Publications by authors named "Scott E Baker"

92 Publications

Multi-Omics Driven Metabolic Network Reconstruction and Analysis of Lignocellulosic Carbon Utilization in .

Front Bioeng Biotechnol 2020 8;8:612832. Epub 2021 Jan 8.

Department of Energy, Agile BioFoundry, Emeryville, CA, United States.

An oleaginous yeast is a promising host for converting lignocellulosic biomass to bioproducts and biofuels. In this work, we performed multi-omics analysis of lignocellulosic carbon utilization in and reconstructed the genome-scale metabolic network of . High-quality metabolic network models for model organisms and orthologous protein mapping were used to build a draft metabolic network reconstruction. The reconstruction was manually curated to build a metabolic model using functional annotation and multi-omics data including transcriptomics, proteomics, metabolomics, and RB-TDNA sequencing. The multi-omics data and metabolic model were used to investigate metabolism including lipid accumulation and lignocellulosic carbon utilization. The developed metabolic model was validated against high-throughput growth phenotyping and gene fitness data, and further refined to resolve the inconsistencies between prediction and data. We believe that this is the most complete and accurate metabolic network model available for to date.
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http://dx.doi.org/10.3389/fbioe.2020.612832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873862PMC
January 2021

High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in KT2440.

Front Bioeng Biotechnol 2020 2;8:603488. Epub 2020 Dec 2.

Department of Energy, Agile BioFoundry, Emeryville, CA, United States.

Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339 peptides representing 132 enzymes in KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for the majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.
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http://dx.doi.org/10.3389/fbioe.2020.603488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793925PMC
December 2020

Comparative Genomic Analysis of Ochratoxin A Biosynthetic Cluster in Producing Fungi: New Evidence of a Cyclase Gene Involvement.

Front Microbiol 2020 18;11:581309. Epub 2020 Dec 18.

Functional and Systems Biology Group, Environmental Molecular Sciences Division, Pacific Northwest National Laboratory, Richland, WA, United States.

The widespread use of Next-Generation Sequencing has opened a new era in the study of biological systems by significantly increasing the catalog of fungal genomes sequences and identifying gene clusters for known secondary metabolites as well as novel cryptic ones. However, most of these clusters still need to be examined in detail to completely understand the pathway steps and the regulation of the biosynthesis of metabolites. Genome sequencing approach led to the identification of the biosynthetic genes cluster of ochratoxin A (OTA) in a number of producing fungal species. Ochratoxin A is a potent pentaketide nephrotoxin produced by and species and found as widely contaminant in food, beverages and feed. The increasing availability of several new genome sequences of OTA producer species in JGI Mycocosm and/or GenBank databanks led us to analyze and update the gene cluster structure in 19 and 2 OTA producing species, resulting in a well conserved organization of OTA core genes among the species. Furthermore, our comparative genome analyses evidenced the presence of an additional gene, previously undescribed, located between the polyketide and non-ribosomal synthase genes in the cluster of all the species analyzed. The presence of a SnoaL cyclase domain in the sequence of this gene supports its putative role in the polyketide cyclization reaction during the initial steps of the OTA biosynthesis pathway. The phylogenetic analysis showed a clustering of OTA SnoaL domains in accordance with the phylogeny of OTA producing species at species and section levels. The characterization of this new OTA gene, its putative role and its expression evidence in three important representative producing species, are reported here for the first time.
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http://dx.doi.org/10.3389/fmicb.2020.581309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775548PMC
December 2020

A comparative genomics study of 23 Aspergillus species from section Flavi.

Nat Commun 2020 02 27;11(1):1106. Epub 2020 Feb 27.

Department of Biotechnology and Bioengineering, Technical University of Denmark, Søltoft Plads 223, 2800, Kongens Lyngby, Denmark.

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.
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http://dx.doi.org/10.1038/s41467-019-14051-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046712PMC
February 2020

Colonies of the fungus Aspergillus niger are highly differentiated to adapt to local carbon source variation.

