Giancarlo Perrone Francois Piumi Peter J Punt Arthur F J Ram Ana Ramón Stefan Rauscher Eric Record Diego Mauricio Riaño-Pachón Vincent Robert Julian Röhrig Roberto Ruller Asaf Salamov Nadhira S Salih Rob A Samson Erzsébet Sándor Manuel Sanguinetti Tabea Schütze Kristina Sepčić Ekaterina Shelest Gavin Sherlock Vicky Sophianopoulou Fabio M Squina Hui Sun Antonia Susca Richard B Todd Adrian Tsang Shiela E Unkles Nathalie van de Wiele Diana van Rossen-Uffink Juliana Velasco de Castro Oliveira Tammi C Vesth Jaap Visser Jae-Hyuk Yu Miaomiao Zhou Mikael R Andersen David B Archer Scott E Baker Isabelle Benoit Axel A Brakhage Gerhard H Braus Reinhard Fischer Jens C Frisvad Gustavo H Goldman Jos Houbraken Berl Oakley István Pócsi Claudio Scazzocchio Bernhard Seiboth Patricia A vanKuyk Jennifer Wortman Paul S Dyer Igor V Grigoriev

Genome Biol 2017 02 14;18(1):28. Epub 2017 Feb 14.

US Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA, 94598, USA.

Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus.

Results: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli.

Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.
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http://dx.doi.org/10.1186/s13059-017-1151-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5307856PMC
February 2017

An Evolutionarily Conserved Transcriptional Activator-Repressor Module Controls Expression of Genes for D-Galacturonic Acid Utilization in Aspergillus niger.

Genetics 2017 01 9;205(1):169-183. Epub 2016 Nov 9.

Molecular Microbiology and Biotechnology, Leiden University, Institute of Biology Leiden, 2333 BE Leiden, The Netherlands

The expression of genes encoding extracellular polymer-degrading enzymes and the metabolic pathways required for carbon utilization in fungi are tightly controlled. The control is mediated by transcription factors that are activated by the presence of specific inducers, which are often monomers or monomeric derivatives of the polymers. A D-galacturonic acid-specific transcription factor named GaaR was recently identified and shown to be an activator for the expression of genes involved in galacturonic acid utilization in Botrytis cinerea and Aspergillus niger Using a forward genetic screen, we isolated A. niger mutants that constitutively express GaaR-controlled genes. Reasoning that mutations in the gaaR gene would lead to a constitutively activated transcription factor, the gaaR gene in 11 of the constitutive mutants was sequenced, but no mutations in gaaR were found. Full genome sequencing of five constitutive mutants revealed allelic mutations in one particular gene encoding a previously uncharacterized protein (NRRL3_08194). The protein encoded by NRRL3_08194 shows homology to the repressor of the quinate utilization pathway identified previously in Neurospora crassa (qa-1S) and Aspergillus nidulans (QutR). Deletion of NRRL3_08194 in combination with RNA-seq analysis showed that the NRRL3_08194 deletion mutant constitutively expresses genes involved in galacturonic acid utilization. Interestingly, NRRL3_08194 is located next to gaaR (NRRL3_08195) in the genome. The homology to the quinate repressor, the chromosomal clustering, and the constitutive phenotype of the isolated mutants suggest that NRRL3_08194 is likely to encode a repressor, which we name GaaX. The GaaR-GaaX module and its chromosomal organization is conserved among ascomycetes filamentous fungi, resembling the quinate utilization activator-repressor module in amino acid sequence and chromosomal organization.
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http://dx.doi.org/10.1534/genetics.116.194050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223501PMC
January 2017

Rewiring a secondary metabolite pathway towards itaconic acid production in Aspergillus niger.

Microb Cell Fact 2016 Jul 28;15(1):130. Epub 2016 Jul 28.

Dutch DNA Biotech B.V, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.

Background: The industrially relevant filamentous fungus Aspergillus niger is widely used in industry for its secretion capabilities of enzymes and organic acids. Biotechnologically produced organic acids promise to be an attractive alternative for the chemical industry to replace petrochemicals. Itaconic acid (IA) has been identified as one of the top twelve building block chemicals which have high potential to be produced by biotechnological means. The IA biosynthesis cluster (cadA, mttA and mfsA) has been elucidated in its natural producer Aspergillus terreus and transferred to A. niger to enable IA production. Here we report the rewiring of a secondary metabolite pathway towards further improved IA production through the overexpression of a putative cytosolic citrate synthase citB in a A. niger strain carrying the IA biosynthesis cluster.

