PROCESS FOR PREPARING PHENYLACETIC ACID

Abstract

A process for preparing phenylacetic acid is provided. The process essentially includes the steps of providing one or more yeast strains belonging to the genus Yarrowia and mutants thereof, providing a culture medium including phenylalanine, transforming phenylalanine into phenylacetic acid by fermentation of the one or more yeast strains in the culture medium, the phenylacetic acid being contained in a fermentation broth obtained by the fermentation, and isolating the phenylacetic acid from the fermentation broth.

Claims

1. A process for preparing phenylacetic acid comprising the steps of: providing one or more yeast strains belonging to the genus Yarrowia and mutants thereof; providing a culture medium comprising phenylalanine; transforming phenylalanine into phenylacetic acid by fermentation of said one or more yeast strains in said culture medium, said phenylacetic acid being contained in a fermentation broth obtained by said fermentation; isolating the phenylacetic acid from said fermentation broth.

2. The process according to claim 1, wherein said phenylalanine is (L)-phenylalanine.

3. The process according to claim 1, wherein said fermentation is a ‘one pot’ fermentation.

4. The process according to claim 1, wherein said fermentation is a single fermentation and phenylalanine is transformed directly into phenylacetic acid.

5. The process according to claim 1, wherein the phenylalanine contained in said culture medium has a concentration between 10 and 15 g/L.

6. The process according to claim 1, wherein said fermentation is carried out: at a temperature between 20 and 32° C.; at a pH between 5.5 and 7.5; over a time period between 4 and 10 days.

7. The process according to claim 1, wherein said fermentation is carried out under aerobic conditions: in a flask, by using a volume of fermentation broth smaller than one fifth of the volume of the flask and by stirring at a speed higher than 80 rpm, or in a fermenter, by using an airflow such that it ensures an oxygen partial pressure higher than 10% over the entire duration of fermentation.

8. The process according to claim 7, wherein said fermentation in the fermenter is carried out by using an airflow greater than 0.2 v/v/min and by stirring at a speed higher than 100 rpm.

9. The process according to claim 1, wherein said yeast strains belonging to the genus Yarrowia and mutants thereof are non-genetically modified strains.

10. The process according to claim 1, wherein said yeast strains belonging to the genus Yarrowia are strains belonging to the species Yarrowia lipolytica and mutants thereof.

11. The process according to claim 1, wherein said yeast strains belonging to the genus Yarrowia are strains belonging to the species Yarrowia deformans or Yarrowia yakushimensis or Yarrowia bubula and mutants thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0039] These and other features and advantages of the present invention will become more apparent from the following description of preferred embodiments given by way of non-limiting examples with reference to the accompanying drawings, in which:

[0040] FIG. 1 is a representation of the known Ehrlic metabolic pathway through which (L)-phenylalanine is transformed into phenylacetaldehyde, phenylethanol and phenylacetic acid;

[0041] FIG. 2 is a representation of the direct transformation of (L)-phenylalanine into phenylacetic acid according to the invention.

DESCRIPTION OF EMBODIMENTS

[0042] A process for preparing phenylacetic acid from phenylalanine, and, in particular, natural phenylacetic acid from natural (L)-phenylalanine is described below. The process provides for a single fermentation of yeast strains belonging exclusively to the genus Yarrowia in a culture medium containing phenylalanine, preferably natural (L)-phenylalanine, at a concentration preferably lower than 20 g/L, more preferably between 10 and 15 g/L.

[0043] In particular, known strains belonging to the genus Yarrowia and mutants thereof, and preferably strains belonging to the species Yarrowia lipolytica and mutants thereof, are used. Preferably, instead, genetically modified microorganisms (GMO), even when belonging to the genus Yarrowia, are not used.

[0044] The fermentation of yeast strains according to the invention is a fermentation of the ‘one pot’ type and is conducted under aerobic conditions.

[0045] This fermentation enables the direct transformation of the amino acid (L)-phenylalanine into phenylacetic acid.

[0046] The conditions under which the process is carried out are the following: [0047] reaction temperature between 20 and 32° C., more preferably between 26 and 30° C.; [0048] pH in a range between 5.5 and 7.5, more preferably of 6.5; [0049] fermentation duration between 4 and 10 days, more preferably 7 days.

[0050] In addition, the formation of phenylacetic acid by means of said fermentation is suitably obtained under conditions of efficient stirring and oxygenation. These conditions can be guaranteed in accordance with one of the following alternative experimental conditions:

a) Fermentation in a flask:
Use of a volume of fermentation broth preferably smaller than one fifth of the flask volume and stirring at a speed preferably higher than 80 rpm.
b) Fermentation in a fermenter (bioreactor):
Use of an airflow such that it ensures a oxygen partial pressure (pO.sub.2) higher than 10% (relative to a maximum value of pO.sub.2 that can be obtained with an airflow within the fermenter containing the culture medium before the addition of Yarrowia) over the entire duration of fermentation. This oxygen pressure is suitably guaranteed by means of an airflow greater than 0.2 v/v/min and by stirring at a speed higher than 100 rpm.

