Search NASA⌕ Search

Engineering topics

de Vries, Ronald P.

Publications and source records attributed to de Vries, Ronald P..

15 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Exploring the Structural, Biochemical, and Functional Diversity of Glycoside Hydrolase Family 12 from Penicillium subrubescens

Glycoside hydrolases (GHs) play an essential role in plant biomass degradation and modification for the sustainable production of biochemicals. The filamentous Ascomycete fungus Penicillium subrubescens contains a higher number of GH12 candidates compared to related species. Therefore, we aimed to compare P. subrubescens GH12s for their ability and substrate specificity for plant cell wall polysaccharide degradation and species’ potential as a source of novel enzymes for plant biomass valorization. Our re-evaluated phylogenetic analysis of fungal GH12 members showed that the P. subrubescens GH12s were located in different (new) clades. Biochemical characterization marked PsEglA as an endoglucanase and four other P. subrubescens GH12s (i.e., PsXegA–D) as xyloglucanases. Interestingly, structural features of PsXegD and PsXegE were more comparable to those of Basidiomycete GH12 xyloglucanases with a unique open substrate-binding cleft. PsUegA displayed dual xyloglucanase and endoglucanase activity and also showed distinct structural features. Comparative transcriptome analysis supported the functional diversity of P. subrubescens GH12s in plant biomass degradation. The gene encoding PsUegA was expressed under diverse conditions, suggesting a scouting role for this enzyme.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Comparative genomic analysis of thermophilic fungi reveals convergent evolutionary adaptations and gene losses

Thermophily is a trait scattered across the fungal tree of life, with its highest prevalence within three fungal families (Chaetomiaceae, Thermoascaceae, and Trichocomaceae), as well as some members of the phylum Mucoromycota. We examined 37 thermophilic and thermotolerant species and 42 mesophilic species for this study and identified thermophily as the ancestral state of all three prominent families of thermophilic fungi. Thermophilic fungal genomes were found to encode various thermostable enzymes, including carbohydrate-active enzymes such as endoxylanases, which are useful for many industrial applications. At the same time, the overall gene counts, especially in gene families responsible for microbial defense such as secondary metabolism, are reduced in thermophiles compared to mesophiles. We also found a reduction in the core genome size of thermophiles in both the Chaetomiaceae family and the Eurotiomycetes class. The Gene Ontology terms lost in thermophilic fungi include primary metabolism, transporters, UV response, and O-methyltransferases. Comparative genomics analysis also revealed higher GC content in the third base of codons (GC3) and a lower effective number of codons in fungal thermophiles than in both thermotolerant and mesophilic fungi. Furthermore, using the Support Vector Machine classifier, we identified several Pfam domains capable of discriminating between genomes of thermophiles and mesophiles with 94% accuracy. Using AlphaFold2 to predict protein structures of endoxylanases (GH10), we built a similarity network based on the structures. We found that the number of disulfide bonds appears important for protein structure, and the network clusters based on protein structures correlate with the optimal activity temperature. Thus, comparative genomics offers new insights into the biology, adaptation, and evolutionary history of thermophilic fungi while providing a parts list for bioengineering applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Genomic Analysis of Aspergillus Section Terrei Reveals a High Potential in Secondary Metabolite Production and Plant Biomass Degradation

Aspergillus terreus has attracted interest due to its application in industrial biotechnology, particularly for the production of itaconic acid and bioactive secondary metabolites. As related species also seem to possess a prosperous secondary metabolism, they are of high interest for genome mining and exploitation. Here, we present draft genome sequences for six species from Aspergillus section Terrei and one species from Aspergillus section Nidulantes. Whole-genome phylogeny confirmed that section Terrei is monophyletic. Genome analyses identified between 70 and 108 key secondary metabolism genes in each of the genomes of section Terrei, the highest rate found in the genus Aspergillus so far. The respective enzymes fall into 167 distinct families with most of them corresponding to potentially unique compounds or compound families. Moreover, 53% of the families were only found in a single species, which supports the suitability of species from section Terrei for further genome mining. Intriguingly, this analysis, combined with heterologous gene expression and metabolite identification, suggested that species from section Terrei use a strategy for UV protection different to other species from the genus Aspergillus. Section Terrei contains a complete plant polysaccharide degrading potential and an even higher cellulolytic potential than other Aspergilli, possibly facilitating additional applications for these species in biotechnology.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Genome evolution and transcriptome plasticity is associated with adaptation to monocot and dicot plants in Colletotrichum fungi

