Strategy for directing combinatorial genome engineering in Escherichia coli

Abstract

We describe a directed genome-engineering approach that combines genome-wide methods for mapping genes to traits [Warner JR, Reeder PJ, Karimpour-Fard A, Woodruff LBA, Gill RT (2010) Nat Biotechnol 28:856-862] with strategies for rapidly creating combinatorial ribosomal binding site (RBS) mutation libraries containing billions of targeted modifications [Wang HH, et al. (2009) Nature 460:894-898]. This approach should prove broadly applicable to various efforts focused on improving production of fuels, chemicals, and pharmaceuticals, among other products. We used barcoded promoter mutation libraries to map the effect of increased or decreased expression of nearly every gene in Escherichia coli onto growth in several model environments (cellulosic hydrolysate, low pH, and high acetate). Based on these data, we created and evaluated RBS mutant libraries (containing greater than 100,000,000 targeted mutations), targeting the genes identified to most affect growth. On laboratory timescales, we successfully identified a broad range of mutations (>25 growth-enhancing mutations confirmed), which improved growth rate 10-200% for several different conditions. Although successful, our efforts to identify superior combinations of growth-enhancing genes emphasized the importance of epistatic interactions among the targeted genes (synergistic, antagonistic) for taking full advantage of this approach to directed genome engineering.

Fig. 1.

Overview of strategy. (A) The TRMR library (middle circle) is generated by introducing mutations into WT E. coli (inner circle). A selection is performed yielding data on high-fitness mutants (outer circle). (B) A few mutants are chosen to be targets for further study. (C) A recursive multiplex recombineering library is constructed, generating a large diversity of clones with one or more mutations. (D) A selection is performed on the library to yield the most tolerant clones.

Fig. 2.

(A–C) Circle plots showing the result of TRMR analyses for acetate (16 g/L), hydrolysate (18–20%), and low pH. Clone fitness is mapped over the E. coli genome. Peak location represents location of clone in E. coli genome; peak size is relative to fitness. Colors denote the type of mutation in the clones: red spikes indicate an up mutation, blue spikes are down mutations. [B is adapted from Warner et al. (1).] (D–F) Growth studies of individual TRMR mutants. (D) Twenty-four-hour growth in 16 g/L acetate. (E) Fourteen-hour growth in 27.5% hydrolysate. (F) Twelve-hour growth in pH 5.0 M9 media. Error bars represent 1 SD.

Fig. 3.

Model of recursive multiplex recombineering library growth. (A) Construction of the library. Recombination efficiencies of 2–10% were routinely achieved, as quantified using an oligo that restores operational sequence of the galK gene in the SIMD70 strain (oligo 478 in ref. 19). Shown is the theoretical population distribution of the library where recombination efficiency is 5.0%. After 13 rounds of recombination, single, double, and triple mutants represent 35%, 11%, and 2%, of the total library populations, respectively. (B–E) Four cases of varying epistasis in a growth selection. WT growth rate was set to 0.05 1/h (typical for 40% hydrolysate growth). Mutations were modeled to be either beneficial (35% increase in growth rate over control; 10% of mutations) or neutral (no change in growth rate; 90% of mutations). (B) Synergistic: combinations of mutations increase in growth 10% more than additive. (C) Additive: benefits of individual mutations are additive. (D) Less-than-additive: combinations 10% less than additive. (E) Antagonistic: combinations of mutations reduce growth rate by 15% compared with individual mutations.

Fig. 4.

Recursive multiplex recombineering hydrolysate selection isolates. (A) Relative growth over 44 h of 10 isolated clones taken from sample plates after the initial selection. (B) Mutations in RBS of tolerant clones. (C) Relative growth over 20 h of four mutants isolated after the second selection compared with WT and H1 G and I (isolated from the first selection). (D) Mutations in RBS of the second combinatorial library clones isolated after the second selection. Error bars represent 1 SD.