An ADH toolbox for raspberry ketone production from natural resources via a biocatalytic cascade

Abstract

Raspberry ketone is a widely used flavor compound in food and cosmetic industry. Several processes for its biocatalytic production have already been described, but either with the use of genetically modified organisms (GMOs) or incomplete conversion of the variety of precursors that are available in nature. Such natural precursors are rhododendrol glycosides with different proportions of (R)- and (S)-rhododendrol depending on the origin. After hydrolysis of these rhododendrol glycosides, the formed rhododendrol enantiomers have to be oxidized to obtain the final product raspberry ketone. To be able to achieve a high conversion with different starting material, we assembled an alcohol dehydrogenase toolbox that can be accessed depending on the optical purity of the intermediate rhododendrol. This is demonstrated by converting racemic rhododendrol using a combination of (R)- and (S)-selective alcohol dehydrogenases together with a universal cofactor recycling system. Furthermore, we conducted a biocatalytic cascade reaction starting from naturally derived rhododendrol glycosides by the use of a glucosidase and an alcohol dehydrogenase to produce raspberry ketone in high yield. Key points • LB-ADH, LK-ADH and LS-ADH oxidize (R)-rhododendrol • RR-ADH and ADH1E oxidize (S)-rhododendrol • Raspberry ketone production via glucosidase and alcohol dehydrogenases from a toolbox Graphical abstract

Detection of Raspberry Ketone after Percutaneous Absorption of Rhododendrol-Containing Cosmetics and Its Mechanism of Formation

“…The metabolism from RD to RK, which is considered to be related to leukoderma, may also be another cause of leukoderma. It has been reported that (R)-RD [19,21] in plants is converted to RK by ADH [22]. As ADH is a ubiquitously expressed enzyme that is also present in human skin [23], it is possible that RD, a secondary alcohol, is oxidized to RK.…”

Synthesis of Modified Poly(vinyl Alcohol)s and Their Degradation Using an Enzymatic Cascade

“…After cleaving the ester bonds, the hydroxyl groups are oxidized to ketones by an alcohol dehydrogenase from Lactobacillus kefir (LK‐ADH) utilizing the cofactor NADP + with formation of NADPH (Scheme 2, step 2) [13a] . These ketones are then further oxidized to esters by the cyclohexanone monooxygenase (CHMO) variant M15 originally from the organism Acinetobacter calcoaceticus .…”

“…All enzymes used are well characterized and either easily recombinantly expressible in Escherichia coli or commercially available. [13] The enzyme cascade consists of four steps (Scheme 2) for modified PVA but can also be applied for unmodified PVA, starting at step 2. In step 1, ester bonds in the derivatized side chains are hydrolyzed by the commercially available lipase TL-IM resulting in unmodified PVA.…”

Biomacromolecule-Assisted Screening for Reaction Discovery and Catalyst Optimization

“…Biocatalysis has taken on ever increasing importance for pharmaceutical process groups, − including the design of enzyme cascades ,− to mediate key steps in building ever more complex molecular frameworks. ,,, Biocatalysis is becoming more and more versatile, and high-throughput assay systems have rendered directed evolution approaches more practical. Modern protein engineering methods allow for the combination of directed evolution approaches with in silico design of biocatalysts to enhance properties such as stability, substrate specificity, and enantioselectivity.…”