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  1. AU="Santos, Christine Nicole S"
  2. AU="Severi, Kristen E"
  3. AU="Robotti, Marzia"
  4. AU="Kaspar, Charles W"
  5. AU="Wallach, E E"
  6. AU="Temnikov, P"
  7. AU="Gomez-Verjan, Juan Carlos"
  8. AU="Mayle, Francis E."
  9. AU="Rhoades, Elizabeth"
  10. AU="Riaz, Huma"
  11. AU="Eliseu, Gabriel"
  12. AU="Hill, Lori R"
  13. AU="Boppana, Suresh B"

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  1. Buch: Microbial metabolic engineering

    Santos, Christine Nicole S. / Ajikumar, Parayil Kumaran

    methods and protocols

    (Methods in molecular biology ; 1927 ; Springer protocols)

    2019  

    Verfasserangabe edited by Christine Nicole S. Santos and Parayil Kumaran Ajikumar
    Serientitel Methods in molecular biology ; 1927
    Springer protocols
    Überordnung
    Sprache Englisch
    Umfang x, 252 Seiten, Illustrationen
    Verlag Humana Press
    Erscheinungsort New York, NY
    Erscheinungsland Vereinigte Staaten
    Dokumenttyp Buch
    HBZ-ID HT019995897
    ISBN 978-1-4939-9141-9 ; 9781493991426 ; 1-4939-9141-8 ; 1493991426
    Datenquelle Katalog ZB MED Medizin, Gesundheit

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    SignaturLeihstatusStandort
    Zs A 7042 -1927- verfügbar in Königswinter Königswinter

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  2. Artikel ; Online: Targeted Mass Spectrometry-Based Proteomics Tools for Strain Optimization.

    Tseng, Hsien-Chung / Santos, Christine Nicole S

    Methods in molecular biology (Clifton, N.J.)

    2019  Band 1927, Seite(n) 191–201

    Abstract: The goal of strain optimization is to create high-performance strains producing compounds of interest at a high titer, yield, and volumetric productivity. The effectiveness of strain optimization relies on methodologies used to aid optimization of native ...

    Abstract The goal of strain optimization is to create high-performance strains producing compounds of interest at a high titer, yield, and volumetric productivity. The effectiveness of strain optimization relies on methodologies used to aid optimization of native or novel pathways within cells. Many different factors, including mRNA abundance, protein abundance, and enzyme activity/stability, will contribute to the strain performance, which is not often evident by simply monitoring product titers. To this end, targeted proteomics tools utilizing LC-MS-MS (liquid chromatography coupled with tandem mass spectrometry) have recently been developed and can monitor protein levels at great sensitivities. Here, we describe all relevant aspects when developing proteomics tools for strain optimization.
    Mesh-Begriff(e) Biotechnology ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Data Analysis ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Metabolic Engineering ; Proteomics/methods ; Tandem Mass Spectrometry
    Sprache Englisch
    Erscheinungsdatum 2019-02-20
    Erscheinungsland United States
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-4939-9142-6_13
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  3. Artikel ; Online: Engineering complex biological systems in bacteria through recombinase-assisted genome engineering.

    Santos, Christine Nicole S / Yoshikuni, Yasuo

    Nature protocols

    2014  Band 9, Heft 6, Seite(n) 1320–1336

    Abstract: Here we describe an advanced paradigm for the design, construction and stable implementation of complex biological systems in microbial organisms. This engineering strategy was previously applied to the development of an Escherichia coli-based platform, ... ...

