Engineered metabolic pathways constructed from enzymes heterologous to the production host often suffer from flux imbalances, as they typically lack the regulatory mechanisms characteristic of natural metabolism. In an attempt to increase the effective concentration of each component of a pathway of interest, we built synthetic protein scaffolds that spatially recruit metabolic enzymes in a designable manner. Scaffolds bearing interaction domains from metazoan signaling proteins specifically accrue pathway enzymes tagged with their cognate peptide ligands. The natural modularity of these domains enabled us to optimize the stoichiometry of three mevalonate biosynthetic enzymes recruited to a synthetic complex and thereby achieve 77-fold improvement in product titer with low enzyme expression and reduced metabolic load. One of the same scaffolds was used to triple the yield of glucaric acid, despite high titers (0.5 g/l) without the synthetic complex. These strategies should prove generalizeable to other metabolic pathways and programmable for fine-tuning pathway flux.

Metabolic engineering of microorganisms to produce desirable products on an industrial scale can result in unbalanced cellular metabolic networks that reduce productivity and yield. Metabolic fluxes can be rebalanced using dynamic pathway regulation, but few broadly applicable tools are available to achieve this. We present a pathway-independent genetic control module that can be used to dynamically regulate the expression of target genes. We applied our module to identify the optimal point to redirect glycolytic flux into heterologous engineered pathways in Escherichia coli, resulting in 5.5-fold increased titres of myo-inositol and titers of glucaric acid that improved from unmeasurable quantities to >0.8 g/L. Scaled-up production in benchtop bioreactors resulted in almost 10-fold and 5-fold increases in titers of myo-inositol and glucaric acid. We also used our module to control flux into aromatic amino acid biosynthesis to increase titers of shikimate in E. coli from unmeasurable quantities to >100 mg/L.

Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expression of a recombinant carboxylic acid reductase in the RARE strain and addition of benzoate during growth, benzaldehyde remained in the culture after 24 h, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expression of a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.

Genome engineering methods in E. coli allow for easy to perform manipulations of the chromosome in vivo with the assistance of the λ-Red recombinase system. These methods generally rely on the insertion of an antibiotic resistance cassette followed by removal of the same cassette, resulting in a two-step procedure for genomic manipulations. Here we describe a method and plasmid system that can edit the genome of E. coli without chromosomal markers. This system, known as Scarless Cas9 Assisted Recombineering (no-SCAR), uses λ-Red to facilitate genomic integration of donor DNA and double stranded DNA cleavage by Cas9 to counterselect against wild-type cells. We show that point mutations, gene deletions, and short sequence insertions were efficiently performed in several genomic loci in a single-step with regards to the chromosome and did not leave behind scar sites. The single-guide RNA encoding plasmid can be easily cured due to its temperature sensitive origin of replication, allowing for iterative chromosomal manipulations of the same strain, as is often required in metabolic engineering. In addition, we demonstrate the ability to efficiently cure the second plasmid in the system by targeting with Cas9, leaving the cells plasmid-free.

A common strategy of metabolic engineering is to increase the endogenous supply of precursor metabolites to improve pathway productivity. The ability to further enhance heterologous production of a desired compound may be limited by the inherent capacity of the imported pathway to accommodate high precursor supply. Here, we present engineered diterpenoid biosynthesis as a case where insufficient downstream pathway capacity limits highlevel levopimaradiene production in Escherichia coli. To increase levopimaradiene synthesis, we amplified the flux toward isopentenyl diphosphate and dimethylallyl diphosphate precursors and reprogrammed the rate-limiting downstream pathway by generating combinatorial mutations in geranylgeranyl diphosphate synthase and levopimaradiene synthase. The mutant library contained pathway variants that not only increased diterpenoid production but also tuned the selectivity toward levopimaradiene. The most productive pathway, combining precursor flux amplification and mutant synthases, conferred approximately 2,600-fold increase in levopimaradiene levels. A maximum titer of approximately 700 mg∕L was subsequently obtained by cultivation in a benchscale bioreactor. The present study highlights the importance of engineering proteins along with pathways as a key strategy in achieving microbial biosynthesis and overproduction of pharmaceutical and chemical products.GGPP synthase | levopimaradiene synthase | metabolic engineering | Escherichia coli | molecular reprogramming M etabolic engineering is the enabling technology for the manipulation of organisms to synthesize high-value compounds of both natural and heterologous origin (1-4). In the case of heterologous production, well-characterized microorganisms are used as production hosts because targeted optimization can be performed using widely available genetic tools and synthetic biology frameworks (5, 6). One important application of engineered microbial systems is geared toward the synthesis of terpenoid natural products (7-9). Terpenoids represent one of the largest classes of secondary metabolites that includes pharmaceuticals, cosmetics, and potential biofuels candidates (8,(10)(11)(12). Metabolic engineering approaches to produce terpenoids in microbial systems such as Escherichia coli and yeast have commonly focused on increasing the precursor flux into the heterologous terpenoid pathway by rerouting endogenous isoprenoid metabolism (13-15). These engineering strategies have relied heavily on changing the enzyme concentrations in the product pathway.Many properties of a metabolic pathway, however, are not limited solely by the enzyme concentration, as is particularly true for the terpenoid pathway. In nature, terpenoid biosynthesis is regulated at multiple metabolic branch points to create large structural and functional diversity (16-18). In the major metabolic branch point in terpenoid biosynthesis, the prenyl transferases and terpenoid synthases catalyze the formation of a wide range of structurally diverse acyclic and cyclic terp...

