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.

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.

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.

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.

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.

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.

4-4' Diaponeurosporenic Acid, the C30 Carotenoid Pigment in Endophytic Pseudomonas Mendocina with Squalene Cyclase Activity.

Even though organisms with squalene hopene cyclase activity involved in hopanoid synthesis has been reported earlier, their existence along with carotenoid synthesis is rarely reported. Here, we report the existence of hopanoid and C30 carotenoid biosynthetic pathway in Pseudomonas mendocina, the squalene hopene cyclase producing endophyte of the medicinal plant Murraya koenigii. The enzyme squalene hopene cyclase from Pseudomonas mendocina is involved in the synthesis of dehydrosqualene-mediated alternate pathway for carotenoid biosynthesis. The hopanoids are involved in membrane stability and integrity, and the carotene chromophores are involved in the photo protection of the cell. The orange-colored C30 carotenoid pigment 4-4' diaponeurosporenic acid in the extracellular extract of Pseudomonas mendocina with squalene cyclase activity was detected by the combination of UV/Vis spectrometry, FTIR, and Mass Spectrometry. 4-4' diaponeurosporenic acid could be traced as the end product of the carotenoid pathway and belonged to the xanthophyll group of carotenoids.

In Vitro Enzymatic Conversion of Glibenclamide Using Squalene Hopene Cyclase from Pseudomonas mendocina Expressed in E. coli BL21 (DE3).

Squalene hopene cyclases catalyse the conversion of a linear substrate squalene to a cyclic product with high stereo-selectivity.The enzyme squalene hopene cyclase from Pseudomonas mendocina expressed in E. coli BL21 (DE3) was evaluated for its synthetic drug transforming ability. Nine synthetic drugs were selected as substrates for biotransformation reactions by the enzyme. The homology modelling of the protein and docking of the selected ligands were performed using GOLD suite docking software. The drug which showed maximum binding with the active-site residues of the enzyme was selected for biotransformation studies. On transformation with the enzyme, Glibenclamide, the selected antidiabetic drug alone showed significant changes in the FT/IR spectra; hence, it was selected for LCMS analysis to confirm the transformations. From the chromatogram and MS spectra, the mono-oxygenation of the product due to the enzymatic activity was confirmed. The drug transforming ability of the purified SHC could be used as an ideal tool for the generation of new and active substrate derivatives.

Introduction of a green algal squalene synthase enhances squalene accumulation in a strain of Synechocystis sp. PCC 6803

Squalene is a triterpene which is produced as a precursor for a wide range of terpenoid compounds in many organisms. It has commercial use in food and cosmetics but could also be used as a feedstock for production of chemicals and fuels, if generated sustainably on a large scale. We have engineered a cyanobacterium, Synechocystis sp. PCC 6803, for production of squalene from CO2. In this organism, squalene is produced via the methylerythritol-phosphate (MEP) pathway for terpenoid biosynthesis, and consumed by the enzyme squalene hopene cyclase (Shc) for generation of hopanoids. The gene encoding Shc in Synechocystis was inactivated (Δshc) by insertion of a gene encoding a squalene synthase from the green alga Botryococcus braunii, under control of an inducible promoter. We could demonstrate elevated squalene generation in cells where the algal enzyme was induced. Heterologous overexpression of genes upstream in the MEP pathway further enhanced the production of squalene, to a level three times higher than the Δshc background strain. During growth in flat panel bioreactors, a squalene titer of 5.1 ​mg/L of culture was reached.

In silico characterization and over-expression of squalene hopene cyclase from Pseudomonas mendocina.

Pseudomonas mendocina was identified as a novel endophytic isolate of Murraya koenigii with squalene cyclase activity. The PCR amplification of squalene hopene cyclase (shc) gene from the isolate Pseudomonas mendocina with the primers PA1/PA2 showed a band at 1980bp specific for the enzyme squalene hopene cyclase. The in silico translation of the squalene hopene cyclase gene showed 96% sequence similarity with squalene hopene cyclase of Pseudomonas agarici (WP-060782422). Docking studies of the template and the modeled protein with the ligand squalene showed that the main interacting residues were Asp376 and Asp377. Squalene hopene cyclase template 1 sqc.1A sequence from Alicyclobacillus acidocaldaruis was used as the template for docking experiments. The gene coding for squalene hopene cyclase from Pseudomonas mendocina has been cloned in pET-28a vector to produce recombinant vector and was expressed in E.coli BL21 (DE3) expression system. Squalene hopene cyclase enzyme was isolated, purified and the molecular weight was confirmed by SDS-PAGE as 75KDa.