Results: We have previously shown that expression of cadA from A. terreus results in itaconic acid production in A. niger AB1.13, albeit at low levels. This low-level production is boosted fivefold by the overexpression of mttA and mfsA in itaconic acid producing AB1.13 CAD background strains. Controlled batch cultivations with AB1.13 CAD + MFS + MTT strains showed increased production of itaconic acid compared with AB1.13 CAD strain. Moreover, preliminary RNA-Seq analysis of an itaconic acid producing AB1.13 CAD strain has led to the identification of the putative cytosolic citrate synthase citB which was induced in an IA producing strain. We have overexpressed citB in a AB1.13 CAD + MFS + MTT strain and by doing so hypothesize to have targeted itaconic acid production to the cytosolic compartment. By overexpressing citB in AB1.13 CAD + MFS + MTT strains in controlled batch cultivations we have achieved highly increased titers of up to 26.2 g/L IA with a productivity of 0.35 g/L/h while no CA was produced.

Conclusions: Expression of the IA biosynthesis cluster in Aspergillus niger AB1.13 strain enables IA production. Moreover, in the AB1.13 CAD strain IA production resulted in overexpression of a putative cytosolic citrate synthase citB. Upon overexpression of citB we have achieved titers of up to 26.2 g/L IA with a productivity of 0.35 g/L/h in controlled batch cultivations. By overexpressing citB we have also diminished side product formation and optimized the production pathway towards IA.
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http://dx.doi.org/10.1186/s12934-016-0527-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965889PMC
July 2016

The unconventional secretion of PepN is independent of a functional autophagy machinery in the filamentous fungus Aspergillus niger.

FEMS Microbiol Lett 2016 08 8;363(15). Epub 2016 Jun 8.

Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands

During unconventional protein secretion (UPS), proteins do not pass through the classical endoplasmic reticulum (ER)-Golgi-dependent pathway, but are transported to the cell membrane via alternative routes. One type of UPS is dependent on several autophagy-related (Atg) proteins in yeast and mammalian cells, but mechanisms for unconventional secretion are largely unknown for filamentous fungi. In this study, we investigated whether the autophagy machinery is used for UPS in the filamentous fungus Aspergillus niger An aspartic protease, which we called PepN, was identified as being likely to be secreted unconventionally, as this protein is highly abundant in culture filtrates during carbon starvation while it lacks a conventional N-terminal secretion sequence. We analysed the presence of PepN in the culture filtrates of carbon starved wild-type, atg1 and atg8 deletion mutant strains by Western blot analysis and by secretome analysis using nanoLC-ESI-MS/MS (wild-type and atg8 deletion mutant). Besides the presence of carbohydrate-active enzymes and other types of proteases, PepN was abundantly found in culture filtrates of both wild-type and atg deletion strains, indicating that the secretion of PepN is independent of the autophagy machinery in A. niger and hence most likely occurs via a different mechanism.
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http://dx.doi.org/10.1093/femsle/fnw152DOI Listing
August 2016

Characterizing MttA as a mitochondrial cis-aconitic acid transporter by metabolic engineering.

Metab Eng 2016 May 10;35:95-104. Epub 2016 Feb 10.

Austrian Centre of Industrial Biotechnology (ACIB GmbH), Muthgasse 18, Vienna, Austria; Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria; CD Laboratory for Biotechnology of Glycerol, Muthgasse 18, Vienna, Austria. Electronic address:

The mitochondrial carrier protein MttA is involved in the biosynthesis of itaconic acid in Aspergillus terreus. In this paper, the transport specificity of MttA is analyzed making use of different metabolically engineered Aspergillus niger strains. Furthermore, the mitochondrial localization of this protein is confirmed using fluorescence microscopy. It was found that MttA preferentially transports cis-aconitic acid over citric acid and does not transport itaconic acid. The expression of MttA in selected A. niger strains results in secretion of aconitic acid. MttA can be used in further strain engineering strategies to transport cis-aconitic acid to the cytosol to produce itaconic acid or related metabolites. The microbial production of aconitic acid (9g/L) is achieved in strains expressing this transport protein. Thus, metabolic engineering can be used for both the in vivo characterization of transport protein function like MttA and to make use of this protein by creating aconitic acid producing strains.
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http://dx.doi.org/10.1016/j.ymben.2016.02.003DOI Listing
May 2016

Identification of a Classical Mutant in the Industrial Host Aspergillus niger by Systems Genetics: LaeA Is Required for Citric Acid Production and Regulates the Formation of Some Secondary Metabolites.

