Widespread production of biomass-derived fuels and chemicals will require cost-effective processes for breaking down cellulose and hemicellulose into their constituent sugars. Here, we report laboratory-scale production of soluble carbohydrates from corn stover, hardwood, and softwood at high yields (70 to 90%) in a solvent mixture of biomass-derived γ-valerolactone (GVL), water, and dilute acid (0.05 weight percent H2SO4). GVL promotes thermocatalytic saccharification through complete solubilization of the biomass, including the lignin fraction. The carbohydrates can be recovered and concentrated (up to 127 grams per liter) by extraction from GVL into an aqueous phase by addition of NaCl or liquid CO2. This strategy is well suited for catalytic upgrading to furans or fermentative upgrading to ethanol at high titers and near theoretical yield. We estimate through preliminary techno-economic modeling that the overall process could be cost-competitive for ethanol production, with biomass pretreatment followed by enzymatic hydrolysis.

Pseudomonas putida is a promising bacterial host for producing natural products, such as polyketides and nonribosomal peptides. In these types of projects, researchers need a genetic toolbox consisting of plasmids, characterized promoters, and techniques for rapidly editing the genome. Past reports described constitutive promoter libraries, a suite of broad host range plasmids that replicate in P. putida, and genome-editing methods. To augment those tools, we have characterized a set of inducible promoters and discovered that IPTG-inducible promoter systems have poor dynamic range due to overexpression of the LacI repressor. By replacing the promoter driving lacI expression with weaker promoters, we increased the fold induction of an IPTG-inducible promoter in P. putida KT2440 to 80-fold. Upon discovering that gene expression from a plasmid was unpredictable when using a high-copy mutant of the BBR1 origin, we determined the copy numbers of several broad host range origins and found that plasmid copy numbers are significantly higher in P. putida KT2440 than in the synthetic biology workhorse, Escherichia coli. Lastly, we developed a λRed/Cas9 recombineering method in P. putida KT2440 using the genetic tools that we characterized. This method enabled the creation of scarless mutations without the need for performing classic two-step integration and marker removal protocols that depend on selection and counterselection genes. With the method, we generated four scarless deletions, three of which we were unable to create using a previously established genome-editing technique.

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Enzyme fusions have been widely used as a tool in metabolic engineering to increase pathway efficiency by reducing substrate loss and accumulation of toxic intermediates. Alternatively, enzymes can be colocalized through attachment to a synthetic scaffold via noncovalent interactions. Here we describe the use of affibodies for enzyme tagging and scaffolding. The scaffolding is based on the recognition of affibodies to their anti-idiotypic partners in vivo, and was first employed for colocalization of farnesyl diphosphate synthase and farnesene synthase in S. cerevisiae. Different parameters were modulated to improve the system, and the enzyme:scaffold ratio was most critical for its functionality. Ultimately, the yield of farnesene on glucose Y could be improved by 135% in fed-batch cultivations using a 2-site affibody scaffold. The scaffolding strategy was then extended to a three-enzyme polyhydroxybutyrate (PHB) pathway, heterologously expressed in E. coli. Within a narrow range of enzyme and scaffold induction, the affibody tagging and scaffolding increased PHB production 7-fold. This work demonstrates how the versatile affibody can be used for metabolic engineering purposes.

