Genome-Wide Mapping of Furfural Tolerance Genes in Escherichia coli

Abstract

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) Nat. Method.) approach to map, in parallel, the effect of increased dosage for >105 different fragments of the Escherichia coli genome onto furfural tolerance (furfural is a key toxin of lignocellulosic hydrolysate). Only 268 of >4,000 E. coli 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 thyA, lpcA, or groESL individually increased growth in the presence of furfural. Overexpression of lpcA, but not groESL or thyA, 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.

Polyamine Transporters and Polyamines Increase Furfural Tolerance during Xylose Fermentation with Ethanologenic Escherichia coli Strain LY180

“…Genes have been described that increase the resistance of bacterial and yeast biocatalysts to furfural and hydroxymethylfurfural (5,7,(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). However, to date, none have completely solved the toxicity problem.…”

“…Both enzymes reduce furfural (aldehyde) to the less toxic furfuryl alcohol. Recently, a new genomic tool, multiscale analysis of library enrichments (SCALE), was developed and identified four genes in E. coli that increase furfural tolerance (15): thyA (DNA biosynthesis), lpcA (lipopolysaccharide biosynthesis), groES, and groEL (GroES-EL chaperonin complex). Increased expression of the groES, groEL, grpE, and clpB genes (21,22) has also been shown to increase ethanol tolerance.…”

Identification of Genes Conferring Tolerance to Lignocellulose-Derived Inhibitors by Functional Selections in Soil Metagenomes

“…In contrast, ThyX produces THF as a direct product of dTMP synthesis but oxidizes NADPH during this reaction, obviating the need for DHF reduction but not NADPH oxidation (60,71,72). Because both thymidylate synthases result in oxidized NADPH and yet both confer furfural tolerance when overexpressed, these enzymes likely ameliorate furfural toxicity independently of the compound's effect on NADPH levels (59,70,73,74).…”

Engineering membrane and cell-wall programs for tolerance to toxic chemicals: Beyond solo genes

“…The lpcA gene, coding the first committed step in the LPS inner core precursor pathway (Fig. 2b), has been selected for using the scalar analysis of library enrichments (SCALES) method against acetate, furfural, and ethanol[38,63,64]. SCALES selection against furfural, a hydrophobic lignocellulosic hydrolysate byproduct known to cause membrane disruption[6], also showed that high copy number of genes related to synthesis of the outer membrane glycolipid ECA (enterobacterial common antigen, rffA , rffC , rffD , rffE , rffG , wzxE ) and high copy number of the division and cell-wall synthesis (dcw) gene cluster ( murEF-mraY-murD-ftsW-murGC-ddlB-ftsQ , Fig 2.)…”