G3 (Bethesda) 2015 Nov 13;6(1):193-204. Epub 2015 Nov 13.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, 2333 BE, Leiden, The Netherlands

The asexual filamentous fungus Aspergillus niger is an important industrial cell factory for citric acid production. In this study, we genetically characterized a UV-generated A. niger mutant that was originally isolated as a nonacidifying mutant, which is a desirable trait for industrial enzyme production. Physiological analysis showed that this mutant did not secrete large amounts of citric acid and oxalic acid, thus explaining the nonacidifying phenotype. As traditional complementation approaches to characterize the mutant genotype were unsuccessful, we used bulk segregant analysis in combination with high-throughput genome sequencing to identify the mutation responsible for the nonacidifying phenotype. Since A. niger has no sexual cycle, parasexual genetics was used to generate haploid segregants derived from diploids by loss of whole chromosomes. We found that the nonacidifying phenotype was caused by a point mutation in the laeA gene. LaeA encodes a putative methyltransferase-domain protein, which we show here to be required for citric acid production in an A. niger lab strain (N402) and in other citric acid production strains. The unexpected link between LaeA and citric acid production could provide new insights into the transcriptional control mechanisms related to citric acid production in A. niger. Interestingly, the secondary metabolite profile of a ΔlaeA strain differed from the wild-type strain, showing both decreased and increased metabolite levels, indicating that LaeA is also involved in regulating the production of secondary metabolites. Finally, we show that our systems genetics approach is a powerful tool to identify trait mutations.
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http://dx.doi.org/10.1534/g3.115.024067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704718PMC
November 2015

Genome mining and functional genomics for siderophore production in Aspergillus niger.

Brief Funct Genomics 2014 Nov 25;13(6):482-92. Epub 2014 Jul 25.

Iron is an essential metal for many organisms, but the biologically relevant form of iron is scarce because of rapid oxidation resulting in low solubility. Simultaneously, excessive accumulation of iron is toxic. Consequently, iron uptake is a highly controlled process. In most fungal species, siderophores play a central role in iron handling. Siderophores are small iron-specific chelators that can be secreted to scavenge environmental iron or bind intracellular iron with high affinity. A second high-affinity iron uptake mechanism is reductive iron assimilation (RIA). As shown in Aspergillus fumigatus and Aspergillus nidulans, synthesis of siderophores in Aspergilli is predominantly under control of the transcription factors SreA and HapX, which are connected by a negative transcriptional feedback loop. Abolishing this fine-tuned regulation corroborates iron homeostasis, including heme biosynthesis, which could be biotechnologically of interest, e.g. the heterologous production of heme-dependent peroxidases. Aspergillus niger genome inspection identified orthologues of several genes relevant for RIA and siderophore metabolism, as well as sreA and hapX. Interestingly, genes related to synthesis of the common fungal extracellular siderophore triacetylfusarinine C were absent. Reverse-phase high-performance liquid chromatography (HPLC) confirmed the absence of triacetylfusarinine C, and demonstrated that the major secreted siderophores of A. niger are coprogen B and ferrichrome, which is also the dominant intracellular siderophore. In A. niger wild type grown under iron-replete conditions, the expression of genes involved in coprogen biosynthesis and RIA was low in the exponential growth phase but significantly induced during ascospore germination. Deletion of sreA in A. niger resulted in elevated iron uptake and increased cellular ferrichrome accumulation. Increased sensitivity toward phleomycin and high iron concentration reflected the toxic effects of excessive iron uptake. Moreover, SreA-deficiency resulted in increased accumulation of heme intermediates, but no significant increase in heme content. Together with the upregulation of several heme biosynthesis genes, these results reveal a complex heme regulatory mechanism.
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http://dx.doi.org/10.1093/bfgp/elu026DOI Listing
November 2014

Aspergillus niger RhaR, a regulator involved in L-rhamnose release and catabolism.

