Biocatalytic Process for (-)-Ambrox Production Using Squalene Hopene Cyclase

Alicyclobacillus acidocaldarius Squalene Hopene Cyclase was evolved to a biocatalyst suitable for (-)-Ambrox production at industrial scale. One round of random mutagenesis led to the identification of three variants with (E,E)-homofarnesol conversion properties improved about 1.5- to 10-fold over that of the wild type enzyme. Eight distinct amino acid mutations were identified overall; only one mutation was at the active site of the enzyme. Each of the three variants contained only two or three mutations over the 631 amino acids of the Alicyclobacillus acidocaldarius Squalene Hopene Cyclase polypeptide chain. Mutations responsible for improved (E,E)-homofarnesol conversion were identified. Investigations on reaction conditions led to the selection of one variant, with which reaction parameters were optimized towards process-relevant conditions. A whole cell biotransformation process is presented in which Escherichia coli cells producing an improved Squalene Hopene Cyclase variant allows the conversion of 125 g/L (E,E)-homofarnesol in <= 72 hours. The developed process for the production of the fragrance ingredient (-)-Ambrox as Ambrofix (R) expands the biocatalysis toolbox by setting out a general basis for biocatalytic Squalene Hopene Cyclase cyclization reactions at industrial scale.