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Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in

Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1, a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1, ISU1, GRE3, and IRA2, increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products.

Imidazolium ionic liquids (IILs) have a range of biotechnological applications, including as pretreatment solvents that extract cellulose from plant biomass for microbial fermentation into sustainable bioenergy. However, residual levels of IILs, such as 1-ethyl-3-methylimidazolium chloride ([C2C1im]Cl), are toxic to biofuel-producing microbes, including the yeast Saccharomyces cerevisiae. S. cerevisiae strains isolated from diverse ecological niches differ in genomic sequence and in phenotypes potentially beneficial for industrial applications, including tolerance to inhibitory compounds present in hydrolyzed plant feedstocks. We evaluated >100 genome-sequenced S. cerevisiae strains for tolerance to [C2C1im]Cl and identified one strain with exceptional tolerance. By screening a library of genomic DNA fragments from the [C2C1im]Cl-tolerant strain for improved IIL tolerance, we identified SGE1, which encodes a plasma membrane multidrug efflux pump, and a previously uncharacterized gene that we named ionic liquid tolerance 1 (ILT1), which encodes a predicted membrane protein. Analyses of SGE1 sequences from our panel of S. cerevisiae strains together with growth phenotypes implicated two single nucleotide polymorphisms (SNPs) that associated with IIL tolerance and sensitivity. We confirmed these phenotypic effects by transferring the SGE1 SNPs into a [C2C1im]Cl-sensitive yeast strain using CRISPR/Cas9 genome editing. Further studies indicated that these SNPs affect Sge1 protein stability and cell surface localization, influencing the amount of toxic IILs that cells can pump out of the cytoplasm. Our results highlight the general potential for discovering useful biotechnological functions from untapped natural sequence variation and provide functional insight into emergent SGE1 alleles with reduced capacities to protect against IIL toxicity.

Imidazolium ionic liquids (IILs) have a range of biotechnological applications, including as pretreatment solvents that extract cellulose from plant biomass for microbial fermentation into sustainable bioenergy. However, residual levels of IILs, such as 1-ethyl-3-methylimidazolium chloride ([C2C1im]Cl), are toxic to biofuel-producing microbes, including the yeast Saccharomyces cerevisiae. S. cerevisiae strains isolated from diverse ecological niches differ in genomic sequence and in phenotypes potentially beneficial for industrial applications, including tolerance to inhibitory compounds present in hydrolyzed plant feedstocks. We evaluated >100 genome-sequenced S. cerevisiae strains for tolerance to [C2C1im]Cl and identified one strain with exceptional tolerance. By screening a library of genomic DNA fragments from the [C2C1im]Cl-tolerant strain for improved IIL tolerance, we identified SGE1, which encodes a plasma membrane multidrug efflux pump, and a previously uncharacterized gene that we named ionic liquid tolerance 1 (ILT1), which encodes a predicted membrane protein. Analyses of SGE1 sequences from our panel of S. cerevisiae strains together with growth phenotypes implicated two single nucleotide polymorphisms (SNPs) that associated with IIL tolerance and sensitivity. We confirmed these phenotypic effects by transferring the SGE1 SNPs into a [C2C1im]Cl-sensitive yeast strain using CRISPR/Cas9 genome editing. Further studies indicated that these SNPs affect Sge1 protein stability and cell surface localization, influencing the amount of toxic IILs that cells can pump out of the cytoplasm. Our results highlight the general potential for discovering useful biotechnological functions from untapped natural sequence variation and provide functional insight into emergent SGE1 alleles with reduced capacities to protect against IIL toxicity.

Currently microbial conversion of lignocellulose-derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, we developed Escherichia coli strains that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome-scale models and adaptive laboratory evolution. We designed and constructed E. coli strains that co-utilize glucose and xylose, and adaptively evolved them to improve glucose and xylose utilization. Whole-genome resequencing of the evolved strains found relevant mutations in metabolic and regulatory genes, and we investigated the mutations’ involvement in sugar co-utilization. The developed strains showed significantly improved co-conversion of sugars in lignocellulosic biomass hydrolysates, and they provide a promising platform for producing next-generation biofuels.

The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and rpsQ recapitulated most of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. Ethanol induced miscoding errors during protein synthesis, from which the evolved rpsQ allele protected cells by increasing ribosome accuracy. Ribosome profiling and RNAseq analyses established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through direct effects on ribosomes and RNA polymerase conformations are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ help protect these central dogma processes in the presence of ethanol.

The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.