Publications by authors named "Jon K Magnuson"

38 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

Gene family expansions and transcriptome signatures uncover fungal adaptations to wood decay.

Environ Microbiol 2021 Feb 4. Epub 2021 Feb 4.

INRAE, Aix Marseille Univ, UMR1163, Biodiversité et Biotechnologie Fongiques, Marseille, 13009, France.

Because they comprise some of the most efficient wood-decayers, Polyporales fungi impact carbon cycling in forest environment. Despite continuous discoveries on the enzymatic machinery involved in wood decomposition, the vision on their evolutionary adaptation to wood decay and genome diversity remains incomplete. We combined the genome sequence information from 50 Polyporales species, including 26 newly sequenced genomes and sought for genomic and functional adaptations to wood decay through the analysis of genome composition and transcriptome responses to different carbon sources. The genomes of Polyporales from different phylogenetic clades showed poor conservation in macrosynteny, indicative of genome rearrangements. We observed different gene family expansion/contraction histories for plant cell wall degrading enzymes in core polyporoids and phlebioids and captured expansions for genes involved in signalling and regulation in the lineages of white rotters. Furthermore, we identified conserved cupredoxins, thaumatin-like proteins and lytic polysaccharide monooxygenases with a yet uncharacterized appended module as new candidate players in wood decomposition. Given the current need for enzymatic toolkits dedicated to the transformation of renewable carbon sources, the observed genomic diversity among Polyporales strengthens the relevance of mining Polyporales biodiversity to understand the molecular mechanisms of wood decay.
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http://dx.doi.org/10.1111/1462-2920.15423DOI Listing
February 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

Conversion of poplar biomass into high-energy density tricyclic sesquiterpene jet fuel blendstocks.

Microb Cell Fact 2020 Nov 12;19(1):208. Epub 2020 Nov 12.

Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, 94608, USA.

Background: In an effort to ensure future energy security, reduce greenhouse gas emissions and create domestic jobs, the US has invested in technologies to develop sustainable biofuels and bioproducts from renewable carbon sources such as lignocellulosic biomass. Bio-derived jet fuel is of particular interest as aviation is less amenable to electrification compared to other modes of transportation and synthetic biology provides the ability to tailor fuel properties to enhance performance. Specific energy and energy density are important properties in determining the attractiveness of potential bio-derived jet fuels. For example, increased energy content can give the industry options such as longer range, higher load or reduced takeoff weight. Energy-dense sesquiterpenes have been identified as potential next-generation jet fuels that can be renewably produced from lignocellulosic biomass.

Results: We developed a biomass deconstruction and conversion process that enabled the production of two tricyclic sesquiterpenes, epi-isozizaene and prespatane, from the woody biomass poplar using the versatile basidiomycete Rhodosporidium toruloides. We demonstrated terpene production at both bench and bioreactor scales, with prespatane titers reaching 1173.6 mg/L when grown in poplar hydrolysate in a 2 L bioreactor. Additionally, we examined the theoretical fuel properties of prespatane and epi-isozizaene in their hydrogenated states as blending options for jet fuel, and compared them to aviation fuel, Jet A.

Conclusion: Our findings indicate that prespatane and epi-isozizaene in their hydrogenated states would be attractive blending options in Jet A or other lower density renewable jet fuels as they would improve viscosity and increase their energy density. Saturated epi-isozizaene and saturated prespatane have energy densities that are 16.6 and 18.8% higher than Jet A, respectively. These results highlight the potential of R. toruloides as a production host for the sustainable and scalable production of bio-derived jet fuel blends, and this is the first report of prespatane as an alternative jet fuel.
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http://dx.doi.org/10.1186/s12934-020-01456-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659065PMC
November 2020

Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design.

Metab Eng Commun 2020 Dec 19;11:e00139. Epub 2020 Jul 19.

Biosciences Division, Oak Ridge National Laboratory, P.O. Box 2008, MS6342 Oak Ridge, Tennessee, 37831-6342, USA.

