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.

Deep sequencing methods for protein engineering and design

“…Thus, variants enabling higher flux through and enzyme permit faster growth rates and become enriched in the population. Klesmith et al performed deep mutational scanning of levoglucosan kinase, where levoglucosan was fed as the carbon source [28]. Similarly, Wrenbeck et al performed deep mutational scanning on amiE, an aliphatic amidase from Pseudomonas aeruginosa , by feeding amides as the nitrogen source [E. E. Wrenbeck et al, unpublished].…”

“…For example, Klesmith et al performed deep mutational scanning of levoglucosan kinase to identify mutations that improved fitness through improved flux of levoglucosan conversion. They characterized a set of beneficial mutations for activity and thermodynamic stability and used this information to generate designs, one of which had greater than 24-fold improvement in activity and 7°C increase in apparent melting temperature [28]. An alternative approach is to generate multiple fitness landscapes under different conditions (concentration and identity of substrate, temperature, etc.)…”

High-throughput evaluation of synthetic metabolic pathways

“…A new approach called FluxScan was recently reported 80 that maps the sequence determinants of flux in living cells (Fig. 2b).…”

“…The frequency change of each variant can be calculated and converted to a flux value. To demonstrate this method, Klesmith et al determined the effect of flux for over 8,000 single-point mutants in a pyrolysis oil catabolic pathway 80 . One designed pathway incorporating 15 beneficial mutations identified from FluxScan supported a 15-fold improvement in growth rate on levoglucosan, a chief pyrolysis oil constituent.…”

“…While these examples highlight gene expression on the genomic scale, the problem also applies to expression and gene variants on the individual pathway level. An example of this is from the aforementioned FluxScan study where the enrichment of individual mutations from the enzyme levoglucosan kinase was strongly dependent on the biophysical properties of the starting enzyme variant from each selection 80 . Being able to measure the pathway phenotype from different expression elements and individual gene variants under different growth conditions should allow the elucidation of pathway sequences that are optimal over a range of diverse conditions and determine why other sequences fail when these conditions change.…”