    Abstract Here we describe an advanced paradigm for the design, construction and stable implementation of complex biological systems in microbial organisms. This engineering strategy was previously applied to the development of an Escherichia coli-based platform, which enabled the use of brown macroalgae as a feedstock for the production of biofuels and renewable chemicals. In this approach, functional genetic modules are first designed in silico and constructed on a bacterial artificial chromosome (BAC) by using a recombineering-based inchworm extension technique. Stable integration into the recipient chromosome is then mediated through the use of recombinase-assisted genome engineering (RAGE). The flexibility, simplicity and speed of this method enable a comprehensive optimization of several different parameters, including module configuration, strain background, integration locus, gene copy number and intermodule compatibility. This paradigm therefore has the potential to markedly expedite most strain-engineering endeavors. Once a biological system has been designed and constructed on a BAC, its implementation and optimization in a recipient host can be carried out in as little as 1 week.
    Mesh-Begriff(e) Bacteria/genetics ; Chromosomes, Artificial, Bacterial/genetics ; Genetic Engineering/methods ; Genome, Bacterial/genetics ; Metabolic Networks and Pathways/genetics ; Recombinases/metabolism
    Chemische Substanzen Recombinases
    Sprache Englisch
    Erscheinungsdatum 2014-05-15
    Erscheinungsland England
    Dokumenttyp Journal Article
    ZDB-ID 2244966-8
    ISSN 1750-2799 ; 1754-2189
    ISSN (online) 1750-2799
    ISSN 1754-2189
    DOI 10.1038/nprot.2014.084
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  4. Artikel ; Online: Implementation of stable and complex biological systems through recombinase-assisted genome engineering.

    Santos, Christine Nicole S / Regitsky, Drew D / Yoshikuni, Yasuo

    Nature communications

    2013  Band 4, Seite(n) 2503

    Abstract: Evaluating the performance of engineered biological systems with high accuracy and precision is nearly impossible with the use of plasmids due to phenotypic noise generated by genetic instability and natural population dynamics. Minimizing this ... ...

    Abstract Evaluating the performance of engineered biological systems with high accuracy and precision is nearly impossible with the use of plasmids due to phenotypic noise generated by genetic instability and natural population dynamics. Minimizing this uncertainty therefore requires a paradigm shift towards engineering at the genomic level. Here, we introduce an advanced design principle for the stable installment and implementation of complex biological systems through recombinase-assisted genome engineering (RAGE). We apply this concept to the development of a robust strain of Escherichia coli capable of producing ethanol directly from brown macroalgae. RAGE significantly expedites the optimal implementation of a 34 kb heterologous pathway for alginate metabolism based on genetic background, integration locus, copy number and compatibility with two other pathway modules (alginate degradation and ethanol production). The resulting strain achieves a ~40% higher titre than its plasmid-based counterpart and enables substantial improvements in titre (~330%) and productivity (~1,200%) after 50 generations.
    Mesh-Begriff(e) Alginates/metabolism ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Ethanol/metabolism ; Genetic Engineering ; Genome, Bacterial ; Glucuronic Acid/metabolism ; Hexuronic Acids/metabolism ; Metabolic Networks and Pathways ; Mutagenesis, Insertional ; Phaeophyceae/chemistry ; Plasmids ; Recombinases/genetics ; Recombinases/metabolism
    Chemische Substanzen Alginates ; Escherichia coli Proteins ; Hexuronic Acids ; Recombinases ; Ethanol (3K9958V90M) ; Glucuronic Acid (8A5D83Q4RW)
    Sprache Englisch
    Erscheinungsdatum 2013-08-27
    Erscheinungsland England
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/ncomms3503
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  5. Artikel ; Online: Melanin-based high-throughput screen for L-tyrosine production in Escherichia coli.

    Santos, Christine Nicole S / Stephanopoulos, Gregory

    Applied and environmental microbiology

    2008  Band 74, Heft 4, Seite(n) 1190–1197

    Abstract: We present the development of a simple, high-throughput screen for identifying bacterial strains capable of L-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link L-tyrosine production in ... ...

