Natural Variation in the Multidrug Efflux Pump SGE1 Underlies Ionic Liquid Tolerance in Yeast

Abstract

Imidazolium ionic liquids (IILs) have a range of biotechnological applications, including as pretreatment solvents that extract cellulose from plant biomass for microbial fermentation into sustainable bioenergy. However, residual levels of IILs, such as 1-ethyl-3-methylimidazolium chloride ([C2C1im]Cl), are toxic to biofuel-producing microbes, including the yeast Saccharomyces cerevisiae. S. cerevisiae strains isolated from diverse ecological niches differ in genomic sequence and in phenotypes potentially beneficial for industrial applications, including tolerance to inhibitory compounds present in hydrolyzed plant feedstocks. We evaluated >100 genome-sequenced S. cerevisiae strains for tolerance to [C2C1im]Cl and identified one strain with exceptional tolerance. By screening a library of genomic DNA fragments from the [C2C1im]Cl-tolerant strain for improved IIL tolerance, we identified SGE1, which encodes a plasma membrane multidrug efflux pump, and a previously uncharacterized gene that we named ionic liquid tolerance 1 (ILT1), which encodes a predicted membrane protein. Analyses of SGE1 sequences from our panel of S. cerevisiae strains together with growth phenotypes implicated two single nucleotide polymorphisms (SNPs) that associated with IIL tolerance and sensitivity. We confirmed these phenotypic effects by transferring the SGE1 SNPs into a [C2C1im]Cl-sensitive yeast strain using CRISPR/Cas9 genome editing. Further studies indicated that these SNPs affect Sge1 protein stability and cell surface localization, influencing the amount of toxic IILs that cells can pump out of the cytoplasm. Our results highlight the general potential for discovering useful biotechnological functions from untapped natural sequence variation and provide functional insight into emergent SGE1 alleles with reduced capacities to protect against IIL toxicity.

Guanidine Riboswitch-Regulated Efflux Transporters Protect Bacteria against Ionic Liquid Toxicity

“…A library of IIL efflux pumps. Our goal at the outset of this investigation was to identify new ionic liquid tolerance mechanisms beyond the MFS transporters we previously identified (16,17). Our results include five SMR pumps, two from the B. cereus group, two from B. licheniformis and one from E. coli, that protected E. coli from the IILs tested.…”

“…cereus smr genes protect E. coli. Functional genomics is a powerful technique that we have recently used specifically to identify IIL tolerance mechanisms (16,17). In this approach, large donor DNA regions (ϳ35 kb) from the tolerant organism are delivered to E. coli or S. cerevisiae by fosmid cloning, and cells are then immediately screened for their ability to grow on IIL-containing media.…”

“…Although SGE1 is known to protect yeast against QACs, such as crystal violet (18), that share some chemical features with imidazolium cations, laboratory strains and many environmental isolates of S. cerevisiae are highly IIL sensitive (IIL S ). Just as the eilA gene can grant IIL tolerance to E. coli (17), introduction of the environmental SGE1 variant confers tolerance to the IIL S lab strain of S. cerevisiae (16). We also identified a second yeast IIL tolerance gene, ILT1, which encodes an uncharacterized membrane protein (16).…”

“…Two of these genes form a genetic cassette encoding a multidrug efflux pump of the major facilitator superfamily (MFS) and an autoregulatory component, a TetR-type repressor of the MFS gene (eilA and eilR, respectively). Moreover, in an environmental strain of the yeast Saccharomyces cerevisiae, we discovered that tolerance to IILs can be attributed to two single nucleotide polymorphisms (SNPs) in the MFS efflux pump encoded by SGE1 (16). Although SGE1 is known to protect yeast against QACs, such as crystal violet (18), that share some chemical features with imidazolium cations, laboratory strains and many environmental isolates of S. cerevisiae are highly IIL sensitive (IIL S ).…”

“…Just as the eilA gene can grant IIL tolerance to E. coli (17), introduction of the environmental SGE1 variant confers tolerance to the IIL S lab strain of S. cerevisiae (16). We also identified a second yeast IIL tolerance gene, ILT1, which encodes an uncharacterized membrane protein (16). Apart from these natural isolates, adaptive lab evolution was recently used to generate IIL T mutants of E. coli.…”