Rerouting carbon flux for optimized biosynthesis of mesaconate in Escherichia coli.

Mesaconate, a branched unsaturated dicarboxylic acid, has drawn great interest because of its versatile applications. In this work, we optimized the fermentation efficiency of Escherichia coli to produce mesaconate from glucose. We first drove the carbon flux to 2-ketoglutarate by overexpressing genes involved in TCA precursor pathway and anaplerotic pathways. Then, to increase the pool of phosphoenolpyruvate (PEP), an upstream precursor for 2-ketoglutarate, the phosphotransferase system (PTS) of E. coli was inactivated by deleting glucose PTS permease and the import of glucose was altered by overexpressing galactose/H+ symporter GalP. Further, production optimization was achieved by deleting a class I fumarase (FumA) to block the hydration of mesaconate. Finally, we overexpressed PEP synthase (PpsA) to increase the availability of phosphoenolpyruvate and accelerate the production of mesaconate. These genetic modifications led to mesaconate production with a titer of 23.1 g L-1 and a yield of 0.46 g g-1 glucose (64% of the theoretical maximum). This work demonstrates the possibility of engineering a highly efficient bacteria strain that converts glucose into mesaconate with promising titer, rate, and yield.

Engineering of a Highly Efficient Escherichia coli Strain for Mevalonate Fermentation through Chromosomal Integration.

Chromosomal integration of heterologous metabolic pathways is optimal for industrially relevant fermentation, as plasmid-based fermentation causes extra metabolic burden and genetic instabilities. In this work, chromosomal integration was adapted for the production of mevalonate, which can be readily converted into ß-methyl-δ-valerolactone, a monomer for the production of mechanically tunable polyesters. The mevalonate pathway, driven by a constitutive promoter, was integrated into the chromosome of Escherichia coli to replace the native fermentation gene adhE or ldhA The engineered strains (CMEV-1 and CMEV-2) did not require inducer or antibiotic and showed slightly higher maximal productivities (0.38 to ∼0.43 g/liter/h) and yields (67.8 to ∼71.4% of the maximum theoretical yield) than those of the plasmid-based fermentation. Since the glycolysis pathway is the first module for mevalonate synthesis, atpFH deletion was employed to improve the glycolytic rate and the production rate of mevalonate. Shake flask fermentation results showed that the deletion of atpFH in CMEV-1 resulted in a 2.1-fold increase in the maximum productivity. Furthermore, enhancement of the downstream pathway by integrating two copies of the mevalonate pathway genes into the chromosome further improved the mevalonate yield. Finally, our fed-batch fermentation showed that, with deletion of the atpFH and sucA genes and integration of two copies of the mevalonate pathway genes into the chromosome, the engineered strain CMEV-7 exhibited both high maximal productivity (∼1.01 g/liter/h) and high yield (86.1% of the maximum theoretical yield, 30 g/liter mevalonate from 61 g/liter glucose after 48 h in a shake flask). IMPORTANCE: Metabolic engineering has succeeded in producing various chemicals. However, few of these chemicals are commercially competitive with the conventional petroleum-derived materials. In this work, chromosomal integration of the heterologous pathway and subsequent optimization strategies ensure stable and efficient (i.e., high-titer, high-yield, and high-productivity) production of mevalonate, which demonstrates the potential for scale-up fermentation. Among the optimization strategies, we demonstrated that enhancement of the glycolytic flux significantly improved the productivity. This result provides an example of how to tune the carbon flux for the optimal production of exogenous chemicals.

Engineering nonphosphorylative metabolism to synthesize mesaconate from lignocellulosic sugars in Escherichia coli.

