10 results on '"Kim, Joonhoon"'
Search Results
2. PeakDecoder enables machine learning-based metabolite annotation and accurate profiling in multidimensional mass spectrometry measurements.
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Bilbao, Aivett, Munoz, Nathalie, Kim, Joonhoon, Orton, Daniel J., Gao, Yuqian, Poorey, Kunal, Pomraning, Kyle R., Weitz, Karl, Burnet, Meagan, Nicora, Carrie D., Wilton, Rosemarie, Deng, Shuang, Dai, Ziyu, Oksen, Ethan, Gee, Aaron, Fasani, Rick A., Tsalenko, Anya, Tanjore, Deepti, Gardner, James, and Smith, Richard D.
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ION mobility spectroscopy ,MASS spectrometry ,MASS measurement ,ASPERGILLUS niger ,PSEUDOMONAS putida ,ERROR rates - Abstract
Multidimensional measurements using state-of-the-art separations and mass spectrometry provide advantages in untargeted metabolomics analyses for studying biological and environmental bio-chemical processes. However, the lack of rapid analytical methods and robust algorithms for these heterogeneous data has limited its application. Here, we develop and evaluate a sensitive and high-throughput analytical and computational workflow to enable accurate metabolite profiling. Our workflow combines liquid chromatography, ion mobility spectrometry and data-independent acquisition mass spectrometry with PeakDecoder, a machine learning-based algorithm that learns to distinguish true co-elution and co-mobility from raw data and calculates metabolite identification error rates. We apply PeakDecoder for metabolite profiling of various engineered strains of Aspergillus pseudoterreus, Aspergillus niger, Pseudomonas putida and Rhodosporidium toruloides. Results, validated manually and against selected reaction monitoring and gas-chromatography platforms, show that 2683 features could be confidently annotated and quantified across 116 microbial sample runs using a library built from 64 standards. Alternative algorithms exploiting advantages of multidimensional mass spectrometry in untargeted metabolomics are needed. Here, the authors develop and demonstrate PeakDecoder for confident and accurate metabolite profiling in 116 microbial sample runs and using a library built from 64 standards. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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3. Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species.
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Dai, Ziyu, Pomraning, Kyle R., Deng, Shuang, Kim, Joonhoon, Campbell, Kristen B., Robles, Ana L., Hofstad, Beth A., Munoz, Nathalie, Gao, Yuqian, Lemmon, Teresa, Swita, Marie S., Zucker, Jeremy D., Kim, Young-Mo, Burnum-Johnson, Kristin E., and Magnuson, Jon K.
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ASPERGILLUS ,ASPERGILLUS niger ,PYRUVATE carboxylase ,SUCCINATE dehydrogenase ,CORN stover ,PYRUVATES ,LIGNOCELLULOSE ,ASPARTATE aminotransferase - Abstract
Background: Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. Results: In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol
−1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol−1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol−1 sugars and resulted in a final titer of 36.0 g/L 3-HP. Conclusions: The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane. [ABSTRACT FROM AUTHOR]- Published
- 2023
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4. Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass.
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Kirby, James, Geiselman, Gina M., Yaegashi, Junko, Kim, Joonhoon, Zhuang, Xun, Tran-Gyamfi, Mary Bao, Prahl, Jan-Philip, Sundstrom, Eric R., Gao, Yuqian, Munoz, Nathalie, Burnum-Johnson, Kristin E., Benites, Veronica T., Baidoo, Edward E. K., Fuhrmann, Anna, Seibel, Katharina, Webb-Robertson, Bobbie-Jo M., Zucker, Jeremy, Nicora, Carrie D., Tanjore, Deepti, and Magnuson, Jon K.
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TERPENES ,MONOTERPENES ,CLIMATE change mitigation ,MEVALONATE kinase ,BIOMASS ,GENES ,ENGINEERING - Abstract
Background: Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates. Results: This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement. Conclusions: This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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5. Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in <italic>Aspergillus niger</italic>.
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Reilly, Morgann C., Kim, Joonhoon, Lynn, Jed, Simmons, Blake A., Gladden, John M., Magnuson, Jon K., and Baker, Scott E.
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ASPERGILLUS niger ,FUNGAL genetics ,FUNGAL enzymes ,NUCLEOTIDE sequencing ,PLANT biomass ,MUTAGENESIS - Abstract
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 inA. 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. [ABSTRACT FROM AUTHOR]- Published
- 2018
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6. Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification.
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Keating, David H., Yaoping Zhang, Ong, Irene M., McIlwain, Sean, Morales, Eduardo H., Grass, Jeffrey A., Tremaine, Mary, Bothfeld, William, Higbee, Alan, Ulbrich, Arne, Balloon, Allison J., Westphall, Michael S., Aldrich, Josh, Lipton, Mary S., Kim, Joonhoon, Moskvin, Oleg V., Bukhman, Yury V., Coon, Joshua J., Kiley, Patricia J., and Bates, Donna M.
