# Chemoenzymatic Synthesis of Norisoprenoid Aroma Compounds via C–H Activation by Engineered P450BM3

## TL;DR

Researchers developed a new chemoenzymatic method to efficiently produce norisoprenoid aroma compounds using engineered enzymes, offering a milder and more scalable alternative to traditional chemical methods.

## Contribution

The study introduces engineered P450BM3 enzymes for C–H activation in chemoenzymatic synthesis of norisoprenoids, achieving high conversion and scalability.

## Key findings

- Engineered P450BM3 variants achieved high conversion rates and regioselectivity for synthesizing damascenones.
- Alternative oxidation routes yielded tabanone isomers despite rearrangement challenges.
- Enzymatic oxidation reached sp3 aliphatic positions, expanding product diversity and scalability.

## Abstract

Norisoprenoid compounds
such as the damascones, damascenones, and
megastigmatrienones are widely used in the flavor and fragrance industry.
Their low natural abundance and the limitations of traditional synthetic
routes, such as high energy demands, use of toxic reagents, and challenges
in isomeric selectivity, hinder production under mild conditions.
Here, we report chemoenzymatic synthesis routes to these compounds
using engineered P450BM3 variants for late-stage C–H
activation. Screening of α- and β-damascone with a panel
of 96 P450BM3 variants revealed high conversion rates and
regioselectivities for the intermediates for acid-catalyzed dehydration
to form γ- and β-damascenone, respectively. Megastigmatriene
oxidation did not give tabanone due to rearrangement to β-ionol,
but alternative routes via α-ionol and α-ionone oxidation
yielded a mixture of tabanone isomers. Beyond allylic oxidation, the
enzyme collection also oxidized these norisoprenoids at the less reactive
sp3 aliphatic positions, expanding the diversity of accessible
products. Scalability of enzymatic oxidation was demonstrated by the
high titer (7.3 g/L), conversion (95%), and total turnover number
(9500) for β-damascone oxidation. The findings demonstrate the
power of chemoenzymatic strategies in accessing complex norisoprenoids
in fewer steps than chemical synthesis routes and lay the groundwork
for scalable biotechnological production processes.