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

Abstract

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.

Engineering Escherichia coli to grow constitutively on D-xylose using the carbon-efficient Weimberg pathway

“…For example, upstream enzymes from the Weimberg pathway have been coupled with a decarboxylase from Pseudomonas putida or Lactobacillus lactis and native E. coli alcohol dehydrogenases or aldehyde dehydrogenases to produce d -1,2,4-butanetriol, d -1,4-butanediol and 3,4-dihydroxybutyric acid from d -xylose [ 10–16 ]. The complete pathway has also been used to produce glutaric acid and mesaconic acid using E. coli as the host strains, or to improve Corynebacterium glutamicum d -xylose utilization [ 17–21 ]. However, these systems relied on inducible expression, using an expensive inducer (IPTG), which increases the process costs.…”

“…However, these systems relied on inducible expression, using an expensive inducer (IPTG), which increases the process costs. Furthermore, the engineered E. coli strains were dependent on glucose for growth in minimal media [ 17, 19 ], so that the benefit of using d -xylose to manufacture the chemicals is offset by the use of food-grade glucose to produce the E. coli biocatalyst. The engineered C. glutamicum strains could grow on d -xylose alone, but the growth rates were only a fraction of those achieved by the progenitor strains using the PPP [ 18, 20 ].…”

Application of Nonphosphorylative Metabolism as an Alternative for Utilization of Lignocellulosic Biomass

“…Compared to the glycolysis pathway and the tricarboxylic acid (TCA) cycle, nonphosphorylative metabolism ensures high maximum theoretical yields, because xylose or arabinose flows to C5 compounds without excretion of CO 2 (Weimberg, 1961 ; Stephens et al, 2007 ). High experimental yields have also been achieved when we employed nonphosphorylative metabolism to produce mesaconate and glutamate in E. coli (Bai et al, 2016 ). This is probably because in E. coli , nonphosphorylative metabolism is isolated from the endogenous metabolic pathways, and consequently, its intermediates cannot be competitively consumed by the intrinsic metabolic network but flow into the final products.…”

“…This wastes energy and generate by-products, such as acetate. A different mesaconate pathway was constructed so that nonphosphorylative metabolism converted xylose/arabinose into glutamate, and then mesaconate was produced by the same glutamate catabolic pathway from C. tetanomorphum (Bai et al, 2016 ). For the new engineered strains, glucose was specifically used to support bacterial growth and xylose/arabinose was used to produce mesaconate through nonphosphorylative metabolism.…”

“…Fermentation results showed that mesaconate yield was more than 0.4 mol/mol xylose/arabinose without any optimization. Subsequent optimization strategies including screening nonphosphorylative operons with high activities, overexpression of xylose/arabinose transport system ( araE ), and deletion of sucA , ensure an even higher yield (0.85 mol/mol xylose/arabinose) (Bai et al, 2016 ). This work also suggested that the transport system is the rate-limiting step for nonphosphorylative metabolism-dependent mesaconate production.…”