Dicarboxylic acids are attractive biosynthetic targets due to their broad applications and their challenging manufacturing process from fossil fuel feedstock. Mesaconate is a branched, unsaturated dicarboxylic acid that can be used as a co-monomer to produce hydrogels and fire-retardant materials. In this study, we engineered nonphosphorylative metabolism to produce mesaconate from d-xylose and l-arabinose. This nonphosphorylative metabolism is orthogonal to the intrinsic pentose metabolism in Escherichia coli and has fewer enzymatic steps and a higher theoretical yield to TCA cycle intermediates than the pentose phosphate pathway. Here mesaconate production was enabled from the d-xylose pathway and the l-arabinose pathway. To enhance the transportation of d-xylose and l-arabinose, pentose transporters were examined. We identified the pentose/proton symporter, AraE, as the most effective transporter for both d-xylose and l-arabinose in mesaconate production process. Further production optimization was achieved by operon screening and metabolic engineering. These efforts led to the engineered strains that produced 12.5g/l and 13.2g/l mesaconate after 48h from 20g/l of d-xylose and l-arabinose, respectively. Finally, the engineered strain overexpressing both l-arabinose and d-xylose operons produced 14.7g/l mesaconate from a 1:1 d-xylose and l-arabinose mixture with a yield of 85% of the theoretical maximum. (0.87g/g). This work demonstrates an effective system that converts pentoses into a value-added chemical, mesaconate, with promising titer, rate, and yield.

Diffusion of Ellipsoids in Bacterial Suspensions.

Active fluids such as swarming bacteria and motile colloids exhibit exotic properties different from conventional equilibrium materials. As a peculiar example, a spherical tracer immersed inside active fluids shows an enhanced translational diffusion, orders of magnitude stronger than its intrinsic Brownian motion. Here, rather than spherical tracers, we investigate the diffusion of isolated ellipsoids in a quasi-two-dimensional bacterial bath. Our study shows a nonlinear enhancement of both translational and rotational diffusions of ellipsoids. More importantly, we uncover an anomalous coupling between particles' translation and rotation that is strictly prohibited in Brownian diffusion. The coupling reveals a counterintuitive anisotropic particle diffusion, where an ellipsoid diffuses fastest along its minor axis in its body frame. Combining experiments with theoretical modeling, we show that such an anomalous diffusive behavior arises from the generic straining flow of swimming bacteria. Our work illustrates an unexpected feature of active fluids and deepens our understanding of transport processes in microbiological systems.

Engineering nonphosphorylative metabolism to generate lignocellulose-derived products.

Conversion of lignocellulosic biomass into value-added products provides important environmental and economic benefits. Here we report the engineering of an unconventional metabolism for the production of tricarboxylic acid (TCA)-cycle derivatives from D-xylose, L-arabinose and D-galacturonate. We designed a growth-based selection platform to identify several gene clusters functional in Escherichia coli that can perform this nonphosphorylative assimilation of sugars into the TCA cycle in less than six steps. To demonstrate the application of this new metabolic platform, we built artificial biosynthetic pathways to 1,4-butanediol (BDO) with a theoretical molar yield of 100%. By screening and engineering downstream pathway enzymes, 2-ketoacid decarboxylases and alcohol dehydrogenases, we constructed E. coli strains capable of producing BDO from D-xylose, L-arabinose and D-galacturonate. The titers, rates and yields were higher than those previously reported using conventional pathways. This work demonstrates the potential of nonphosphorylative metabolism for biomanufacturing with improved biosynthetic efficiencies.

Engineered biosynthesis of medium-chain esters in Escherichia coli.

Medium-chain esters such as isobutyl acetate (IBAc) and isoamyl acetate (IAAc) are high-volume solvents, flavors and fragrances. In this work, we engineered synthetic metabolic pathways in Escherichia coli for the total biosynthesis of IBAc and IAAc directly from glucose. Our pathways harnessed the power of natural amino acid biosynthesis. In particular, the native valine and leucine pathways in E. coli were utilized to supply the precursors. Then alcohol acyltransferases from various organisms were investigated on their capability to catalyze esterification reactions. It was discovered that ATF1 from Saccharomyces cerevisiae was the best enzyme for the formation of both IBAc and IAAc in E. coli. In vitro biochemical characterization of ATF1 confirmed the fermentation results and provided rational guidance for future enzyme engineering. We also performed strain improvement by removing byproduct pathways (Δldh, ΔpoxB, Δpta) and increased the production of both target chemicals. Then the best IBAc producing strain was used for scale-up fermentation in a 1.3-L benchtop bioreactor. 36g/L of IBAc was produced after 72h fermentation. This work demonstrates the feasibility of total biosynthesis of medium-chain esters as renewable chemicals.