Identification of a lichen depside polyketide synthase gene by heterologous expression in Saccharomyces cerevisiae.

Lichen-forming fungi produce a variety of secondary metabolites including bioactive polyketides. Advances in DNA and RNA sequencing have led to a growing database of new lichen gene clusters encoding polyketide synthases (PKS) and associated ancillary activities. Definitive assignment of a PKS gene to a metabolic product has been challenging in the lichen field due to a lack of established gene knockout or heterologous gene expression systems. Here, we report the reconstitution of a non-reducing PKS gene from the lichen Pseudevernia furfuracea and successful heterologous expression of the synthetic lichen PKS gene in engineered Saccharomyces cerevisiae. We show that P. furfuracea PFUR17_02294 produces lecanoric acid, the depside dimer of orsellinic acid, at 360 mg/L in small-scale yeast cultures. Our results unequivocally identify PFUR17_02294 as a lecanoric acid synthase and establish that a single lichen PKS synthesizes two phenolic rings and joins them by an ester linkage to form the depside product.

Comparative systems analysis of the secretome of the opportunistic pathogen Aspergillus fumigatus and other Aspergillus species.

Aspergillus fumigatus and multiple other Aspergillus species cause a wide range of lung infections, collectively termed aspergillosis. Aspergilli are ubiquitous in environment with healthy immune systems routinely eliminating inhaled conidia, however, Aspergilli can become an opportunistic pathogen in immune-compromised patients. The aspergillosis mortality rate and emergence of drug-resistance reveals an urgent need to identify novel targets. Secreted and cell membrane proteins play a critical role in fungal-host interactions and pathogenesis. Using a computational pipeline integrating data from high-throughput experiments and bioinformatic predictions, we have identified secreted and cell membrane proteins in ten Aspergillus species known to cause aspergillosis. Small secreted and effector-like proteins similar to agents of fungal-plant pathogenesis were also identified within each secretome. A comparison with humans revealed that at least 70% of Aspergillus secretomes have no sequence similarity with the human proteome. An analysis of antigenic qualities of Aspergillus proteins revealed that the secretome is significantly more antigenic than cell membrane proteins or the complete proteome. Finally, overlaying an expression dataset, four A. fumigatus proteins upregulated during infection and with available structures, were found to be structurally similar to known drug target proteins in other organisms, and were able to dock in silico with the respective drug.

Network reconstruction and systems analysis of plant cell wall deconstruction by Neurospora crassa.

BACKGROUND: Plant biomass degradation by fungal-derived enzymes is rapidly expanding in economic importance as a clean and efficient source for biofuels. The ability to rationally engineer filamentous fungi would facilitate biotechnological applications for degradation of plant cell wall polysaccharides. However, incomplete knowledge of biomolecular networks responsible for plant cell wall deconstruction impedes experimental efforts in this direction. RESULTS: To expand this knowledge base, a detailed network of reactions important for deconstruction of plant cell wall polysaccharides into simple sugars was constructed for the filamentous fungus Neurospora crassa. To reconstruct this network, information was integrated from five heterogeneous data types: functional genomics, transcriptomics, proteomics, genetics, and biochemical characterizations. The combined information was encapsulated into a feature matrix and the evidence weighted to assign annotation confidence scores for each gene within the network. Comparative analyses of RNA-seq and ChIP-seq data shed light on the regulation of the plant cell wall degradation network, leading to a novel hypothesis for degradation of the hemicellulose mannan. The transcription factor CLR-2 was subsequently experimentally shown to play a key role in the mannan degradation pathway of N. crassa. CONCLUSIONS: Here we built a network that serves as a scaffold for integration of diverse experimental datasets. This approach led to the elucidation of regulatory design principles for plant cell wall deconstruction by filamentous fungi and a novel function for the transcription factor CLR-2. This expanding network will aid in efforts to rationally engineer industrially relevant hyper-production strains.

Direct target network of the Neurospora crassa plant cell wall deconstruction regulators CLR-1, CLR-2, and XLR-1.

