Harnessing the Structure and Dynamics of the Squalene‐Hopene Cyclase for (−)‐Ambroxide Production**

Abstract

Terpene cyclases offer enormous synthetic potential, given their unique ability to forge complex hydrocarbon scaffolds from achiral precursors within a single cationic rearrangement cascade. Harnessing their synthetic power, however, has proved to be challenging owing to their generally low catalytic performance. In this study, we unveiled the catalytic potential of the squalene-hopene cyclase (SHC) by harnessing its structure and dynamics. First, we synergistically tailored the active site and entrance tunnel of the enzyme to generate a 397-fold improved (À )-ambroxide synthase. Our computational investigations explain how the introduced mutations work in concert to improve substrate acquisition, flow, and chaperoning. Kinetics, however, showed terpene-induced inactivation of the membranebound SHC to be the major turnover limitation in vivo. Merging this insight with the improved and stereoselective catalysis of the enzyme, we applied a feeding strategy to exceed 10 5 total turnovers. We believe that our results may bridge the gap for broader application of SHCs in synthetic chemistry.

A Career in Catalysis: Bernhard Hauer

“…Encouraged by these engineering campaigns, the Hauer group next faced again the intricate cyclization of homofarnesol (15) toward the high-value fragrance ingredient (−)-ambroxan (16). 71 Since no chemical catalyst was able to sufficiently control the stereoselective outcome of this reaction, the SHC equipped with its strongly confined active site served as a potent solution in this regard, which was already recognized by Neumann and Simon as early as 1986. 47 Since the product 16 was the only isomer that crystallized during biotransformation, the authors concluded that the absolute cyclization selectivity toward this single isomer should enhance catalysis because of a shift in chemical equilibrium.…”

“…(D) Strategies used during the target-oriented engineering campaigns. Adapted with permission from ref . Copyright 2023 Wiley-VCH Verlag GmbH & Co. KGAA.…”

“…Encouraged by these engineering campaigns, the Hauer group next faced again the intricate cyclization of homofarnesol ( 15 ) toward the high-value fragrance ingredient (−)-ambroxan ( 16 ) . Since no chemical catalyst was able to sufficiently control the stereoselective outcome of this reaction, the SHC equipped with its strongly confined active site served as a potent solution in this regard, which was already recognized by Neumann and Simon as early as 1986 .…”

The Scent of Change: Sustainable Fragrances Through Industrial Biotechnology

“…In a follow-up study, the Hauer group sought to improve the biocatalytic synthesis of (À )-ambrox 1. [41] The researchers re-engineered the active site and entrance tunnel of AacSHC to achieve a 397-fold activity improvement. This study aimed to address the limitations in SHC catalysis through a combination of directed evolution and computational studies.…”

“…This knowledge was then harnessed for the synthesis of ( S )‐γ‐dihydroionone, allowing the researchers to produce the complementary fragrance with high enantiomeric excess (>99.9 % ee). In a follow‐up study, the Hauer group sought to improve the biocatalytic synthesis of (−)‐ambrox 1 [41] . The researchers re‐engineered the active site and entrance tunnel of AacSHC to achieve a 397‐fold activity improvement.…”