The development of strains for industrial production of fuels and chemicals will require the integration of heterologous genes and pathways into the chromosome. Finding the most appropriate integration site to maximize strain performance is an essential part of the strain design process. We characterized seven chromosomal loci in KT2440 for integration of a fluorescent protein expression construct. Insertion in five of the loci did not affect growth rate, but fluorescence varied by up to 27-fold. Three sites displaying a diversity of phenotypes with the fluorescent reporter were also chosen for the integration of a gene encoding a muconate importer. Depending on the integration locus, expression of the importer varied by approximately 3-fold and produced significant phenotypic differences. This work demonstrates the impact of the integration location on host viability, gene expression, and overall strain performance.
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http://dx.doi.org/10.1016/j.mec.2020.e00139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398981PMC
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

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.

Microb Cell Fact 2020 Feb 5;19(1):24. Epub 2020 Feb 5.

Department of Energy, Agile BioFoundry, Emeryville, CA, 94608, USA.

Background: Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials.

Results: The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L.

Conclusion: This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism.
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http://dx.doi.org/10.1186/s12934-020-1293-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003354PMC
February 2020

Transcriptomic analysis of the oleaginous yeast during lipid accumulation on enzymatically treated corn stover hydrolysate.

Biotechnol Biofuels 2019 26;12:162. Epub 2019 Jun 26.

1Pacific Northwest National Laboratory, Richland, WA USA.

Background: Efficient and economically viable production of biofuels from lignocellulosic biomass is dependent on mechanical and chemical pretreatment and enzymatic hydrolysis of plant material. These processing steps yield simple sugars as well as plant-derived and process-added organic acids, sugar-derived dehydration products, aldehydes, phenolics and other compounds that inhibit the growth of many microorganisms. is an oleaginous yeast capable of robust growth on a variety of sugars and lipid accumulation on pretreated lignocellulosic substrates making it attractive as an industrial producer of biofuels. Here, we examined gene expression during batch growth and lipid accumulation in a 20-L bioreactor with either a blend of pure glucose and xylose or pretreated corn stover (PCS) that had been enzymatically hydrolyzed as the carbon sources.

Results: We monitored sugar and ammonium utilization as well as biomass accumulation and found that growth of is inhibited with PCS hydrolysate as the carbon source. Both acetic acid and furfural are present at concentrations toxic to in PCS hydrolysate. We quantified gene expression at seven time-points for each carbon source during batch growth and found that gene expression is similar at physiologically equivalent points. Analysis of promoter regions revealed that gene expression during the transition to lipid accumulation is regulated by carbon and nitrogen catabolite repression, regardless of carbon source and is associated with decreased expression of the translation machinery and suppression of the cell cycle. We identified 73 differentially expressed genes during growth phase in the bioreactor that may be involved in detoxification of corn stover hydrolysate.

Conclusions: Growth of is inhibited by compounds present in PCS hydrolysate. Here, we monitored key metabolites to establish physiologically equivalent comparisons during a batch bioreactor run comparing PCS hydrolysate and purified sugars. 's response to PCS hydrolysate is primarily at the beginning of the run during growth phase when inhibitory compounds are presumably at their highest concentration and inducing the general detoxification response by . Differentially expressed genes identified herein during growth phase will aid in the improvement of industrial strains capable of robust growth on substrates containing various growth inhibitory compounds.
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http://dx.doi.org/10.1186/s13068-019-1510-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593508PMC
June 2019

Conversion of depolymerized sugars and aromatics from engineered feedstocks by two oleaginous red yeasts.

Bioresour Technol 2019 Aug 22;286:121365. Epub 2019 Apr 22.

