Production of Glucaric Acid from Hemicellulose Substrate by Rosettasome Enzyme Assemblies

Abstract

Hemicellulose biomass is a complex polymer with many different chemical constituents that can be utilized as industrial feedstocks. These molecules can be released from the polymer and transformed into value-added chemicals through multistep enzymatic pathways. Some bacteria produce cellulosomes which are assemblies composed of lignocellulolytic enzymes tethered to a large protein scaffold. Rosettasomes are artificial engineered ring scaffolds designed to mimic the bacterial cellulosome. Both cellulosomes and rosettasomes have been shown to facilitate much higher rates of biomass hydrolysis compared to the same enzymes free in solution. We investigated whether tethering enzymes involved in both biomass hydrolysis and oxidative transformation to glucaric acid onto a rosettasome scaffold would result in an analogous production enhancement in a combined hydrolysis and bioconversion metabolic pathway. Three different enzymes were used to hydrolyze birchwood hemicellulose and convert the substituents to glucaric acid, a top-12 DOE value added chemical feedstock derived from biomass. It was demonstrated that colocalizing the three different enzymes to the synthetic scaffold resulted in up to 40 % higher levels of product compared to uncomplexed enzymes.

Enzymatic production of 4-O-methyl d-glucaric acid from hardwood xylan

“…The conversion yield was not reported by Lee et al [15], who proposed the in vitro pathway with three enzymes including endo-xylanase, α-glucuronidase, and uronate dehydrogenase to produce (methyl)glucaric acid from birchwood xylan. The authors reported a maximum production of 0.7 mM (methyl)glucaric acid from 1% birchwood xylan after more than 2 h, as quantified by NADH absorbance at 340 nm [15]. Provided that a similar xylan source was used, this would be equivalent to a conversion yield of ~ 20%.…”

“…Furthermore, fermentation approaches can complicate product recovery, due to the presence of medium components and other metabolites/intermediates. A cell-free approach to produce 4-O-methyl d-glucaric acid (or methyl glucaric acid, for short) from glucuronoxylan was reported [15], where three enzymes including an endo-xylanase (EC 3.2.1.8), α-glucuronidase (EC 3.2.1.139), and uronate dehydrogenase (EC 1.1.1.203) were used in a cocktail or co-localized on a scaffold. The xylanase cleaved glucuronoxylan to various xylo-oligosaccharides, of which some contained MeGlcA.…”

“…The α-glucuronidase then removed MeGlcA residues that were attached to the non-reducing end of short xylooligosaccharides. The released MeGlcA was finally converted to methyl glucaric acid by the dehydrogenase [15]. Notably, this approach requires a continuous supply of an exogenous cofactor (NAD + ) and the separation of methyl glucaric acid from other soluble xylo-oligosaccharides.…”

Cell-based and cell-free biocatalysis for the production of d-glucaric acid

“…4 a). The pathway was different from the previously reported pathway constructed in recombinant E. coli , and used three enzymes: xylanase (Xyn) from Flavobacterium sp., glcuronidase (AG) from a rumen metagenomic library, and Udh from P. mendocina [ 67 ]. These three enzymes were co-localized into a scaffold and the final product GA increased by 20% compared with the case when the three enzymes were free in solution.…”

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Fig. 4 In vitro cascade for production of GA. Four different methods of synthesizing GA in vitro are represented by different colored text and arrows [ 64 , 67 – 69 ]. a Pink text indicates the GA synthesis route starting from sucrose.

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