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).

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.

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A microbial production platform has been developed in Escherichia coli to synthesize d-glyceric acid from d-galacturonate. The expression of uronate dehydrogenase (udh) from Pseudomonas syringae and galactarolactone isomerase (gli) from Agrobacterium fabrum, along with the inactivation of garK, encoding for glycerate kinase, enables d-glyceric acid accumulation by utilizing the endogenous expression of galactarate dehydratase (garD), 5-keto-4-deoxy-D-glucarate aldolase (garL), and 2-hydroxy-3-oxopropionate reductase (garR). Optimization of carbon flux through the elimination of competing metabolic pathways led to the development of a ΔgarKΔhyiΔglxKΔuxaC mutant strain that produced 4.8 g/l of d-glyceric acid from d-galacturonate, with an 83% molar yield. Cultivation in a minimal medium produced similar yields and demonstrated that galactose or glycerol serve as possible carbon co-feeds for industrial production. This novel platform represents an alternative for the production of d-glyceric acid, an industrially relevant chemical, that addresses current challenges in using acetic acid bacteria for its synthesis: increasing yield, enantio-purity and biological stability.

Chemoenzymatic dynamic kinetic resolution (DKR) of rac-1-phenyl ethanol into R-1-phenylethanol acetate was investigated with emphasis on the minimization of side reactions. The organometallic hydrogen transfer (racemization) catalyst was varied, and this was observed to alter the rate and extent of oxidation of the alcohol to form ketone side products. The performance of highly active catalyst [(pentamethylcyclopentadienyl)IrCl(2)(1-benzyl,3-methyl-imidazol-2-ylidene)] was found to depend on the batch of lipase B used. The interaction between the bio- and chemo-catalysts was reduced by employing physical entrapment of the enzyme in silica using a sol-gel process. The nature of the gelation method was found to be important, with an alkaline method preferred, as an acidic method was found to initiate a further side reaction, the acid catalyzed dehydration of the secondary alcohol. The acidic gel was found to be a heterogeneous solid acid.