Bioatalytic pathway to Ambrox

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Conversion obtained at 125g/L substrate with the best variant from first directed evolution round (215G2) versus wild type enzyme

Ambrox is the fragrance of Ambergris, a rare and expensive fragrance which comes in its natural form from sperm whale bile secretions.

Alicyclobacillus acidocaldarius Squalene Hopene Cyclase was evolved for (-)-Ambrox production. After one round of directed evolution (EvosightTM), 3 variants were identified with an activity improvement by a factor 1.6 to 3.5. An optimization of the reaction conditions through a Design of Experiments (DoE) approach for the best performing variant allowed a factor 10 improvement

Reference: Eichorn et al., Biocatalytic Process for (-)-Ambrox Production Using Squalene Hopene Cyclase, Adv. Synth. Catal. 10.1002/adsc.201800132

green route to green notes

green notes scheme

“Green notes” result from C6 aldehydes and derivatives naturally generated by plants for herbivore defense (for example, chemical signaling to attract herbivore insects predators). However, these pathways cannot be used as such for commercial processes.
The pathway can be reconstituted using 13-lipoxygenase from soybean flour, 13-hydroxyperoxyde lyase (CYP74 family) from guava, and baker’s yeast for isomerization and derivatization of the C6 aldehydes. However, the 13-hydroxyperoxyde lyase from guava exhibited a too low activity and had to be improved.

Reference: F. Brühlmann et al. / Journal of Biotechnology 163 (2013) 339– 345

Proteus proprietary directed evolution technology L-ShufflingTM was used in order to improve the activity of 13-hydroxyperxyde lyase from guava. Homologous enzymes used to generate sequence diversity were 13-HPL from apple, orange and tobacco and 9-HPL from melon.

  • 65,000 variants were screened during 4 rounds of directed evolution
  • The best variant exhibited an activity improvement by a factor 15. Interestingly, the thermostability of the best variant was improved by a factor 48 at 50°C.
  • The process is used at commercial scale by Firmenich.