Squalene-hopene Cyclase Research Articles

Sustainable (-)-Ambrox Production: Chemistry Meets Biocatalysis.

(-)-Ambrox, the most prominent olfactive component of ambergris, is one of the most widely used biodegradable fragrance ingredients. It is traditionally produced from the diterpene sclareol chemically modified and cyclized into (-)-ambrox. The availability of the new feedstock (E)-β-farnesene produced by fermentation opened new routes to (E,E)-homofarnesol as a precursor to (-)-ambrox. Combining the chemical transformation of (E)-β-farnesene to (E,E)-homofarnesol and its enzymatic cyclization with an engineered Squalene Hopene Cyclase provided a new sustainable route for the production of (-)-ambrox at industrial scale. Compared to the traditional synthesis from sclareol, the new and innovative route from (E)-β-farnesene improves atom and step economy, reduces waste production, solvent and energy consumption.

Biocatalytic stereocontrolled head-to-tail cyclizations of unbiased terpenes as a tool in chemoenzymatic synthesis

Terpene synthesis stands at the forefront of modern synthetic chemistry and represents the state-of-the-art in the chemist’s toolbox. Notwithstanding, these endeavors are inherently tied to the current availability of natural cyclic building blocks. Addressing this limitation, the stereocontrolled cyclization of abundant unbiased linear terpenes emerges as a valuable tool, which is still difficult to achieve with chemical catalysts. In this study, we showcase the remarkable capabilities of squalene-hopene cyclases (SHCs) in the chemoenzymatic synthesis of head-to-tail-fused terpenes. By combining engineered SHCs and a practical reaction setup, we generate ten chiral scaffolds with >99% ee and de, at up to decagram scale. Our mechanistic insights suggest how cyclodextrin encapsulation of terpenes may influence the performance of the membrane-bound enzyme. Moreover, we transform the chiral templates to valuable (mero)-terpenes using interdisciplinary synthetic methods, including a catalytic ring-contraction of enol-ethers facilitated by cooperative iodine/lipase catalysis.

Ambrox through the Looking Glass: Chemoenzymatic Synthesis and GC‐Olfactometric Analysis of 15 Ambrox Stereoisomers

AbstractAll eight theoretical stereoisomers of (10S)‐Ambrox have been synthesized by enzymatic polycyclization of the four geometric isomers of homofarnesol with selected squalene hopene cyclases. This includes the highly strained (+)‐(8S,9S)‐Ambrox, an isomer historically considered unlikely to exist. The enantiomeric (10R)‐series has been prepared by a combination of diastereoselective synthesis and preparative chiral HPLC. Thus, for the first time, the synthesis and sensory properties of all but one stereoisomers of Ambrox are presented. The results solve a long standing peradventure: the commercial product (−)‐Ambrox exhibits by far the strongest odour, the previously described 9‐epi‐Ambrox is 26 times weaker. The enantiomer difference between (−)‐and (+)‐Ambrox was also found much higher than in previous reports (1000 vs. 8 times). The (8R)‐configuration was identified as the single most important structural feature for high odour strength.

Controlling Monoterpene Isomerization by Guiding Challenging Carbocation Rearrangement Reactions in Engineered Squalene-Hopene Cyclases.

The interconversion of monoterpenes is facilitated by a complex network of carbocation rearrangement pathways. Controlling these isomerization pathways is challenging when using common Brønsted and Lewis acid catalysts, which often produce product mixtures that are difficult to separate. In contrast, natural monoterpene cyclases exhibit high control over the carbocation rearrangement reactions but are reliant on phosphorylated substrates. In this study, we present engineered squalene-hopene cyclases from Alicyclobacillus acidocaldarius (AacSHC) that catalyze the challenging isomerization of monoterpenes with unprecedented precision. Starting from a promiscuous isomerization of (+)-β-pinene, we first demonstrate noticeable shifts in the product distribution solely by introducing single point mutations. Furthermore, we showcase the tuneable cation steering by enhancing (+)-borneol selectivity from 1 % to >90 % (>99 % de) aided by iterative saturation mutagenesis. Our combined experimental and computational data suggest that the reorganization of key aromatic residues leads to the restructuring of the water network that facilitates the selective termination of the secondary isobornyl cation. This work expands our mechanistic understanding of carbocation rearrangements and sets the stage for target-oriented skeletal reorganization of broadly abundant terpenes.

