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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 (<0.1 h-1 or none) was evolutionarily optimized to rates of up to 0.25 h-1 on xylose and 0.52 h-1 on galactose. Whole-genome sequencing, transcriptomic analysis, and growth screens revealed significant roles of kguT encoding a 2-ketogluconate operon repressor and 2-ketogluconate transporter, and gtsABCD encoding an ATP-binding cassette (ABC) sugar transporting system in xylose and galactose growth conditions, respectively. Finally, we expressed the heterologous indigoidine production pathway in the evolved and unevolved engineered strains and successfully produced 3.2 g/L and 2.2 g/L from 10 g/L of either xylose or galactose in the evolved strains whereas the unevolved strains did not produce any detectable product. Thus, the generated KT2440 strains have the potential for broad application as optimized platform chassis to develop efficient microorganism-based biomass-utilizing bioprocesses.

Although the use of ionic liquids (ILs) for the pretreatment of lignocellulosic biomass has been limited due to high costs, recent efforts to develop low-cost protic ILs show promise for achieving cost-effectiveness for biorefineries. However, an additional challenge remains in that ILs present in biomass hydrolysates are toxic to most microbial hosts, resulting in poor growth phenotypes. To address this issue, we applied an adaptive laboratory evolution (ALE) approach for tolerizingPseudomonas putidaKT2440, an industrially relevant bacterial host, to two low-cost ILs (triethanolammonium acetate [TEOH][OAc] and triethylammonium hydrogen sulfate [TEA][HS]). After continuous cultivations with gradually increased IL levels, we obtained evolved strains showing significant improvements in their growth performance under high concentrations of the ILs (maximum 4% [TEOH][OAc] and 8% [TEA][HS], in w/v) at which the wild-type strain cannot grow. Sequencing of evolved strains revealed multiple regions where mutations were associated with improved performance in minimal media conditions (relA,gacS,oprB/PP_1446,fleQ,tktA, anduvrY/PP_4100) and in IL-specific conditions (PP_5350, PP_4929/emrE,oprD, and PP_5324). We further validated the causality of the PP_5350 andemrEgenes for improved IL toleranceviareverse engineering and transcriptomic analysis. A common mutation in the PP_5350 gene, encoding a RpiR family transcriptional regulator, was shown to significantly upregulate the glyoxylate cycle for efficient acetate catabolism. In addition, it was suggested that theemrEgene encodes an efflux pump which can export [TEA][HS]. Finally, the cultivation of two of the best performing evolved strains with IL-treated biomass hydrolysates demonstrated their considerable potential to be used as platform strains. Taken as a whole, this work provides strains for utilization of IL-treated biomass and a mechanistic understanding that could be further leveraged to develop efficient microbial bio

Although the use of ionic liquids (ILs) for the pretreatment of lignocellulosic biomass has been limited due to high costs, recent efforts to develop low-cost protic ILs show promise for achieving cost-effectiveness for biorefineries. However, an additional challenge remains in that ILs present in biomass hydrolysates are toxic to most microbial hosts, resulting in poor growth phenotypes. To address this issue, we applied an adaptive laboratory evolution (ALE) approach for tolerizingPseudomonas putidaKT2440, an industrially relevant bacterial host, to two low-cost ILs (triethanolammonium acetate [TEOH][OAc] and triethylammonium hydrogen sulfate [TEA][HS]). After continuous cultivations with gradually increased IL levels, we obtained evolved strains showing significant improvements in their growth performance under high concentrations of the ILs (maximum 4% [TEOH][OAc] and 8% [TEA][HS], in w/v) at which the wild-type strain cannot grow. Sequencing of evolved strains revealed multiple regions where mutations were associated with improved performance in minimal media conditions (relA,gacS,oprB/PP_1446,fleQ,tktA, anduvrY/PP_4100) and in IL-specific conditions (PP_5350, PP_4929/emrE,oprD, and PP_5324). We further validated the causality of the PP_5350 andemrEgenes for improved IL toleranceviareverse engineering and transcriptomic analysis. A common mutation in the PP_5350 gene, encoding a RpiR family transcriptional regulator, was shown to significantly upregulate the glyoxylate cycle for efficient acetate catabolism. In addition, it was suggested that theemrEgene encodes an efflux pump which can export [TEA][HS]. Finally, the cultivation of two of the best performing evolved strains with IL-treated biomass hydrolysates demonstrated their considerable potential to be used as platform strains. Taken as a whole, this work provides strains for utilization of IL-treated biomass and a mechanistic understanding that could be further leveraged to develop efficient microbial bio

QCR

A corrosion-resistant coating which can be applied directly to a surface as a self-priming corrosion-resistant topcoat comprising an alkyd resin binder and a combination of pigments consisting essentially of an aluminum triphosphate, zinc salts of benzoic acids, and an alkaline earth metal phosphate such as zinc-barium phosphate. In addition, the coating contains up to about 40 parts by weight of a titanium dioxide pigment, up to about 2.0 parts by weight of an oil-soluble surface active or wetting agent and up to about 40 parts by weight of at least one organic solvent., Supersedes PAT-APPL-472 375-95, AD-D017 970.

The Freedom Flats deposit is a shallow-level, epithermal volcanic-hosted gold deposit. The "blind" deposit was discovered in 1986 during a pediment drilling programme and was intersected in a drill hole that contained 108 m grading 0.078 ounces/t Au. The orebody was mined-out in 1989, produced 1 280 000 t of ore grading 0.153 ounces/ton Au (195 000 ounces Au), and was the highest grade and most productive of the deposits mined in the Borealis district. The deposit formed at relatively shallow depths based on the types of alteration, trace element zoning and mineralogy, and the hydrothermal fluids were rich in silica, iron and sulphur. Pre-mineral structure was important in controlling mineralisation, but the specific structures are obscured by complex post-mineral faulting. Other important ore controls were stratigraphy and relative vertical and lateral position within the hydrothermal feeder., The Freedom Flats deposit is a shallow-level, epithermal volcanic-hosted gold deposit. The "blind" deposit was discovered in 1986 during a pediment drilling programme and was intersected in a drill hole that contained 108 m grading 0.078 ounces/t Au. The orebody was mined-out in 1989, produced 1 280 000 t of ore grading 0.153 ounces/ton Au (195 000 ounces Au), and was the highest grade and most productive of the deposits mined in the Borealis district. The deposit formed at relatively shallow depths based on the types of alteration, trace element zoning and mineralogy, and the hydrothermal fluids were rich in silica, iron and sulphur. Pre-mineral structure was important in controlling mineralisation, but the specific structures are obscured by complex post-mineral faulting. Other important ore controls were stratigraphy and relative vertical and lateral position within the hydrothermal feeder.