# Patent

## Title
Genetically transformed pepper plants and methods for their production

## Abstract

This application is a continuation of application Ser. No. 07/686,940, filed Apr. 18, 1991, now abandoned.

## Background
The present invention relates to a process for producing vanillin by bioconversion of benzenoid precursors.

Vanillin is currently obtained either by chemical synthesis, or in the natural state by extraction from vanilla pods.

The object of the present invention is to obtain a process for producing vanillin which is simple to industrialize and which makes it possible to obtain a vanillin which may be considered under the regulations in force, in particular in the EEC countries, to be natural. Such is the case with the products obtained by bioconversion of natural precursors by means of microorganisms.

To this end, the subject of the present invention is a process for producing vanillin, wherein culturing of a basidiomycete fungus of the genus Pycnoporus or of their variants and mutants is performed in a culture medium to which a benzenoid precursor of vanillin has been added, and wherein the vanillin produced is recovered after bioconversion of said precursor, the Pycnoporus strain being selected for its metabolic property of degrading said precursors with accumulation of vanillin in the medium.

As a benzenoid precursor, derivatives at the 1-position of 4-hydroxy-3-methoxybenzene, such as vanillic acid or ferulic acid, will be used in particular.

In an especially suitable implementation, the fungus of the species Pycnoporus cinnabarinus will be used, in particular the strains CNCM Nos. I-937 and I-938 or their variants and mutants which possess the metabolic property of producing a high level of degradation of benzenoid precursors of vanillin such as ferulic acid, with accumulation of vanillin in the medium.

Advantageously, precursors originating from natural plant substrates, preferably rich in said precursors, will be used. There may be mentioned ferulic acid, which is present in the cell membranes of many monocotyledons and dicotyledons such as, for example, sugar-beet pulp, a byproduct of the sugar industry, or aleurone grains of wheat or of different cereals. The natural sources of ferulic acids are hence relatively abundant. Another benzenoid precursor of natural origin which is useable according to the invention is isoeugenol.

Moreover, compounds such as para-hydroxybenzoic acid, para-hydroxybenzaldehyde, vanillyl alcohol and stilbenes, inter alia, may be used.

In a preferred embodiment of the process according to the invention, the fungus is first cultured in an agitated liquid medium, and the precursor is added only after a large biomass, possessing optimal enzymatic capacity for bioconversion of said precursor, is obtained.

Thus, advantageously, in the case of culture of the fungus Pycnoporus cinnabarinus, the precursor is added when the necessary biomass is obtained, namely 3 days under the culture conditions of the medium of Appendix 2, described in detail below.

When the precursor is added at the start of culturing, bioconversion to vanillin is observed but with much lower yields.

In a particular embodiment of the process according to the invention, the inoculum via which the fungus is inoculated into the culture medium consists of mycelium fragments or spores, or precultures of these inocula.

A quantity of precursors ranging up to 50 g/ml per culture medium may be introduced.

In the process according to the invention, at least 15 mycelium fragments per 100 ml of culture medium and 10.sup.5 to 10.sup.7 spores/ml of medium will be used. The pH of the culture medium can be between 2.5 and 6.5. The incubation temperature of the cultures can be between 25.degree. and 40.degree. C.

Advantageously, the vanillin is recovered when the precursor is no longer detectable in the culture medium and before its bioconversion to vanillyl alcohol.

To recover the vanillin, it is possible, according to processes known to those skilled in the art, to extract it from the culture media with solvents, followed by a distillation; it is also possible to perform a fractional crystallization or a preparative industrial chromatography directly on the culture media.

According to the process according to the invention, the precursor may be introduced sequentially or in continuous fashion, and the vanillin may likewise be recovered sequentially or in continuous fashion.

Under the culture conditions of the medium of Appendix 2 described in detail below, the vanillin is recovered 4 or 5 days after the introduction of the precursor.

Other features and advantages of the present invention will become apparent in the light of the detailed description which follows.

In the description, reference is made to FIGS. 1 to 5.

FIG. 1 shows the bioconversion of vanillic acid added during the inoculation.

FIG. 2 shows the bioconversion of vanillic acid added at 3 days of culture.

FIG. 3 shows the bioconversion of vanillic acid to vanillin and vanillyl alcohol.

FIG. 4 shows the bioconversion of ferulic acid added with the inoculation.

FIG. 5 shows the bioconversion of ferulic acid added at 3 days of culture.

The filamentous basidiomycete fungus is cultured beforehand in an agitated liquid medium in order to obtain a large biomass possessing the enzymatic capacity needed for bioconversion, as well as a sufficient yield. After a few days of culture, the sterile precursor is added aseptically. Disappearance of the precursor (ferulic acid or vanillic acid) and bioconversion to vanillyl alcohol and vanillic acid (in the case of ferulic acid) are monitored by HPLC analysis.

