Synthesis of Cbz-protected 3-aminopiperidine and 3-aminoazepane using a multi-enzyme cascade consisting of galactose oxidase and imine reductase variants.
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.
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.
Promiscuous activity of a glycosyltransferase was exploited to polymerise glucose from UDP-glucose via the generation of β-1,4-glycosidic linkages. The biocatalyst was incorporated into biocatalytic cascades and chemo-enzymatic strategies to synthesise...
Oxygenase enzymes generate reactive intermediates at their active sites to effect controlled functionalizations of inert C–H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during turnover in the absence of substrate. Herein we use a combination of stopped-flow spectroscopy, targeted mutagenesis, DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy (HERFD-XAS), and electron paramagnetic resonance spectroscopy (EPR) to capture two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a spin singlet (S = 0) CuII-(histidyl radical) is generated at the histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII-tyrosyl radical pair, thereby restoring the histidine and the histidine brace active site to its resting state to facilitate resumption of the catalytic cycle through reduction. This process gives the enzyme the capacity to repair any damage to the active site histidine residues ‘on the fly’, highlighting how enzymes protect themselves from deleterious side reactions during uncoupled turnover.
(2R,4R)‐Pentanediol is an interesting precursor for the synthesis of chiral ligands. A ketoreductase (KRED) was employed for the asymmetric reduction of acetylacetone to this diol. Biocatalysis often suffers from low concentrations of hydrophobic substrates and low stability of the enzyme in unconventional media. Here, we present an engineered KRED variant applicable in a neat substrate system, including upscaling to the multi‐liter scale and downstream processing (DSP). Our engineered KRED applied in a neat substrate system is a powerful technique for the synthesis of chiral diols yielding product concentrations of 208 g L−1.
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