Environ Microbiol 2020 03 6;22(3):1154-1166. Epub 2020 Jan 6.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.

Saprobic fungi, such as Aspergillus niger, grow as colonies consisting of a network of branching and fusing hyphae that are often considered to be relatively uniform entities in which nutrients can freely move through the hyphae. In nature, different parts of a colony are often exposed to different nutrients. We have investigated, using a multi-omics approach, adaptation of A. niger colonies to spatially separated and compositionally different plant biomass substrates. This demonstrated a high level of intra-colony differentiation, which closely matched the locally available substrate. The part of the colony exposed to pectin-rich sugar beet pulp and to xylan-rich wheat bran showed high pectinolytic and high xylanolytic transcript and protein levels respectively. This study therefore exemplifies the high ability of fungal colonies to differentiate and adapt to local conditions, ensuring efficient use of the available nutrients, rather than maintaining a uniform physiology throughout the colony.
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http://dx.doi.org/10.1111/1462-2920.14907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065180PMC
March 2020

A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable.

PLoS One 2019 17;14(1):e0210243. Epub 2019 Jan 17.

Joint Bioenergy Institute, Emeryville, CA, United States of America.

Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "pop-out" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210243PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336261PMC
October 2019

Circadian Proteomic Analysis Uncovers Mechanisms of Post-Transcriptional Regulation in Metabolic Pathways.

Cell Syst 2018 12 12;7(6):613-626.e5. Epub 2018 Dec 12.

Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA. Electronic address:

Transcriptional and translational feedback loops in fungi and animals drive circadian rhythms in transcript levels that provide output from the clock, but post-transcriptional mechanisms also contribute. To determine the extent and underlying source of this regulation, we applied newly developed analytical tools to a long-duration, deeply sampled, circadian proteomics time course comprising half of the proteome. We found a quarter of expressed proteins are clock regulated, but >40% of these do not arise from clock-regulated transcripts, and our analysis predicts that these protein rhythms arise from oscillations in translational rates. Our data highlighted the impact of the clock on metabolic regulation, with central carbon metabolism reflecting both transcriptional and post-transcriptional control and opposing metabolic pathways showing peak activities at different times of day. The transcription factor CSP-1 plays a role in this metabolic regulation, contributing to the rhythmicity and phase of clock-regulated proteins.
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http://dx.doi.org/10.1016/j.cels.2018.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433121PMC
December 2018

Regulation of Yeast-to-Hyphae Transition in Yarrowia lipolytica.

mSphere 2018 12 5;3(6). Epub 2018 Dec 5.

Environmental Molecular Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA

The yeast undergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All the mutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5'-CCCCT-3') and upregulation of genes with cell cycle box (5'-ACGCG-3') motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p in We confirmed that the (Yl) ortholog is required for hyphal growth and found that overexpression of Yl enables hyphal growth in strains. The cell cycle box is bound by the Mbp1p/Swi6p complex in to regulate G-to-S phase progression. We found that overexpression of either the Yl or Yl homologs decreased hyphal growth and that deletion of either Yl or Yl promotes hyphal growth in strains. A second forward genetic screen for reversion to hyphal growth was performed with the mutant to identify additional genetic factors regulating hyphal growth in Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Yl and Yl and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Yl, Yl, and Yl Together, these results demonstrate that transitions to hyphal growth in response to stress through multiple signaling pathways. Many yeasts undergo a morphological transition from yeast-to-hyphal growth in response to environmental conditions. We used forward and reverse genetic techniques to identify genes regulating this transition in We confirmed that the transcription factor Yl is required for the transition to hyphal growth and found that signaling by the histidine kinases Yl and Yl as well as the MAP kinases of the HOG pathway (Yl, Yl, and Yl) regulates the transition to hyphal growth. These results suggest that transitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response.
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http://dx.doi.org/10.1128/mSphere.00541-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282006PMC
December 2018

Correction to: Cloning and Expression of Heterologous Cellulases and Enzymes in Aspergillus niger.

