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Metabolic Engineering

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https://www.readbyqxmd.com/read/28212815/systematic-testing-of-enzyme-perturbation-sensitivities-via-graded-dcas9-modulation-in-saccharomyces-cerevisiae
#1
Matthew Deaner, Hal S Alper
Dissecting genotype-phenotype relationships in a high-throughput and scalable manner is still an unresolved problem facing metabolic engineers. While the RNA-guided nuclease Cas9 has been repurposed as a programmable transcription regulator, its application has typically been limited to binary on/off regulation and thus misses informative and potentially optimal intermediate levels of gene expression. In this work, we establish a rapid method for fine-tuned, graded expression of pathway enzymes via dCas9 regulation by varying sgRNA target location as the dominant parameter...
February 14, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28192215/review-of-metabolic-pathways-activated-in-cancer-cells-as-determined-through-isotopic-labeling-and-network-analysis
#2
Wentao Dong, Mark A Keibler, Gregory Stephanopoulos
Cancer metabolism has emerged as an indispensable part of contemporary cancer research. During the past 10 years, the use of stable isotopic tracers and network analysis have unveiled a number of metabolic pathways activated in cancer cells. Here, we review such pathways along with the particular tracers and labeling observations that led to the discovery of their rewiring in cancer cells. The list of such pathways comprises the reductive metabolism of glutamine, altered glycolysis, serine and glycine metabolism, mutant isocitrate dehydrogenase (IDH) induced reprogramming and the onset of acetate metabolism...
February 10, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28188893/ultra-deep-next-generation-mitochondrial-genome-sequencing-reveals-widespread-heteroplasmy-in-chinese-hamster-ovary-cells
#3
Paul S Kelly, Colin Clarke, Alan Costello, Craig Monger, Justine Meiller, Heena Dhiman, Nicole Borth, Michael J Betenbaugh, Martin Clynes, Niall Barron
Recent sequencing of the Chinese hamster ovary (CHO) cell and Chinese hamster genomes has dramatically advanced our ability to understand the biology of these mammalian cell factories. In this study, we focus on the powerhouse of the CHO cell, the mitochondrion. Utilizing a high-resolution next generation sequencing approach we sequenced the Chinese hamster mitochondrial genome for the first time and surveyed the mutational landscape of CHO cell mitochondrial DNA (mtDNA). Depths of coverage ranging from ~3,319X to 8,056X enabled accurate identification of low frequency mutations (>1%) revealing that mtDNA heteroplasmy is widespread in CHO cells...
February 7, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28163219/probing-the-metabolic-phenotype-of-breast-cancer-cells-by-multiple-tracer-stable-isotope-resolved-metabolomics
#4
Andrew N Lane, Julie Tan, Yali Wang, Jun Yan, Richard M Higashi, Teresa W-M Fan
Breast cancers vary by their origin and specific set of genetic lesions, which gives rise to distinct phenotypes and differential response to targeted and untargeted chemotherapies. To explore the functional differences of different breast cell types, we performed Stable Isotope Resolved Metabolomics (SIRM) studies of one primary breast (HMEC) and three breast cancer cells (MCF-7, MDAMB-231, and ZR75-1) having distinct genotypes and growth characteristics, using (13)C6-glucose, (13)C-1+2-glucose, (13)C5,(15)N2-Gln, (13)C3-glycerol, and (13)C8-octanoate as tracers...
February 2, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28159643/metabolic-flexibility-of-a-butyrate-pathway-mutant-of-clostridium-acetobutylicum
#5
Minyeong Yoo, Christian Croux, Isabelle Meynial-Salles, Philippe Soucaille
Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Δbuk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Δbuk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell...
January 31, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28122259/application-of-13-c-flux-analysis-to-identify-high-productivity-cho-metabolic-phenotypes
#6
Neil Templeton, Kevin D Smith, Allison G McAtee-Pereira, Haimanti Dorai, Michael J Betenbaugh, Steven E Lang, Jamey D Young
Industrial bioprocesses place high demands on the energy metabolism of host cells to meet biosynthetic requirements for maximal protein expression. Identifying metabolic phenotypes that promote high expression is therefore a major goal of the biotech industry. We conducted a series of (13)C flux analysis studies to examine the metabolic response to IgG expression during early stationary phase of CHO cell cultures grown in 3L fed-batch bioreactors. We examined eight clones expressing four different IgGs and compared with three non-expressing host-cell controls...
January 23, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28119139/towards-improved-butanol-production-through-targeted-genetic-modification-of-clostridium-pasteurianum
#7
Katrin M Schwarz, Alexander Grosse-Honebrink, Kamila Derecka, Carlo Rotta, Ying Zhang, Nigel P Minton
Declining fossil fuel reserves, coupled with environmental concerns over their continued extraction and exploitation have led to strenuous efforts to identify renewable routes to energy and fuels. One attractive option is to convert glycerol, a by-product of the biodiesel industry, into n-butanol, an industrially important chemical and potential liquid transportation fuel, using Clostridium pasteurianum. Under certain growth conditions this Clostridium species has been shown to predominantly produce n-butanol, together with ethanol and 1, 3-propanediol, when grown on glycerol...
