Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene Cyclase Variants

Abstract

Squalene-hopene cyclases (SHCs) catalyze the polycyclization of squalene into a mixture of hopene and hopanol. Recently, amino-acid residues lining the catalytic cavity of the SHC from Alicyclobacillus acidocaldarius were replaced by small and large hydrophobic amino acids. The alteration of leucine 607 to phenylalanine resulted in increased enzymatic activity towards the formation of an intermolecular farnesyl-farnesyl ether product from farnesol. Furthermore, the addition of small-chain alcohols acting as nucleophiles led to the formation of non-natural ether-linked terpenoids and, thus, to significant alteration of the product pattern relative to that obtained with the wild type. It is proposed that the mutation of leucine at position 607 may facilitate premature quenching of the intermediate by small alcohol nucleophiles. This mutagenesis-based study opens the field for further intermolecular bond-forming reactions and the generation of non-natural products.

Enzymatic Friedel‐Crafts Alkylation Using Squalene‐Hopene Cyclases

“…The substrate spectrum of SHCs, and in particular of the enzyme from Alicyclobacillus acidocaldarius ( Aac SHC), has been addressed several times in the past [35–37] . In this light, several hot‐spot residues in the active‐site pocket of Aac SHC including W169, I261, Y420, V448, G600, F605, L607 and Y609 were identified that resulted in variants with increased activity or shifted product selectivity in cyclizations of smaller linear terpenes, [38–40] Prins‐type reactions, [29,41,42] bond‐forming reactions, [43] and isomerization reactions [36] . In very recent work, the combination of mutations at residues G600, Y420, Y609 and L607 resulted in variants for stereoselective directed cationic‐cyclization reactions to produce highly valuable flavours and fragrances [44] …”

Biocatalysis for terpene-based polymers

“…Recently, metabolic engineering and promiscuous TCs were used to generate unprecedented cyclic C 11 terpenes from methyl-GPP, exemplified here for 2-methyllimonene 9 [29]. The exciting possibility of interception of highly reactive carbocations generated during TC catalysis, by additional nucleophiles beyond water, have been demonstrated (corresponding to Nu = R-OH in Figure 1, top right) [30].…”

Asymmetric Cation‐Olefin Monocyclization by Engineered Squalene–Hopene Cyclases

“…Importantly, SHCs have proven to be highly evolvable: Engineered SHC variants with not more than three mutations enabled a viable industrial‐scale process to obtain Ambrofix TM , [14, 15] as well as dramatically increased activity and altered chemo‐ and stereoselectivity of cyclization reactions with mono‐ and sesquiterpenoids, such as geraniol, [11] farnesol, [16] or citronellal [17] . A limitation of SHCs, however, is their strict ( S )‐enantioselectivity at the stereocenter formed after the first cyclization of all polyisoprenoids tested so far (an overview of products is given in reviews [18, 19] ).…”