Methods Mol Biol 2018 ;1796:E1

Joint BioEnergy Institute, Emeryville, CA, USA.

The author's family name were incorrectly published in the original version. This has been corrected to read as.
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http://dx.doi.org/10.1007/978-1-4939-7877-9_22DOI Listing
January 2018

Investigation of inter- and intraspecies variation through genome sequencing of Aspergillus section Nigri.

Nat Genet 2018 12 22;50(12):1688-1695. Epub 2018 Oct 22.

Department of Biotechnology and Bioengineering, Technical University of Denmark, Kongens Lyngby, Denmark.

Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter- and intraspecies genomic variation. We further predicted 17,903 carbohydrate-active enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.
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http://dx.doi.org/10.1038/s41588-018-0246-1DOI Listing
December 2018

Dichomitus squalens partially tailors its molecular responses to the composition of solid wood.

Environ Microbiol 2018 11 18;20(11):4141-4156. Epub 2018 Oct 18.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute and Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands.

White-rot fungi, such as Dichomitus squalens, degrade all wood components and inhabit mixed-wood forests containing both soft- and hardwood species. In this study, we evaluated how D. squalens responded to the compositional differences in softwood [guaiacyl (G) lignin and higher mannan content] and hardwood [syringyl/guaiacyl (S/G) lignin and higher xylan content] using semi-natural solid cultures. Spruce (softwood) and birch (hardwood) sticks were degraded by D. squalens as measured by oxidation of the lignins using 2D-NMR. The fungal response as measured by transcriptomics, proteomics and enzyme activities showed a partial tailoring to wood composition. Mannanolytic transcripts and proteins were more abundant in spruce cultures, while a proportionally higher xylanolytic activity was detected in birch cultures. Both wood types induced manganese peroxidases to a much higher level than laccases, but higher transcript and protein levels of the manganese peroxidases were observed on the G-lignin rich spruce. Overall, the molecular responses demonstrated a stronger adaptation to the spruce rather than birch composition, possibly because D. squalens is mainly found degrading softwoods in nature, which supports the ability of the solid wood cultures to reflect the natural environment.
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http://dx.doi.org/10.1111/1462-2920.14416DOI Listing
November 2018

Protein hyperproduction in fungi by design.

Authors:
Scott E Baker

Appl Microbiol Biotechnol 2018 Oct 4;102(20):8621-8628. Epub 2018 Aug 4.

Department of Energy Joint BioEnergy Institute, Emeryville, CA, 94608, USA.

The secretion of enzymes used by fungi to digest their environment has been exploited by humans for centuries for food and beverage production. More than a century after the first biotechnology patent, we know that the enzyme cocktails secreted by these amazing organisms have tremendous use across a number of industrial processes. Secreting the maximum titer of enzymes is critical to the economic feasibility of these processes. Traditional mutagenesis and screening approaches have generated the vast majority of strains used by industry for the production of enzymes. Until the emergence of economical next generation DNA sequencing platforms, the majority of the genes mutated in these screens remained uncharacterized at the sequence level. In addition, mutagenesis comes with a cost to an organism's fitness, making tractable rational strain design approaches an attractive alternative. As an alternative to traditional mutagenesis and screening, controlled manipulation of multiple genes involved in processes that impact the ability of a fungus to sense its environment, regulate transcription of enzyme-encoding genes, and efficiently secrete these proteins will allow for rational design of improved fungal protein production strains.
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http://dx.doi.org/10.1007/s00253-018-9265-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153651PMC
October 2018

Cloning and Expression of Heterologous Cellulases and Enzymes in Aspergillus niger.

Methods Mol Biol 2018 ;1796:123-133

Joint BioEnergy Institute, Emeryville, CA, USA.

