Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli.

The field of metabolic engineering has the potential to produce a wide variety of chemicals in both an inexpensive and ecologically-friendly manner. Heterologous expression of novel combinations of enzymes promises to provide new or improved synthetic routes towards a substantially increased diversity of small molecules. Recently, we constructed a synthetic pathway to produce d-glucaric acid, a molecule that has been deemed a "top-value added chemical" from biomass, starting from glucose. Limiting flux through the pathway is the second recombinant step, catalyzed by myo-inositol oxygenase (MIOX), whose activity is strongly influenced by the concentration of the myo-inositol substrate. To synthetically increase the effective concentration of myo-inositol, polypeptide scaffolds were built from protein-protein interaction domains to co-localize all three pathway enzymes in a designable complex as previously described (Dueber et al., 2009). Glucaric acid titer was found to be strongly affected by the number of scaffold interaction domains targeting upstream Ino1 enzymes, whereas the effect of increased numbers of MIOX-targeted domains was much less significant. We determined that the scaffolds directly increased the specific MIOX activity and that glucaric acid titers were strongly correlated with MIOX activity. Overall, we observed an approximately 5-fold improvement in product titers over the non-scaffolded control, and a 50% improvement over the previously reported highest titers. These results further validate the utility of these synthetic scaffolds as a tool for metabolic engineering.

Integrated bioprocessing for the pH-dependent production of 4-valerolactone from levulinate in Pseudomonas putida KT2440.

Enzymes are powerful biocatalysts capable of performing specific chemical transformations under mild conditions, yet as catalysts they remain subject to the laws of thermodynamics, namely, that they cannot catalyze chemical reactions beyond equilibrium. Here we report the phenomenon and application of using extracytosolic enzymes and medium conditions, such as pH, to catalyze metabolic pathways beyond their intracellular catalytic limitations. This methodology, termed "integrated bioprocessing" because it integrates intracellular and extracytosolic catalysis, was applied to a lactonization reaction in Pseudomonas putida for the economical and high-titer biosynthesis of 4-valerolactone from the inexpensive and renewable source levulinic acid. Mutant paraoxonase I (PON1) was expressed in P. putida, shown to export from the cytosol in Escherichia coli and P. putida using an N-terminal sequence, and demonstrated to catalyze the extracytosolic and pH-dependent lactonization of 4-hydroxyvalerate to 4-valerolactone. With this production system, the titer of 4-valerolactone was enhanced substantially in acidic medium using extracytosolically expressed lactonase versus an intracellular lactonase: from <0.2 g liter(-1) to 2.1 +/- 0.4 g liter(-1) at the shake flask scale. Based on these results, the production of 4-hydroxyvalerate and 4-valerolactone was examined in a 2-liter bioreactor, and titers of 27.1 g liter(-1) and 8.2 g liter(-1) for the two respective compounds were achieved. These results illustrate the utility of integrated bioprocessing as a strategy for enabling production from novel metabolic pathways and enhancing product titers.

Enzymatic assay of D-glucuronate using uronate dehydrogenase.

D-Glucuronate is a key metabolite in the process of detoxification of xenobiotics and in a recently constructed synthetic pathway to produce D-glucaric acid, a "top value-added chemical" from biomass. A simple and specific assay of D-glucuronate would be useful for studying these processes, but existing assays are either time-consuming or nonspecific. Using uronate dehydrogenase cloned from Agrobacterium tumefaciens, we developed an assay for D-glucuronate with a detection limit of 5 microM. This method was shown to be more suitable for a system with many interfering compounds than previous methods and was also applied to assays for myo-inositol oxygenase activity.

Engineering alternative butanol production platforms in heterologous bacteria.