A systematic overexpression approach reveals native targets to increase squalene production in Synechocystis sp. PCC 6803.

Cyanobacteria are a promising platform for the production of the triterpene squalene (C30), a precursor for all plant and animal sterols, and a highly attractive intermediate towards triterpenoids, a large group of secondary plant metabolites. Synechocystis sp. PCC 6803 natively produces squalene from CO2 through the MEP pathway. Based on the predictions of a constraint-based metabolic model, we took a systematic overexpression approach to quantify native Synechocystis gene's impact on squalene production in a squalene-hopene cyclase gene knock-out strain (Δshc). Our in silico analysis revealed an increased flux through the Calvin-Benson-Bassham cycle in the Δshc mutant compared to the wildtype, including the pentose phosphate pathway, as well as lower glycolysis, while the tricarboxylic acid cycle predicted to be downregulated. Further, all enzymes of the MEP pathway and terpenoid synthesis, as well as enzymes from the central carbon metabolism, Gap2, Tpi and PyrK, were predicted to positively contribute to squalene production upon their overexpression. Each identified target gene was integrated into the genome of Synechocystis Δshc under the control of the rhamnose-inducible promoter Prha. Squalene production was increased in an inducer concentration dependent manner through the overexpression of most predicted genes, which are genes of the MEP pathway, ispH, ispE, and idi, leading to the greatest improvements. Moreover, we were able to overexpress the native squalene synthase gene (sqs) in Synechocystis Δshc, which reached the highest production titer of 13.72 mg l-1 reported for squalene in Synechocystis sp. PCC 6803 so far, thereby providing a promising and sustainable platform for triterpene production.

Cover Picture: Harnessing the Structure and Dynamics of the Squalene‐Hopene Cyclase for (−)‐Ambroxide Production (Angew. Chem. Int. Ed. 22/2023)

Ambergris is a highly valued natural product in the fragrance industry and is also known as the “floating gold” due to its biosynthesis in the intestines of sperm whales. Owing to this scarce natural resource, the stereoselective synthesis of its most prominent olfactory component, (−)-Ambroxide, via alternative routes is attractive. In their Research Article (e202301607), Sílvia Osuna, Bernhard Hauer et al. demonstrate the catalytic potential of an engineered squalene-hopene cyclase in the intricate cyclization of the precursor homofarnesol. The artwork was designed by Verena Resch, Graz.

Harnessing the Structure and Dynamics of the Squalene-Hopene Cyclase for (-)-Ambroxide Production.

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 (-)-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 membrane-bound 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 105 total turnovers. We believe that our results may bridge the gap for broader application of SHCs in synthetic chemistry.

Squalene hopene cyclases and oxido squalene cyclases: potential targets for regulating cyclisation reactions

Squalene hopene cyclases (SHC) convert squalene, the linear triterpene to fused ring product hopanoid by the cationic cyclization mechanism. The main function of hopanoids, a class of pentacyclic triterpenoids in bacteria involves the maintenance of membrane fluidity and stability. 2, 3-oxido squalene cyclases are functional analogues of SHC in eukaryotes and both these enzymes have fascinated researchers for the high stereo selectivity, complexity, and efficiency they possess. The peculiar property of the enzyme squalene hopene cyclase to accommodate substrates other than its natural substrate can be exploited for the use of these enzymes in an industrial perspective. Here, we present an extensive overview of the enzyme squalene hopene cyclase with emphasis on the cloning and overexpression strategies. An attempt has been made to explore recent research trends around squalene cyclase mediated cyclization reactions of flavour and pharmaceutical significance by using non-natural molecules as substrates.

Alternative Active Site Confinement in Squalene–Hopene Cyclase Enforces Substrate Preorganization for Cyclization