Cellulases and other enzymes are needed for saccharification of plant biomass in the biorefinery industry. Expression, characterization, and eventual large-scale production of known and novel cellulases requires the ability to express and secrete heterologous enzymes in relevant protein production platforms like Aspergillus niger. A method for cloning and expression of genes for these desirable enzymes in A. niger is presented in this Chapter.
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http://dx.doi.org/10.1007/978-1-4939-7877-9_10DOI Listing
February 2019

Blocking hexose entry into glycolysis activates alternative metabolic conversion of these sugars and upregulates pentose metabolism in Aspergillus nidulans.

BMC Genomics 2018 03 22;19(1):214. Epub 2018 Mar 22.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584, CT, Utrecht, The Netherlands.

Background: Plant biomass is the most abundant carbon source for many fungal species. In the biobased industry fungi, are used to produce lignocellulolytic enzymes to degrade agricultural waste biomass. Here we evaluated if it would be possible to create an Aspergillus nidulans strain that releases, but does not metabolize hexoses from plant biomass. For this purpose, metabolic mutants were generated that were impaired in glycolysis, by using hexokinase (hxkA) and glucokinase (glkA) negative strains. To prevent repression of enzyme production due to the hexose accumulation, strains were generated that combined these mutations with a deletion in creA, the repressor involved in regulating preferential use of different carbon catabolic pathways.

Results: Phenotypic analysis revealed reduced growth for the hxkA1 glkA4 mutant on wheat bran. However, hexoses did not accumulate during growth of the mutants on wheat bran, suggesting that glucose metabolism is re-routed towards alternative carbon catabolic pathways. The creAΔ4 mutation in combination with preventing initial phosphorylation in glycolysis resulted in better growth than the hxkA/glkA mutant and an increased expression of pentose catabolic and pentose phosphate pathway genes. This indicates that the reduced ability to use hexoses as carbon sources created a shift towards the pentose fraction of wheat bran as a major carbon source to support growth.

Conclusion: Blocking the direct entry of hexoses to glycolysis activates alternative metabolic conversion of these sugars in A. nidulans during growth on plant biomass, but also upregulates conversion of other sugars, such as pentoses.
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http://dx.doi.org/10.1186/s12864-018-4609-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5863803PMC
March 2018

Linking secondary metabolites to gene clusters through genome sequencing of six diverse species.

Proc Natl Acad Sci U S A 2018 01 9;115(4):E753-E761. Epub 2018 Jan 9.

Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Lyngby, Denmark;

The fungal genus of is highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse species (, , , and ) have been whole-genome PacBio sequenced to provide genetic references in three sections. and also were sequenced for SM elucidation. Thirteen genomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular, was compared with the pathogenic species This suggests that can produce most of the same allergens, virulence, and pathogenicity factors as , suggesting that could be as pathogenic as Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol in , , and , respectively, and novofumigatonin, -cycloechinulin, and -aszonalenins in Our study delivers six fungal genomes, showing the large diversity found in the genus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports of pathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.
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http://dx.doi.org/10.1073/pnas.1715954115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789934PMC
January 2018

Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger.

Appl Microbiol Biotechnol 2018 Feb 6;102(4):1797-1807. Epub 2018 Jan 6.

Joint BioEnergy Institute, Emeryville, CA, 94608, USA.

Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers of heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. This strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.
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http://dx.doi.org/10.1007/s00253-017-8717-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5794824PMC
February 2018

Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger.

PLoS One 2017 27;12(12):e0189604. Epub 2017 Dec 27.

Joint BioEnergy Institute (JBEI), Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, California, United States of America.

Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme production host before they could be considered a viable alternative to current commercial cellulases. Aspergillus niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. niger and Escherichia coli. This comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. niger is equivalent, suggesting that A. niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189604PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744941PMC
January 2018

Multimodal microfluidic platform for controlled culture and analysis of unicellular organisms.

