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

Abstract

Plant cell walls contain a renewable, nearly limitless supply of sugar that could be used to support microbial production of commodity chemicals and biofuels. Imidazolium ionic liquid (IIL) solvents are among the best reagents for gaining access to the sugars in this otherwise recalcitrant biomass. However, the sugars from IIL-treated biomass are inevitably contaminated with residual IILs that inhibit growth in bacteria and yeast, blocking biochemical production by these organisms. IIL toxicity is, therefore, a critical roadblock in many industrial biosynthetic pathways. Although several IIL-tolerant (IILT) bacterial and yeast isolates have been identified in nature, few genetic mechanisms have been identified. In this study, we identified two IILT Bacillus isolates as well as a spontaneous IILT Escherichia coli lab strain that are tolerant to high levels of two widely used IILs. We demonstrate that all three IILT strains contain one or more pumps of the small multidrug resistance (SMR) family, and two of these strains contain mutations that affect an adjacent regulatory guanidine riboswitch. Furthermore, we show that the regulation of E. coli sugE by the guanidine II riboswitch can be exploited to promote IIL tolerance by the simple addition of guanidine to the medium. Our results demonstrate the critical role that transporter genes play in IIL tolerance in their native bacterial hosts. The study presented here is another step in engineering IIL tolerance into industrial strains toward overcoming this key gap in biofuels and industrial biochemical production processes. IMPORTANCE This study identifies bacteria that are tolerant to ionic liquid solvents used in the production of biofuels and industrial biochemicals. For industrial microbiology, it is essential to find less-harmful reagents and microbes that are resistant to their cytotoxic effects. We identified a family of small multidrug resistance efflux transporters, which are responsible for the tolerance of these strains. We also found that this resistance can be caused by mutations in the sequences of guanidine-specific riboswitches that regulate these efflux pumps. Extending this knowledge, we demonstrated that guanidine itself can promote ionic liquid tolerance. Our findings will inform genetic engineering strategies that improve conversion of cellulosic sugars into biofuels and biochemicals in processes where low concentrations of ionic liquids surpass bacterial tolerance.

Generation of ionic liquid tolerant Pseudomonas putida KT2440 strains via adaptive laboratory evolution

“…In this manner, from the literature, it was found that E. coli K-12 MG1655 also has a similar EmrE and can export imidazolium ILs. 57 However, we found that the EmrE gene of KT2440 shares only 47% identity with its E. coli homolog (Fig. S8) and one isolate (TA13_F83_I1) from an IL mix TALE experiment acquired a frame-shift mutation in its emrE, which likely disrupts its activity.…”

Biochemical Validation of a Fourth Guanidine Riboswitch Class in Bacteria

“…Perhaps with the addition of the genes known to associate with guanidine-IV riboswitches, the expanded "super-regulon" of guanidinecontrolled pathways will provide some hints at the natural source(s) of this molecule. Regardless, advances are already being made on biological processes controlled by guanidine riboswitches, 21,39,40 and guanidine aptamers appear to be promising RNA structures for use in synthetic biology. 41 It is also interesting to consider why the guanidine-IV riboswitch class was not encountered in previous bioinformatics searches for novel ncRNA motifs.…”

Former orphan riboswitches reveal unexplored areas of bacterial metabolism, signaling, and gene control processes

“…However, guanidine is toxic to B. subtilis cells at high concentrations , and the small multi-drug resistance (SMR) transporters associated with guanidine riboswitches appear to expel guanidine to reduce its toxicity (Kermani et al 2018;Higgins et al 2019).…”