One‐Pot Biocatalytic In Vivo Methylation‐Hydroamination of Bioderived Lignin Monomers to Generate a Key Precursor to L‐DOPA

Abstract

Electron‐rich phenolic substrates can be derived from the depolymerisation of lignin feedstocks. Direct biotransformations of the hydroxycinnamic acid monomers obtained can be exploited to produce high‐value chemicals, such as α‐amino acids, however the reaction is often hampered by the chemical autooxidation in alkaline or harsh reaction media. Regioselective O‐methyltransferases (OMTs) are ubiquitous enzymes in natural secondary metabolic pathways utilising an expensive co‐substrate S‐adenosyl‐l‐methionine (SAM) as the methylating reagent altering the physicochemical properties of the hydroxycinnamic acids. In this study, we engineered an OMT to accept a variety of electron‐rich phenolic substrates, modified a commercial E. coli strain BL21 (DE3) to regenerate SAM in vivo, and combined it with an engineered ammonia lyase to partake in a one‐pot, two whole cell enzyme cascade to produce the l‐DOPA precursor l‐veratrylglycine from lignin‐derived ferulic acid.

Enzymatic Oxy‐ and Amino‐Functionalization in Biocatalytic Cascade Synthesis: Recent Advances and Future Perspectives

“…Recently, Galman et al proposed a biocatalytic in vivo cascade for the synthesis of the L-DOPA precursor Lveratrylglycine (130) by combining an OMT and an engineered PAL (Scheme 8z). [193] As previously mentioned, OMTs need SAM as a cofactor to perform catalysis. Therefore, the authors used an engineered E. coli BL21 (DE3) strain to regenerate SAM in vivo, thereby reducing the overall process costs.…”

“…Some examples of whole-cell biocatalysts give another solution to this issue, utilizing the cellular NAD-(P)H and ATP source or tuning the natural cofactor production in vivo to enhance the overall cascade outcome. [193] Albeit whole-cell biocatalysts are less prohibitive and time-consuming than their in vitro counterparts, other systems were developed in the past years mimicking cellular factories via cell-free compartmentalized biocatalysis. [196] Various methodologies have been developed in the recent years to construct artificial cells via enzymes encapsulation by water-in-oil droplets, transfer to liposomes for the synthesis of ethanol from lactate, [201] using metalorganic framework nanoparticles as nanoreactors, mainly based on zeolitic imidazole frameworks, [202,203] or adopting DNA origami structures (1D, 2D or 3D) to spatially separate the enzymes, [204] improving substrate channelling and the overall cascade rate and stability.…”

Lignin Valorization: Production of High Value-Added Compounds by Engineered Microorganisms

“…Currently, L-DOPA is produced via chemical synthesis and extraction from plants; however, these production methods are not sustainable [133]. Galman et al developed a two-stage one-pot process to produce biobased L-veratrylglycine, a precursor of L-DOPA [123]. In the first step, ferulic acid is methylated to 3,4-dimethoxycinnamic acid: the plasmid pET28 harboring the gene encoding a variant of O-methyltransferase EjOMT enzyme (I133S/L138V/L342V) from Eisenia japonica and the plasmid p-ACYCDuet-1 carry-ing the gene encoding S-adenosylhomocysteine nucleosidase MntN, S-ribosylhomocysteine lyase LuxS and a variant of SAM synthetase MetK enzyme (I303V) from E. coli were transferred into the E. coli BL21(DE3) strain.…”

“…To increase the methionine availability, the endogenous transcriptional repressor MetJ was deleted from the E. coli's genome. The engineered strain (at 0.7% w/v), when incubated in M9 medium supplemented with 5 mM D,L-methionine and 3 mM ferulic acid at 30 • C, produced 0.6 g/L of 3,4-dimethoxycinnamic acid (99% conversion) in 48 h. In the second step, an engineered E. coli BL21(DE3) strain overexpressing a variant of the ammonia lyase AL-11 enzyme (Q84V) was added to the reaction mixture (0.8% w/v) together with 4 M ammonium carbamate: after an additional 18 h of incubation at 30 • C, the complete conversion in L-veratrylglycine into L-DOPA (>99% ee) was obtained (Table 4) [123].…”

The 55th Bürgenstock Conference under the Banner of Sustainability**

“…[79] Furthermore, many developed enzymes can be applied in cascades together with carboxylic acid reductases, transaminases, amine oxidases, alcohol dehydrogenases or alcohol oxidases enabling the direct conversion of available starting materials, e.g., carboxylic acids or alcohols, into chiral amines with high selectivities and activities. [80] Such cascades can be executed efficiently with the use of flow chemistry thanks to the use of immobilized biocatalysts, [81] facilitating their practical use in an industrial context. [82] On Thursday afternoon, the 55 th Bürgenstock Conference came to an end.…”