Colletotrichum fungi infect a wide diversity of monocot and dicot hosts, causing diseases on almost all economically important plants worldwide. Colletotrichum is also a suitable model for studying gene family evolution on a fine scale to uncover events in the genome associated with biological changes. Here we present the genome sequences of 30 Colletotrichum species covering the diversity within the genus. Evolutionary analyses revealed that the Colletotrichum ancestor diverged in the late Cretaceous in parallel with the diversification of flowering plants. We provide evidence of independent host jumps from dicots to monocots during the evolution of Colletotrichum, coinciding with a progressive shrinking of the plant cell wall degradative arsenal and expansions in lineage-specific gene families. Comparative transcriptomics of 4 species adapted to different hosts revealed similarity in gene content but high diversity in the modulation of their transcription profiles on different plant substrates. Combining genomics and transcriptomics, we identified a set of core genes such as specific transcription factors, putatively involved in plant cell wall degradation. These results indicate that the ancestral Colletotrichum were associated with dicot plants and certain branches progressively adapted to different monocot hosts, reshaping the gene content and its regulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Comparative Genomics and Transcriptomics Analyses Reveal Divergent Plant Biomass-Degrading Strategies in Fungi

Plant biomass is one of the most abundant renewable carbon sources, which holds great potential for replacing current fossil-based production of fuels and chemicals. In nature, fungi can efficiently degrade plant polysaccharides by secreting a broad range of carbohydrate-active enzymes (CAZymes), such as cellulases, hemicellulases, and pectinases. Due to the crucial role of plant biomass-degrading (PBD) CAZymes in fungal growth and related biotechnology applications, investigation of their genomic diversity and transcriptional dynamics has attracted increasing attention. In this project, we systematically compared the genome content of PBD CAZymes in six taxonomically distant species, Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium, and Dichomitus squalens, as well as their transcriptome profiles during growth on nine monosaccharides. Considerable genomic variation and remarkable transcriptomic diversity of CAZymes were identified, implying the preferred carbon source of these fungi and their different methods of transcription regulation. In addition, the specific carbon utilization ability inferred from genomics and transcriptomics was compared with fungal growth profiles on corresponding sugars, to improve our understanding of the conversion process. This study enhances our understanding of genomic and transcriptomic diversity of fungal plant polysaccharide-degrading enzymes and provides new insights into designing enzyme mixtures and metabolic engineering of fungi for related industrial applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The Amylolytic Regulator AmyR of Aspergillus niger Is Involved in Sucrose and Inulin Utilization in a Culture-Condition-Dependent Manner

Filamentous fungi degrade complex plant material to its monomeric building blocks, which have many biotechnological applications. Transcription factors play a key role in plant biomass degradation, but little is known about their interactions in the regulation of polysaccharide degradation. Here, we deepened the knowledge about the storage polysaccharide regulators AmyR and InuR in Aspergillus niger. AmyR controls starch degradation, while InuR is involved in sucrose and inulin utilization. In our study, the phenotypes of A. niger parental, ΔamyR, ΔinuR and ΔamyRΔinuR strains were assessed in both solid and liquid media containing sucrose or inulin as carbon source to evaluate the roles of AmyR and InuR and the effect of culture conditions on their functions. In correlation with previous studies, our data showed that AmyR has a minor contribution to sucrose and inulin utilization when InuR is active. In contrast, growth profiles and transcriptomic data showed that the deletion of amyR in the ΔinuR background strain resulted in more pronounced growth reduction on both substrates, mainly evidenced by data originating from solid cultures. Overall, our results show that submerged cultures do not always reflect the role of transcription factors in the natural growth condition, which is better represented on solid substrates. Importance: The type of growth has critical implications in enzyme production by filamentous fungi, a process that is controlled by transcription factors. Submerged cultures are the preferred setups in laboratory and industry and are often used for studying the physiology of fungi. In this study, we showed that the genetic response of A. niger to starch and inulin was highly affected by the culture condition, since the transcriptomic response obtained in a liquid environment did not fully match the behavior of the fungus in a solid environment. These results have direct implications in enzyme production and would help industry choose the best approaches to produce specific CAZymes for industrial purposes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The transcriptional activator ClrB is crucial for the degradation of soybean hulls and guar gum in Aspergillus niger

