Bioprospecting of Native Efflux Pumps To Enhance Furfural Tolerance in Ethanologenic Escherichia coli

Abstract

Efficient microbial conversion of lignocellulose into valuable products is often hindered by the presence of furfural, a dehydration product of pentoses in hemicellulose sugar syrups derived from woody biomass. For a cost-effective lignocellulose microbial conversion, robust biocatalysts are needed that can tolerate toxic inhibitors while maintaining optimal metabolic activities. A comprehensive plasmid-based library encoding native multidrug resistance (MDR) efflux pumps, porins, and select exporters fromEscherichia coliwas screened for furfural tolerance in an ethanologenicE. colistrain. Small multidrug resistance (SMR) pumps, such as SugE and MdtJI, as well as a lactate/glycolate:H+symporter, LldP, conferred furfural tolerance in liquid culture tests. Expression of the SMR pump potentially increased furfural efflux and cellular viability upon furfural assault, suggesting novel activities for SMR pumps as furfural efflux proteins. Furthermore, induced expression ofmdtJIenhanced ethanol fermentative production of LY180 in the presence of furfural or 5-hydroxymethylfurfural, further demonstrating the applications of SMR pumps. This work describes an effective approach to identify useful efflux systems with desired activities for nonnative toxic chemicals and provides a platform to further enhance furfural efflux by protein engineering and mutagenesis.IMPORTANCELignocellulosic biomass, especially agricultural residues, represents an important potential feedstock for microbial production of renewable fuels and chemicals. During the deconstruction of hemicellulose by thermochemical processes, side products that inhibit cell growth and production, such as furan aldehydes, are generated, limiting cost-effective lignocellulose conversion. Here, we developed a new approach to increase cellular tolerance by expressing multidrug resistance (MDR) pumps with putative efflux activities for furan aldehydes. The developed plasmid library and screening methods may facilitate new discoveries of MDR pumps for diverse toxic chemicals important for microbial conversion.

Transporter engineering in microbial cell factories: the ins, the outs, and the in-betweens

“…However, pretreatment of biomass and waste stream into fermentable sugars releases growth inhibitory compounds. Tolerance against these inhibitors can be achieved by engineering their efflux [ 9 ]. Furthermore, substrate uptake is a relevant strategy for the production of some specialty chemicals that require specific precursor molecules; for example, the production of fluorinated compounds requires efficient uptake of fluoride ions from the medium.…”

Deletion of pgi gene in E. coli increases tolerance to furfural and 5-hydroxymethyl furfural in media containing glucose–xylose mixture

“…Different cellular components involved in maintaining physiological homeostasis have been involved in conferring tolerance against furfural [ 10 , 11 ]. Components of DNA repair [ 10 , 12 ], cell membrane and chaperones [ 11 ], and MDR efflux pumps [ 13 ] have been reported to confer tolerance against furfural. Causal mechanisms for toxicity of furfural to microbial cell have been suggested to be altered redox ratios [ 14 ], accumulation of ROS [ 15 ] and reduced biosynthesis of sulfur containing macromolecules [ 16 ].…”

Deletion of Pgi Gene in E. coli Increases Tolerance to Furfural and 5-Hydroxymethyl Furfural in Media Containing Glucose-Xylose Mixture

“…Different cellular components involved in maintaining physiological homeostasis have been involved in conferring tolerance against furfural [6,7]. Components of DNA repair [6,8], cell membrane and chaperones [7] and MDR e ux pumps [9] have been reported to confer tolerance against furfural. Causal mechanisms for toxicity of furfural to microbial cell have been suggested to be altered redox ratios [10], accumulation of ROS [11] and reduced biosynthesis of sulfur containing macromolecules [12].…”