We recently reported an onenzymatic biomass deconstruction process for producing carbohydrates using homogeneous mixtures of g-valerolactone (GVL) and water as as olvent. Ak ey step in this process is the separation of the GVL from the aqueous phase, enabling GVL recycling and the production of ac oncentrated aqueous carbohydrate solution.I nt his study, we demonstrate that phenolic solvents-sec-butylphenol, nonylphenol, and lignin-derived propylg uaiacol-are effective at separating GVL from the aqueous phase using only small amountso fs olvent (0.5 gp er go ft he originalw ater,G VL, and sugar hydrolysate). Furthermore, using nonylphenol, we produced ah ydrolysate that supported robustg rowth and high yields of ethanol (0.49 gE tOH per gg lucose) at an industrially relevant concentration (50.8 gL À1 EtOH). These resultss uggest that using phenolic solvents could be an interesting solution for separating and/or detoxifying aqueous carbohydrate solutions produced using GVL-based biomass deconstruction processes.Lignocellulosic biomass is emerging as ap otentialr enewable feedstock to replace crude oil as as ource of fuels and commodity chemicals. For this reason,t argeted upgrading routes are being sought to convert biomass to these valuablep roducts. In this context, soluble carbohydrates are an attractive intermediate for biomass upgrading. By weight, structuralp olysaccharides typically represent between 60 and 80 wt %o f lignocellulosic biomass.I na ddition, many chemical [1][2][3][4] and biological [5][6][7][8] processes exist for upgrading carbohydrates to fuels and chemicals.Different strategies exist for deconstructing biomass hemicellulose (a polymer of C 5 sugars; mostly xylose) and cellulose (a polymer of cellobiose, ad imer of glucose) to their soluble counterparts. Concentrated mineral acids such as sulfuric or hydrochloric acid have been used to hydrolyze hemicellulose and cellulose to soluble oligomers almost quantitatively. [9][10][11][12][13] Recently,atwo-stage process consisting of at hermochemical or pretreatment stage followed by enzymatic hydrolysis has been one of the most prominently researched biomass depolymerization methods. [14][15][16][17] Both of these methods can achieve soluble carbohydrate yields upwards of 90 %a nd produce concentrated solutionso fc arbohydrates (> 100 gL À1 ). [10,13,18] Ionic liquids are also attractive solvents for cellulose dissolution and soluble carbohydrate production. [19,20] Although these processes can be used to produce sugars from biomass,t he cost of producing and/or recovering the enzyme, mineral acid, or solvent still represents as ignificant portion of the final process. [21][22][23] For this reason,a lternate systems using significantly less catalyst have also been explored, including dilute acid hydrolysis [24,25] or hydrothermalb iomass depolymerization. [13,[26][27][28] However,a tm ost temperatures (below 510-570 K) and low acid contents( >3%), the rate of sugar degradation and dehydration to furans or other degradation products is of the sam...

No abstract

Escherichia coli strain LS5218 is a useful host for the production of fatty acid derived products, but the genetics underlying this utility have not been fully investigated. Here, we report the genome sequence of LS5218 and a list of large mutations and single nucleotide permutations (SNPs) relative to E. coli K-12 strain MG1655. We discuss how genetic differences may affect the physiological differences between LS5218 and MG1655. We find that LS5218 is more closely related to E. coli strain NCM3722 and suspect that small genetic differences between K-12 derived strains may have a significant impact on metabolic engineering efforts.

This work presents a microfluidic chamber array that generates soluble gradients using sequentially pulsed fluid delivery (SPFD). SPFD produces stable gradients by delivering flow pulses to either side of a chamber. The pulses on each side contain different signal concentrations, and they alternate in sequence, providing the driving force to establish a gradient via diffusion. The device, herein, is significant because it demonstrates the potential to simultaneously meet four important needs that can accelerate and enhance the study of cellular responses to signal gradients. These needs are (i) a scalable chamber array, (ii) low complexity fabrication, (iii) a non-shearing microenvironment, and (iv) gradients with low (near zero) background concentrations. The ability to meet all four needs distinguishes the SPFD device from flow-based and diffusion-based designs, which can only achieve a subset of such needs. Gradients are characterized using fluorescence measurements, which reveal the ability to change the curvature of concentration profiles by simple adjustments to pulsing sequence and flow rate. Preliminary experiments with MDA-MB-231 cancer cells demonstrate cell viability and indicate migrational and morphological responses to a fetal bovine serum gradient. Improved and expanded versions of this technology could form the basis of high-throughput screening tools to study cell migration, development, and cancer.