Alternative microbial hosts have been engineered as biocatalysts for butanol biosynthesis. The butanol synthetic pathway of Clostridium acetobutylicum was first re-constructed in Escherichia coli to establish a baseline for comparison to other hosts. Whereas polycistronic expression of the pathway genes resulted in the production of 34 mg/L butanol, individual expression of pathway genes elevated titers to 200 mg/L. Improved titers were achieved by co-expression of Saccharomyces cerevisiae formate dehydrogenase while overexpression of E. coli glyceraldehyde 3-phosphate dehydrogenase to elevate glycolytic flux improved titers to 580 mg/L. Pseudomonas putida and Bacillus subtilis were also explored as alternative production hosts. Polycistronic expression of butanol biosynthetic genes yielded butanol titers of 120 and 24 mg/L from P. putida and B. subtilis, respectively. Production in the obligate aerobe P. putida was dependent upon expression of bcd-etfAB. These results demonstrate the potential of engineering butanol biosynthesis in a variety of heterologous microorganisms, including those cultivated aerobically.

Metabolic engineering of Escherichia coli for enhanced production of (R)- and (S)-3-hydroxybutyrate.

Synthetic metabolic pathways have been constructed for the production of enantiopure (R)- and (S)-3-hydroxybutyrate (3HB) from glucose in recombinant Escherichia coli strains. To promote maximal activity, we profiled three thiolase homologs (BktB, Thl, and PhaA) and two coenzyme A (CoA) removal mechanisms (Ptb-Buk and TesB). Two enantioselective 3HB-CoA dehydrogenases, PhaB, producing the (R)-enantiomer, and Hbd, producing the (S)-enantiomer, were utilized to control the 3HB chirality across two E. coli backgrounds, BL21Star(DE3) and MG1655(DE3), representing E. coli B- and K-12-derived strains, respectively. MG1655(DE3) was found to be superior for the production of each 3HB stereoisomer, although the recombinant enzymes exhibited lower in vitro specific activities than BL21Star(DE3). Hbd in vitro activity was significantly higher than PhaB activity in both strains. The engineered strains achieved titers of enantiopure (R)-3HB and (S)-3HB as high as 2.92 g liter(-1) and 2.08 g liter(-1), respectively, in shake flask cultures within 2 days. The NADPH/NADP+ ratio was found to be two- to three-fold higher than the NADH/NAD+ ratio under the culture conditions examined, presumably affecting in vivo activities of PhaB and Hbd and resulting in greater production of (R)-3HB than (S)-3HB. To the best of our knowledge, this study reports the highest (S)-3HB titer achieved in shake flask E. coli cultures to date.

Synthetic metabolism: engineering biology at the protein and pathway scales.

Biocatalysis has become a powerful tool for the synthesis of high-value compounds, particularly so in the case of highly functionalized and/or stereoactive products. Nature has supplied thousands of enzymes and assembled them into numerous metabolic pathways. Although these native pathways can be use to produce natural bioproducts, there are many valuable and useful compounds that have no known natural biochemical route. Consequently, there is a need for both unnatural metabolic pathways and novel enzymatic activities upon which these pathways can be built. Here, we review the theoretical and experimental strategies for engineering synthetic metabolic pathways at the protein and pathway scales, and highlight the challenges that this subfield of synthetic biology currently faces.

Production of glucaric acid from a synthetic pathway in recombinant Escherichia coli.

A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a "top value-added chemical" from biomass.

High-titer production of monomeric hydroxyvalerates from levulinic acid in Pseudomonas putida.

Hydroxyacids represent an important class of compounds that see application in the production of polyesters, biodegradable plastics and antibiotics, and that serve as useful chiral synthetic building blocks for other fine chemicals and pharmaceuticals. An economical, high-titer method for the production of 4-hydroxyvalerate (4HV) and 3-hydroxyvalerate (3HV) from the inexpensive and renewable carbon source levulinic acid was developed. These hydroxyvalerates were produced by periodically feeding levulinate to Pseudomonas putida KT2440 expressing a recombinant thioesterase II (tesB) gene from Escherichia coli K12. The titer of 4HV in shake flask culture reached 13.9+/-1.2 g L(-1) from P. putida tesB(+) cultured at 32 degrees C in LB medium periodically supplemented with glucose and levulinate. The highest 3HV titer obtained was 5.3+/-0.1 g L(-1) in M9 minimal medium supplemented with glucose and levulinate.