Appl Microbiol Biotechnol 2014 Jun 28;98(12):5531-40. Epub 2014 Feb 28.

Microbiology & Kluyver Centre for Genomics of Industrial Fermentation, Utrecht University, Utrecht, The Netherlands.

The genome of the filamentous fungus Aspergillus niger is rich in genes encoding pectinases, a broad class of enzymes that have been extensively studied due to their use in industrial applications. The sequencing of the A. niger genome provided more knowledge concerning the individual pectinolytic genes, but little is known about the regulatory genes involved in pectin degradation. Understanding regulation of the pectinolytic genes provides a tool to optimize the production of pectinases in this industrially important fungus. This study describes the identification and characterization of one of the activators of pectinase-encoding genes, RhaR. Inactivation of the gene encoding this regulator resulted in down-regulation of genes involved in the release of L-rhamnose from the pectin substructure rhamnogalacturonan I, as well as catabolism of this monosaccharide. The rhaR disruptant was unable to grow on L-rhamnose, but only a small reduction in growth on pectin was observed. This is likely caused by the presence of a second, so far unknown regulator that responds to the presence of D-galacturonic acid.
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http://dx.doi.org/10.1007/s00253-014-5607-9DOI Listing
June 2014

Identifying inhibitory compounds in lignocellulosic biomass hydrolysates using an exometabolomics approach.

BMC Biotechnol 2014 Mar 21;14:22. Epub 2014 Mar 21.

TNO Microbiology & Systems Biology, Utrechtsweg 48, Zeist 3704 HE, The Netherlands.

Background: Inhibitors are formed that reduce the fermentation performance of fermenting yeast during the pretreatment process of lignocellulosic biomass. An exometabolomics approach was applied to systematically identify inhibitors in lignocellulosic biomass hydrolysates.

Results: We studied the composition and fermentability of 24 different biomass hydrolysates. To create diversity, the 24 hydrolysates were prepared from six different biomass types, namely sugar cane bagasse, corn stover, wheat straw, barley straw, willow wood chips and oak sawdust, and with four different pretreatment methods, i.e. dilute acid, mild alkaline, alkaline/peracetic acid and concentrated acid. Their composition and that of fermentation samples generated with these hydrolysates were analyzed with two GC-MS methods. Either ethyl acetate extraction or ethyl chloroformate derivatization was used before conducting GC-MS to prevent sugars are overloaded in the chromatograms, which obscure the detection of less abundant compounds. Using multivariate PLS-2CV and nPLS-2CV data analysis models, potential inhibitors were identified through establishing relationship between fermentability and composition of the hydrolysates. These identified compounds were tested for their effects on the growth of the model yeast, Saccharomyces. cerevisiae CEN.PK 113-7D, confirming that the majority of the identified compounds were indeed inhibitors.

Conclusion: Inhibitory compounds in lignocellulosic biomass hydrolysates were successfully identified using a non-targeted systematic approach: metabolomics. The identified inhibitors include both known ones, such as furfural, HMF and vanillin, and novel inhibitors, namely sorbic acid and phenylacetaldehyde.
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http://dx.doi.org/10.1186/1472-6750-14-22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3998114PMC
March 2014

Molecular genetic analysis of vesicular transport in Aspergillus niger reveals partial conservation of the molecular mechanism of exocytosis in fungi.

Microbiology (Reading) 2014 Feb 2;160(Pt 2):316-329. Epub 2013 Dec 2.

Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA Delft, The Netherlands.

