title: Comprehensive Sequence-Flux Mapping of a Levoglucosan Utilization Pathway in E. coli.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Justin R
authors.initials: JR
authors.lastname: Klesmith
authors.affiliation: Bioscience Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
authors.forename: John-Paul
authors.initials: JP
authors.lastname: Bacik
authors.affiliation: Bioscience Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
authors.forename: Ryszard
authors.initials: R
authors.lastname: Michalczyk
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University , East Lansing, Michigan 48824, United States.|Department of Biosystems and Agricultural Engineering, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Whitehead
mesh_terms: D055162:Biocatalysis; D018533:Biomass; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D005947:Glucose / Q000031:analogs & derivatives* / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology
chemical_list: C014989:1,6-anhydro-beta-glucopyranose; D010770:Phosphotransferases; D005947:Glucose
keywords: biomass conversion; deep mutational scanning; fast pyrolysis; metabolic engineering; protein design