[0051] The molar yields of the transformation of (L)-phenylalanine into phenylacetic acid according to the process of the invention range from 50 to 85%, depending on the yeast strain used.

[0052] The molar yields of the transformation of racemic phenylalanine (DL-phenylalanine) into phenylacetic acid according to the process of the invention range from 25 to 80%, depending on the yeast strain used.

[0053] At the end of fermentation, the natural phenylacetic acid is conveniently isolated by means of extraction and distillation/crystallization methods.

[0054] The process of the invention is suitably conducted by using yeasts belonging to the genus Yarrowia.

[0055] According to the procedures described above, some illustrative and non-limiting examples of the process according to the invention are provided below.

Esempio 1

[0056] In a fermenter, there are loaded 2 L of a liquid culture medium having the following composition: yeast extract (3 g/L), malt extract (3 g/L), soybean peptone (5 g/L), glucose (10 g/L) and (L)-phenylalanine (12.5 g/L).

[0057] The fermenter is then sterilized at 121° C. for 15 minutes and the pH is adjusted at 6.50 by adding solutions of ammonia (10% w/v) or acetic acid (10% w/v).

[0058] The fermenter is subsequently stirred at 250 rpm at 28° C. and Yarrowia lipolytica (about 5 of wet yeast obtained by centrifugation of an active culture at about 3500×g) suspended in about 10 mL of saline is then added thereto. In particular, the strain used in this example is Yarrowia lipolytica DSM 8218, belonging to the International Collection DSMZ (German Collection of Microorganisms and Cell Cultures GmbH).

[0059] A continuous airflow (about 1 L/min) is then introduced and fermentation is effected for 7 days, while continuing stirring the fermenter (at about 250 rpm).

[0060] The fermentation (biotransformation) is then stopped by adding an acid (for example, citric acid) until a pH of 4.0-4.5 is reached.

[0061] At this time, the fermentation broth is filtered through Celite and extracted with ethyl acetate (4×200 mL).

[0062] The combined organic phases resulting from the preceding extraction are then dried over anhydrous sodium sulfate and are subsequently concentrated under reduced pressure.

[0063] The residue (17.4 g) obtained from the preceding step crystallizes at room temperature and consists of quasi-pure phenylacetic acid.

[0064] Subsequent distillation of the residue under reduced pressure gives a yield of 15.0 g of phenylacetic acid (titrated at 97% by GC), molar yield 71%.

Example 2

[0065] In a one liter flask, there are loaded 100 mL of a liquid culture medium having the following composition: yeast extract (3 g/L), malt extract (3 g/L), soybean peptone (5 g/L), glucose (10 g/L) and (L)-phenylalanine (12.5 g/L).

[0066] The flask is closed with a cellulose plug and is then sterilized at 121° C. for 15 minutes. After cooling, an inoculum of Yarrowia lipolytica (about 0.5 g of wet yeast obtained by centrifugation of an active culture at about 3500×g) suspended in about 5 mL of saline is added thereto. In this example, too, the strain used is Yarrowia lipolytica DSM 8218.

[0067] The flask is stirred at 150 rpm at 28° C. for 7 days.

[0068] The fermentation (biotransformation) is then stopped by adding an acid (for example, citric acid) until a pH of 4.0-4.5 is reached.

[0069] At this time, the fermentation broth is filtered through Celite and extracted with ethyl acetate (3×60 mL).

[0070] The combined organic phases resulting from the preceding extraction are then dried over anhydrous sodium sulfate and are subsequently concentrated under reduced pressure.

[0071] The residue (1.5 g) obtained from the preceding step is distilled under reduced pressure to give 0.72 g of phenylacetic acid (titrated at 96% by GC), molar yield 67%.

[0072] By way of illustrative and non-limiting example, further examples of fungal species of the genus Yarrowia that are used according to the present invention and the ID number of the relevant strain are provided in Table 1 here below. For each of these examples, the type of transformation process (in a fermenter or in a flask) as well as the corresponding molar yield of phenylacetic acid are indicated. All the experimental process conditions are the same as those described in Example 1 above (in case of fermentation in a fermenter) and in Example 2 above (in case of fermentation in a flask).

TABLE-US-00001 TABLE 1 Species Strain Type of fermentation Yield Yarrowia lipolytica DSM 70562 Fermenter 75% Yarrowia deformans DSM 70561 Fermenter 72% Yarrowia yakushimensis CBS 10252 Flask 24% Yarrowia bubula CBS 12934 Flask 66%

[0073] The strains identified with the code DSM belong, as mentioned above, to the Collection DSMZ (German Collection of Microorganisms and Cell Cultures GmbH), whereas those identified with the code CBS belong to the International Collection CBS Knaw (Westerdijk Fungal Biodiversity Institute).