    Abstract We present the development of a simple, high-throughput screen for identifying bacterial strains capable of L-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link L-tyrosine production in Escherichia coli with the synthesis of the black and diffusible pigment melanin. Although melanin was initially produced only at low levels in morpholinepropanesulfonic acid (MOPS) minimal medium, phosphate supplementation was found to be sufficient for increasing both the rates of synthesis and the final titers of melanin. Furthermore, a strong linear correlation between extracellular L-tyrosine content and melanin formation was observed by use of this new medium formulation. A selection strategy that utilizes these findings has been developed and has been shown to be effective in screening large combinatorial libraries in the search for L-tyrosine-overproducing strains.
    Mesh-Begriff(e) Culture Media/chemistry ; DNA Primers/genetics ; Escherichia coli/metabolism ; Gene Library ; Melanins/biosynthesis ; Melanins/genetics ; Morpholines/chemistry ; Mutagenesis ; Tyrosine/biosynthesis
    Chemische Substanzen Culture Media ; DNA Primers ; Melanins ; Morpholines ; morpholinopropane sulfonic acid (273BP63NV3) ; Tyrosine (42HK56048U)
    Sprache Englisch
    Erscheinungsdatum 2008-02
    Erscheinungsland United States
    Dokumenttyp Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 223011-2
    ISSN 1098-5336 ; 0099-2240
    ISSN (online) 1098-5336
    ISSN 0099-2240
    DOI 10.1128/AEM.02448-07
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  6. Artikel: Combinatorial engineering of microbes for optimizing cellular phenotype.

    Santos, Christine Nicole S / Stephanopoulos, Gregory

    Current opinion in chemical biology

    2008  Band 12, Heft 2, Seite(n) 168–176

    Abstract: Although random mutagenesis and screening and evolutionary engineering have long been the gold standards for strain improvement in industry, the development of more sophisticated recombinant DNA tools has led to the introduction of alternate methods for ... ...

    Abstract Although random mutagenesis and screening and evolutionary engineering have long been the gold standards for strain improvement in industry, the development of more sophisticated recombinant DNA tools has led to the introduction of alternate methods for engineering strain diversity. Here, we summarize several combinatorial cell optimization methods developed in recent years, many of which are more amenable to phenotypic transfer and more efficient in probing greater dimensions of the available phenotypic space. They include tools that enable the fine-tuning of pathway expression (synthetic promoter libraries, tunable intergenic regions (TIGRs)), methods for generating randomized knockout and overexpression libraries, and more global techniques (artificial transcription factor engineering, global transcription machinery engineering, ribosome engineering, and genome shuffling) for eliciting complex, multigenic cellular properties.
    Mesh-Begriff(e) Cells/metabolism ; DNA Shuffling ; Gene Library ; Genetic Engineering/methods ; Microbiology ; Phenotype
    Sprache Englisch
    Erscheinungsdatum 2008-04
    Erscheinungsland England
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
    ZDB-ID 1439176-4
    ISSN 1879-0402 ; 1367-5931
    ISSN (online) 1879-0402
    ISSN 1367-5931
    DOI 10.1016/j.cbpa.2008.01.017
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  7. Artikel ; Online: Rational, combinatorial, and genomic approaches for engineering L-tyrosine production in Escherichia coli.

    Santos, Christine Nicole S / Xiao, Wenhai / Stephanopoulos, Gregory

    Proceedings of the National Academy of Sciences of the United States of America

    2012  Band 109, Heft 34, Seite(n) 13538–13543

    Abstract: Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use of combinatorial approaches exploiting phenotypic ... ...

