Polyamine Transporters and Polyamines Increase Furfural Tolerance during Xylose Fermentation with Ethanologenic Escherichia coli Strain LY180

Abstract

Expression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenic Escherichia coli LY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression of yqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion of potE in EMFR9 lowered furfural tolerance to that of the parent. Deletion of potE and puuP in LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural.

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

“…In addition, increased IPTG concentrations were observed to influence cellular furfural sensitivity even for the cells with the empty vector ( Fig. 2C), which was consistent with previous results (39).…”

Mutation in galP improved fermentation of mixed sugars to succinate using engineered Escherichia coli AS1600a and AM1 mineral salts medium

“…Cell mass was estimated from OD 550nm (0.33 mg of cell dry weight/mL Â OD 550nm ) using a Bausch & Lomb Spectronic 70 spectrophotometer (Jantama et al, 2008b). OD was not measured in hydrolysate fermentations due to color (Geddes et al, 2014(Geddes et al, , 2015. Sugars, furans, and organic acids were analyzed by two high-performance liquid chromatography (HPLC) systems (Agilent Technologies 1200) as described previously (Geddes et al, 2014).…”

“…OD was not measured in hydrolysate fermentations due to color (Geddes et al, 2014(Geddes et al, , 2015. Sugars, furans, and organic acids were analyzed by two high-performance liquid chromatography (HPLC) systems (Agilent Technologies 1200) as described previously (Geddes et al, 2014). Sugars and furan were analyzed using a BioRad (Hercules, CA) Aminex HPX-87P ion exclusion column (80°C; nano-pure water as the mobile phase, 0.6 mL/min).…”

Unique k-mers as Strain-Specific Barcodes for Phylogenetic Analysis and Natural Microbiome Profiling

“…Thirteen of them encode enzymes of metabolic pathways (aes, icd, pabB, trpA, trpB, fumC, mdh, purA, aspC, fadD, uidA, aroE, mtlD); ten belong to replication, repair, or transcription systems (polB, gyrA, recA, dinB, dnaG, mutS, arcA, cyaA, grpE, rpoS), and the remaining four control the production of two symporters (putP, lysP), kinase (adk) and protease (clpX). The sequences of these genes were obtained from 124 E. coli genomes (listed in Supplementary Table S1), including 59 complete genomes, whose phylogroups were previously identified in original papers [45,52,[55][56][57][58][59][60][61][62][63][64][65][66][67][68][69] (shown in bold in Supplementary Table S1). The remaining 65 genomes unassigned to phylogroups and containing orthologs of discriminatory genes were added to increase the resolution of phylogenetic trees.…”