Two basidiomycetes of the species Pycnoporus cinnabarinus deposited at the Collection Nationale de Culture de Microorganisme (National Collection of Microorganism Cultures) of the Pasteur Institute (CNCM) under Nos. I-937 and I-938 on Apr. 10, 1990.

Three types of inoculum are possible: they are, respectively, spores or mycelium fragments or a preculture of these.

250 ml of sporulation medium (composition given in Appendix 1) are inoculated in a Roux flask with mycelium fragments from culture on an agar medium. Incubation is carried out at 37.degree. C. for three weeks. During this period, the mycelium develops on the agar surface, producing conidia (spores). These spores are detached from the surface of the solid culture by the agitation of glass beads immersed in sterile aqueous physiological solution. The spore suspension thereby obtained is filtered through glass wool in order to remove all large mycelial fragments. Counting is carried out on a Thoma cell. The inoculum of the cultures may then be quantified using an aliquot of known volume of this solution. Appendix 1: Composition of the solid sporulation medium ______________________________________ Yeast extract             3      g/l Malt extract              3      g/l Bacto peptone             5      g/l Glucose                   10     g/l Agar                      15     g/l Biotin, 0.5 ml/l of a 0.001% sterile solution Solution of salts (1)     20     ml/l (1) Solution of salts Na.sub.3 citrate.2H.sub.2 O 125    g/l KH.sub.2 PO.sub.4 (anhydrous) 250    g/l NH.sub.4 NO.sub.3 (anhydrous) 100    g/l MgSO.sub.4.7H.sub.2 O     10     g/l Solution of trace elements (2) 5      ml/l (2) Solution of trace elements Citric ac.H.sub.2 O       50     g/l Zinc sulfate              50     g/l Copper sulfate            2.5    g/l Manganese sulfate         0.5    g/l Boric ac.                 0.5    g/l Na.sub.2 MoO.sub.4.2H.sub.2 O 0.5    g/l Fe(NH.sub.4).sub.2 (SO.sub.4).sub.2.6H.sub.2 O 10     g/l ______________________________________

A solid medium (20 g/l of malt extract +15 g/l of agar +2 g/l of yeast extract) in a Petri dish is inoculated centrally with a mycelium fragment from culture on agar medium. Incubation is carried out at 37.degree. C. until the mycelium completely covers the surface. Fragments of this mycelium are then used as an inoculum; the mycelium fragments are cut out with a hollow punch approximately 4 mm in diameter.

An incubation of the spores or mycelium fragments is carried out in a volume of medium (5 to 10% of the final volume of the culture) and then used as an inoculum.

100 ml of medium conventionally containing, in particular, an animal or vegetable carbon source, inorganic salts and vitamins (a detailed composition of the culture medium used is given in Appendix 2) are used in an Erlenmeyer of capacity 500 ml. Inoculation is performed either with mycelium fragments (15 to 30 per culture) or with spores on the basis of 2.times.10.sup.5 spores/ml of medium. The cultures are incubated at 37.degree. C. and subjected to a rotary agitation at 120 rpm with an amplitude of 6 cm. Appendix 2: Composition of the culture medium ______________________________________ Maltose              20        g/l Diammonium tartrate  1.8415    g/l KH.sub.2 PO.sub.4    0.2       g/l CaCl.sub.2.2H.sub.2 O 0.0132    g/l MgSO.sub.4.7H.sub.2 O 0.5       g/l Yeast extract        0.5       g/l Thiamine hydrochloride 2.5       mg/l Sterilization by autoclaving: 120.degree. C. - 20 min. ______________________________________

After 72 h of culture, sterile ferulic acid or sterile vanillic acid is added in the proportion of 0.3 g/l of culture. The ferulic acid and vanillic acid are used : NH.sub.4 * salt) and sterilized by filtration (porosity: 0.2 .mu.m).

Every day, once the culture has been supplemented with precursor, 1.5 ml of each culture is withdrawn under sterile conditions. This aliquot is filtered through a hydroprobic membrane (porosity:0.2 .mu.m) pretreated with methanol and an HPLC analysis of the filtrate is carried out.

This measurement necessitates "sacrifice" of the culture; the results obtained under these conditions do not correspond to a true picture of the reaction rate, since the same culture is not monitored throughout the study.

The 100 ml cultures are filtered through a previously tared glass fiber membrane. The biomass collected is placed for 24 h at 100.degree. C. The corresponding dry matter content is determined by weighing.