Biomicrofluidics 2017 Sep 19;11(5):054104. Epub 2017 Sep 19.

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory Richland, Washington 99354, USA.

Modern live-cell imaging approaches permit real-time visualization of biological processes, yet limitations exist for unicellular organism isolation, culturing, and long-term imaging that preclude fully understanding how cells sense and respond to environmental perturbations and the link between single-cell variability and whole-population dynamics. Here, we present a microfluidic platform that provides fine control over the local environment with the capacity to replace media components at any experimental time point, and provides both perfused and compartmentalized cultivation conditions depending on the valve configuration. The functionality and flexibility of the platform were validated using both bacteria and yeast having different sizes, motility, and growth media. The demonstrated ability to track the growth and dynamics of both motile and non-motile prokaryotic and eukaryotic organisms emphasizes the versatility of the devices, which should enable studies in bioenergy and environmental research.
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http://dx.doi.org/10.1063/1.4986533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608609PMC
September 2017

Omics Analyses of Trichoderma reesei CBS999.97 and QM6a Indicate the Relevance of Female Fertility to Carbohydrate-Active Enzyme and Transporter Levels.

Appl Environ Microbiol 2017 Nov 31;83(22). Epub 2017 Oct 31.

TU Wien, Insitute of Chemical Engineering, Research Area Molecular Biotechnology, Vienna, Austria

The filamentous fungus is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids (for example proline or alanine), which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis. is a filamentous fungus with a high potential for secretion of plant cell wall-degrading enzymes. We sequenced the genome of the fully fertile field isolate CBS999.97 and analyzed its gene regulation characteristics in comparison with the commonly used laboratory wild-type strain QM6a, which is not female fertile. Additionally, we also evaluated fully fertile strains with genotypes very close to that of QM6a in order to distinguish between strain-specific and fertility-specific characteristics. We found that QM6a and CBS999.97 clearly differ in their growth patterns on different carbon sources, CAZyme gene regulation, and secondary metabolism. Importantly, we found altered regulation of 90 genes associated with female fertility, including CAZyme genes and transporter genes, but only minor mating type-dependent differences. Hence, when using sexual crossing in research and for strain improvement, it is important to consider female fertile and female sterile strains for comparison with QM6a and to achieve optimal performance.
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http://dx.doi.org/10.1128/AEM.01578-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666144PMC
November 2017

Genome sequencing and transcriptome analysis of QM9978 strain reveals a distal chromosome translocation to be responsible for loss of expression and loss of cellulase induction.

Biotechnol Biofuels 2017 7;10:209. Epub 2017 Sep 7.

IFP Energies Nouvelles, 1-4 Avenue de Bois-Préau, 92852 Rueil-Malmaison, France.

Background: The hydrolysis of biomass to simple sugars used for the production of biofuels in biorefineries requires the action of cellulolytic enzyme mixtures. During the last 50 years, the ascomycete , the main source of industrial cellulase and hemicellulase cocktails, has been subjected to several rounds of classical mutagenesis with the aim to obtain higher production levels. During these random genetic events, strains unable to produce cellulases were generated. Here, whole genome sequencing and transcriptomic analyses of the cellulase-negative strain QM9978 were used for the identification of mutations underlying this cellulase-negative phenotype.

Results: Sequence comparison of the cellulase-negative strain QM9978 to the reference strain QM6a identified a total of 43 mutations, of which 33 were located either close to or in coding regions. From those, we identified 23 single-nucleotide variants, nine InDels, and one translocation. The translocation occurred between chromosomes V and VII, is located upstream of the putative transcription factor , and abolishes its expression in QM9978 as detected during the transcriptomic analyses. Ectopic expression of under the control of its native promoter as well as overexpression of under the control of a strong constitutive promoter restored cellulase expression in QM9978, thus confirming that the translocation event is the reason for the cellulase-negative phenotype. Gene deletion of in the moderate producer strain QM9414 and in the high producer strain Rut-C30 reduced cellulase expression in both cases. Overexpression of in QM9414 and Rut-C30 had no effect on cellulase production, most likely because is already expressed at an optimal level under normal conditions.