UNLABELLED: Fungal deconstruction of the plant cell requires a complex orchestration of a wide array of intracellular and extracellular enzymes. In Neurospora crassa, CLR-1, CLR-2, and XLR-1 have been identified as key transcription factors regulating plant cell wall degradation in response to soluble sugars. The XLR-1 regulon was defined using a constitutively active mutant allele, resulting in hemicellulase gene expression and secretion under noninducing conditions. To define genes directly regulated by CLR-1, CLR-2, and XLR-1, we performed chromatin immunoprecipitation and next-generation sequencing (ChIPseq) on epitope-tagged constructs of these three transcription factors. When N. crassa is exposed to plant cell wall material, CLR-1, CLR-2, and XLR-1 individually bind to the promoters of the most strongly induced genes in their respective regulons. These include promoters of genes encoding cellulases for CLR-1 and CLR-2 (CLR-1/CLR-2) and promoters of genes encoding hemicellulases for XLR-1. CLR-1 bound to its regulon under noninducing conditions; however, this binding alone did not translate into gene expression and enzyme secretion. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest paralog in Saccharomyces cerevisiae, Gal4p. Coimmunoprecipitation studies showed that CLR-1 and CLR-2 act in a homocomplex but not as a CLR-1/CLR-2 heterocomplex. IMPORTANCE: Understanding fungal regulation of complex plant cell wall deconstruction pathways in response to multiple environmental signals via interconnected transcriptional circuits provides insight into fungus/plant interactions and eukaryotic nutrient sensing. Coordinated optimization of these regulatory networks is likely required for optimal microbial enzyme production.

Deletion of homologs of the SREBP pathway results in hyper-production of cellulases in Neurospora crassa and Trichoderma reesei.

BACKGROUND: The filamentous fungus Neurospora crassa efficiently utilizes plant biomass and is a model organism for genetic, molecular and cellular biology studies. Here, a set of 567 single-gene deletion strains was assessed for cellulolytic activity as compared to the wild-type parental strain. Mutant strains included were those carrying a deletion in: (1) genes encoding proteins homologous to those implicated in the Saccharomyces cerevisiae secretion apparatus; (2) genes that are homologous to those known to differ between the Trichoderma reesei hyper-secreting strain RUT-C30 and its ancestral wild-type strain; (3) genes encoding proteins identified in the secretome of N. crassa when cultured on plant biomass and (4) genes encoding proteins predicted to traverse the secretory pathway. RESULTS: The 567 single-gene deletion collection was cultured on crystalline cellulose and a comparison of levels of secreted protein and cellulase activity relative to the wild-type strain resulted in the identification of seven hyper-production and 18 hypo-production strains. Some of these deleted genes encoded proteins that are likely to act in transcription, protein synthesis and intracellular trafficking, but many encoded fungal-specific proteins of undetermined function. Characterization of several mutants peripherally linked to protein processing or secretion showed that the hyper- or hypo-production phenotypes were primarily a response to cellulose. The altered secretome of these strains was not limited to the production of cellulolytic enzymes, yet was part of the cellulosic response driven by the cellulase transcription factor CLR-2. Mutants implicated the loss of the SREBP pathway, which has been found to regulate ergosterol biosynthesis genes in response to hypoxic conditions, resulted in a hyper-production phenotype. Deletion of two SREBP pathway components in T. reesei also conferred a hyper-production phenotype under cellulolytic conditions. CONCLUSIONS: These studies demonstrate the utility of screening the publicly available N. crassa single-gene deletion strain collection for a particular phenotype. Mutants in a predicted E3 ligase and its target SREBP transcription factor played an unanticipated role in protein production under cellulolytic conditions. Furthermore, phenotypes similar to those observed in N. crassa were seen following the targeted deletion of orthologous SREBP pathway loci in T. reesei, a fungal species commonly used in industrial enzyme production.

Conserved and essential transcription factors for cellulase gene expression in ascomycete fungi.

Rational engineering of filamentous fungi for improved cellulase production is hampered by our incomplete knowledge of transcriptional regulatory networks. We therefore used the model filamentous fungus Neurospora crassa to search for uncharacterized transcription factors associated with cellulose deconstruction. A screen of a N. crassa transcription factor deletion collection identified two uncharacterized zinc binuclear cluster transcription factors (clr-1 and clr-2) that were required for growth and enzymatic activity on cellulose, but were not required for growth or hemicellulase activity on xylan. Transcriptional profiling with next-generation sequencing methods refined our understanding of the N. crassa transcriptional response to cellulose and demonstrated that clr-1 and clr-2 were required for the bulk of that response, including induction of all major cellulase and some major hemicellulase genes. Functional CLR-1 was necessary for expression of clr-2 and efficient cellobiose utilization. Phylogenetic analyses showed that CLR-1 and CLR-2 are conserved in the genomes of most filamentous ascomycete fungi capable of degrading cellulose. In Aspergillus nidulans, a strain carrying a deletion of the clr-2 homolog (clrB) failed to induce cellulase gene expression and lacked cellulolytic activity on Avicel. Further manipulation of this control system in industrial production strains may significantly improve yields of cellulases for cellulosic biofuel production.