Joint BioEnergy Institute, 5885 Hollis St, Emeryville, CA 94608, USA; Sandia National Laboratories, 7011 East Ave, Livermore, CA 94551, USA. Electronic address:

One of the requirements for efficient biological conversion of lignocellulose to bioproducts is the compatibility of biological catalysts with the processes employed to solubilize and depolymerize the lignocellulosic components. The red yeasts Rhodosporidium toruloides and Rhodotorula mucilaginosa were evaluated for their ability to assimilate sugars and aromatic compounds extracted from two engineered lines of Arabidopsis thaliana with modified lignin or the wild-type using ionic liquid, acid or alkaline pretreatments. Differential amounts of monomeric sugars, organic acids and, in the case of the engineered lines, either 4-hydroxybenzoic or protocatechuic acid were additionally released from the biomass and found to be tolerated and consumed by both microorganisms. Genetically-engineered strains of the two red yeasts successfully converted the depolymerized products into the biofuel precursor bisabolene when cultivated on hydrolysates or synthetic media containing specific sugars, acids and aromatics found in the hydrolysates.
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http://dx.doi.org/10.1016/j.biortech.2019.121365DOI Listing
August 2019

Multiplexed CRISPR-Cas9-Based Genome Editing of .

mSphere 2019 03 20;4(2). Epub 2019 Mar 20.

Energy Biosciences Institute, Berkeley, California, USA

Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as and Significant advancements in the past few years have bolstered ' engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of exhibiting successful targeted disruption of the native gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, , with much greater success; editing efficiencies of deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both and in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism. Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast's production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in : CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in .
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http://dx.doi.org/10.1128/mSphere.00099-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429044PMC
March 2019

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

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

Deletion of the KU70 homologue facilitates gene targeting in Lipomyces starkeyi strain NRRL Y-11558.

Curr Genet 2019 Feb 18;65(1):269-282. Epub 2018 Aug 18.

Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.

The objective of this study was to disrupt the non-homologous end-joining (NHEJ) pathway gene (Lsku70Δ) and evaluate the effects of selected gene deletions related to glycogen synthesis (LsGSY1) and lipid degradation (LsMFE1, LsPEX10, and LsTGL4) on lipid production in the oleaginous yeast Lipomyces starkeyi. Disruption of the NHEJ pathway to reduce the rate of non-homologous recombination is a common approach used to overcome low-efficiency targeted deletion or insertion in various organisms. Here, the homologue of the LsKU70 gene was identified and disrupted in L. starkeyi NRRL Y-11558. The LsGSY1, LsMFE1, LsPEX10, LsTGL4, and LsURA3 genes were then replaced with a resistance marker in the Lsku70Δ strain and several site-specific insertions were assessed for targeted over-expression of selected genes. The targeted disruption efficiency of five selected genes (LsGSY1, LsMFE1, LsPEX10, LsTGL4, and LsURA3) was increased from 0 to 10% in the parent to 50-100% of transformants screened in the Lsku70Δ strain with 0.8-1.4 kb homologous flanking sequences, while the efficiency of site-specific gene insertion with the β-glucuronidase reporter gene was 100% in the locus near the 3'-end coding (LsKU70) and non-coding (LsGSY1, LsMFE1, and LsPEX10) regions. Disruption of LsKU70 in isolation and in conjunction with LsGSY1, LsMFE1, LsPEX10, or LsTGL4 did not affect lipid production in L. starkeyi. Furthermore, β-glucuronidase reporter gene activity was similar in strains containing site-specific targeted insertions. Therefore, over-expression of genes related to lipid synthesis at targeted loci can be further examined for improvement of total lipid production in L. starkeyi.
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http://dx.doi.org/10.1007/s00294-018-0875-zDOI Listing
February 2019

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

Draft Genome Sequence of Aspergillus oryzae ATCC 12892.

Genome Announc 2018 May 3;6(18). Epub 2018 May 3.

Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, Washington, USA

The draft genome sequence of ATCC 12892 is presented here. produces 3-nitropropionic acid, which has been investigated with regard to understanding the biosynthesis of nitroorganic compounds.
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http://dx.doi.org/10.1128/genomeA.00251-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940936PMC
May 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

a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts.

Biotechnol Biofuels 2017 23;10:241. Epub 2017 Oct 23.

Joint BioEnergy Institute, 5885 Hollis St, Emeryville, CA 94608 USA.

Background: Economical conversion of lignocellulosic biomass into biofuels and bioproducts is central to the establishment of a robust bioeconomy. This requires a conversion host that is able to both efficiently assimilate the major lignocellulose-derived carbon sources and divert their metabolites toward specific bioproducts.