Confinement of an enzyme’s active site is critical to the efficiency of chemical reactions and has been recognized as an important tool for catalysis to accelerate reactions by proximity and via specific interactions with the substrate to control the course of the reaction. Despite these benefits, a strongly confined active site is inherently limited to compounds that resemble the native substrate, with only small deviations tolerated. To unlock the potential of modifying the terpene scaffold as building blocks of interest with branched isoprene/terpene motifs, the present study demonstrates a simple structure-guided strategy to create alternative confinement in the squalene–hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC). This strategy aims for proximity and shape complementarity between the protein and substrate to enforce preorganization of geranyl acetone analogs with deviant isoprene patterns. Screening of AacSHC variants affecting the substrate tunnel allowed us to detect starting activity with the AacSHC G600M variant. Structural analysis led to the identification of reduced tunnel radii that resulted in steric interactions and proximity by a decrease in the average distances between the double bond of the substrate’s terminal isoprene unit and the catalytic site. Iterative saturation mutagenesis with SHC variants and screening with the geranyl acetone analog dihydropseudoirone were used to generate complementarity and demonstrated further evolvability. The final variant showed a 1174-fold increase in total turnover number and 111-fold increased catalytic efficiency compared to the wild-type. This approach to create alternative active site confinement demonstrates great potential to overcome limitations in the engineering of biocatalysts and generation of interesting building blocks.

From Ambergris to (-)-Ambrox: Chemistry Meets Biocatalysis for Sustainable (-)-Ambrox Production.

(-)-Ambrox, the most prominent olfactive component of ambergris is one of the most widely used biodegradable fragrance ingredients. Traditionally it is produced from the diterpene sclareol, modified and cyclized into (-)-ambrox by classical chemistry steps. The availability of the new feedstock (E)-β-farnesene produced by fermentation opened new pathways to (E,E)-homofarnesol as a precursor to (-)-ambrox. Combining chemical transformation of (E)-β-farnesene to (E,E)-homofarnesol and its enzymatic cyclization at the industrial scale to (-)-ambrox with an engineered squalene hopene cyclase illustrates the potential of biotechnology for a more sustainable process, thus meeting the increasing consumers' demand for sustainably produced high quality perfumery and consumer goods. This review traces back to the origin of ambergris and the search for the source of its mysterious odor, leading to the discovery of (-)-ambrox as its main olfactive principle. It discusses the plethora of ways explored for its synthesis from diverse starting materials and presents the development of a process with significantly improved carbon efficiency for the industrial production of (-)-ambrox as 100% renewable Ambrofix.

Expanding the Cation Cage: Squalene-Hopene Cyclase-Mediated Enantioselective Semipinacol Rearrangement

Squalene-hopene cyclases (SHCs) are the biocatalytic pendant to asymmetric Brønsted-acid catalysis and thus comprise enormous synthetic potential. Nevertheless, their substrate scope is currently limited to terpenes. Herein, we present how to tailor the SHC’s cation cage for an enantioselective semipinacol rearrangement of bicyclic allylic alcohols to produce valuable oxa-carbon spirocyclic compounds. Exploiting the subtle divergence of SHC active sites combined with structure-guided semirational engineering, we designed a biocatalyst with a high catalytic performance of ∼4500 TTN and excellent enantioselectivity of 99.5% enantiomeric excess (ee). In silico studies suggest that a broadened active site is pivotal for catalysis. This intricate cationic rearrangement is easily scalable, employing lyophilized cell powder in water. Furthermore, our substrate scope studies demonstrate the acceptance of diverse ring-sized substrates but also reveal that the naturally confined active site limits the function as a general “semipinacolase.” This study showcases the ability to harness the SHC’s cation cage to tap into the broader field of asymmetric Brønsted-acid catalysis.

Chemoenzymatic synthesis of (+)-isoagatholactone, (+)-spongian-16-one, and 3-deoxychavalone A via biocatalytic polyene cyclization.

The stereoselective cyclization of geranylgeraniol catalysed by squalene-hopene cyclase (SHC) was investigated. By use of this transformation, spongiane diterpenoids (+)-isoagatholactone and (+)-spongian-16-one, and meroterpenoid 3-deoxychavalone A were synthesized in a concise and redox-economic manner. This work showcases the application of SHC-catalysed cyclization as a key step in terpenoid synthesis.

Spheroplasts preparation boosts the catalytic potential of a squalene-hopene cyclase

Squalene-hopene cyclases are a highly valuable and attractive class of membrane-bound enzymes as sustainable biotechnological tools to produce aromas and bioactive compounds at industrial scale. However, their application as whole-cell biocatalysts suffer from the outer cell membrane acting as a diffusion barrier for the highly hydrophobic substrate/product, while the use of purified enzymes leads to dramatic loss of stability. Here we present an unexplored strategy for biocatalysis: the application of squalene-hopene-cyclase spheroplasts. By removing the outer cell membrane, we produce stable and substrate-accessible biocatalysts. These spheroplasts exhibit up to 100-fold higher activity than their whole-cell counterparts for the biotransformations of squalene, geranyl acetone, farnesol, and farnesyl acetone. Their catalytic ability is also higher than the purified enzyme for all high molecular weight terpenes. In addition, we introduce a concept for the carrier-free immobilization of spheroplasts via crosslinking, crosslinked spheroplasts. The crosslinked spheroplasts maintain the same catalytic activity of the spheroplasts, offering additional advantages such as recycling and reuse. These timely solutions contribute not only to harness the catalytic potential of the squalene-hopene cyclases, but also to make biocatalytic processes even greener and more cost-efficient.

