BackgroundThere is a need to replace petroleum-derived with sustainable feedstocks for chemical production. Certain biomass feedstocks can meet this need as abundant, diverse, and renewable resources. Specific ionic liquids (ILs) can play a role in this process as promising candidates for chemical pretreatment and deconstruction of plant-based biomass feedstocks as they efficiently release carbohydrates which can be fermented. However, the most efficient pretreatment ILs are highly toxic to biological systems, such as microbial fermentations, and hinder subsequent bioprocessing of fermentative sugars obtained from IL-treated biomass.MethodsTo generate strains capable of tolerating residual ILs present in treated feedstocks, a tolerance adaptive laboratory evolution (TALE) approach was developed and utilized to improve growth of two different Escherichia coli strains, DH1 and K-12 MG1655, in the presence of two different ionic liquids, 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and 1-butyl-3-methylimidazolium chloride ([C4C1Im]Cl). For multiple parallel replicate populations of E. coli, cells were repeatedly passed to select for improved fitness over the course of approximately 40 days. Clonal isolates were screened and the best performing isolates were subjected to whole genome sequencing.ResultsThe most prevalent mutations in tolerant clones occurred in transport processes related to the functions of mdtJI, a multidrug efflux pump, and yhdP, an uncharacterized transporter. Additional mutations were enriched in processes such as transcriptional regulation and nucleotide biosynthesis. Finally, the best-performing strains were compared to previously characterized tolerant strains and showed superior performance in tolerance of different IL and media combinations (i.e., cross tolerance) with robust growth at 8.5% (w/v) and detectable growth up to 11.9% (w/v) [C2C1Im][OAc].ConclusionThe generated strains thus represent the best performing platform strains available for bioproduction utilizing IL-treated renewable substrates, and the TALE method was highly successful in overcoming the general issue of substrate toxicity and has great promise for use in tolerance engineering.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0819-1) contains supplementary material, which is available to authorized users.

Pseudomonas putida KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to aromatic catabolism and toxicity tolerance of P. putida will be required to achieve industrial relevance. Here, tolerance adaptive laboratory evolution (TALE) was employed with increasing concentrations of the hydroxycinnamic acids p -coumaric acid ( p CA) and ferulic acid (FA) individually and in combination ( p CA ​+ ​FA). The TALE experiments led to evolved P. putida strains with increased tolerance to the targeted acids as compared to wild type. Specifically, a 37 ​h decrease in lag phase in 20 ​g/L p CA and a 2.4-fold increase in growth rate in 30 ​g/L FA was observed. Whole genome sequencing of intermediate and endpoint evolved P. putida populations revealed several expected and non-intuitive genetic targets underlying these aromatic catabolic and toxicity tolerance enhancements. PP_3350 and ttgB were among the most frequently mutated genes, and the beneficial contributions of these mutations were verified via gene knockouts. Deletion of PP_3350, encoding a hypothetical protein, recapitulated improved toxicity tolerance to high concentrations of p CA, but not an improved growth rate in high concentrations of FA. Deletion of ttgB, part of the TtgABC efflux pump, severely inhibited growth in p CA ​+ ​FA TALE-derived strains but did not affect growth in p CA ​+ ​FA in a wild type background, suggesting epistatic interactions. Genes involved in flagellar movement and transcriptional regulation were often mutated in the TALE experiments on multiple substrates, reinforcing ideas of a minimal and deregulated cell as optimal for domesticated growth. Overall, this work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving P. putida for microbial lignin valorization.

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Most microbial species, including model eukaryote Saccharomyces cerevisiae, possess genetic capability to utilize many alternative nutrient sources. Yet, it remains an open question whether these manifest into assimilatory phenotypes. Despite possessing all necessary pathways, S. cerevisiae grows poorly or not at all when glycerol is the sole carbon source. Here we discover, through multiple evolved lineages, genetic determinants underlying glycerol catabolism and the associated fitness trade-offs. Most evolved lineages adapted through mutations in the HOG pathway, but showed hampered osmotolerance. In the other lineages, we find that only three mutations cause the improved phenotype. One of these contributes counter-intuitively by decoupling the TCA cycle from oxidative phosphorylation, and thereby hampers ethanol utilization. Transcriptomics, proteomics and metabolomics analysis of the re-engineered strains affirmed the causality of the three mutations at molecular level. Introduction of these mutations resulted in improved glycerol utilization also in industrial strains. Our findings not only have a direct relevance for improving glycerol-based bioprocesses, but also illustrate how a metabolic pathway can remain unexploited due to fitness trade-offs in other, ecologically important, traits.

Toxicity from the external presence or internal production of compounds can reduce the growth and viability of microbial cell factories and compromise productivity. Aromatic compounds are generally toxic for microorganisms, which makes their production in microbial hosts challenging. Here we use adaptive laboratory evolution to generate Saccharomyces cerevisiae mutants tolerant to two aromatic acids, coumaric acid and ferulic acid. The evolution experiments were performed at low pH (3.5) to reproduce conditions typical of industrial processes. Mutant strains tolerant to levels of aromatic acids near the solubility limit were then analyzed by whole genome sequencing, which revealed prevalent point mutations in a transcriptional activator (Aro80) that is responsible for regulating the use of aromatic amino acids as the nitrogen source. Among the genes regulated by Aro80, ESBP6 was found to be responsible for increasing tolerance to aromatic acids by exporting them out of the cell. Further examination of the native function of Esbp6 revealed that this transporter can excrete fusel acids (byproducts of aromatic amino acid catabolism) and this role is shared with at least one additional transporter native to S. cerevisiae (Pdr12). Besides conferring tolerance to aromatic acids, ESBP6 overexpression was also shown to significantly improve the secretion in coumaric acid production strains. Overall, we showed that regulating the activity of transporters is a major mechanism to improve tolerance to aromatic acids. These findings can be used to modulate the intracellular concentration of aromatic compounds to optimize the excretion of such products while keeping precursor molecules inside the cell.

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Background Sucrose is an attractive industrial carbon source due to its abundance and the fact that it can be cheaply generated from sources such as sugarcane. However, only a few characterized Escherichia coli strains are able to metabolize sucrose, and those that can are typically slow growing or pathogenic strains. Methods To generate a platform strain capable of efficiently utilizing sucrose with a high growth rate, adaptive laboratory evolution (ALE) was utilized to evolve engineered E. coli K-12 MG1655 strains containing the sucrose utilizing csc genes ( cscB , cscK , cscA ) alongside the native sucrose consuming E. coli W. Results Evolved K-12 clones displayed an increase in growth and sucrose uptake rates of 1.72- and 1.40-fold on sugarcane juice as compared to the original engineered strains, respectively, while E. coli W clones showed a 1.4-fold increase in sucrose uptake rate without a significant increase in growth rate. Whole genome sequencing of evolved clones and populations revealed that two genetic regions were frequently mutated in the K-12 strains; the global transcription regulatory genes rpoB and rpoC, and the metabolic region related to a pyrimidine biosynthetic deficiency in K-12 attributed to pyrE expression. These two mutated regions have been characterized to confer a similar benefit when glucose is the main carbon source, and reverse engineering revealed the same causal advantages on M9 sucrose. Additionally, the most prevalent mutation found in the evolved E. coli W lineages was the inactivation of the cscR gene, the transcriptional repression of sucrose uptake genes. Conclusion The generated K-12 and W platform strains, and the specific sets of mutations that enable their phenotypes, are available as valuable tools for sucrose-based industrial bioproduction in the facile E. coli chassis. Electronic supplementary material The online version of this article (10.1186/s12934-019-1165-2) contains supplementary material, which is available to authorized users.

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