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 ß-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.

The utilization of gum tragacanth to improve the growth of Rhodotorula aurantiaca and the production of gamma-decalactone in large scale.

The production of gamma-decalactone and 4-hydroxydecanoic acid by the psychrophilic yeast R. aurantiaca was studied. The effect of both compounds on the growth of R. aurantiaca was also investigated and our results show that gamma-decalactone must be one of the limiting factors for its production. The addition of gum tragacanth to the medium at concentrations of 3 and 4 g/l seems to be an adequate strategy to enhance gamma-decalactone production and to reduce its toxicity towards the cell. The production of gamma-decalactone and 4-hydroxydecanoic acid was significantly higher in 20-l bioreactor than in 100-l bioreactor. By using 20 g/l of castor oil, 6.5 and 4.5 g/l of gamma-decalactone were extracted after acidification at pH 2.0 and distillation at 100 degrees C for 45 min in 20- and 100-l bioreactors, respectively. We propose a process at industrial scale using a psychrophilic yeast to produce naturally gamma-decalactone from castor oil which acts also as a detoxifying agent; moreover the process was improved by adding a natural gum.

The use of Macronet resins to recover gamma-decalactone produced by Rhodotorula aurantiaca from the culture broth.

During the biotransformation of castor oil into gamma-decalactone, R. aurantiaca produced both the lactone form and its precursor (4-hydroxydecanoic acid). After six days of culture, a maximum yield of gamma-decalactone of 6.5 g/l was obtained. The parameters of gamma-decalactone adsorption on three Macronet resins (MN-202, MN-102 and MN-100) were investigated in water. Adsorption isotherms of gamma-decalactone for the three Macronet resins were linear. The trapping of gamma-decalactone produced by R. aurantiaca on these resins was then carried out. gamma-Decalactone was effectively retained by all the studied Macronet resins. The resin MN-202 trapped gamma-decalactone more efficiently than MN-102 and MN-100. The percentages of gamma-decalactone adsorbed on the resins MN-202, MN-102 and MN-100 were, respectively, 85, 75 and 81%, whereas around 70% of the adsorbed gamma-decalactone was then desorbed. We propose an industrial process that uses Macronet resins to extract gamma-decalactone from culture broth of R. aurantiaca.

Production of gamma-decalactone by a psychrophilic and a mesophilic strain of the yeast Rhodotorula aurantiaca.

Among 18 psychrophilic strains isolated near the Antarctic Station, the psychrophilic strain Rhodotorula aurantiaca A19 was selected for its ability of growth and gamma-decalactone production at low temperatures. The effects of temperature, initial pH, and castor oil concentration on the growth and gamma-decalactone production by a psychrophilic and a mesophilic strain of R. aurantiaca were investigated. The highest gamma-decalactone production in flasks (5.8 g/l) was obtained with the strain A19 at 14 degrees C and initial pH 7.0 in medium containing 20 g/l castor oil. On the other hand, these factors did not affect the production of gamma-decalactone by the mesophilic strain. In fermentor, a gamma-decalactone concentration of 6.6 g/l was reached with the strain A19, whereas a maximum of 0.1 g/l was obtained with the mesophilic strain. Our results suggest that the ability to synthesize gamma-decalactone is a particularity of the strain A19, since the mesophilic strain (no. 30645) produced small amounts, and the other (no. 31354) did not exhibit this property. It is, to our knowledge, the first report of gamma-decalactone production by R. aurantiaca and furthermore by a psychrophilic yeast strain. Moreover, the amount of gamma-decalactone obtained in fermentor with the strain 19 was on the order of concentrations usually described in patents.