Production of cholesterol-like molecules impacts Escherichia coli robustness, production capacity, and vesicle trafficking

The economic viability of bioprocesses is constrained by the limited range of operating conditions that can be tolerated by the cell factory. Engineering of the microbial cell membrane is one strategy that can increase robustness and thus alter this range. In this work, we targeted cellular components that contribute to maintenance of appropriate membrane function, such as: flotillin-like proteins, membrane structural proteins, and membrane lipids. Specifically, we exploited the promiscuity of squalene hopene cyclase (SHC) to produce polycyclic terpenoids with properties analogous to cholesterol. Strains producing these cholesterol-like molecules were visualized by AFM and height features were observed. Production of these cholesterol-like molecules was associated with increased tolerance towards a diversity of chemicals, particularly alcohols, and membrane trafficking processes such as lipid droplet accumulation and production of extracellular vesicles. This engineering approach improved the production titers for wax-esters and ethanol by 80- and 10-fold, respectively. Expression of SHC resulted in the production of steroids. Strains engineered to also express truncated squalene synthase (tERG9) produced diplopterol and generally did not perform as well. Increased expression of several membrane-associated proteins, such as YqiK, was observed to impact vesicle trafficking and further improve tolerance relative to SHC alone, but did not improve bio-production. Deletion of YbbJ increased lipid droplet accumulation as well as production of intracellular wax esters. This work serves as a proof of concept for engineering strategies targeting membrane physiology and trafficking to expand the production capacity of microbial cell factories.

High-Level Squalene Production from Methane Using a Metabolically Engineered Methylomonas sp. DH-1 Strain

Methane is an inexpensive and sustainable raw material with high potential bioconversion into high-value-added compounds. We report the development of a metabolically engineered type I methanotroph, the Methylomonas sp. DH-1 strain, capable of producing squalene from methane. First, a base strain equipped with a Cre-lox-based automated genetic engineering system was constructed in the genome for efficient strain development. Second, the pds-ald-crtN2 gene cluster, which encodes phytoene desaturase, aldehyde dehydrogenase, and diapolycopene oxygenase, was deleted to increase the pool of presqualene diphosphate, an immediate precursor of squalene. Third, the activity of squalene–hopene cyclase was prohibited using ferulenol to concentrate the squalene. Finally, the squalene synthase regulated by a strong promoter was further overexpressed to carry strong flux toward squalene. The final engineered strain produced 8.4 ± 1.2 mg/L of squalene with the content and productivity of 12.0 ± 1.0 mg/g DCW and 58.7 ± 8.5 μg/(L/h), respectively. The fed-batch fermentation of the final engineered strain produced 31.3 mg/L of squalene with the content and productivity of 39.3 mg/g DCW and 746.2 μg/(L/h), respectively, by supplementing KNO3. The final engineered strain exhibited remarkable performance in the bioconversion of methane into squalene with the CH4 consumption rate, yield, and CH4 conversion rate values of 3.09 g/L, 10.2 mg of squalene per gram of methane, and 1.19%, respectively.

Titelbild: Asymmetric Cation‐Olefin Monocyclization by Engineered Squalene–Hopene Cyclases (Angew. Chem. 50/2021)

Aus Pottwalen gewonnener Ambra wird wegen seiner besonderen Geruchseigenschaften geschätzt. Heute sind synthetische Wege zum enthaltenen (S)-y-Dihydroionon und seinem optischen Antipoden, der einen blumigen Geruch verströmt, für die Duftstoffindustrie von Interesse. Im Forschungsartikel auf S. 26284 setzen Rebecca Buller et al. gentechnisch veränderte Squalen-Hopen-Zyklasen für die Monozyklisierung von geometrischen Geranylaceton-Isomeren zu y-Dihydroionon-Antipoden ein.

