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

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.
Authors:
 [1] ;  [2] ;  [2] ;  [1]
  1. Michigan State Univ., East Lansing, MI (United States)
  2. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Publication Date:
OSTI Identifier:
1258435
Report Number(s):
LA-UR--15-27108
Journal ID: ISSN 2161-5063
Grant/Contract Number:
AC52-06NA25396
Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 4; Journal Issue: 11; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society
Research Org:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 09 BIOMASS FUELS Biological Science; Energy Sciences