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 electronrich 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.…”

“…Recently, Galman et al. proposed a biocatalytic in vivo cascade for the synthesis of the L–DOPA precursor L‐veratrylglycine ( 130 ) by combining an OMT and an engineered PAL (Scheme 8z) [193] . As previously mentioned, OMTs need SAM as a cofactor to perform catalysis.…”

“…Improving the use of cofactor‐free enzymes and their exploitation in biocatalytic cascades could dramatically reduce the costs of the overall synthesis. 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 cost‐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] .…”

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

“…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 overall cascade outcome. [193] Albeit whole-cell biocatalysts are less costprohibitive 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.…”