The filamentous fungus Aspergillus niger is an industrially exploited protein expression platform, well known for its capacity to secrete high levels of proteins. To study the process of protein secretion in A. niger, we established a GFP-v-SNARE reporter strain in which the trafficking and dynamics of secretory vesicles can be followed in vivo. The biological role of putative A. niger orthologues of seven secretion-specific genes, known to function in key aspects of the protein secretion machinery in Saccharomyces cerevisiae, was analysed by constructing respective gene deletion mutants in the GFP-v-SNARE reporter strain. Comparison of the deletion phenotype of conserved proteins functioning in the secretory pathway revealed common features but also interesting differences between S. cerevisiae and A. niger. Deletion of the S. cerevisiae Sec2p orthologue in A. niger (SecB), encoding a guanine exchange factor for the GTPase Sec4p (SrgA in A. niger), did not have an obvious phenotype, while SEC2 deletion in S. cerevisiae is lethal. Similarly, deletion of the A. niger orthologue of the S. cerevisiae exocyst subunit Sec3p (SecC) did not result in a lethal phenotype as in S. cerevisiae, although severe growth reduction of A. niger was observed. Deletion of secA, secH and ssoA (encoding SecA, SecH and SsoA the A. niger orthologues of S. cerevisiae Sec1p, Sec8p and Sso1/2p, respectively) showed that these genes are essential for A. niger, similar to the situation in S. cerevisiae. These data demonstrate that the orchestration of exocyst-mediated vesicle transport is only partially conserved in S. cerevisiae and A. niger.
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http://dx.doi.org/10.1099/mic.0.074252-0DOI Listing
February 2014

The role of coproporphyrinogen III oxidase and ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger.

Appl Microbiol Biotechnol 2013 Nov 11;97(22):9773-85. Epub 2013 Oct 11.

Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

Heme is a suggested limiting factor in peroxidase production by Aspergillus spp., which are well-known suitable hosts for heterologous protein production. In this study, the role of genes coding for coproporphyrinogen III oxidase (hemF) and ferrochelatase (hemH) was analyzed by means of deletion and overexpression to obtain more insight in fungal heme biosynthesis and regulation. These enzymes represent steps in the heme biosynthetic pathway downstream of the siroheme branch and are suggested to play a role in regulation of the pathway. Based on genome mining, both enzymes deviate in cellular localization and protein domain structure from their Saccharomyces cerevisiae counterparts. The lethal phenotype of deletion of hemF or hemH could be remediated by heme supplementation confirming that Aspergillus niger is capable of hemin uptake. Nevertheless, both gene deletion mutants showed an extremely impaired growth even with hemin supplementation which could be slightly improved by media modifications and the use of hemoglobin as heme source. The hyphae of the mutant strains displayed pinkish coloration and red autofluorescence under UV indicative of cellular porphyrin accumulation. HPLC analysis confirmed accumulation of specific porphyrins, thereby confirming the function of the two proteins in heme biosynthesis. Overexpression of hemH, but not hemF or the aminolevulinic acid synthase encoding hemA, modestly increased the cellular heme content, which was apparently insufficient to increase activity of endogenous peroxidase and cytochrome P450 enzyme activities. Overexpression of all three genes increased the cellular accumulation of porphyrin intermediates suggesting regulatory mechanisms operating in the final steps of the fungal heme biosynthesis pathway.
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http://dx.doi.org/10.1007/s00253-013-5274-2DOI Listing
November 2013

Pichia anomala 29X: a resistant strain for lignocellulosic biomass hydrolysate fermentation.

FEMS Yeast Res 2013 Nov 12;13(7):609-17. Epub 2013 Aug 12.

TNO Microbiology & Systems Biology, Zeist, The Netherlands; Netherlands Metabolomics Centre (NMC), Leiden, The Netherlands.

To efficiently use lignocellulosic biomass hydrolysates as fermentation media for bioethanol production, besides being capable of producing significant amount of ethanol, the fermenting host should also meet the following two requirements: (1) resistant to the inhibitory compounds formed during biomass pretreatment process, (2) capable of utilizing C5 sugars, such as xylose, as carbon source. In our laboratory, a screening was conducted on microorganisms collected from environmental sources for their tolerance to hydrolysate inhibitors. A unique resistant strain was selected and identified as Pichia anomala (Wickerhamomyces anomalus), deposited as CBS 132101. The strain is able to produce ethanol in various biomass hydrolysates, both with and without oxygen. Besides, the strain could assimilate xylose and use nitrate as N source. These physiological characteristics make P. anomala an interesting strain for bioethanol production from lignocellulosic biomass hydrolysates.
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http://dx.doi.org/10.1111/1567-1364.12062DOI Listing
November 2013