Production of d-Glyceric acid from d-Galacturonate in Escherichia coli

Abstract

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.

Substrate‐activated expression of a biosynthetic pathway in Escherichia coli

“…To characterize this system, we conducted fermentations with the MG1655(DE3) ΔgarK Δhyi ΔglxK ΔuxaC strain used previously. [30] We verified that the control circuit modulates transcription in response to galacturonate by quantifying the relative mRNA levels of udh, the second gene in the operon, over the course of a fermentation using qRT-PCR (Figure 5A). For all samples, the fold change of mRNA was analyzed relative to P1OO levels before galaturonate addition.…”

“…The biosensor controlled pathway and constitutive pathway plasmids (pP1OO-gli-udh and pP1XX-gliudh in SI Table S4, respectively) were constructed with the PCR products of P1OO-f and P1OO-r on template pP1OO-gfp or P1XX-gfp, and gli-f and gli-r, plus udh-f and udh-r on the previously constructed pathway plasmid, [30] using NEBuilder HiFi DNA Assembly Cloning Kit (NEB). RBS sequences for the operon (SI Table S3), calculated using the Operon Calculator function of the RBS Calculator, [42] were included in the primers (SI Table S2).…”

“…We demonstrated this application with the D-glyceric acid production pathway previously developed in our group. [30] The pathway utilizes enzymes encoded by two heterologous genes, uronate dehydrogenase (udh) and galactarolactone isomerase (gli), to generate the intermediate Dgalactarate. D-galactarate is converted to D-glyceric acid by enzymes encoded by the endogenous genes galactarate dehydratase (garD), 5-keto-4-deoxy-D-glucarate aldolase (garL), and 2-hydroxy-3oxopropionate reductase (garR) (Figure 4A, SI Table S1).…”

“…We demonstrated our approach with a heterologous biosynthetic pathway to produce D-glyceric acid from galacturonate that was previously developed in our lab. [30] D-glyceric acid holds promise due to its biological activity and for its applications in surfactants and polymers. [31] Furthermore, the pathway's galacturonate feed is the main component of pectin, a ubiquitous plant polysaccharide abundantly found in fruit and vegetable food waste streams, [32] that could serve as a renewable feedstock for the process.…”

Probiotic Combination CBLEB Alleviates Streptococcus pneumoniae Infection Through Immune Regulation in Immunocompromised Rats

“…We found that CBLEB alleviates the faecal enrichment of 1-octadecanol, 2-propenoic acid, 2.4-di-tert-butylphenol, D-lyxose, glyceric acid and m-cresol, as well as the depletion of (Z,Z)-9,12-octadecadienoic acid in immunocompromised SP-infection rats. Among these metabolites, 1-octadecanol, glyceric acid and (Z,Z)-9,12-octadecadienoic acid are commonly found in animals, plants and microbes and can also be used in cosmetics, medicines or food additives; [30][31][32] D-lyxose, a rare pentose found in bacterial glycolipids, is not usually utilized by microbes and is a precursor to antitumour and immunostimulatory α-galactosylceramide agents; 33 and 2-propenoic acid, 2.4-di-tert-butylphenol and m-cresol are compounds with low toxicity and antioxidant functions. [34][35][36] Nevertheless, the effects of these compounds on the lung and gut are not fully understood.…”