January 21, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28111249/metabolic-engineering-of-clostridium-autoethanogenum-for-selective-alcohol-production
#8
Fungmin Liew, Anne M Henstra, Michael Kӧpke, Klaus Winzer, Sean D Simpson, Nigel P Minton
Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%)...
January 19, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28111248/improving-key-enzyme-activity-in-phenylpropanoid-pathway-with-a-designed-biosensor
#9
Dandan Xiong, Shikun Lu, Jieyuan Wu, Chaoning Liang, Wei Wang, Wenzhao Wang, Jian-Ming Jin, Shuang-Yan Tang
Overexpressing key enzymes of biosynthetic pathways for overproduction of value-added products usually imposes metabolic burdens on cells, which can be circumvented by improving the key enzyme activities. p-Coumarate: CoA ligase (4CL) is a critical enzyme in the phenylpropanoid pathway that synthesizes various natural products. To screen for 4CL with improved activity, a biosensor of resveratrol whose biosynthetic pathway involves 4CL was designed by engineering the TtgR regulatory protein. The biosensor exhibited good specificity and robustness, allowing rapid and sensitive selection of resveratrol hyper-producers...
January 19, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28110078/escherichia-coli-hgt-engineered-for-high-glucose-throughput-even-under-slowly-growing-or-resting-conditions
#10
Annette Michalowski, Martin Siemann-Herzberg, Ralf Takors
Aerobic production-scale processes are constrained by the technical limitations of maximum oxygen transfer and heat removal. Consequently, microbial activity is often controlled via limited nutrient feeding to maintain it within technical operability. Here, we present an alternative approach based on a newly engineered Escherichia coli strain. This E. coli HGT (high glucose throughput) strain was engineered by modulating the stringent response regulation program and decreasing the activity of pyruvate dehydrogenase...
January 18, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28088541/secretory-pathway-optimization-of-cho-producer-cells-by-co-engineering-of-the-mitosrna-1978-target-genes-cers2-and-tbc1d20
#11
Lisa A Pieper, Michaela Strotbek, Till Wenger, Martin Gamer, Monilola A Olayioye, Angelika Hausser
Chinese Hamster Ovary (CHO) cells are the most commonly used host for the production of biopharmaceuticals. Although transcription and translation engineering strategies have been employed to generate high-producer cell clones, the secretory pathway still remains a bottleneck in cellular productivity. In this study we show that ectopic expression of a human mitochondrial genome-encoded small RNA (mitosRNA-1978) in an IgG expressing CHO cell line strongly improved specific productivity by functioning in a microRNA-like fashion...
January 11, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28088540/multiplexed-site-specific-genome-engineering-for-overproducing-bioactive-secondary-metabolites-in-actinomycetes
#12
Lei Li, Guosong Zheng, Jun Chen, Mei Ge, Weihong Jiang, Yinhua Lu
Actinomycetes produce a large variety of pharmaceutically active compounds, yet production titers often require to be improved for discovery, development and large-scale manufacturing. Here, we describe a new technique, multiplexed site-specific genome engineering (MSGE) via the 'one integrase-multiple attB sites' concept, for the stable integration of secondary metabolite biosynthetic gene clusters (BGCs). Using MSGE, we achieved five-copy chromosomal integration of the pristinamycin II (PII) BGC in Streptomyces pristinaespiralis, resulting in the highest reported PII titers in flask and batch fermentations (2...
January 11, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28087332/exo-mfa-a-13c-metabolic-flux-analysis-framework-to-dissect-tumor-microenvironment-secreted-exosome-contributions-towards-cancer-cell-metabolism
#13
Abhinav Achreja, Hongyun Zhao, Lifeng Yang, Tae Hyun Yun, Juan Marini, Deepak Nagrath
Dissecting the pleiotropic roles of tumor micro-environment (TME) on cancer progression has been brought to the foreground of research on cancer pathology. Extracellular vesicles such as exosomes, transport proteins, lipids, and nucleic acids, to mediate intercellular communication between TME components and have emerged as candidates for anti-cancer therapy. We previously reported that cancer-associated fibroblast (CAF) derived exosomes (CDEs) contain metabolites in their cargo that are utilized by cancer cells for central carbon metabolism and promote cancer growth...
January 11, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28089747/metabolic-engineering-of-a-diazotrophic-bacterium-improves-ammonium-release-and-biofertilization-of-plants-and-microalgae
#14
Rafael Ambrosio, Juan Cesar Federico Ortiz-Marquez, Leonardo Curatti
The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. However, with the exception of the symbiotic rhizobia-legumes system, progress towards a more extensive realization of this goal has been slow. In this study we manipulated the endogenous regulation of both nitrogen fixation and assimilation in the aerobic bacterium Azotobacter vinelandii. Substituting an exogenously inducible promoter for the native promoter of glutamine synthetase produced conditional lethal mutant strains unable to grow diazotrophically in the absence of the inducer...