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ESCHERICHIA coli ,LIGNOCELLULOSE ,ENZYME inhibitors ,RNA sequencing ,PROTEOMICS ,ETHANOL ,BIOMASS energy - Abstract
Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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7. Large-Scale Bi-Level Strain Design Approaches and Mixed-Integer Programming Solution Techniques.
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Kim, Joonhoon, Reed, Jennifer L., and Maravelias, Christos T.
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INTEGER programming ,MATHEMATICAL models ,METABOLISM ,GENOMES ,PROBLEM solving ,GENETIC algorithms - Abstract
The use of computational models in metabolic engineering has been increasing as more genome-scale metabolic models and computational approaches become available. Various computational approaches have been developed to predict how genetic perturbations affect metabolic behavior at a systems level, and have been successfully used to engineer microbial strains with improved primary or secondary metabolite production. However, identification of metabolic engineering strategies involving a large number of perturbations is currently limited by computational resources due to the size of genome-scale models and the combinatorial nature of the problem. In this study, we present (i) two new bi-level strain design approaches using mixed-integer programming (MIP), and (ii) general solution techniques that improve the performance of MIP-based bi-level approaches. The first approach (SimOptStrain) simultaneously considers gene deletion and non-native reaction addition, while the second approach (BiMOMA) uses minimization of metabolic adjustment to predict knockout behavior in a MIP-based bi-level problem for the first time. Our general MIP solution techniques significantly reduced the CPU times needed to find optimal strategies when applied to an existing strain design approach (OptORF) (e.g., from ∼10 days to ∼5 minutes for metabolic engineering strategies with 4 gene deletions), and identified strategies for producing compounds where previous studies could not (e.g., malate and serine). Additionally, we found novel strategies using SimOptStrain with higher predicted production levels (for succinate and glycerol) than could have been found using an existing approach that considers network additions and deletions in sequential steps rather than simultaneously. Finally, using BiMOMA we found novel strategies involving large numbers of modifications (for pyruvate and glutamate), which sequential search and genetic algorithms were unable to find. The approaches and solution techniques developed here will facilitate the strain design process and extend the scope of its application to metabolic engineering. [ABSTRACT FROM AUTHOR]
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- 2011
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8. Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose‐Derived Sugars.
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Kim, Joonhoon, Tremaine, Mary, Grass, Jeffrey A., Purdy, Hugh M., Landick, Robert, Kiley, Patricia J., and Reed, Jennifer L.
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- 2019
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9. A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides.
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Nora, Luísa Czamanski, Wehrs, Maren, Kim, Joonhoon, Cheng, Jan-Fang, Tarver, Angela, Simmons, Blake A., Magnuson, Jon, Harmon-Smith, Miranda, Silva-Rocha, Rafael, Gladden, John M., Mukhopadhyay, Aindrila, Skerker, Jeffrey M., and Kirby, James
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ROBUST control ,TRANSLOCATOR proteins ,GLYCERALDEHYDEPHOSPHATE dehydrogenase ,VALUE engineering ,PROMOTERS (Genetics) ,GERMPLASM ,GENETIC engineering - Abstract
Background: Rhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides. Results: This data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. Promoter expression strength was determined by measurement of EGFP and mRuby2 reporters by flow cytometry. A total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) were found, and are expected to be of potential value for genetic engineering of R. toruloides. Conclusions: A set of robust and constitutive promoters to facilitate genetic engineering of R. toruloides is presented here, ranging from a promoter previously used for this purpose (P7, glyceraldehyde 3-phosphate dehydrogenase, GAPDH) to stronger monodirectional (e.g., P15, mitochondrial adenine nucleotide translocator, ANT) and bidirectional (e.g., P9 and P9R, histones H3 and H4, respectively) promoters. We also identified promoters that may be useful for specific applications such as late-stage expression (e.g., P3, voltage-dependent anion channel protein 2, VDAC2). This set of characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies. [ABSTRACT FROM AUTHOR]
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- 2019
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10. Transcriptomic analysis of the oleaginous yeast Lipomyces starkeyi during lipid accumulation on enzymatically treated corn stover hydrolysate.
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Pomraning, Kyle R., Collett, James R., Kim, Joonhoon, Panisko, Ellen A., Culley, David E., Dai, Ziyu, Deng, Shuang, Hofstad, Beth A., Butcher, Mark G., and Magnuson, Jon K.
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CORN stover ,LIGNOCELLULOSE ,CATABOLITE repression ,ORGANIC acids ,LIPIDS ,YEAST - Abstract
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. Lipomyces starkeyi 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 L. starkeyi is inhibited with PCS hydrolysate as the carbon source. Both acetic acid and furfural are present at concentrations toxic to L. starkeyi 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 L. starkeyi 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. L. starkeyi'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 L. starkeyi. 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. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
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