Biochemistry of lactone formation in yeast and fungi and its utilisation for the production of flavour and fragrance compounds

The consumers’ demand for natural flavour and fragrances rises. To be natural, compounds have to result from the extraction of natural materials and/or to be transformed by natural means such as the use of enzymes or whole cells. Fungi are able to transform some fatty acids into lactones that can thus be natural. Although some parts of this subject have been reviewed several times, the present article proposes to review the different pathways utilised, the metabolic engineering strategies and some current concerns on the reactor application of the transformation including scaling up data. The main enzymatic steps are hydroxylation and β-oxidation in the traditional way, and lactone desaturation or Baeyer–Villiger oxidation. Although the pathway to produce γ-decalactone is rather well known, metabolic engineering strategies may result in significant improvements in the productivity. For the production of other lactones, a key step is the hydroxylation of fatty acids. Beside the biotransformation, increasing the production of the various lactones requires from biotechnologists to solve two main problems which are the toxicity of lactones toward the producing cell and the aeration of the emulsified reactor as the biochemical pathway is very sensitive to the level of available oxygen. The strategies employed to resolve these problems will be presented.     

Hirsutinolides,glaucolides and sesquiterpene lactone from Vernonia species

The investigation of nine Vernonia species afforded in addition to known sesquiterpenes 28 new ones. The structures were elucidated by high field ¹H NMR spectroscopy and the configurations were determined by NOE difference spectroscopy and, in one case, by X-ray analysis. The results indicated that configurations of several previously reported sesquiterpene lactones have had to be revised. In addition to known types two new ones, the jalcaguaianolides and the vernojalcanolides, are described. Furthermore some unusual reaction products are presented which, in part, led to some natural occurring lactones.     

Germacranolides,a guaianolide with a β-lactone ring and further constituents from Grazielia species

An investigation of three Grazielia species afforded, in addition to known compounds, several new sesquiterpene lactones, eight germacranolides, two melampolides and one guaianolide as well as three diterpenes, two geranylgeraniol derivatives and a labda-diene. Three of the sesquiterpene lactones had an additional β-lactone ring, one an acid function, and two others unusual ester functions. The structures were elucidated by spectroscopic methods and some chemical transformations. The chemotaxonomic situation is discussed briefly.     

Preparation of Optically Active γ- and δ-Lactones by Microbiological Reduction of the Corresponding Keto Acids

It was found, by means of simple screening methods, that several yeasts (Saccharomyces and Candida), molds (Cladosporium), and bacteria (Sarcina) are able to reduce keto acids to hydroxy acids, which are easily converted into lactones. Chemical analysis showed that some of the microorganisms (Saccharomyces and Candida) produce dextrorotatory lactones and others (Cladosporium and Sarcina) produce levorotatory lactones. High yields of dextrorotatory (both γ- and δ-lactones) were obtained by using Saccharomyces cerevisiae. The physical properties of the carefully distilled lactones obtained indicated high purity and high optical purity.     

Conformations of methyl 2,5-di-O-acetyl-beta-D-glucofuranosidurono-6,3-lactone and 1,2,5-tri-O-acetyl-beta-D-glucofuranurono-6,3-lactone in the crystal structure and in solution

The single-crystal X-ray diffraction and high-resolution 1H and 13C NMR spectral data for methyl 2,5-di-O-acetyl-beta-D-glucofuranosidurono-6,3-lactone and 1,2,5-tri-O-acetyl-beta-D-glucofuranurono-6,3-lactone are reported. The lactones were synthesized as byproducts of reactions carried out to obtain methyl 1,2,3,4-tetra-O-acetyl-D-glucopyranuronate. The conformations of these lactones in the crystal structure and in solution are discussed. A 1T2-like conformation was found to be the preferred form for these lactones in both the crystal lattice and in solution.     

Fingerprint profile of Ginkgo biloba nutritional supplements by LC/ESI-MS/MS

Ginkgo biloba is one of the most popular herb nutrition supplements, with terpene lactones and flavonoids being the two major active components. A fingerprint profile method was developed using a capillary HPLC/MS method which can identify more than 70 components from the G. biloba product. The method allows the flavonoids and terpene lactones to be detected simultaneously and information of both the parent ion and its fragmentation can be obtained in just one HPLC/MS run. Targeted post-acquisition analysis allows mass spectrometric information regarding the identification of flavonoid components to be easily distinguished from other data, however the same approach for terpene lactones was less successful due to dimer formation and requires further development. The fingerprint profiles of five commercial G. biloba nutritional supplements were obtained and compared; variation of some components among the samples was observed and fortification could be detected. In the quality control analysis of the G. biloba product this method could be viewed as complementary to specific quantitative analysis of some bioactive components of the herb.     