Example using the strain No. I-937 (the same results were obtained with the strain No. I-938.) PRECURSOR ADDED DURING THE INOCULATION (see FIG. 1) Age of the Biomass (in g Vanillic acid culture   of dry matter precursor  Vanillin (in d)    per liter)   (in mg/l)  (in mg/l) ______________________________________ 0                      310 1         0.088        314 2         0.389        228        6.8 3         0.737        18         9.2 4         0.911        0.3        10.7 ______________________________________

A rapid and total consumption of vanillic acid is observed in 3-4 days of culture. The microbial biomass increases up to the last day of analysis. Vanillin synthesis is detected from day two onwards, and the quantity of vanillin accumulated is rising up to the last day of the study. The maximum molar yield of conversion to vanillin is low: 3.8%. PRECURSOR ADDED ON DAY THREE OF CULTURE (see FIG. 2) Age of the Biomass (in g Vanillic acid culture   of dry matter precursor  Vanillin (in d)    per liter)   (in mg/l)  (in mg/l) ______________________________________ 3                      286 4         0.782        184.5      25.6 5         0.798        0          90.8 6         0.869        0          78 7         0.872        0          58 ______________________________________

The consumption of vanillic acid is more rapid than in the above case: in 2 days, the precursor concentration has fallen to 0 mg/l. The microbial biomass increases little after the time of introduction of vanillic acid. Vanillin synthesis is virtually immediate, and a maximum content is attained 2 days after the supplementation. Beyond this point, vanillin disappears very rapidly from the medium. In this case, the maximum molar yield of conversion to vanillin is much higher than before: 35.1%. Alcohol PRECURSOR ADDED ON DAY THREE OF CULTURE (see FIG. 3) Age of the Vanillic acid          Vanillyl culture   precursor     Vanillin Alcohol (in d)    (in mg/l)     (in mg/l) (in mg/l) ______________________________________ 3         300 4         191           35.7     5.1 5         10.3          81.4     38.9 6         1.2           39.4     68.2 7         2.1           23.4     76.5 ______________________________________

The conditions of bioconversion are the same as before; the concentrations of vanillic acid and vanillin are thus similar, the vanillin concentration is maximal when the precursor concentration has fallen to 0 mg/l. The results of assay of vanillyl alcohol suggest that the vanillin is converted to its alcohol. In effect, the appearance of the alcohol is retarded by approximately 24 h relative to that of vanillin, and its concentration increases considerably when the vanillin disappears from the culture medium. One of the means of optimization of the production would be to block this reduction, which leads to the formation of vanillyl alcohol to the detriment of vanillin. The maximum molar yield of conversion to vanillin is comparable to that above: 31%. PRECURSOR ADDED DURING THE INOCULATION (see FIG. 4) Age of the    Biomass in Ferulic acid Vanillic culture g of dry   precursor   acid   Vanillin (in d) matter per l) (in mg/l)   (in mg/l) (in mg/l) ______________________________________ 0                 294 1      0.095      287 2      0.314      133.7 3      0.484      15.7        4.2    0.5 4      0.590      7.4         1.1    0.35 ______________________________________

As in the case of vanillic acid added during the inoculation, the biomass increases considerably up to the last day of the study. Degradation of the ferulic acid is complete after 4-5 days. A synthesis of vanillic acid and of vanillin is observed, but the yields are rather low (maximum molar yield of conversion to vanillin: 0.2%). PRECURSOR ADDED ON DAY THREE OF CULTURE (see FIG. 5) Age of the    Biomass in Ferulic acid Vanillic culture g of dry   precursor   acid   Vanillin (in d) matter per l) (in mg/l)   (in mg/l) (in mg/l) ______________________________________ 3                 289 4      0.609      241 5      0.789      41.5               11.5 6      1.023      0.9         7.7    46.2 7      1.099      0.85        4.1    45.4 ______________________________________

In this case of supplementation at 3 days of culture, the ferulic acid disappears completely from the medium in 3 days. Simultaneous syntheses of vanillic acid and of vanillin are observed. As with vanillic acid, the quantity of vanillin is maximal when the precursor concentration becomes zero. The maximum molar yield of conversion to vanillin is: 20.5%.

## Claims

1. A process for the production of vanillin, said process comprising and assimilable sources of carbon and nitrogen and Pycnoporus cinnabarinus capable of producing vanillin from a benzenoid precursor of vanillin; of Pycnoporus cinnabarinus CNCM No. I-937 and Pycnoporus cinnabarinus CNCM No. I-938, and wherein said benzenoid precursor is selected from the group consisting of vanillic acid and ferulic acid.

2. The process of claim 1, wherein said benzenoid precursor is vanillic acid.

3. The process of claim 1, wherein said benzenoid precursor is ferulic acid.

4. The process of claim 1, wherein said precursor is added to the culture medium after three days of culture of Pycnoporus cinnabarinus.

5. The process of claim 1, wherein said Pycnoporus cinnabarinus is provided in a form selected from the group consisting of

6. The process of claim 1, wherein said vanillin is recovered when said precursor is no longer detectable in the culture medium and before bioconversion of said vanillin to vanillyl alcohol.

7. The process of claim 1, wherein said precursor is introduced and said vanillin is recovered periodically.

8. The process of claim 1, wherein said precursor is introduced and said vanillin is recovered continuously.

9. The process of claim 4, wherein the amount of vanillin recovered is 40-90 mg/l.

10. The process of claim 4, wherein the amount of vanillin recovered is 70-90 mg/l.