Development of a Native Escherichia coli Induction System for Ionic Liquid Tolerance

Abstract

The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR’, PydfO’, and PydfA’, were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA’ and PmarR’ based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA’ and PmarR’ in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR’-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR’ can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.

Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters

“…4), which could enhance the tolerance and yields of metabolically engineered chemical production strains [4, 8, 73, 74]. In all three fungal strains, we find a number of Drug:Proton antiporter proteins (DHA) (TCDB 2.A.1.2, 2.A.1.3; 55 proteins in total).…”

Ionic liquid-tolerant microorganisms and microbial communities for lignocellulose conversion to bioproducts

“…Moreover, due to the lack of well-mixed conditions in high solid systems, the mechanisms and pathways that microorganisms use to tolerate IL may be different between aqueous and high solid systems . Although IL tolerant mechanisms have not been fully explored, a few studies have applied transcriptomic analyses to begin to elucidate the molecular mechanisms of IL tolerance , and have successfully demonstrated an engineered IL-tolerant microorganism with potential applications in converting IL treated biomass into advanced biofuels (Bokinsky et al 2011;Dickinson et al 2016;Frederix et al 2014;Ruegg et al 2014). These recent studies demonstrate the potential opportunities for discovering genes from environmental communities that confer IL tolerance, and engineering industrial microorganisms for bioprocesses that include IL pretreatment.…”

“…With the identification of an adjacent gene, eilR (locus tag Entcl_2353), encoding a repressor protein, an autoregulation mechanism of the IL efflux pump was demonstrated . The concept of condition-dependent regulation of IL tolerance genes was later demonstrated (Frederix et al 2014). Native E. coli IL-inducible promoters (PmarR', PydfO', and PydfA') were evaluated for their ability to provide a dynamic control system for regulating YbjJ, which appears to encode another efflux pump (Frederix et al 2016).…”

Tolerance engineering in bacteria for the production of advanced biofuels and chemicals

“…This prediction was successfully tested using the heterologous MFS pump EilA (Box 3). Expression of this pump using native E. coli promoters that respond to ionic liquid as a stressor resulted in a strain that has greater fitness relative to the pump regulated by an IPTG-induced P lac promoter [96]. Dynamic expression of genes involved in tolerance is an important route to explore, to constrain the required gene expression to only when the tolerance phenotype is required, and to minimize the number of inducible promoters being used in strain engineering.…”