Tunable Production of (R)- or (S)-Citronellal from Geraniol via a Bienzymatic Cascade Using a Copper Radical Alcohol Oxidase and Old Yellow Enzyme

Abstract

Biocatalytic pathways for the synthesis of (−)-menthol, the most sold flavor worldwide, are highly sought-after. To access the key intermediate ( R )-citronellal used in current major industrial production routes, we established a one-pot bienzymatic cascade from inexpensive geraniol, overcoming the problematic biocatalytic reduction of the mixture of ( E/Z )-isomers in citral by harnessing a copper radical oxidase ( Cgr AlcOx) and an old yellow enzyme (OYE). The cascade using OYE2 delivered 95.1% conversion to ( R )-citronellal with 95.9% ee , a 62 mg scale-up affording high yield and similar optical purity. An alternative OYE, GluER, gave ( S )-citronellal from geraniol with 95.3% conversion and 99.2% ee .

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

“…The Ts OYE was produced and purified identically to methods previously described 69 . Briefly, E. coli BL21(DE3) strains harboring the gene for Ts OYE (grown at 37 °C, 180 rpm) were induced at OD 600 of ~0.6 with IPTG (0.1 mM, 30 min, 4 °C), resuspended in MOPS-NaOH (20 mM, pH 7.0), disrupted using a Multi Shot Cell Disruption System (two cycles) and clarified by centrifugation (17,500 × g , 30 min, 4 °C).…”

Rational engineering of AA5_2 copper radical oxidases to probe the molecular determinants governing their substrate selectivity

“…In 2021, the alcohol oxidase from the plant pathogen Colletotrichum graminicola ( Cgr AlcOx) was explored for its capacity to convert industrially‐relevant long‐chain aliphatic alcohols into fatty aldehydes, which constitute major compounds in flavours and fragrances [17]. This enzyme was also used in a cascade with ene ‐reductases for the synthesis of ( R )‐citronellal, a key intermediate in the production process of (−)‐menthol [29].…”

“…CROs from the AA5_2 subfamily are attractive biocatalysts for biotechnology and green chemistry. Indeed, they have gained increasing attention for their ability to functionalize polysaccharides [26] and for their oxidizing properties on small organic molecules [17,20,[27][28][29]. For instance, the archetypal FgrGalOx and its engineered variants have been used in glycoprotein labelling [30,31], biosensor development [32], polysaccharide functionalization [33], oxidation of diverse carbohydrates or other alcohols and more recently in the synthesis of Islatravir, an anti-HIV drug [34].…”

Co‐Immobilization of GOD & HRP on Y‐Shaped DNA Scaffold and the Regulation of Inter‐Enzyme Distance

“…By accelerating the diffusion of intermediate products and increasing its concentration around the downstream enzyme, it has high catalytic reaction efficiency and strong practical operability due to the reduction of side reactions and the elimination of the need to separate intermediate products. [5,6,7,8] In vivo, cells conduct modular co-localization and regionalization management for the multienzymes cascade system, which can realize efficient cascade catalysis among multienzymes. For example, multiple enzymes participating in the respiratory chain are colocalized on the inner membrane of mitochondria, and multiple cellulases/hemicellulases in bacteria combine to form a cellulosome structure, [9] etc.…”

“…By accelerating the diffusion of intermediate products and increasing its concentration around the downstream enzyme, it has high catalytic reaction efficiency and strong practical operability due to the reduction of side reactions and the elimination of the need to separate intermediate products. [ 5,6,7,8 ]…”