De novo biosynthetic pathways: rational design of microbial chemical factories.

Increasing interest in the production of organic compounds from non-petroleum-derived feedstocks, especially biomass, is a significant driver for the construction of new recombinant microorganisms for this purpose. As a discipline, Metabolic Engineering has provided a framework for the development of such systems. Efforts have traditionally been focused, first, on the optimization of natural producers, later progressing towards re-construction of natural pathways in heterologous hosts. To maximize the potential of microbes for biosynthetic purposes, new tools and methodologies within Metabolic Engineering are needed for the proposition and construction of de novo designed pathways. This review will focus on recent advances towards the design and assembly of biosynthetic pathways, and provide a Synthetic Biology perspective for the construction of microbial chemical factories.

Identification and characterization of IS1 transposition in plasmid amplification mutants of E. coli clones producing DNA vaccines.

Merck Research Laboratories has developed a highly productive Escherichia coli fermentation process to produce plasmid DNA for use as vaccines. The process consists of a fed-batch fermentation in a chemically defined medium. Initiation of the feed stream precedes a growth-limited phase in which plasmid DNA is amplified. The fermentation is only maximally productive for a small fraction of E. coli transformants designated as high-producers, while the predominant low-producer population does not amplify plasmid DNA. In experiments undertaken to probe this phenomenon, transposition of the 768-bp E. coli insertion sequence IS1 into an HIV DNA vaccine vector was observed in several low-producer clones. IS1 was found to insert in or near the neomycin resistance gene in nearly a dozen unique sites from within a single population of plasmid molecules. The fraction of IS1-containing plasmids within several clones was determined by quantitative polymerase chain reaction and was found to increase with increasing cultivation time in the chemically defined medium. Because transposition into an antibiotic-resistance gene is unlikely to affect plasmid amplification, the genomes of high- and low-producers of three different HIV DNA vaccine vectors were subsequently profiled by restriction fragment length polymorphism analysis. In all three cases, IS1 insertional mutations were found in the genomes of the predominant low-producers, while the genomes of the high-producers were indistinguishable from untransformed cells. The insertions reside on similarly sized fragments for two of the low-producer clones, and the fragment size is smaller for the third clone. The third clone also produces much less plasmid DNA than a typical low-producer. The results suggest the presence of an IS1 insertional mutation that affects plasmid replication and amplification, possibly in a position-dependent manner.

Metabolic engineering of a novel propionate-independent pathway for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in recombinant Salmonella enterica serovar typhimurium.

A pathway was metabolically engineered to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable thermoplastic with proven commercial applications, from a single, unrelated carbon source. An expression system was developed in which a prpC strain of Salmonella enterica serovar Typhimurium, with a mutation in the ability to metabolize propionyl coenzyme A (propionyl-CoA), served as the host for a plasmid harboring the Acinetobacter polyhydroxyalkanoate synthesis operon (phaBCA) and a second plasmid with the Escherichia coli sbm and ygfG genes under an independent promoter. The sbm and ygfG genes encode a novel (2R)-methylmalonyl-CoA mutase and a (2R)-methylmalonyl-CoA decarboxylase, respectively, which convert succinyl-CoA, derived from the tricarboxylic acid cycle, to propionyl-CoA, an essential precursor of 3-hydroxyvalerate (HV). The S. enterica system accumulated PHBV with significant HV incorporation when the organism was grown aerobically with glycerol as the sole carbon source. It was possible to vary the average HV fraction in the copolymer by adjusting the arabinose or cyanocobalamin (precursor of coenzyme B12) concentration in the medium.