Genome-Wide Mapping of Furfural Tolerance Genes in <i>Escherichia coli</i>

<div><p>Advances in genomics have improved the ability to map complex genotype-to-phenotype relationships, like those required for engineering chemical tolerance. Here, we have applied the multiSCale Analysis of Library Enrichments (SCALEs; Lynch et al. (2007) <i>Nat. Method</i>.) approach to map, in parallel, the effect of increased dosage for >10⁵ different fragments of the <i>Escherichia coli</i> genome onto furfural tolerance (furfural is a key toxin of lignocellulosic hydrolysate). Only 268 of >4,000 <i>E. coli</i> genes (∼6%) were enriched after growth selections in the presence of furfural. Several of the enriched genes were cloned and tested individually for their effect on furfural tolerance. Overexpression of <i>thyA</i>, <i>lpcA</i>, or <i>groESL</i> individually increased growth in the presence of furfural. Overexpression of <i>lpcA</i>, but not <i>groESL</i> or <i>thyA</i>, resulted in increased furfural reduction rate, a previously identified mechanism underlying furfural tolerance. We additionally show that plasmid-based expression of functional LpcA or GroESL is required to confer furfural tolerance. This study identifies new furfural tolerant genes, which can be applied in future strain design efforts focused on the production of fuels and chemicals from lignocellulosic hydrolysate.</p></div

Phenotypic analysis of tolerant clones for furfural reduction and DNA mutation frequency.

<p>A) Samples were collected for measuring furfural in growth curve cultures with 0.75 g l⁻¹ furfural initial concentration. Furfural concentrations were normalized to cell number (optical density) for each value and disappearance rate was calculated during the transition from lag to exponential phase (n = 3). B) Frequency of rifampin resistance of cells treated with furfural (n = 4). Error bars represent one standard error. Double asterisks denote <i>p</i><0.05.</p

Growth curve analysis of tolerant clones grown in minimal medium with 0.75⁻¹ furfural.

<p>A) Seed cultures were inoculated into furfural at the same initial density and grown for 24 hours. Optical density was recorded every 3–6 hours. Error bars represent one standard error (n = 3). Double asterisks denote <i>p</i><0.05.</p

Mutational studies on <i>lpcA</i> and <i>groESL</i> clones.

<p>Mutations were introduced onto the plasmids within the coding sequence targeted for (A) <i>lpcA</i> or (B) <i>groESL</i>. Cultures were grown in 0.75 g l⁻¹ furfural for 20 hr. (n = 3; error bars represent standard error). Percentage improvement was calculated as the difference of the test strain subtracted from the control, divided by the control.</p

Gene(s) cloned for confirmation studies.

<p>*<i>groESL</i> operon was cloned into a single plasmid.</p

Overview of furfural selection and SCALEs analysis.

<p>A) 1, 2, 4, and 8 kb fragments were prepared from <i>E. coli</i> genomic DNA and ligated into pSMART-LCK vector. Each sized genomic library was transformed into BW25113 Δ<i>recA</i>::FRT host cells, recovered, mixed together, and then grown on minimal medium plates (control) or solid minimal medium with 0.75 g l⁻¹ furfural. Cells were harvested from the plates and microarrays (square boxes) were run with plasmid extracts from both the furfural and control plates in order to determine individual gene fitness scores (<i>W</i>). The fitness vs. position plot illustrates how different clones (stacked rectangles) can contribute to an individual gene's fitness. The red “triangle” has contribution from various sized clones, but is centered around a specific locus, whereas the blue “rectangle” represents a high fitness score from the presence of one single sized clone (e.g., requiring a large operon where smaller library sizes would not be found). B) Genome plot depicting clone fitnesses for the different library sizes. Loci corresponding to the top gene fitness scores are labeled. C) Histogram of log-transformed gene fitness scores, where increased fitness corresponds to ln(<i>W</i>)>0.</p