Low-cost plant substrates, such as soybean hulls, are used for various industrial applications. Filamentous fungi are important producers of Carbohydrate Active enZymes (CAZymes) required for the degradation of these plant biomass substrates. CAZyme production is tightly regulated by several transcriptional activators and repressors. One such transcriptional activator is CLR-2/ClrB/ManR, which has been identified as a regulator of cellulase and mannanase production in several fungi. However, the regulatory network governing the expression of cellulase and mannanase encoding genes has been reported to differ between fungal species. Previous studies showed that Aspergillus niger ClrB is involved in the regulation of (hemi-)cellulose degradation, although its regulon has not yet been identified. To reveal its regulon, we cultivated an A. niger ΔclrB mutant and control strain on guar gum (a galactomannan-rich substrate) and soybean hulls (containing galactomannan, xylan, xyloglucan, pectin and cellulose) to identify the genes that are regulated by ClrB. Gene expression data and growth profiling showed that ClrB is indispensable for growth on cellulose and galactomannan and highly contributes to growth on xyloglucan in this fungus. Therefore, we show that A. niger ClrB is crucial for the utilization of guar gum and the agricultural substrate, soybean hulls. Moreover, we show that mannobiose is most likely the physiological inducer of ClrB in A. niger and not cellobiose, which is considered to be the inducer of N. crassa CLR-2 and A. nidulans ClrB.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Unraveling the regulation of sugar beet pulp utilization in the industrially relevant fungus Aspergillus niger

Efficient utilization of agro-industrial waste, such as sugar beet pulp, is crucial for the bio-based economy. The fungus Aspergillus niger possesses a wide array of enzymes that degrade complex plant biomass substrates, and several regulators have been reported to play a role in their production. The role of the regulators GaaR, AraR, and RhaR in sugar beet pectin degradation has previously been reported. However, genetic regulation of the degradation of sugar beet pulp has not been assessed in detail. In this study, we generated a set of single and combinatorial deletion mutants targeting the pectinolytic regulators GaaR, AraR, RhaR, and GalX as well as the (hemi-)cellulolytic regulators XlnR and ClrB to address their relative contribution to the utilization of sugar beet pulp. We show that A. niger has a flexible regulatory network, adapting to the utilization of (hemi-)cellulose at early timepoints when pectin degradation is impaired.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

GalR, GalX and AraR co-regulate $\tiny {D}$-galactose and $\tiny{L}$-arabinose utilization in Aspergillus nidulans

Filamentous fungi produce a wide variety of enzymes in order to efficiently degrade plant cell wall polysaccharides. The production of these enzymes is controlled by transcriptional regulators, which also control the catabolic pathways that convert the released monosaccharides. Two transcriptional regulators, GalX and GalR, control d-galactose utilization in the model filamentous fungus Aspergillus nidulans, while the arabinanolytic regulator AraR regulates l-arabinose catabolism. d-Galactose and l-arabinose are commonly found together in polysaccharides, such as arabinogalactan, xylan and rhamnogalacturonan I. Therefore, the catabolic pathways that convert d-galactose and l-arabinose are often also likely to be active simultaneously. In this study, we investigated the interaction between GalX, GalR and AraR in d-galactose and l-arabinose catabolism. For this, we generated single, double and triple mutants of the three regulators, and analysed their growth and enzyme and gene expression profiles. Our results clearly demonstrated that GalX, GalR and AraR co-regulate d-galactose catabolism in A. nidulans. GalX has a prominent role on the regulation of genes of d-galactose oxido-reductive pathway, while AraR can compensate for the absence of GalR and/or GalX.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Detailed analysis of the D-galactose catabolic pathways in Aspergillus niger reveals complexity at both metabolic and regulatory level