Evaluation of Cultivar Resistance to Soybean Cyst Nematode with a Quantitative Polymerase Chain Reaction Assay.

Heterodera glycines, the soybean cyst nematode, is a major pathogen of soybean. Effective management of this pathogen is contingent on the use of resistant cultivars; thus, screening for resistant cultivars is essential. The purpose of this research was to develop a method to assess infection of soybean roots by H. glycines with real-time quantitative polymerase chain reaction (qPCR). This method will serve as a prelude to differentiation of resistance levels in soybean cultivars. A reproducible inoculation method was developed by means of a sand column to provide active second-stage juveniles (J2). Two-day-old soybean roots were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours after infestation, the roots were surface-sterilized and genomic DNA (gDNA) was extracted. For the qPCR assay, a primer pair for the single copy gene HgSNO, which codes for a protein involved in the production of vitamin B6, was selected for H. glycines gDNA amplification within soybean roots. Compatible 'Lee 74', incompatible 'Peking', and cultivars with different levels of resistance to H. glycines were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours postinfestation, infected seedlings were transplanted into pasteurized soil. Subsequently, they were harvested at 1, 7, 10, 14, and 21 days postinfestation for gDNA extraction. With the qPCR assay, the time needed to differentiate highly resistant cultivars from the rest was reduced. Quantification of H. glycines infection by traditional means (numbers of females produced in 30 days) is a time-consuming practice. This qPCR assay has the potential to replace the traditional Female Index-based screening and improve precision in determining infection levels.

Evidence for horizontally transferred genes involved in the biosynthesis of vitamin B(1), B(5), and B(7) in Heterodera glycines.

Heterodera glycines is a nematode that is highly adapted to manipulate and parasitize plant hosts. The molecular players involved in these interactions have only recently begun to be identified. Here, the sequencing of the second stage juvenile transcriptome, followed by a bioinformatic screen for novel genes, identified seven new genes involved in biosynthesis and salvage of vitamins B1, B5, and B7. With no confirmed reports in the literature, each of these biosynthesis pathways is believed to have been lost in multicellular animals. However, eukaryotic-like introns in the genomic sequences of the genes confirmed eukaryotic origin and nematode-specific splice leaders found on five of the cDNAs confirmed their nematode origin. Two of the genes were found to be flanked by known nematode sequences and quantitative polymerase chain reactions on individual nematodes showed similar and consistent amplification between the vitamin B biosynthesis genes and other known H. glycines genes. This further confirmed their presence in the nematode genome. Similarity to bacterial sequences at the amino acid level suggested a prokaryotic ancestry and phylogenetic analysis of the genes supported a likely horizontal gene transfer event, suggesting H. glycines re-appropriated the genes from the prokaryotic kingdom. This finding complements the previous discovery of a vitamin B6 biosynthesis pathway within the nematode. However, unlike the complete vitamin B6 pathway, many of these vitamin B pathways appear to be missing the initial enzymes required for full de novo biosynthesis, suggesting that initial substrates in the pathways are obtained exogenously. These partial vitamin B biosynthesis enzymes have recently been identified in other single-celled eukaryotic parasites and on rhizobia symbiosis plasmids, indicating that they may play an important role in host-parasite interactions and survival within the plant environment.

Analysis of a horizontally transferred pathway involved in vitamin B6 biosynthesis from the soybean cyst nematode Heterodera glycines.

Heterodera glycines is an obligate plant parasite capable of biochemically and developmentally altering its host's cells in order to create a specialized feeding cell. Although the exact mechanism of feeding cell morphogenesis remains a mystery, the nematode's ability to manipulate the plant is thought to be due in part to horizontal gene transfers (HGTs). A bioinformatic screen of the nematode genome has revealed homologues of the genes SNZ and SNO, which comprise a metabolic pathway for the de novo biosynthesis of pyridoxal 5'-phosphate, the active form of vitamin B(6) (VB(6)). Analysis of the 2 genes, HgSNZ and HgSNO, show that they contain nematode-like introns, generate polyadenylated mRNAs, and map to the soybean cyst nematode genetic linkage map, indicating that they are part of the nematode genome. However, gene synteny, protein homology, and phylogenetic evidence suggest prokaryotic origin. This would represent the first case of the HGT of a complete pathway into a nematode or terrestrial animal. VB(6) acts as a cofactor in over 140 different enzymes, and recent studies point toward an important role as a potent quencher of reactive oxygen species. With H. glycines' penchant for acquiring parasitism genes through HGT along with the absence of this pathway in other land-based animals suggests a specific need for VB(6) which may involve the parasite-host interaction.