11β,13-Dihydroguaianolides from Artemisia douglasiana and a thiophene acetylene from A. schmidtiana

The aerial parts of Artemisia douglasiana afforded, in addition to some known sesquiterpene lactones, 22 new closely related guaianolides. A new thiophene acetylene was isolated from the aerial parts of A. schmidtiana together with some known sesquiterpene lactones. The structures were elucidated by high field ¹H NMR spectroscopy.     

The fumigant and repellent activity of aliphatic lactones against Pediculus humanus capitis (Anoplura: Pediculidae)

New alternative insecticides are necessary for the chemical control of head lice. In this study the fumigant knockdown time 50% (KT50) and repellency index (RI) of three aliphatic lactones was compared with two essential oils and DDVP, against permethrin-resistance Pediculus humanus capitis from Argentina. In the fumigant assay, none of the lactones were effective compared to the highest activity of eucalyptus (KT50 15.53 m). In the repellency test, the three lactones were equally or more effective (RI ranging from 60.50 to 76.68) than the positive control (piperonal). These lactones are promising as head lice repellents.     

A new type of germacranolide from Vernonia species

From several South African Vernonia species, a considerable number of sesquiterpene lactones has been isolated. Together with known compounds, seven members of a new type with an enol lactone ring have been found, their structures being elucidated by spectroscopic methods and some chemical transformations. Furthermore two new derivatives of costunolide are present in two species, and three new guaianolides have been found. From some species, however, no lactones could be isolated. Together with a new bisabolene derivative, a known eremophilone has been isolated, probably for the first time, from a member of the tribe Vernonieae.     

Long-chain acyl-homoserine lactone quorum-sensing regulation of Rhodobacter capsulatus gene transfer agent production

Many proteobacteria use acyl-homoserine lactones as quorum-sensing signals. Traditionally, biological detection systems have been used to identify bacteria that produce acyl-homoserine lactones, although the specificities of these detection systems can limit discovery. We used a sensitive approach that did not require a bioassay to detect production of long-acyl-chain homoserine lactone production by Rhodobacter capsulatus and Paracoccus denitrificans. These long-chain acyl-homoserine lactones are not readily detected by standard bioassays. The most abundant acyl-homoserine lactone was N-hexadecanoyl-homoserine lactone. The long-chain acyl-homoserine lactones were concentrated in cells but were also found in the culture fluid. An R. capsulatus gene responsible for long-chain acyl-homoserine lactone synthesis was identified. A mutation in this gene, which we named gtaI, resulted in decreased production of the R. capsulatus gene transfer agent, and gene transfer agent production was restored by exogenous addition of N-hexadecanoyl-homoserine lactone. Thus, long-chain acyl-homoserine lactones serve as quorum-sensing signals to enhance genetic exchange in R. capsulatus.     

Allenic germacranolides,bourbonene derived lactones and other constituents from Vernonia species

An investigation of seven Vernonia species, mainly from northern Brazil, afforded in addition to known compounds three new unusual sesquiterpene lactones with an allenic group. Intensive NMR studies fully established the structures. Furthermore, two lactones derived from bourbonene, two new glaucolides, a new furansesquiterpene, a nerolidol derivative and two other sesquiterpenes derived from eudesmane were isolated. The biogenetic relationships of the sesquiterpene lactones present in Veronia species as well as the chemotaxonomic aspects are discussed.     

Sesquiterpene lactones and their precursors as chemosystematic markers in the tribe Cichorieae of the Asteraceae

This review summarizes all reports on sesquiterpene lactones and their immediate precursors from the Cichorieae (Lactuceae) tribe of the Asteraceae. A total of 360 compounds have been reported from this tribe. The reported substances belong to three classes of sesquiterpenoids: guaianolides (243 compounds), eudesmanolides (73 compounds), and germacranolides (44 compounds). Sources of these compounds encompass 139 taxa from 31 different genera. The distribution of these lactones within the tribe Cichorieae is discussed in a chemosystematic context. Moreover, some general ideas about the interpretation of chemosystematic data are discussed.     

A chemotaxonomic survey of kaurene derivatives in the genus Alepidea (Apiaceae)

Lipophilic extracts from the roots of 16 species of Alepidea were studied by GC-MS for the presence of kaurenoic acids, dehydrokaurenoic acids, kaurenic lactones, hydroxykaurenoic acids and other kaurene derivatives. Various mixtures of these compounds are present in yields of up to 11.8% dry wt. in the rhizomes and roots. Two different isomers of kaurenoic acid occur in Alepidea, the one as a major compound in all the species, the other as a major compound only in some of them. The first isomer is clearly a useful chemical marker for the genus Alepidea, but the apparent chemical dichotomy is not obviously correlated with morphological patterns. Alepidea amatymbica, however, is part of a morphologically isolated group within the genus and also differs from other species in its chemical complexity and in the unique presence of kaurenoic lactones.     