23Alternative microbial hosts have been engineered as biocatalysts for butanol biosynthesis. 24The butanol synthetic pathway of Clostridium acetobutylicum was first re-constructed in 25

1The field of metabolic engineering has the potential to produce a wide variety of 2 chemicals in both an inexpensive and ecologically-friendly manner. Heterologous 3 expression of novel combinations of enzymes promises to provide new or improved 4 synthetic routes towards a substantially increased diversity of small molecules. Recently, 5we constructed a synthetic pathway to produce D-glucaric acid, a molecule that has been 6 deemed a "top-value added chemical" from biomass, starting from glucose. Limiting 7 flux through the pathway is the second recombinant step, catalyzed by myo-inositol 8 oxygenase (MIOX), whose activity is strongly influenced by the concentration of the 9 myo-inositol substrate. To synthetically increase the effective concentration of myo-10 inositol, polypeptide scaffolds were built from protein-protein interaction domains to co-11 localize all three pathway enzymes in a designable complex as previously described 12 (Dueber et al., 2009). Glucaric acid titer was found to be strongly affected by the number 13 of scaffold interaction domains targeting upstream Ino1 enzymes, whereas the effect of 14 increased numbers of MIOX-targeted domains was much less significant. We 15 determined that the scaffolds directly increased the specific MIOX activity and that 16 glucaric acid titers were strongly correlated with MIOX activity. Overall, we observed 17 an approximately 5-fold improvement in product titers over the non-scaffolded control, 18 and a 50% improvement over the previously reported highest titers. These results further 19 validate the utility of these synthetic scaffolds as a tool for metabolic engineering. 20 21

Microbial production of value-added chemicals from biomass is a sustainable alternative to chemical synthesis. To improve product titer, yield, and selectivity, the pathways engineered into microbes must be optimized. One strategy for optimization is dynamic pathway regulation, which modulates expression of pathway-relevant enzymes over the course of fermentation. Metabolic engineers have used dynamic regulation to redirect endogenous flux toward product formation, balance the production and consumption rates of key intermediates, and suppress production of toxic intermediates until later in the fermentation. Most cases, however, have utilized a single strategy for dynamically regulating pathway fluxes. Here we layer two orthogonal, autonomous, and tunable dynamic regulation strategies to independently modulate expression of two different enzymes to improve production of D-glucaric acid from a heterologous pathway. The first strategy uses a previously described pathway-independent quorum sensing system to dynamically knock down glycolytic flux and redirect carbon into production of glucaric acid, thereby switching cells from "growth" to "production" mode. The second strategy, developed in this work, uses a biosensor for -inositol (MI), an intermediate in the glucaric acid production pathway, to induce expression of a downstream enzyme upon sufficient buildup of MI. The latter, pathway-dependent strategy leads to a 2.5-fold increase in titer when used in isolation and a fourfold increase when added to a strain employing the former, pathway-independent regulatory system. The dual-regulation strain produces nearly 2 g/L glucaric acid, representing the highest glucaric acid titer reported to date in K-12 strains.