Results: In this study, the carotenogenic yeast was examined for its ability to convert lignocellulose into two non-native sesquiterpenes with biofuel (bisabolene) and pharmaceutical (amorphadiene) applications. We found that can efficiently convert a mixture of glucose and xylose from hydrolyzed lignocellulose into these bioproducts, and unlike many conventional production hosts, its growth and productivity were enhanced in lignocellulosic hydrolysates relative to purified substrates. This organism was demonstrated to have superior growth in corn stover hydrolysates prepared by two different pretreatment methods, one using a novel biocompatible ionic liquid (IL) choline α-ketoglutarate, which produced 261 mg/L of bisabolene at bench scale, and the other using an alkaline pretreatment, which produced 680 mg/L of bisabolene in a high-gravity fed-batch bioreactor. Interestingly, was also observed to assimilate -coumaric acid liberated from acylated grass lignin in the IL hydrolysate, a finding we verified with purified substrates. was also able to consume several additional compounds with aromatic motifs similar to lignin monomers, suggesting that this organism may have the metabolic potential to convert depolymerized lignin streams alongside lignocellulosic sugars.

Conclusions: This study highlights the natural compatibility of with bioprocess conditions relevant to lignocellulosic biorefineries and demonstrates its ability to produce non-native terpenes.
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http://dx.doi.org/10.1186/s13068-017-0927-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651578PMC
October 2017

Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species.

Appl Microbiol Biotechnol 2017 Aug 19;101(15):6099-6110. Epub 2017 Jun 19.

Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species.
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http://dx.doi.org/10.1007/s00253-017-8357-7DOI Listing
August 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

Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger.

Fungal Genet Biol 2013 Dec 25;61:120-32. Epub 2013 Sep 25.

Fungal Biotechnology Team, Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA 99352, United States. Electronic address:

Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl α-1,3-mannosyltransferase (also known as "asparagine-linked glycosylation 3", or ALG3) is involved in early N-linked glycan synthesis and thus is essential for formation of N-linked protein glycosylation. In this study, we examined the effects of alg3 gene deletion (alg3Δ) on growth, development, pigment production, protein secretion and recombinant Trichoderma reesei cellobiohydrolase (rCel7A) expressed in Aspergillus niger. The alg3Δ delayed spore germination in liquid cultures of complete medium (CM), potato dextrose (PD), minimal medium (MM) and CM with addition of cAMP (CM+cAMP), and resulted in significant reduction of hyphal growth on CM, potato dextrose agar (PDA), and CM+cAMP and spore production on CM. The alg3Δ also led to a significant accumulation of red pigment on both liquid and solid CM cultures. The relative abundances of 54 of the total 215 proteins identified in the secretome were significantly altered as a result of alg3Δ, 63% of which were secreted at higher levels in alg3Δ strain than the parent. The rCel7A expressed in the alg3Δ mutant was smaller in size than that expressed in both wild-type and parental strains, but still larger than T. reesei Cel7A. The circular dichroism (CD)-melt scans indicated that change in glycosylation of rCel7A does not appear to impact the secondary structure or folding. Enzyme assays of Cel7A and rCel7A on nanocrystalline cellulose and bleached kraft pulp demonstrated that the rCel7As have improved activities on hydrolyzing the nanocrystalline cellulose. Overall, the results suggest that alg3 is critical for growth, sporulation, pigment production, and protein secretion in A. niger, and demonstrate the feasibility of this alternative approach to evaluate the roles of N-linked glycosylation in glycoprotein secretion and function.
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http://dx.doi.org/10.1016/j.fgb.2013.09.004DOI Listing
December 2013

Post-genomic approaches to understanding interactions between fungi and their environment.

IMA Fungus 2011 Jun 24;2(1):81-6. Epub 2011 May 24.

Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands;

Fungi inhabit every natural and anthropogenic environment on Earth. They have highly varied life-styles including saprobes (using only dead biomass as a nutrient source), pathogens (feeding on living biomass), and symbionts (co-existing with other organisms). These distinctions are not absolute as many species employ several life styles (e.g. saprobe and opportunistic pathogen, saprobe and mycorrhiza). To efficiently survive in these different and often changing environments, fungi need to be able to modify their physiology and in some cases will even modify their local environment. Understanding the interaction between fungi and their environments has been a topic of study for many decades. However, recently these studies have reached a new dimension. The availability of fungal genomes and development of post-genomic technologies for fungi, such as transcriptomics, proteomics and metabolomics, have enabled more detailed studies into this topic resulting in new insights. Based on a Special Interest Group session held during IMC9, this paper provides examples of the recent advances in using (post-)genomic approaches to better understand fungal interactions with their environments.
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http://dx.doi.org/10.5598/imafungus.2011.02.01.11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3317359PMC
June 2011

Proteomic and functional analysis of the cellulase system expressed by Postia placenta during brown rot of solid wood.

Appl Environ Microbiol 2011 Nov 23;77(22):7933-41. Epub 2011 Sep 23.

Eco-Friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services, Jinju 660-360, Republic of Korea.

Brown rot basidiomycetes have an important ecological role in lignocellulose recycling and are notable for their rapid degradation of wood polymers via oxidative and hydrolytic mechanisms. However, most of these fungi apparently lack processive (exo-acting) cellulases, such as cellobiohydrolases, which are generally required for efficient cellulolysis. The recent sequencing of the Postia placenta genome now permits a proteomic approach to this longstanding conundrum. We grew P. placenta on solid aspen wood, extracted proteins from the biodegrading substrate, and analyzed tryptic digests by shotgun liquid chromatography-tandem mass spectrometry. Comparison of the data with the predicted P. placenta proteome revealed the presence of 34 likely glycoside hydrolases, but only four of these--two in glycoside hydrolase family 5, one in family 10, and one in family 12--have sequences that suggested possible activity on cellulose. We expressed these enzymes heterologously and determined that they all exhibited endoglucanase activity on phosphoric acid-swollen cellulose. They also slowly hydrolyzed filter paper, a more crystalline substrate, but the soluble/insoluble reducing sugar ratios they produced classify them as nonprocessive. Computer simulations indicated that these enzymes produced soluble/insoluble ratios on reduced phosphoric acid-swollen cellulose that were higher than expected for random hydrolysis, which suggests that they could possess limited exo activity, but they are at best 10-fold less processive than cellobiohydrolases. It appears likely that P. placenta employs a combination of oxidative mechanisms and endo-acting cellulases to degrade cellulose efficiently in the absence of a significant processive component.
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http://dx.doi.org/10.1128/AEM.05496-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208998PMC
November 2011

Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88.

Genome Res 2011 Jun 4;21(6):885-97. Epub 2011 May 4.

Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark.

The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.
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http://dx.doi.org/10.1101/gr.112169.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3106321PMC
June 2011

Genome sequence of the model mushroom Schizophyllum commune.

Nat Biotechnol 2010 Sep 11;28(9):957-63. Epub 2010 Jul 11.

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

Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species's unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals.
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http://dx.doi.org/10.1038/nbt.1643DOI Listing
September 2010

The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA.

BMC Plant Biol 2010 May 7;10:83. Epub 2010 May 7.

Department of Biology, Dalhousie University, Halifax, Canada.

Background: Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of beta-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri.

Results: The D. salina organelle genomes are large, circular-mapping molecules with approximately 60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: approximately 1.5 and approximately 0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA.

Conclusions: These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable plastid transformation system for this model alga, and they will complement the forthcoming D. salina nuclear genome sequence, placing D. salina in a group of a select few photosynthetic eukaryotes for which complete genome sequences from all three genetic compartments are available.
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http://dx.doi.org/10.1186/1471-2229-10-83DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017802PMC
May 2010

Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing.

Proc Natl Acad Sci U S A 2009 Sep 2;106(38):16151-6. Epub 2009 Sep 2.

Institut National de la Santé et de la Recherche Médicale, U784, 46 rue d'Ulm, 75230 Paris Cedex 05, France.

Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.
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http://dx.doi.org/10.1073/pnas.0905848106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752593PMC
September 2009