(-)-Ambrox, the smelling principle in ambergris of sperm whales, is a saturated polycyclic terpenoid ether with multiple tertiary and quaternary centers. Despite these structural features, it is readily biodegradable and thus represents an interesting case to understand the degradation pathway of a complex, saturated polycyclic terpene. We isolated two Sphingomonas spp. strains from activated sewage sludge capable of rapidly mineralizing (-)-Ambrox. The structures of seven isolated metabolites were determined by 2D-NMR, and further metabolite structures were proposed based on their exact masses. The use of two 13C-labeled substrates allowed completing the hypothesis for the biodegradation pathway. Biodegradation starts by oxidation of the α-C(4)-methyl group, followed by hydroxylation of the adjacent C(5)-angular CH-group, which allows opening of ring A by a unique retro-aldol process. β-Oxidation leads to a C11 metabolite, which is further degraded to a C6 δ-lactone. This metabolic fate is conserved among strains. The structure-biodegradation relationship indicates that only natural (-)-Ambrox and related labdanoids were mineralized, but not enantiomers and epimers. For both strains, the complete genome was sequenced; for one strain, a fosmid library was prepared, and the gene for the first oxidation step was cloned. The activity for the following step was located on a fosmid in a non-adjacent genomic region, indicating that this pathway is dispersed over the genome. This detailed study provides unique insights into the complex biodegradation pathway of the poorly studied class of saturated polycyclic terpenoids. IMPORTANCE:For complex, polycyclic natural substrates such as sesquiterpenes and diterpenes, there is little knowledge on biodegradation pathways, except for the elucidated steroid degradation pathways. This study presents key steps in the biodegradation pathway for (-)-Ambrox, a natural constituent of the sperm whale secretion ambergris, which was found to be readily biodegradable, despite a polycyclic ring system with multiple tertiary and quaternary carbon centers. This may be one of the first detailed studies on the biodegradation pathway of a polycyclic natural sesquiterpenoid.

In 2024, the Swiss Industrial Biocatalysis Consortium (SIBC), celebrated its 20 years of bringing together experts from the pharma, flavor and fragrance, fine chemicals, and agrochemicals industries to discuss enzyme technology developments. In this perspective, we share recent examples of how our member organizations utilize biocatalysis in their respective industries. While the motivations for employing enzymatic synthesis and the end goals of various production processes may vary, we aim to emphasize the shared aspects that we are coming across. Over the past 20 years, those synergies have provided us with a fruitful basis for pre-competitive knowledge sharing around biocatalysis as a technology. We look forward to many more years of the SIBC and the surprises that await us through the potential of our enzymes.

All 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. image

(–)-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.

To support perfumers in their creation of olfactive signatures resulting in unique and instantly recognizable perfumes, there is a constant demand for the development of new odorant molecules and of novel processes for their production. Increasing the sustainability of both the molecules and the processes is a crucial activity at Givaudan. Biocatalysis has the potential to positively influence metrics applied at Givaudan that drive and measure our ambition to innovate responsibly, which is summarized in the FiveCarbon Path™. It targets an increased use of renewable carbon, carbon efficiency in synthesis, and the production of powerful and biodegradable odorant molecules while maximizing the use of upcycled carbon available from waste and side streams. This review illustrates with some examples how enzymes selected from the oxidoreductase and isomerase enzyme classes are applied at Givaudan for the preparation of odorant molecules both at laboratory and industrial scale.

(-)-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.

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 ( Aci SHC) 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 Aci SHC 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.

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.

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.

Laccases (EC 1.10.3.2) are multi-copper oxidases that catalyse the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals. Owing to their broad substrate range, copper-containing laccases are versatile biocatalysts, capable of oxidizing numerous natural and non-natural industry-relevant compounds, with water as the sole by-product. In the present study, 10 of the 11 multi-copper oxidases, hitherto considered to be laccases, from fungi, plant and bacterial origin were compared. A substrate screen of 91 natural and non-natural compounds was recorded and revealed a fairly broad but distinctive substrate spectrum amongst the enzymes. Even though the enzymes share conserved active site residues we found that the substrate ranges of the individual enzymes varied considerably. The EC classification is based on the type of chemical reaction performed and the actual name of the enzyme often refers to the physiological substrate. However, for the enzymes studied in this work such classification is not feasible, even more so as their prime substrates or natural functions are mainly unknown. The classification of multi-copper oxidases assigned as laccases remains a challenge. For the sake of simplicity we propose to introduce the term "laccase-like multi-copper oxidase" (LMCO) in addition to the term laccase that we use exclusively for the enzyme originally identified from the sap of the lacquer tree Rhus vernicifera.