    Abstract Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use of combinatorial approaches exploiting phenotypic diversification and screening. Here, we demonstrate the combined use of global transcriptional machinery engineering and a high-throughput L-tyrosine screen towards improving L-tyrosine production in Escherichia coli. This methodology succeeded in generating three strains from two separate mutagenesis libraries (rpoA and rpoD) exhibiting up to a 114% increase in L-tyrosine titer over a rationally engineered parental strain with an already high capacity for production. Subsequent strain characterization through transcriptional analysis and whole genome sequencing allowed complete phenotype reconstruction from well-defined mutations and point to important roles for both the acid stress resistance pathway and the stringent response of E. coli in imparting this phenotype. As such, this study presents one of the first examples in which cell-wide measurements have helped to elucidate the genetic and biochemical underpinnings of an engineered cellular property, leading to the total restoration of metabolite overproduction from specific chromosomal mutations.
    Mesh-Begriff(e) Bioreactors ; Chromosomes/ultrastructure ; Combinatorial Chemistry Techniques ; Drug Resistance ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Gene Deletion ; Genetic Engineering/methods ; Genome ; Genomics ; Kinetics ; Models, Genetic ; Mutation ; Phenotype ; Plasmids/metabolism ; Promoter Regions, Genetic ; Protein Structure, Tertiary ; Transcription, Genetic ; Tyrosine/chemistry ; Tyrosine/genetics
    Chemische Substanzen Tyrosine (42HK56048U)
    Sprache Englisch
    Erscheinungsdatum 2012-08-06
    Erscheinungsland United States
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 209104-5
    ISSN 1091-6490 ; 0027-8424
    ISSN (online) 1091-6490
    ISSN 0027-8424
    DOI 10.1073/pnas.1206346109
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  8. Artikel ; Online: Optimization of a heterologous pathway for the production of flavonoids from glucose.

    Santos, Christine Nicole S / Koffas, Mattheos / Stephanopoulos, Gregory

    Metabolic engineering

    2011  Band 13, Heft 4, Seite(n) 392–400

    Abstract: The development of efficient microbial processes for the production of flavonoids has been a metabolic engineering goal for the past several years, primarily due to the purported health-promoting effects of these compounds. Although significant strides ... ...

    Abstract The development of efficient microbial processes for the production of flavonoids has been a metabolic engineering goal for the past several years, primarily due to the purported health-promoting effects of these compounds. Although significant strides have been made recently in improving strain titers and yields, current fermentation strategies suffer from two major drawbacks-(1) the requirement for expensive phenylpropanoic precursors supplemented into the media and (2) the need for two separate media formulations for biomass/protein generation and flavonoid production. In this study, we detail the construction of a series of strains capable of bypassing both of these problems. A four-step heterologous pathway consisting of the enzymes tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) was assembled within two engineered l-tyrosine Escherichia coli overproducers in order to enable the production of the main flavonoid precursor naringenin directly from glucose. During the course of this investigation, we discovered that extensive optimization of both enzyme sources and relative gene expression levels was required to achieve high quantities of both p-coumaric acid and naringenin accumulation. Once this metabolic balance was achieved, however, such strains were found to be capable of producing 29 mg/l naringenin from glucose and up to 84 mg/l naringenin with the addition of the fatty acid enzyme inhibitor, cerulenin. These results were obtained through cultivation of E. coli in a single minimal medium formulation without additional precursor supplementation, thus paving the way for the development of a simple and economical process for the microbial production of flavonoids directly from glucose.
    Mesh-Begriff(e) Escherichia coli K12/enzymology ; Escherichia coli K12/genetics ; Escherichia coli K12/growth & development ; Flavanones/biosynthesis ; Flavanones/genetics ; Gene Expression ; Glucose/metabolism ; Glucose/pharmacology ; Organisms, Genetically Modified/genetics ; Organisms, Genetically Modified/growth & development ; Organisms, Genetically Modified/metabolism ; Plant Proteins/biosynthesis ; Plant Proteins/genetics ; Pueraria/enzymology ; Pueraria/genetics ; Sweetening Agents/metabolism ; Sweetening Agents/pharmacology
    Chemische Substanzen Flavanones ; Plant Proteins ; Sweetening Agents ; naringenin (HN5425SBF2) ; Glucose (IY9XDZ35W2)
    Sprache Englisch
    Erscheinungsdatum 2011-02-12
    Erscheinungsland Belgium
    Dokumenttyp Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 1470383-x
    ISSN 1096-7184 ; 1096-7176
    ISSN (online) 1096-7184
    ISSN 1096-7176
    DOI 10.1016/j.ymben.2011.02.002
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  9. Artikel: Melanin-Based High-Throughput Screen for L-Tyrosine Production in Escherichia coli

    Santos, Christine Nicole S / Stephanopoulos, Gregory

    Applied and environmental microbiology. 2008 Feb. 15, v. 74, no. 4

    2008  

    Abstract: We present the development of a simple, high-throughput screen for identifying bacterial strains capable of L-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link L-tyrosine production in ... ...

