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 journalArticlepeer-review

43 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)1235-1243
Number of pages9
JournalACS Synthetic Biology
Issue number11
StatePublished - Nov 20 2015

Bibliographical note

Publisher Copyright:
© 2015 American Chemical Society.


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


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