Neue melampolide und cis,cis-germacranolide aus vertretern der subtribus melampodiinae

The investigation of four South American species of the tribe Heliantheae afforded in addition to known compounds 14 new sesquiterpene lactones, mostly belonging to the melampolides, but four being further examples of cis,cis-germacranolides. The stereochemistry is established by intensive ¹H-NMR studies and some chemical transformations. Some of the new lactones were only obtainable as inseparable mixtures of 2-methylbutyric and isovaleric esters. Furthermore a new acetylenic aldehyde was isolated. The chemotaxonomic situation is discussed especially with regard to the position of the genus Smallanthus.     

Characterization of the PON1 active site using modeling simulation, in relation to PON1 lactonase activity

Paraoxonase1 (PON1) is a HDL bound enzyme and many of the anti-atherogenic properties of HDL are attributed to PON1. The enzyme precise mechanism of protective action and its endogenous substrate remain elusive. PON1 hydrolyzes organophosphates, arylesters and lactones, whereas the lactones activity is assumed as the physio/pathological one. This study is aimed to predict the location of the PON1 active site within PON1 crystal structure, and the lactone structure suitability as PON1 ligand, by employing modeling techniques. Based on such calculations the ligands-PON1 interactions were characterized, and relating lactones rate of hydrolysis revealed an inverse correlation with the docking energy of the ligands-PON1 complex, and a direct correlation with the lactone side chain length. In conclusion, this study characterized the PON1 possible active site and proposes a tool which may make it possible to envisage the structure of potential endogenous and exogenous lactones such as the PON1 ligand.     

应用生物法合成内酯化合物

内酯是广泛存在于自然界中具有生物活性的一类化合物。由于大多数内酯化合物具有手性,用化学方法合成不仅过程复杂,而且产率也不高。利用酶反应的特异性,应用生物法合成内酯化合物具有很好的应用前景,其中包括微生物次生代谢合成内酯,脂肪酸生物转化合成内酯和脂肪酶在有机相中催化羟基脂肪酸形成内酯。本文报道这些领域的进展。     

The Evolutionary Origins of Detoxifying Enzymes: THE MAMMALIAN SERUM PARAOXONASES (PONs) RELATE TO BACTERIAL HOMOSERINE LACTONASES*

Serum paraoxonases (PONs) are detoxifying lactonases that were first identified in mammals. Three mammalian families are known, PON1, 2, and 3 that reside primarily in the liver. They catalyze essentially the same reaction, lactone hydrolysis, but differ in their substrate specificity. Although some members are highly specific, others have a broad specificity profile. The evolutionary origins and substrate specificities of PONs therefore remain poorly understood. Here, we report a newly identified family of bacterial PONs, and the reconstruction of the ancestor of the three families of mammalian PONs. Both the mammalian ancestor and the characterized bacterial PONX_OCCAL were found to efficiently hydrolyze N-acyl homoserine lactones that mediate quorum sensing in many bacteria, including pathogenic ones. The mammalian PONs may therefore relate to a newly identified family of bacterial, PON-like “quorum-quenching” lactonases. The appearance of PONs in metazoa is likely to relate to innate immunity rather than detoxification. Unlike the bacterial PON, the mammalian ancestor also hydrolyzes, with low efficiency, lactones other than homoserine lactones, thus preceding the detoxifying functions that diverged later in two of the three mammalian families. The bifunctionality of the mammalian ancestor and the trade-off between the quorum-quenching and detoxifying lactonase activities explain the broad and overlapping specificities of some mammalian PONs versus the singular specificity of others.     

PA-I lectin from Pseudomonas aeruginosa binds acyl homoserine lactones

The study analyses the binding affinities of Pseudomonas aeruginosa PA-I lectin (PA-IL) to three N-acyl homoserine lactones (AHSL), quorum sensing signal molecules responsible for cell-cell communication in bacteria. It shows that like some plant lectins, PA-IL has a dual function and, besides its carbohydrate-binding capacity, can accommodate AHLS. Formation of complexes between PA-IL and AHSL with acyl side chains composed of 4, 6 or 12 methyl groups is characterized by changes in the emissions of two incorporated fluorescent markers, TNS and IAEDANS, both derivatives of naphthalene sulfonic acid. PA-IL shows increasing affinities to lactones with longer aliphatic side chains. The values of the apparent dissociation constants (K(d)), which are similar to the previously determined K(d) for the adenine high affinity binding, and the similar effects of lactones and adenine on the TNS emission indicate one identical binding site for these ligands, which is suggested to represent the central cavity of the oligomeric molecule formed after the association of the four identical subunits of PA-IL. Intramolecular distances between the fluorescent markers and protein Trp residues are determined by fluorescence resonance energy transfer (FRET).