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.

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.

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Chromosomal integration of heterologous metabolic pathways is optimal for industrially relevant fermentation, as plasmidbased 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 ␤-methyl-␦-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 ϳ0.43 g/liter/h) and yields (67.8 to ϳ71.4% 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 fedbatch 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 (ϳ1.01 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). IMPORTANCEMetabolic 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. Mevalonate pathways, important in the production of isoprenoids, are broadly present in eukaryotes, archaea, and some prokaryotes (1-3). Besides a precursor for the production of isoprenoids (2, 3), mevalonate has been used in cosmetics and as a building block for the production of sustainable polymers (4). Recently, it has been demonstrated that mevalonate could be transformed, by simple chemical steps, into a branched lactone ␤-methyl-␦-valerolactone (␤M␦VL), and its copolymerization with lactides results in a new class of polyesters with tunable mechanical properties (5). These potential applications motivate the development of ...

Chemo-enzymatic pathways were developed to prepare optically enriched (+)-β-methyl-δ-valerolactone and (−)-β-methyl-δ-valerolactone. Anhydromevalonolactone, synthesized by the acid-catalyzed dehydration of bioderived mevalonate, was transformed to (+)-β-methyl-δ-valerolactone with 76% ee and 69% conversion using the mutant enoate reductase, YqjM(C26D, I69T). With the same substrate but a different enoate reductase (OYE2), we obtained the other enantiomer ((−)-β-methyl-δ-valerolactone) with higher selectivity and yield (96% ee and a 92% conversion). The enzymedocking program LibDock was used to help explain the origin of the divergent enatntioselectivity of the two reductases, and complementary in vitro experiments were used to determine the turnover number and Michaelis constant for each. Finally, the effect of the enantiopurity of the β-methyl-δ-valerolactone monomer on the properties of the corresponding polyester was investigated. Like atactic poly((±)-β-methyl-δ-valerolactone), the isotactic polymer was determined to be amorphous with a low softening temperature (−52 °C).