Cover Picture: Asymmetric Cation‐Olefin Monocyclization by Engineered Squalene–Hopene Cyclases (Angew. Chem. Int. Ed. 50/2021)

Sperm whale derived ambergris has historically been valued for its distinctive olfactory properties. Today, synthetic routes toward the contained (S)-y-dihydroionone and its optical antipode, emitting a floral orris-type odor, are of interest to the fragrance industry. In their Research Article on page 26080, Rebecca Buller et al. employ engineered squalene–hopene cyclases for the monocyclization of geometric geranylacetone isomers into y-dihydroionone antipodes.

Hopanoid Hopene Locates in the Interior of Membranes and Affects Their Properties.

Hopanoids are proposed as sterol surrogates in some bacteria, and it has been proved that some hopanoids are able to induce a liquid-order phase state in lipid membranes. The members of this group of molecules have diverse structures, and not all of them have been studied in detail yet. Here, we study membranes with the hopanoid hopene (hop-22 (29)-ene or diploptene), which is the product of the cycling of squalene by squalene-hopene cyclase, and thus is present in the first step of hopanoid biosynthesis. Hopene is particularly interesting because it lacks a polar head group, which opens the question of how does this molecule accommodate in a lipid membrane, and what are the effects promoted by its presence. In order to get an insight into this, we prepared monolayers and bilayers of a phospholipid with hopene and studied their properties in comparison with pure phospholipid membranes, and with the sterol cholesterol or the hopanoid diplopterol. Film stiffness, shear viscosity, and bending dynamics were very affected by the presence of hopene, while zeta-potential, generalized polarization of Laurdan, and conductivity were affected moderately by this molecule. The results suggest that at very low percentages, hopene locates parallel to the phospholipid molecules, while the excess of the hopene molecules stays between leaflets, as previously proposed using molecular dynamics simulations.

Asymmetric Cation-Olefin Monocyclization by Engineered Squalene-Hopene Cyclases.

Squalene–hopene cyclases (SHCs) have great potential for the industrial synthesis of enantiopure cyclic terpenoids. A limitation of SHC catalysis has been the enzymes’ strict (S)‐enantioselectivity at the stereocenter formed after the first cyclization step. To gain enantio‐complementary access to valuable monocyclic terpenoids, an SHC‐wild‐type library including 18 novel homologs was set up. A previously not described SHC (AciSHC) was found to synthesize small amounts of monocyclic (R)‐γ‐dihydroionone from (E/Z)‐geranylacetone. Using enzyme and process optimization, the conversion to the desired product was increased to 79 %. Notably, analyzed AciSHC variants could finely differentiate between the geometric geranylacetone isomers: While the (Z)‐isomer yielded the desired monocyclic (R)‐γ‐dihydroionone (>99 % ee), the (E)‐isomer was converted to the (S,S)‐bicyclic ether (>95 % ee). Applying the knowledge gained from the observed stereodivergent and enantioselective transformations to an additional SHC‐substrate pair, access to the complementary (S)‐γ‐dihydroionone (>99.9 % ee) could be obtained.

Asymmetric Cation‐Olefin Monocyclization by Engineered Squalene–Hopene Cyclases

AbstractSqualene–hopene cyclases (SHCs) have great potential for the industrial synthesis of enantiopure cyclic terpenoids. A limitation of SHC catalysis has been the enzymes’ strict (S)‐enantioselectivity at the stereocenter formed after the first cyclization step. To gain enantio‐complementary access to valuable monocyclic terpenoids, an SHC‐wild‐type library including 18 novel homologs was set up. A previously not described SHC (AciSHC) was found to synthesize small amounts of monocyclic (R)‐γ‐dihydroionone from (E/Z)‐geranylacetone. Using enzyme and process optimization, the conversion to the desired product was increased to 79 %. Notably, analyzedAciSHC variants could finely differentiate between the geometric geranylacetone isomers: While the (Z)‐isomer yielded the desired monocyclic (R)‐γ‐dihydroionone (>99 %ee), the (E)‐isomer was converted to the (S,S)‐bicyclic ether (>95 %ee). Applying the knowledge gained from the observed stereodivergent and enantioselective transformations to an additional SHC‐substrate pair, access to the complementary (S)‐γ‐dihydroionone (>99.9 %ee) could be obtained.