The current impetus towards a sustainable bio-based economy has accelerated research to better understand the mechanisms through which filamentous fungi convert plant biomass, a valuable feedstock for biotechnological applications. Several transcription factors have been reported to control the polysaccharide degradation and metabolism of the resulting sugars in fungi. However, little is known about their individual contributions, interactions and crosstalk. D-galactose is a hexose sugar present mainly in hemicellulose and pectin in plant biomass. Here, we study D-galactose conversion by Aspergillus niger and describe the involvement of the arabinanolytic and xylanolytic activators AraR and XlnR, in addition to the D-galactose-responsive regulator GalX. Our results deepen the understanding of the complexity of the filamentous fungal regulatory network for plant biomass degradation and sugar catabolism, and facilitate the generation of more efficient plant biomass-degrading strains for biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Re-routing of Sugar Catabolism Provides a Better Insight Into Fungal Flexibility in Using Plant Biomass-Derived Monomers as Substrates

The filamentous ascomycete Aspergillus niger has received increasing interest as a cell factory, being able to efficiently degrade plant cell wall polysaccharides as well as having an extensive metabolism to convert the released monosaccharides into value added compounds. The pentoses D-xylose and L-arabinose are the most abundant monosaccharides in plant biomass after the hexose D-glucose, being major constituents of xylan, pectin and xyloglucan. In this study, the influence of selected pentose catabolic pathway (PCP) deletion strains on growth on plant biomass and re-routing of sugar catabolism was addressed to gain a better understanding of the flexibility of this fungus in using plant biomass-derived monomers. The transcriptome, metabolome and proteome response of three PCP mutant strains, Δ larA Δ xyrA Δ xyrB , Δ ladA Δ xdhA Δ sdhA and Δ xkiA , grown on wheat bran (WB) and sugar beet pulp (SBP), was evaluated. Our results showed that despite the absolute impact of these PCP mutations on pure pentose sugars, they are not as critical for growth of A. niger on more complex biomass substrates, such as WB and SBP. However, significant phenotypic variation was observed between the two biomass substrates, but also between the different PCP mutants. This shows that the high sugar heterogeneity of these substrates in combination with the high complexity and adaptability of the fungal sugar metabolism allow for activation of alternative strategies to support growth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

CreA-mediated repression of gene expression occurs at low monosaccharide levels during fungal plant biomass conversion in a time and substrate dependent manner

Carbon catabolite repression enables fungi to utilize the most favourable carbon source in the environment, and is mediated by a key regulator, CreA, in most fungi. CreA-mediated regulation has mainly been studied at high monosaccharide concentrations, an uncommon situation in most natural biotopes. In nature, many fungi rely on plant biomass as their major carbon source by producing enzymes to degrade plant cell wall polysaccharides into metabolizable sugars. To determine the role of CreA when fungi grow in more natural conditions and in particular with respect to degradation and conversion of plant cell walls, we compared transcriptomes of a creA deletion and reference strain of the ascomycete Aspergillus niger during growth on sugar beet pulp and wheat bran. Transcriptomics, extracellular sugar concentrations and growth profiling of A. niger on a variety of carbon sources, revealed that also under conditions with low concentrations of free monosaccharides, CreA has a major effect on gene expression in a strong time and substrate composition dependent manner. In addition, we compared the CreA regulon from five fungi during their growth on crude plant biomass or cellulose. It showed that CreA commonly regulated genes related to carbon metabolism, sugar transport and plant cell wall degrading enzymes across different species. We therefore conclude that CreA has a crucial role for fungi also in adapting to low sugar concentrations as occurring in their natural biotopes, which is supported by the presence of CreA orthologs in nearly all fungi.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H 2 O 2 -producing enzymes with H 2 O 2 -consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.

59 BASIC BIOLOGICAL SCIENCES↗