Linear enzyme cascade for the production of (-)-iso-isopulegol.

Biocatalysis has developed enormously in the last decade and now offers solutions for the sustainable production of chiral and highly functionalised asset molecules. Products generated by enzymatic transformations are already being used in the food, feed, chemical, pharmaceutical and cosmetic industry, and the accessible compound panoply is expected to expand even further. In particular, the combination of stereo-selective enzymes in linear cascade reactions is an elegant strategy toward enantiomeric pure compounds, as it reduces the number of isolation and purification steps and avoids accumulation of potentially unstable intermediates. Here, we present the set-up of an enzyme cascade to selectively convert citral to (-)-iso-isopulegol by combining an ene reductase and a squalene hopene cyclase. In the initial reaction step, the ene reductase YqjM from Bacillus subtilis selectively transforms citral to (S)-citronellal, which is subsequently cyclised exclusively to (-)-iso-isopulegol by a mutant of the squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC). With this approach, we can convert citral to an enantiopure precursor for isomenthol derivatives.

(–)-Ambrox Production by Biocatalysis Using Squalene Hopene Cyclase

(–)-Ambrox Production by Biocatalysis Using Squalene Hopene Cyclase

Biocatalytic Process for (−)‐Ambrox Production Using Squalene Hopene Cyclase

AbstractAlicyclobacillus 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® expands the biocatalysis toolbox by setting out a general basis for biocatalytic Squalene Hopene Cyclase cyclization reactions at industrial scale.magnified image

MD Simulations Reveal Complex Water Paths in Squalene-Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site.

Squalene–hopene cyclase catalyzes the cyclization of squalene to hopanoids. A previous study has identified a network of tunnels in the protein, where water molecules have been indicated to move. Blocking these tunnels by site-directed mutagenesis was found to change the activation entropy of the catalytic reaction from positive to negative with a concomitant lowering of the activation enthalpy. As a consequence, some variants are faster and others are slower than the wild type (wt) in vitro under optimal reaction conditions for the wt. In this study, molecular dynamics (MD) simulations have been performed for the wt and the variants to investigate how the mutations affect the protein structure and the water flow in the enzyme, hypothetically influencing the activation parameters. Interestingly, the tunnel-obstructing variants are associated with an increased flow of water in the active site, particularly close to the catalytic residue Asp376. MD simulations with the substrate present in the active site indicate that the distance for the rate-determining proton transfer between Asp376 and the substrate is longer in the tunnel-obstructing protein variants than in the wt. On the basis of the previous experimental results and the current MD results, we propose that the tunnel-obstructing variants, at least partly, could operate by a different catalytic mechanism, where the proton transfer may have contributions from a Grotthuss-like mechanism.

Selectivity in the Cyclization of Citronellal Introduced by Squalene Hopene Cyclase Variants

AbstractThe squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC) is a highly efficient enzyme catalyst for stereoselective Brønsted acid catalysis. We engineered AacSHC to catalyze the selective Prins cyclization of citronellal. Four active site variants were identified for the diastereoselective cyclization of (S)‐citronellal to stereoisomers (−)‐iso‐isopulegol, (+)‐isopulegol and (−)‐neo‐isopulegol, respectively. The replacement of active site residues resulted in two triple variants that catalyzed the transformation of (R)‐citronellal to give the isomers (+)‐neo‐isopulegol and (−)‐isopulegol with up to >99 % de, respectively. The newly designed library of functionally diverse active site geometries exhibits high selective control during citronellal cyclization, leading exclusively to a single diastereomer of the desired isopulegol. Whereas the cyclization of citronellal with chemical catalysts was observed to produce the isopulegol isomer with the lowest energy, the reaction with AacSHC variants proceeded with higher product selectivity. The results of this study show that variants of AacSHC are excellent catalysts for the highly selective formation of isopulegol stereoisomers.