Global regulator engineering significantly improved Escherichia coli tolerances toward inhibitors of lignocellulosic hydrolysates

Abstract

Lignocellulosic biomass is regarded as the most viable source of feedstock for industrial biorefinery, but the harmful inhibitors generated from the indispensable pretreatments prior to fermentation remain a daunting technical hurdle. Using an exogenous regulator, irrE, from the radiation-resistant Deinococcus radiodurans, we previously showed that a novel global regulator engineering (GRE) approach significantly enhanced tolerances of Escherichia coli to alcohol and acetate stresses. In this work, an irrE library was subjected to selection under various stresses of furfural, a typical hydrolysate inhibitor. Three furfural tolerant irrE mutants including F1-37 and F2-1 were successfully obtained. The cells containing these mutants reached OD(600) levels of 4- to 16-fold of that for the pMD18T cells in growth assay under 0.2% (v/v) furfural stress. The cells containing irrE F1-37 and F2-1 also showed considerably reduced intracellular oxygen species (ROS) levels under furfural stress. Moreover, these two irrE mutants were subsequently found to confer significant cross tolerances to two other most common inhibitors, 5-hydroxymethyl-2-furaldehyde (HMF), vanillin, as well as real lignocellulosic hydrolysates. When evaluated in Luria-Bertani (LB) medium supplemented with corn stover cellulosic hydrolysate (prepared with a solid loading of 30%), the cells containing the mutants exhibited lag phases markedly shortened by 24-44 h in comparison with the control cells. This work thus presents a promising step forward to resolve the inhibitor problem for E. coli. From the view of synthetic biology, irrE can be considered as an evolvable "part" for various stresses. Furthermore, this GRE approach can be extended to exploit other exogenous global regulators from extremophiles, and the native counterparts in E. coli, for eliciting industrially useful phenotypes.

Genome-Wide Mapping of Furfural Tolerance Genes in Escherichia coli

“…Much research has been performed over the past decade to uncover mechanisms of furfural toxicity and to engineer furfural tolerance in E. coli [29]–[37], [39], [66]. Here, we used the SCALEs method [13] to not only map fitness effects across the entire E. coli genome, but also to identify and confirm both novel ( lpcA and groESL ) and previously identified ( thyA [31]) furfural tolerance genes.…”

“…Similar toxicity mechanisms and genetic manipulations have been beneficial for engineering E. coli for tolerance to 5-hydroxymethylfurfural [37], a hexose degradation product in hydrolysate. In addition to directly redox related mechanisms, reactive oxygen species (ROS) accumulation has been observed in yeast cells [38] and E. coli [39] when treated with furfural, which is a common phenotype associated with DNA damage [40], as well as more generally with chemotoxicity [41].…”

Comparative genomics and metabolomics analyses of the adaptation mechanism in Ketogulonicigenium vulgare-Bacillus thuringiensis consortium

“…Smooth changes in transcriptional and regulatory levels may have significant global response20. Two species both have the genetic mutations in transcriptional or regulatory genes, such as the genes encoding RNA polymerase-binding protein DksA and signal recognition particle (SRP) in K150 (Fig.…”

Optimizing Metabolite Production Using Periodic Oscillations

“…We grouped all nine sensitive enzymes together into a “PEP-influencing cluster” and focused on optimizing collective expression of this cluster as a unit to mimic natural systems, where large groups of enzymes are co-regulated to produce a specific phenotype [37]. This PEP-influencing cluster represents the theoretical maximum number of enzymes that we hypothesized would significantly influence PEP levels.…”