The potential of biotechnology by means of biocatalysis or biosynthesis in organic synthesis is far from being fully exploited. For this reason a group of life science companies active in pharmaceuticals, flavour and fragrance, vitamin and fine chemicals businesses describe some examples of the use of enzymes in industrial organic synthesis and discuss why enzymes are still the exception rather than the rule in organic synthesis.

The potential of biotechnology by means of biocatalysis or biosynthesis in organic synthesis is far from being fully exploited. For this reason a group of life science companies active in pharmaceuticals, flavour and fragrance, vitamin and fine chemicals businesses describe some examples of the use of enzymes in industrial organic synthesis and discuss why enzymes are still the exception rather than the rule in organic synthesis.

The mechanism and substrate specificity of alkanesulfonate monooxygenase (SsuD) was investigated by combining molecular dynamics simulations, docking, and a comprehensive quantitative structure activity relationships (QSAR) analysis. The FMNH2 dependent monooxygenase undergoes a dynamic conformational change of the active site, passing from a closed to an open state. As a consequence, substrates have access to the active site and the cofactor is then regenerated by the associated oxidoreductase FMN reductase SsuE.. Computational analysis of the interaction of SsuD with FMNH2 based on molecular docking and multiple 20 ns molecular dynamics simulations pointed out that the conformational change is mainly driven by salt bridge formation between Arg297 and Glu20 or Asp111. A set of substrates accepted by SsuD were described by means of ALMOND chemical descriptors and a partial least square (PLS) mathematical model was constructed. The PLS model correlates the structure of substrates and enzyme activity, namely kinetic properties (k cat/K M). Therefore, information coming from the PLS analysis goes beyond the simple ability of the enzyme to recognize the substrate, but includes the factors that affect the capacity of the enzyme to reduce the activation energy of the rate determining step of the reaction. The two principal components of the model are able to describe both steric and electronic factors and, more importantly, their interactions. Indeed, interactions of factors appear to affect significantly the ability of SsuD of transforming efficiently a substrate.

One of the most important objectives of the goals of Millennium Development Goals is to eradicate extreme poverty and hunger. Sufficient nourishment for the whole population of the world is one of the challenges of the present era. According to an estimate plant parasitic nematodes are causing much more damage annually compared to insect pests. A crop yield loss due to these tiny unseen pests in various countries is enormous. They caused projected yield loss of 12.3% ($157 billion dollars) worldwide. Out of which $40.3 million is reported from India. Farmers/growers identified insect pests, and other constraints as production problems but overlooked plant parasitic nematodes. Nematode diseases are difficult to control because of their hidden nature and hence, more often overlooked. Plant parasitic nematodes not only cause damage individually but form disease-complexes with other micro-organism and increased the crop loss. Also the symptoms of nematode damage are not specific, resemble with the symptoms of other pathogens and abiotic stresses such as water and mineral deficiency. Future agricultural growth must come from productivity growth to address the persistent problems of poverty, food insecurity and malnutrition. Recommended measures against nematode diseases include use of clean nematode free planting material, resistant varieties, and crop rotations to suppress nematode infestation. Integrated nematode management can be promoted through farmers-nematologists interactions, local production of bio-control agents and strict chemical pesticide regulations. Farmer's/grower's awareness and skills are equally important in minimizing nematode infestation and yield losses, to sustain the agriculture production.

The FMNH(2)-dependent alkanesulfonate monooxygenase SsuD catalyzes the conversion of alkanesulfonates to the corresponding aldehyde and sulfite. The enzyme allows Escherichia coli to use a wide range of alkanesulfonates as sulfur sources for growth when sulfate or cysteine are not available. The structure of SsuD was solved using the multiwavelength anomalous dispersion method from only four ordered selenium sites per asymmetric unit (one site per 20,800 Da). The final model includes 328 of 380 amino acid residues and was refined to an R-factor of 23.5% (R(free)=27.5%) at 2.3A resolution. The X-ray crystal structure of SsuD shows a homotetrameric state for the enzyme, each subunit being composed of a TIM-barrel fold enlarged by four insertion regions that contribute to intersubunit interactions. SsuD is structurally related to a bacterial luciferase and an archaeal coenzyme F(420)-dependent reductase in spite of a low level of sequence identity with these enzymes. The structural relationship is not limited to the beta-barrel region; it includes most but not all extension regions and shows distinct properties for the SsuD TIM-barrel. A likely substrate-binding site is postulated on the basis of the SsuD structure presented here, results from earlier biochemical studies, and structure relatedness to bacterial luciferase. SsuD is related to other FMNH(2)-dependent monooxygenases that show distant sequence relationship to luciferase. Thus, the structure reported here provides a model for enzymes belonging to this family and suggests that they might all fold as TIM-barrel proteins.

