Multi‐Enzymatic Cascade Reactions for the Synthesis of cis,cis‐Muconic Acid

Abstract

Lignin valorization allows the generation of a number of value-added products such as cis,cismuconic acid (ccMA), which is widely used for the synthesis of chemicals for the production of biodegradable plastic materials. In the present work, we reported the first multi-enzymatic, one-pot bioconversion process of vanillin into ccMA. In details, we used four sequential reactions catalyzed by xanthine oxidase, O-demethylase LigM (and the tetrahydrofolate-regeneration enzyme methyl transferase MetE), decarboxylase AroY (based on the use of E. coli transformed cells) and catechol 1,2-dioxygenase CatA. The optimized lab-scale procedure allowed to reach, for the first time, the conversion of 5 mM vanillin into ccMA in ~30 h with a 90% yield: this achievement represents an improvement in terms of yields and time when compared to the use of a whole-cell system. This multi-enzymatic system represents a sustainable alternative for the production of a high value added product from a renewable resource.

Whole-Cell Bioconversion of Renewable Biomasses-Related Aromatics to cis,cis-Muconic Acid

“…The product was recovered from the reaction mixture by methanol precipitation (15 mg) and identified by NMR and ESI-MS (see Figure S5): the ESI-full scan mass spectrum shows an (M-1)- m / z 141.27 and an intense product ion at m / z 97.05 that resulted from a decarboxylation process. An identical fragmentation pattern was observed for the commercial ccMA dissolved in methanol …”

“…33 Bioconversion of Vanillin into cis,cis-Muconic Acid. Starting from the enzymatic cascade process set up to convert vanillin into ccMA, 12 now, we evaluated the assembling of this pathway in a single-engineered E. coli K-12 MG1655 RARE strain to promote the diffusion of intermediates in the confined intracellular space, thus reducing the mass transfer problem. 10 Compared to the cell-free bioconversion system, 12 the commercial enzyme xanthine oxidase catalyzing the oxidation of vanillin into vanillic acid and the THF cofactor-dependent demethylase LigM catalyzing the demethylation of vanillic acid into protocatechuic acid (PCA) have been substituted by the enzymes vanillin dehydrogenase LigV from Sphingomonas paucimobilis SYK-6 and vanillic acid O-demethylase VanAB from P. putida KT2440 (see Scheme 1; this strain encoding five recombinant enzymes was named E. coli V).…”

“…Starting from the enzymatic cascade process set up to convert vanillin into ccMA, now, we evaluated the assembling of this pathway in a single-engineered E. coli K-12 MG1655 RARE strain to promote the diffusion of intermediates in the confined intracellular space, thus reducing the mass transfer problem .…”

“…Whole-cell biocatalysts offer several advantages, such as multi-step reactions with cofactor regeneration, high catalytic efficiency, and mild conditions, but require tailoring by means of protein and metabolic engineering approaches and a critical evaluation of the results in comparison to cell-free systems . Recently, we used a system biocatalysis approach to develop the first multi-enzymatic, one-pot bioconversion process of vanillin into ccMA based on four sequential reactions catalyzed by the commercial enzyme xanthine oxidase, the recombinant enzyme O -demethylase LigM in the presence of tetrahydrofolate (THF) and of the cofactor-regeneration enzyme methyl transferase MetE, and Escherichia coli cells expressing the decarboxylase AroY and the recombinant enzyme catechol 1,2-dioxygenase C12O . The optimized lab-scale procedure converted 5 mM vanillin into ccMA in ≈30 h with a 95% yield.…”

The Laccase‐Lig Multienzymatic Multistep System in Lignin Valorization

“…The use of biological approaches based on microbial cells and enzymes offers various advantages, as it not only permits the setting of eco-friendly mild aqueous reactions, but also exploits the high selectivity of enzymes toward specific valueadded aromatics suitable for fine chemistry. [17,18] However, degradation yields are low, limiting a proper quantification of the aromatic products. [19] The potential of ligninolytic enzymes (i. e., laccases, peroxidases) has been largely investigated using lignin model compounds as substrates to elucidate their reaction mechanism.…”