January 9, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28062280/biotin-independent-strains-of-escherichia-coli-for-enhanced-streptavidin-production
#15
Markus Jeschek, Maximilian O Bahls, Veronika Schneider, Philippe Marlière, Thomas R Ward, Sven Panke
Biotin is an archetypal vitamin used as cofactor for carboxylation reactions found in all forms of life. However, biotin biosynthesis is an elaborate multi-enzymatic process and metabolically costly. Moreover, many industrially relevant organisms are incapable of biotin synthesis resulting in the requirement to supplement defined media. Here we describe the creation of biotin-independent strains of Escherichia coli and Corynebacterium glutamicum through installation of an optimized malonyl-CoA bypass, which re-routes natural fatty acid synthesis, rendering the previously essential vitamin completely obsolete...
January 3, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28040464/metabolic-engineering-of-clostridium-tyrobutyricum-for-enhanced-butyric-acid-production-from-glucose-and-xylose
#16
Hongxin Fu, Le Yu, Meng Lin, Jufang Wang, Zhilong Xiu, Shang-Tian Yang
Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37...
December 28, 2016: Metabolic Engineering
https://www.readbyqxmd.com/read/28038953/leucine-zipper-mediated-targeting-of-multi-enzyme-cascade-reactions-to-inclusion-bodies-in-escherichia-coli-for-enhanced-production-of-1-butanol
#17
Gui Hwan Han, Wonjae Seong, Yaoyao Fu, Paul K Yoon, Seong Keun Kim, Soo-Jin Yeom, Dae-Hee Lee, Seung-Goo Lee
Metabolons in nature have evolved to facilitate more efficient catalysis of multistep reactions through the co-localization of functionally related enzymes to cellular organelles or membrane structures. To mimic the natural metabolon architecture, we present a novel artificial metabolon that was created by targeting multi-enzyme cascade reactions onto inclusion body (IB) in Escherichia coli. The utility of this system was examined by co-localizing four heterologous enzymes of the 1-butanol pathway onto an IB that was formed in E...
December 27, 2016: Metabolic Engineering
https://www.readbyqxmd.com/read/28038952/metabotypes-of-breast-cancer-cell-lines-revealed-by-non-targeted-metabolomics
#18
Sébastien Dubuis, Franziska Baenke, Nina Scherbichler, Leila T Alexander, Almut Schulze, Nicola Zamboni
We present an analysis of intracellular metabolism by non-targeted, high-throughput metabolomics profiling of 18 breast cell lines. We profiled >900 putatively annotated metabolite ions for >100 samples collected under both normoxic and hypoxic conditions and revealed extensive heterogeneity across all metabolic pathways and cell lines. Cell line-specific metabolome profiles dominated over patterns associated with malignancy or with the clinical nomenclature of breast cancer cells. Such characteristic metabolome profiles were reproducible across different laboratories and experiments and exhibited mild to robust changes with change in experimental conditions...
December 27, 2016: Metabolic Engineering
https://www.readbyqxmd.com/read/28034771/lkb1-promotes-metabolic-flexibility-in-response-to-energy-stress
#19
Seth J Parker, Robert U Svensson, Ajit S Divakaruni, Austin E Lefebvre, Anne N Murphy, Reuben J Shaw, Christian M Metallo
The Liver Kinase B1 (LKB1) tumor suppressor acts as a metabolic energy sensor to regulate AMP-activated protein kinase (AMPK) signaling and is commonly mutated in various cancers, including non-small cell lung cancer (NSCLC). Tumor cells deficient in LKB1 may be uniquely sensitized to metabolic stresses, which may offer a therapeutic window in oncology. To address this question we have explored how functional LKB1 impacts the metabolism of NSCLC cells using (13)C metabolic flux analysis. Isogenic NSCLC cells expressing functional LKB1 exhibited higher flux through oxidative mitochondrial pathways compared to those deficient in LKB1...
December 26, 2016: Metabolic Engineering
https://www.readbyqxmd.com/read/27988388/analysis-of-mitochondrial-metabolism-in-situ-combining-stable-isotope-labeling-with-selective-permeabilization
#20
Yannic Nonnenmacher, Roberta Palorini, Aymeric Fouquier d'Herouël, Lisa Krämer, Meina Neumann-Schaal, Ferdinando Chiaradonna, Alexander Skupin, Andre Wegner, Karsten Hiller
To date, it is well-established that mitochondrial dysfunction does not only play a vital role in cancer but also in other pathological conditions such as neurodegenerative diseases and inflammation. An important tool for the analysis of cellular metabolism is the application of stable isotope labeled substrates, which allow for the tracing of atoms throughout metabolic networks. While such analyses yield very detailed information about intracellular fluxes, the determination of compartment specific fluxes is far more challenging...
December 15, 2016: Metabolic Engineering
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