Comprehensive Sequence-Flux Mapping of a Levoglucosan Utilization Pathway in E. coli

Justin R. Klesmith, John Paul Bacik, Ryszard Michalczyk, Timothy A. Whitehead

    Research output: Research - peer-reviewArticle

    • 7 Citations

    Abstract

    Synthetic metabolic pathways often suffer from low specific productivity, and new methods that quickly assess pathway functionality for many thousands of variants are urgently needed. Here we present an approach that enables the rapid and parallel determination of sequence effects on flux for complete gene-encoding sequences. We show that this method can be used to determine the effects of over 8000 single point mutants of a pyrolysis oil catabolic pathway implanted in Escherichia coli. Experimental sequence-function data sets predicted whether fitness-enhancing mutations to the enzyme levoglucosan kinase resulted from enhanced catalytic efficiency or enzyme stability. A structure of one design incorporating 38 mutations elucidated the structural basis of high fitness mutations. One design incorporating 15 beneficial mutations supported a 15-fold improvement in growth rate and greater than 24-fold improvement in enzyme activity relative to the starting pathway. This technique can be extended to improve a wide variety of designed pathways.

    LanguageEnglish (US)
    Pages1235-1243
    Number of pages9
    JournalACS Synthetic Biology
    Volume4
    Issue number11
    DOIs
    StatePublished - Nov 20 2015

    Profile

    Escherichia coli
    Fluxes
    Enzymes
    1,6-anhydro-beta-glucopyranose
    Mutation
    Gene encoding
    Enzyme activity
    Oils
    Pyrolysis
    Phosphotransferases
    Productivity
    Metabolic Networks and Pathways
    Enzyme Stability
    Sequence Analysis
    Growth
    Genes
    Datasets

    Keywords

    • biomass conversion
    • deep mutational scanning
    • fast pyrolysis
    • metabolic engineering
    • protein design

    ASJC Scopus subject areas

    • Biochemistry, Genetics and Molecular Biology (miscellaneous)
    • Biomedical Engineering

    Cite this

    Comprehensive Sequence-Flux Mapping of a Levoglucosan Utilization Pathway in E. coli. / Klesmith, Justin R.; Bacik, John Paul; Michalczyk, Ryszard; Whitehead, Timothy A.

    In: ACS Synthetic Biology, Vol. 4, No. 11, 20.11.2015, p. 1235-1243.

    Research output: Research - peer-reviewArticle

    Klesmith, Justin R. ; Bacik, John Paul ; Michalczyk, Ryszard ; Whitehead, Timothy A./ Comprehensive Sequence-Flux Mapping of a Levoglucosan Utilization Pathway in E. coli. In: ACS Synthetic Biology. 2015 ; Vol. 4, No. 11. pp. 1235-1243
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