    Abstract We present the development of a simple, high-throughput screen for identifying bacterial strains capable of L-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link L-tyrosine production in Escherichia coli with the synthesis of the black and diffusible pigment melanin. Although melanin was initially produced only at low levels in morpholinepropanesulfonic acid (MOPS) minimal medium, phosphate supplementation was found to be sufficient for increasing both the rates of synthesis and the final titers of melanin. Furthermore, a strong linear correlation between extracellular L-tyrosine content and melanin formation was observed by use of this new medium formulation. A selection strategy that utilizes these findings has been developed and has been shown to be effective in screening large combinatorial libraries in the search for L-tyrosine-overproducing strains.
    Schlagwörter Escherichia coli ; acids ; genes ; melanin ; phosphates ; screening
    Sprache Englisch
    Erscheinungsverlauf 2008-0215
    Umfang p. 1190-1197.
    Erscheinungsort American Society for Microbiology
    Dokumenttyp Artikel
    Anmerkung Includes references
    ZDB-ID 223011-2
    ISSN 1098-5336 ; 0099-2240
    ISSN (online) 1098-5336
    ISSN 0099-2240
    DOI 10.1128/AEM.02448-07
    Datenquelle NAL Katalog (AGRICOLA)

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  10. Artikel: Perspectives of biotechnological production of L-tyrosine and its applications.

    Lütke-Eversloh, Tina / Santos, Christine Nicole S / Stephanopoulos, Gregory

    Applied microbiology and biotechnology

    2007  Band 77, Heft 4, Seite(n) 751–762

    Abstract: The aromatic amino acid L-tyrosine is used as a dietary supplement and has promise as a valuable precursor compound for various industrial and pharmaceutical applications. In contrast to chemical production, biotechnological methods can produce L- ... ...

    Abstract The aromatic amino acid L-tyrosine is used as a dietary supplement and has promise as a valuable precursor compound for various industrial and pharmaceutical applications. In contrast to chemical production, biotechnological methods can produce L-tyrosine from biomass feedstocks under environmentally friendly and near carbon-free conditions. In this minireview, various strategies for synthesizing L-tyrosine by employing biocatalysts are discussed, including initial approaches as well as more recent advances. Whereas early attempts to engineer L-tyrosine-excreting microbes were based on auxotrophic and antimetabolite-resistant mutants, recombinant deoxyribonucleic acid technology and a vastly increasing knowledge of bacterial physiology allowed recently for more targeted genetic manipulations and strain improvements. As an alternative route, L-tyrosine can also be obtained from the conversion of phenol, pyruvate, and ammonia or phenol and serine in reactions catalyzed by the enzyme tyrosine phenol lyase.
    Mesh-Begriff(e) Amino Acids ; Bacteria/enzymology ; Bacteria/genetics ; Bacteria/metabolism ; Bacterial Physiological Phenomena ; Biotechnology/methods ; Genetic Engineering/methods ; Tyrosine/biosynthesis ; Tyrosine Phenol-Lyase/metabolism
    Chemische Substanzen Amino Acids ; Tyrosine (42HK56048U) ; Tyrosine Phenol-Lyase (EC 4.1.99.2)
    Sprache Englisch
    Erscheinungsdatum 2007-12
    Erscheinungsland Germany
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
    ZDB-ID 392453-1
    ISSN 1432-0614 ; 0175-7598 ; 0171-1741
    ISSN (online) 1432-0614
    ISSN 0175-7598 ; 0171-1741
    DOI 10.1007/s00253-007-1243-y
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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