Conclusion: We were able to establish a link between a chromosomal translocation in QM9978 and the cellulase-negative phenotype of the strain. We identified the transcription factor as a key regulator of cellulases in whose expression is absent in QM9978. We propose that in , as in , is involved in cellulase induction, although the exact mechanism remains to be elucidated. The data presented here show an example of a combined genome sequencing and transcriptomic approach to explain a specific trait, in this case the QM9978 cellulase-negative phenotype, and how it helps to better understand the mechanisms during cellulase gene regulation. When focusing on mutations on the single base-pair level, changes on the chromosome level can be easily overlooked and through this work we provide an example that stresses the importance of the big picture of the genomic landscape during analysis of sequencing data.
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http://dx.doi.org/10.1186/s13068-017-0897-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5588705PMC
September 2017

Non-steady state mass action dynamics without rate constants: dynamics of coupled reactions using chemical potentials.

Phys Biol 2017 08 16;14(5):055003. Epub 2017 Aug 16.

Biological Sciences Division, Pacific Northwest National Laboratory, 902 Battelle Blvd, Richland, WA 99352, United States of America. Author to whom any correspondence should be addressed.

Comprehensive and predictive simulation of coupled reaction networks has long been a goal of biology and other fields. Currently, metabolic network models that utilize enzyme mass action kinetics have predictive power but are limited in scope and application by the fact that the determination of enzyme rate constants is laborious and low throughput. We present a statistical thermodynamic formulation of the law of mass action for coupled reactions at both steady states and non-stationary states. The formulation uses chemical potentials instead of rate constants. When used to model deterministic systems, the method corresponds to a rescaling of the time dependent reactions in such a way that steady states can be reached on the same time scale but with significantly fewer computational steps. The relationships between reaction affinities, free energy changes and generalized detailed balance are central to the discussion. The significance for applications in systems biology are discussed as is the concept and assumption of maximum entropy production rate as a biological principle that links thermodynamics to natural selection.
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http://dx.doi.org/10.1088/1478-3975/aa7d80DOI Listing
August 2017

Leucine Biosynthesis Is Involved in Regulating High Lipid Accumulation in .

mBio 2017 06 20;8(3). Epub 2017 Jun 20.

Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden

The yeast is a potent accumulator of lipids, and lipogenesis in this organism can be influenced by a variety of factors, such as genetics and environmental conditions. Using a multifactorial study, we elucidated the effects of both genetic and environmental factors on regulation of lipogenesis in and identified how two opposite regulatory states both result in lipid accumulation. This study involved comparison of a strain overexpressing diacylglycerol acyltransferase () with a control strain grown under either nitrogen or carbon limitation conditions. A strong correlation was observed between the responses on the transcript and protein levels. Combination of overexpression with nitrogen limitation resulted in a high level of lipid accumulation accompanied by downregulation of several amino acid biosynthetic pathways, including that of leucine in particular, and these changes were further correlated with a decrease in metabolic fluxes. This downregulation was supported by the measured decrease in the level of 2-isopropylmalate, an intermediate of leucine biosynthesis. Combining the multi-omics data with putative transcription factor binding motifs uncovered a contradictory role for TORC1 in controlling lipid accumulation, likely mediated through 2-isopropylmalate and a Leu3-like transcription factor. The ubiquitous metabolism of lipids involves refined regulation, and an enriched understanding of this regulation would have wide implications. Various factors can influence lipid metabolism, including the environment and genetics. We demonstrated, using a multi-omics and multifactorial experimental setup, that multiple factors affect lipid accumulation in the yeast Using integrative analysis, we identified novel interactions between nutrient restriction and genetic factors involving regulators that are highly conserved among eukaryotes. Given that lipid metabolism is involved in many diseases but is also vital to the development of microbial cell factories that can provide us with sustainable fuels and oleochemicals, we envision that our report introduces foundational work to further unravel the regulation of lipid accumulation in eukaryal cells.
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http://dx.doi.org/10.1128/mBio.00857-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478895PMC
June 2017

A parts list for fungal cellulosomes revealed by comparative genomics.