Twenty-three Escherichia coli strains were tested for their ability to use taurine, methanesulfonate, L-cysteate and other alkanesulfonates as sole sulfur sources for growth. One strain was unable to use any of the alkanesulfonates offered as sole sulfur sources for growth but grew with sulfate. Seven strains (class I) used alkanesulfonates for this purpose, but not methanesulfonate or L-cysteate. A further seven strains (class II) grew with all compounds tested, except with L-cysteate, and eight strains (class III) utilized all compounds tested as sulfur sources. Sulfur assimilation from methanesulfonate and L-cysteate was absolutely dependent on the ssuEADCB operon that encodes an alkanesulfonate uptake system (SsuABC) and a two-component monooxygenase (SsuDE) involved in the release of sulfite from alkanesulfonates. Long-term exposure of class I strains to methanesulfonate and of class II strains to L-cysteate selected for derivatives that utilized these two sulfur sources as efficiently as sulfate. The nucleotide sequence of the ssuEADCB operon in the methanesulfonate- and L-cysteate-utilizing derivative EC1250Me+ was identical to that in the class I wild-type EC1250. Gain of the ability to utilize methanesulfonate and L-cysteate as sulfur sources thus appears to result from increased expression of ssu genes rather than from a change in the quality of one or several of the Ssu proteins.

In the absence of sulfate and cysteine, Escherichia coli can use aliphatic sulfonates as a source of sulfur for growth. Starvation for sulfate leads to the expression of the tauABCD and ssuEADCB genes. Each of these gene clusters encodes an ABC-type transport system required for uptake of aliphatic sulfonates and a desulfonation enzyme. The TauD protein is an α-ketoglutarate-dependent dioxygenase that preferentially liberates sulfite from taurine (2-aminoethanesulfonic acid). SsuD is a monooxygenase that catalyzes the oxygenolytic desulfonation of a range of aliphatic sulfonates other than taurine. Its cosubstrate is FMNH2, which is provided by SsuE, an NAD(P)H-dependent FMN reductase. In contrast to many other bacteria, E. coli is unable to grow with arylsulfonates or with sulfate esters as sulfur source. The tau and ssu systems thus provide all genes for the utilization of known organosulfur sources by this organism, except the as yet unidentified gene(s) that enable some E. coli strains to grow with methanesulfonate or cysteate as a sulfur source. Expression of the tau and ssu genes requires the LysR-type transcriptional regulatory proteins CysB and Cbl. Synthesis of Cbl itself is under control of the CysB protein, and the CysB protein may therefore be regarded as the master regulator for sulfur assimilation in E. coli, while the Cbl protein functions as an accessory element specific for utilization of sulfur from organosulfur sources.

ABSTRACT The Escherichia coli tauABCD and ssuEADCB gene clusters are required for the utilization of taurine and alkanesulfonates as sulfur sources and are expressed only under conditions of sulfate or cysteine starvation. tauD and ssuD encode an α-ketoglutarate-dependent taurine dioxygenase and a reduced flavin mononucleotide-dependent alkanesulfonate monooxygenase, respectively. These enzymes are responsible for the desulfonation of taurine and alkanesulfonates. The amino acid sequences of SsuABC and TauABC exhibit similarity to those of components of the ATP-binding cassette transporter superfamily, suggesting that two uptake systems for alkanesulfonates are present in E. coli . Chromosomally located in-frame deletions of the tauABC and ssuABC genes were constructed in E. coli strain EC1250, and the growth properties of the mutants were studied to investigate the requirement for the TauABC and SsuABC proteins for growth on alkanesulfonates as sulfur sources. Complementation analysis of in-frame deletion mutants confirmed that the growth phenotypes obtained were the result of the in-frame deletions constructed. The range of substrates transported by these two uptake systems was largely reflected in the substrate specificities of the TauD and SsuD desulfonation systems. However, certain known substrates of TauD were transported exclusively by the SsuABC system. Mutants in which only formation of hybrid transporters was possible were unable to grow with sulfonates, indicating that the individual components of the two transport systems were not functionally exchangeable. The TauABCD and SsuEADCB systems involved in alkanesulfonate uptake and desulfonation thus are complementary to each other at the levels of both transport and desulfonation.

In einer sauerstoffabhängigen Reaktion chloriert die Tryptophan-7-Halogenase PrnA Tryptophan 1 regioselektiv an der 7-Position unter Bildung von 2. Bei der Reinigung der Halogenase stellte sich heraus, dass für die Reaktion neben NADH auch FAD und eine Flavin-Reduktase (Bildung von FADH2) benötigt werden.

In an oxygen-dependant reaction, tryptophan 7-halogenase (PrnA) regioselectively chlorinates tryptophan (1) at the 7-position to yield 2. During purification of the halogenase, a flavin reductase, as second protein component, was detected in addition to NADH and FAD; this second protein is also required for the reaction.