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.
Highlights
The polysaccharides present in lignocellulosic biomass provide an attractive raw material for the production of renewable biofuels
Transcriptomic response of E. coli to [C2mim]Cl The E. coli DH1 toxicity profile to [C2mim]Cl was determined by measuring the optical density of cultures after addition of the ionic liquids (ILs) over a range concentrations (0–400 mM) in mid-log phase. [C2mim]Cl exposure resulted an increase in the lag phase and the doubling time (Figure 1)
PmarR’ and PydfA’ provide good performance by eliminating the burden of pump expression when IL levels are low and expressing pumps highly when IL levels are high. Subtle differences between these two promoters can be detected with sensitive competition assays, like those shown in Conclusion Promoters such as PlacUV5 are commonly used in metabolic and host engineering proof-of-concept studies [40,34]
Summary
The polysaccharides present in lignocellulosic biomass provide an attractive raw material for the production of renewable biofuels. To minimize the costs associated with washing pretreated biomass, and recycling ILs, a residual level (0.2–5wt/ vol%) of IL typically remains in downstream stages of an industrial scale production process [9]. These low levels of IL, contaminating the sugar stream used for cultivation, are toxic to biofuel-producing microorganisms [10,11,12,13]. Residual IL may eventually be utilized in an industrial set-up, in combination with host organisms engineered for IL tolerance, to prevent contamination of the cultures
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