Nat Microbiol 2017 May 30;2:17087. Epub 2017 May 30.

Department of Chemical Engineering, University of California, Santa Barbara, California 93106, USA.

Cellulosomes are large, multiprotein complexes that tether plant biomass-degrading enzymes together for improved hydrolysis. These complexes were first described in anaerobic bacteria, where species-specific dockerin domains mediate the assembly of enzymes onto cohesin motifs interspersed within protein scaffolds. The versatile protein assembly mechanism conferred by the bacterial cohesin-dockerin interaction is now a standard design principle for synthetic biology. For decades, analogous structures have been reported in anaerobic fungi, which are known to assemble by sequence-divergent non-catalytic dockerin domains (NCDDs). However, the components, modular assembly mechanism and functional role of fungal cellulosomes remain unknown. Here, we describe a comprehensive set of proteins critical to fungal cellulosome assembly, including conserved scaffolding proteins unique to the Neocallimastigomycota. High-quality genomes of the anaerobic fungi Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis were assembled with long-read, single-molecule technology. Genomic analysis coupled with proteomic validation revealed an average of 312 NCDD-containing proteins per fungal strain, which were overwhelmingly carbohydrate active enzymes (CAZymes), with 95 large fungal scaffoldins identified across four genera that bind to NCDDs. Fungal dockerin and scaffoldin domains have no similarity to their bacterial counterparts, yet several catalytic domains originated via horizontal gene transfer with gut bacteria. However, the biocatalytic activity of anaerobic fungal cellulosomes is expanded by the inclusion of GH3, GH6 and GH45 enzymes. These findings suggest that the fungal cellulosome is an evolutionarily chimaeric structure-an independently evolved fungal complex that co-opted useful activities from bacterial neighbours within the gut microbiome.
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http://dx.doi.org/10.1038/nmicrobiol.2017.87DOI Listing
May 2017

Erratum to: A molecular genetic toolbox for .

Biotechnol Biofuels 2017 22;10:45. Epub 2017 Feb 22.

Earth and Biological Sciences Directorate, Environmental Molecular Sciences Laboratory, Richland, WA 99354 USA.

[This corrects the article DOI: 10.1186/s13068-016-0687-7.].
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http://dx.doi.org/10.1186/s13068-017-0731-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320769PMC
February 2017

Regulation of Nitrogen Metabolism by GATA Zinc Finger Transcription Factors in .

mSphere 2017 Jan-Feb;2(1). Epub 2017 Feb 15.

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington, USA.

Fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeast , growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism in . Deletion of the GATA transcription factor genes and resulted in nitrogen source-specific growth defects and greater accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion of , which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion of results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, while is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressor , including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism. Nitrogen source is commonly used to control lipid production in industrial fungi. Here we identified regulators of nitrogen catabolite repression in the oleaginous yeast to determine how the nitrogen source regulates lipid metabolism. We show that disruption of both activators and repressors of nitrogen catabolite repression leads to increased lipid accumulation via activation of carbon catabolite repression through an as yet uncharacterized method.
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http://dx.doi.org/10.1128/mSphere.00038-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311114PMC
February 2017

Diverse data supports the transition of filamentous fungal model organisms into the post-genomics era.

Mycology 2017 17;8(2):67-83. Epub 2017 Feb 17.

Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA.

