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While Pseudomonas putida KT2440 has great potential for biomass-converting processes, its inability to utilize the biomass abundant sugars xylose and galactose has limited its applications. In this study, we utilized Adaptive Laboratory Evolution (ALE) to optimize engineered KT2440 with heterologous expression of xylD encoding xylonate dehydratase from Caulobacter crescentus and galETKM encoding UDP-glucose 4-epimerase, galactose-1-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase from Escherichia coli K-12 MG1655. Poor starting strain growth (

Concerns over climate change have necessitated a rethinking of our transportation infrastructure. One possible alternative to carbon-polluting fossil fuels is biofuels produced by engineered microorganisms that use a renewable carbon source. Two biofuels, ethanol and biodiesel, have made inroads in displacing petroleum-based fuels, but their uptake has been limited by the amounts that can be used in conventional engines and by their cost. Advanced biofuels that mimic petroleum-based fuels are not limited by the amounts that can be used in existing transportation infrastructure but have had limited uptake due to costs. In this Review, we discuss engineering metabolic pathways to produce advanced biofuels, challenges with substrate and product toxicity with regard to host microorganisms and methods to engineer tolerance, and the use of functional genomics and machine learning approaches to produce advanced biofuels and prospects for reducing their costs. Biofuels produced by conversion of biomass by engineered microorganisms have the potential to replace fossil fuels and reduce carbon emissions. In this Review, Keasling and colleagues discuss engineering of metabolic pathways to produce advanced biofuels and approaches to reduce metabolite toxicity and cost and increase titre, rate and yield.

There is strong interest in the valorization of lignin to produce valuable products; however, its structural complexity has been a conversion bottleneck. Chemical pretreatment liberates lignin‐derived soluble fractions that may be upgraded by bioconversion. Cholinium ionic liquid pretreatment of sorghum produced soluble, aromatic‐rich fractions that were converted by Pseudomonas putida (P. putida), a promising host for aromatic bioconversion. Growth studies and mutational analysis demonstrated that P. putida growth on these fractions was dependent on aromatic monomers but unknown factors also contributed. Proteomic and metabolomic analyses indicated that these unknown factors were amino acids and residual ionic liquid; the oligomeric aromatic fraction derived from lignin was not converted. A cholinium catabolic pathway was identified, and the deletion of the pathway stopped the ability of P. putida to grow on cholinium ionic liquid. This work demonstrates that aromatic‐rich fractions obtained through pretreatment contain multiple substrates; conversion strategies should account for this complexity.

Background There 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. Methods To 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. Results The 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]. Conclusion The 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 material The online version of this article (10.1186/s12934-017-0819-1) contains supplementary material, which is available to authorized users.

p e te r Singer 's ar t ic le raises five i m p o r t a n t quest ions. The first is w h e t h e r " t h e t rad i t ional e th ic of the sanct i ty o f life" can be d e f e n d e d "in t e rms of pub l i c reasoning." The s e c o n d ques t ion , p r e s e n t bu t no t clear ly a c k n o w l e d g e d , is this: w h a t ough t to be the re la t ionsh ip b e t w e e n the way w e def ine i m p o r t a n t c o n c e p t s and the se t t ing of mora l values and pub l i c pol icy? The th i rd ques t ion is w h e t h e r the deba t e over the def in i t ion of dea th can serve as an i m p o r t a n t e x a m p l e of the e ros ion of the tradit ional ethic . The four th is w h e t h e r " secular" reasoning can offer a b e t t e r p e r s p e c t i v e on the t r ea tmen t of infants w i t h severe disabi l i t ies . The fifth quest ion is one I w a n t to pose : if Pe te r Singer 's w a y of d e t e r m i n i n g h o w w e ough t to value h u m a n life is w r o n g , is t he re a b e t t e r way?