Abstract
BackgroundImidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries. However, residual IILs are toxic to fermentative microbes such as Saccharomyces cerevisiae, making IIL-tolerance a key property for strain engineering. To enable rational engineering, we used chemical genomic profiling to understand the effects of IILs on S. cerevisiae.ResultsWe found that IILs likely target mitochondria as their chemical genomic profiles closely resembled that of the mitochondrial membrane disrupting agent valinomycin. Further, several deletions of genes encoding mitochondrial proteins exhibited increased sensitivity to IIL. High-throughput chemical proteomics confirmed effects of IILs on mitochondrial protein levels. IILs induced abnormal mitochondrial morphology, as well as altered polarization of mitochondrial membrane potential similar to valinomycin. Deletion of the putative serine/threonine kinase PTK2 thought to activate the plasma-membrane proton efflux pump Pma1p conferred a significant IIL-fitness advantage. Conversely, overexpression of PMA1 conferred sensitivity to IILs, suggesting that hydrogen ion efflux may be coupled to influx of the toxic imidazolium cation. PTK2 deletion conferred resistance to multiple IILs, including [EMIM]Cl, [BMIM]Cl, and [EMIM]Ac. An engineered, xylose-converting ptk2∆ S. cerevisiae (Y133-IIL) strain consumed glucose and xylose faster and produced more ethanol in the presence of 1 % [BMIM]Cl than the wild-type PTK2 strain. We propose a model of IIL toxicity and resistance.ConclusionsThis work demonstrates the utility of chemical genomics-guided biodesign for development of superior microbial biocatalysts for the ever-changing landscape of fermentation inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0417-7) contains supplementary material, which is available to authorized users.
Highlights
Imidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries
Among the top 20 sensitive mutants, we found gene ontology (GO) enrichment (p < 0.01) for genes that encode mitochondrial proteins (e.g. ARG2, COQ2, HMI1, IMG2, QCR2, RIM1, SHE9, YPT7); [EMIM]Cl may affect mitochondrial function (Fig. 2a)
The second most significant resistant strain was a deletion mutant of SKY1, which is functionally similar to PTK2 and is a protein kinase that regulates proteins involved in cation homeostasis and polyamine cation uptake [27, 29]
Summary
Imidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries. Dickinson et al Microb Cell Fact (2016) 15:17 rely on solvents like ionic liquids (IL) or γ-valerolactone [5, 6], which are partially retained in the hydrolysates and are not readily tolerated by fermentative microorganisms [7, 8] Despite their toxicity, ILs hold special promise because they can be used either to solubilize crystalline cellulose for enzymatic hydrolysis [9, 10] or to support complete chemical deconstruction without the need for enzymes [6, 11]. ILs hold special promise because they can be used either to solubilize crystalline cellulose for enzymatic hydrolysis [9, 10] or to support complete chemical deconstruction without the need for enzymes [6, 11] Among these ILs, imidazolium ionic liquids (IILs) Increasing microbial tolerance of IILs is one strategy to lower the economic cost of IIL-based conversion processes
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