Filamentous fungi have been important as model organisms since the beginning of modern biological inquiry and have benefitted from open data since the earliest genetic maps were shared. From early origins in simple Mendelian genetics of mating types, parasexual genetics of colony colour, and the foundational demonstration of the segregation of a nutritional requirement, the contribution of research systems utilising filamentous fungi has spanned the biochemical genetics era, through the molecular genetics era, and now are at the very foundation of diverse omics approaches to research and development. Fungal model organisms have come from most major taxonomic groups although Ascomycete filamentous fungi have seen the most major sustained effort. In addition to the published material about filamentous fungi, shared molecular tools have found application in every area of fungal biology. Similarly, shared data has contributed to the success of model systems. The scale of data supporting research with filamentous fungi has grown by 10 to 12 orders of magnitude. From genetic to molecular maps, expression databases, and finally genome resources, the open and collaborative nature of the research communities has assured that the rising tide of data has lifted all of the research systems together.
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http://dx.doi.org/10.1080/21501203.2017.1281849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6059044PMC
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

Fungal Ligninolytic Enzymes and Their Applications.

Microbiol Spectr 2016 12;4(6)

Division of Microbiology and Biotechnology, Department of Food and Environmental Sciences, University of Helsinki, 00014 Helsinki, Finland.

The global push toward an efficient and economical biobased economy has driven research to develop more cost-effective applications for the entirety of plant biomass, including lignocellulosic crops. As discussed elsewhere (Karlsson M, Atanasova L, Funck Jensen D, Zeilinger S, in Heitman J et al. [ed], Tuberculosis and the Tubercle Bacillus, 2nd ed, in press), significant progress has been made in the use of polysaccharide fractions from lignocellulose, cellulose, and various hemicellulose types. However, developing processes for use of the lignin fraction has been more challenging. In this chapter, we discuss characteristics of lignolytic enzymes and the fungi that produce them as well as potential and current uses of lignin-derived products.
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http://dx.doi.org/10.1128/microbiolspec.FUNK-0017-2016DOI Listing
December 2016

A molecular genetic toolbox for .

Biotechnol Biofuels 2017 3;10. Epub 2017 Jan 3.

Earth and Biological Sciences Directorate, Environmental Molecular Sciences Laboratory, Richland, WA 99354 USA ; Department of Energy, Battelle EMSL, 3335 Innovation Blvd, Richland, WA 99354 USA.

Background: is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain.

Results: We have developed a set of genetic and molecular tools in order to expand capabilities of for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in . To complement these strains, we constructed plasmids with -optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the " Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells.

Conclusions: These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in . This work provides the Yarrowia community with tools for cell biology and metabolism research in for further development of biofuels and natural products.
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http://dx.doi.org/10.1186/s13068-016-0687-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210315PMC
January 2017

Mycotoxins: A Fungal Genomics Perspective.

Methods Mol Biol 2017 ;1542:367-379

US Department of Energy, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA.

The chemical and enzymatic diversity in the fungal kingdom is staggering. Large-scale fungal genome sequencing projects are generating a massive catalog of secondary metabolite biosynthetic genes and pathways. Fungal natural products are a boon and bane to man as valuable pharmaceuticals and harmful toxins. Understanding how these chemicals are synthesized will aid the development of new strategies to limit mycotoxin contamination of food and feeds as well as expand drug discovery programs. A survey of work focused on the fumonisin family of mycotoxins highlights technological advances and provides a blueprint for future studies of other fungal natural products. Expressed sequence tags led to the discovery of new fumonisin genes (FUM) and hinted at a role for alternatively spliced transcripts in regulation. Phylogenetic studies of FUM genes uncovered a complex evolutionary history of the FUM cluster, as well as fungi with the potential to synthesize fumonisin or fumonisin-like chemicals. The application of new technologies (e.g., CRISPR) could substantially impact future efforts to harness fungal resources.
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http://dx.doi.org/10.1007/978-1-4939-6707-0_24DOI Listing
January 2018