Comprehensive sequence-flux mapping of a levoglucosan utilization pathway in E. coli

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

doi:10.1021/acssynbio.5b00131

Comprehensive sequence-flux mapping of a levoglucosan utilization pathway in E. coli

Journal Article · Mon Sep 14 00:00:00 EDT 2015 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.5b00131· OSTI ID:1258435

Klesmith, Justin R. ^[1]; Bacik, John -Paul ^[2]; Michalczyk, Ryszard ^[2]; Whitehead, Timothy A. ^[1]

Michigan State Univ., East Lansing, MI (United States)
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

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. Lastly, this technique can be extended to improve a wide variety of designed pathways.

View Accepted Manuscript (DOE)

Research Organization:: Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: AC52-06NA25396

OSTI ID:: 1258435

Report Number(s):: LA-UR--15-27108

Journal Information:: ACS Synthetic Biology, Journal Name: ACS Synthetic Biology Journal Issue: 11 Vol. 4; ISSN 2161-5063

Publisher:: American Chemical SocietyCopyright Statement

Country of Publication:: United States

Language:: English

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Comprehensive sequence-flux mapping of a levoglucosan utilization pathway in E. coli

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