Growth-coupled bioconversion of levulinic acid to butanone.

Common strategies for conversion of lignocellulosic biomass to chemical products center on deconstructing biomass polymers into fermentable sugars. Here, we demonstrate an alternative strategy, a growth-coupled, high-yield bioconversion, by feeding cells a non-sugar substrate, by-passing central metabolism, and linking a key metabolic step to generation of acetyl-CoA that is required for biomass and energy generation. Specifically, we converted levulinic acid (LA), an established degradation product of lignocellulosic biomass, to butanone (a.k.a. methyl-ethyl ketone - MEK), a widely used industrial solvent. Our strategy combines a catabolic pathway from Pseudomonas putida that enables conversion of LA to 3-ketovaleryl-CoA, a CoA transferase that generates 3-ketovalerate and acetyl-CoA, and a decarboxylase that generates 2-butanone. By removing the ability of E. coli to consume LA and supplying excess acetate as a carbon source, we built a strain of E. coli that could convert LA to butanone at high yields, but at the cost of significant acetate consumption. Using flux balance analysis as a guide, we built a strain of E. coli that linked acetate assimilation to production of butanone. This strain was capable of complete bioconversion of LA to butanone with a reduced acetate requirement and increased specific productivity. To demonstrate the bioconversion on real world feedstocks, we produced LA from furfuryl alcohol, a compound readily obtained from biomass. These LA feedstocks were found to contain inhibitors that prevented cell growth and bioconversion of LA to butanone. We used a combination of column chromatography and activated carbon to remove the toxic compounds from the feedstock, resulting in LA that could be completely converted to butanone. This work motivates continued collaboration between chemical and biological catalysis researchers to explore alternative conversion pathways and the technical hurdles that prevent their rapid deployment.

Genetic tools for reliable gene expression and recombineering in Pseudomonas putida.

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.

Genome sequence and analysis of Escherichia coli production strain LS5218.

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.

A metabolic pathway for catabolizing levulinic acid in bacteria.

Microorganisms can catabolize a wide range of organic compounds and therefore have the potential to perform many industrially relevant bioconversions. One barrier to realizing the potential of biorefining strategies lies in our incomplete knowledge of metabolic pathways, including those that can be used to assimilate naturally abundant or easily generated feedstocks. For instance, levulinic acid (LA) is a carbon source that is readily obtainable as a dehydration product of lignocellulosic biomass and can serve as the sole carbon source for some bacteria. Yet, the genetics and structure of LA catabolism have remained unknown. Here, we report the identification and characterization of a seven-gene operon that enables LA catabolism in Pseudomonas putida KT2440. When the pathway was reconstituted with purified proteins, we observed the formation of four acyl-CoA intermediates, including a unique 4-phosphovaleryl-CoA and the previously observed 3-hydroxyvaleryl-CoA product. Using adaptive evolution, we obtained a mutant of Escherichia coli LS5218 with functional deletions of fadE and atoC that was capable of robust growth on LA when it expressed the five enzymes from the P. putida operon. This discovery will enable more efficient use of biomass hydrolysates and metabolic engineering to develop bioconversions using LA as a feedstock.

Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae.

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 YSFar 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.

Solvent-enabled nonenyzmatic sugar production from biomass for chemical and biological upgrading.

We recently reported a nonenzymatic biomass deconstruction process for producing carbohydrates using homogeneous mixtures of γ-valerolactone (GVL) and water as a solvent. A key step in this process is the separation of the GVL from the aqueous phase, enabling GVL recycling and the production of a concentrated aqueous carbohydrate solution. In this study, we demonstrate that phenolic solvents-sec-butylphenol, nonylphenol, and lignin-derived propyl guaiacol-are effective at separating GVL from the aqueous phase using only small amounts of solvent (0.5 g per g of the original water, GVL, and sugar hydrolysate). Furthermore, using nonylphenol, we produced a hydrolysate that supported robust growth and high yields of ethanol (0.49 g EtOH per g glucose) at an industrially relevant concentration (50.8 g L(-1) EtOH). These results suggest that using phenolic solvents could be an interesting solution for separating and/or detoxifying aqueous carbohydrate solutions produced using GVL-based biomass deconstruction processes.

Nonenzymatic sugar production from biomass using biomass-derived γ-valerolactone.

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.

Sequentially pulsed fluid delivery to establish soluble gradients within a scalable microfluidic chamber array.

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.