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: Contribution to journalArticle

  • 12 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
Enzymes
Fluxes
Mutation
Gene encoding
Enzyme activity
Oils
Pyrolysis
Phosphotransferases
Productivity
Enzyme Stability
Metabolic Networks and Pathways
Sequence Analysis
1,6-anhydro-beta-glucopyranose
Growth
Genes

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: Contribution to journalArticle

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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