GB2222176A

GB2222176A - P. putida cells for microbial production of catechols

Info

Publication number: GB2222176A
Application number: GB8818152A
Authority: GB
Inventors: Philip John Geary; Robert John Pryce
Original assignee: Shell Internationale Research Maatschappij BV
Current assignee: Shell Internationale Research Maatschappij BV
Priority date: 1988-07-29
Filing date: 1988-07-29
Publication date: 1990-02-28
Also published as: GB8818152D0

Abstract

Wild type strains of Pseudomonas putida, non-constitutive of the enzymes required for microbial conversion of benzene and substituted benzenes to catechol and substituted catechols, are grown in the presence of non-substrate inducer compounds capable of inducing the missing enzyme or enzymes. The inducer may be pyridine, 3-picoline, alpha -picoline, furan, benzofuran, thiophene, cyclahexane, cyclohexene, cyclohexadiene, cyclohexanol or mesitylene. The substrate for microbial conversion may be fluorobenzene, chlorobenzene, Erifluoromethylbenzene, o-dichlorobenzene, benzonitrile or acetophenone.

Description

MICROBIAL PRODUCTION OF CATECHOLS This invention relates to the microbial production of catechols from benzene and benzene derivatives and to a method of producing cells derived from wild type Pseudomonas putida species for use in such microbial production.

The ability of the organism Pseudomonas putida to metabolise benzene and certain substituted benzenes to their corresponding catechols and further degradation products is known from the work of Gibson et al, Biochemistry, 7(7), 1968, p. 2653; and Biochemistry, 9(7), 1970, p. 1631. Thus, the metabolism is believed to follow the following enzyme catalysed reaction sequence:

In accordance with this metabolic pathway, benzene (I; R = H) is converted by a dioxygenase to cis-1,2-dihydroxycyclohexa-3,5-diene (II; R = H) (sometimes known as "cis-benzene glycol" or "benzene dihydrodiol") which under the action of a diol dehydrogenase is converted to catechol (III; R = H) which is enzymatically converted to further degradation products.A related pathway, where R is methyl, is believed to occur for toluene metabolism using Pseudomonas putida (Gibson et al, Biochemistry, 9(7), 1970, p. 1627).

Our copending European Patent Application, Publication No. 253438 (K 1033) describes certain strains of Pseudomonas putida and mutants thereof which are constitutive of the enzyme or enzymes necessary to convert benzene or its fluorinated analogues to cis-dihydroxycyclohexadiene or catechol or their fluorinated analogues. However for many Pseudomonas species it is necessary to induce one or more of the enzymes necessary for conversion to dihydroxydiene and catechol. This induction is conventionally accomplished by culturing the microorganisms in the presence of a substrate such as benzene or, more commonly, toluene, prior to use of the microorganism to convert a substrate such as fluoro- or chlorobenzene to the corresponding dihydroxydiene or catechol.The introduction of substrates such as benzene and toluene as inducers is undesirable in that the products resulting from microbial conversion of substrates other than the inducer benzene or toluene are contaminated by the dihydroxydienes and/or catechols produced from the inducer benzene or toluene.

It is known from EP-A-250122 that certain mutant strains of Pseudomonas putida, non-constitutive of the dioxygenase necessary for conversion of benzenes to cyclohexadienes, can be subjected to a nonsubstrate inducer compound in order to induce the dioxygenase enzyme necessary for conversion of an aromatic compound to the corresponding cyclic nonaromatic dihydroxy compound. EP-A-250122 is specifically concerned with mutant strains which yield dihydroxydiene products.

According to the present invention we provide a method of modifying a strain of Pseudomonas putida to produce cells containing enzymes capable of converting benzene or a substituted benzene to the corresponding catechol comprising growing a wild type strain of Pseudomonas putida, which strain is non-constitutive of at least one of the enzymes necessary for converting benzene or a substituted benzene to the corresponding catechol but in which strain the missing enzyme or enzymes can be induced, in a culture medium in the presence of a non-substrate inducer compound capable of inducing said missing enzyme or enzymes.

The wild type strain of Pseudomonas putida is suitably Pseudomonas putida NCIB 40035 or Pseudomonas putida NCIB 40034.

NCIB 40035 and 40034 were deposited at the National Collection of Industrial Bacteria, Torrey Research Station, Aberdeen on 21st July 1988. These microorganisms were isolated from refinery soil and soil from the Princess of Wales Conservatory at Kew, U.K.

The non-substrate inducer compound is preferably a cyclic organic compound. Examples of such compounds include pyridine, 3-picoline, a -picoline, furan, benzofuran, thiophene, cyclohexane, cyclohexene, cyclohexadiene, cyclohexanol and mesitylene. Preferred inducers are pyridine and 3-picoline.

The modified cells produced as above may be directly employed in a microbial process for the preparation of a catechol or a substituted catechol from benzene or the corresponding substituted benzene as substrate comprising supplying the substrate to a culture comprising the modified cells in a suitable medium and subsequently recovering the catechol or substituted catechol therefrom.

Preferably the substrate is fluorobenzene or chlorobenzene. However other substrates may be employed, for example benzonitrile, trifluoromethylbenzene, acetophenone and o-dichlorobenzene.

The growth of the modified cells in the presence of the inducer may be carried out in any suitable culture medium, comprising a carbon source such as gluconate. Alternative carbon sources include fructose and succinate. The cells may be isolated or the culture medium used directly in the preparation of the catechol.

The microbial preparation of the catechol may also be carried out in any suitable culture medium or buffer solution containing an energy source, such as ethanol or fructose.

The formation of desired product catechol may be assayed by gas chromatography. Compounds may be recovered from the fermentation broth by any suitable means such as absorption onto granulated charcoal followed by stripping with a suitable solvent or solvent extraction.

The product catechols are difficult to prepare by purely chemical means and are expensive. They are useful intermediates in the preparation of agrochemicals and pharmaceuticals such as those described in GB 2157691A and US 3976695.

The invention will now be further described by way of example.

Gluconate Medium The gluconate medium used in the following Examples contained the following ingredients per litre 25 mM KPo4 buffer pH 7.0: sodium gluconate 3g (NH4)2SO4 2g MnS04.3H20 2.5mg CaCl2. 2H2 0 12.5mg ZnSO4.3H2O 2.5mg to which was added when cold the following separately sterilised ingredients: MgSO4.7H2O 0.4g Bactopeptone 40mg FeS04.7H20 40mg Gas Chromatography Gas chromatographic analyses were carried out on an HP 5790A gas chromatograph with a 5% PMS wide bore and high capacity 25m capillary column operated isothermally at 1300C. Helium (1-2 ml/min) was used as the carrier gas. 0.5 L injections were made.

Standards were prepared in the same solvent as the samples being analysed.

Example 1 Oxidation of Chlorobenzene Cultures of the microorganism NCIB 40035 were grown in gluconate medium (50ml) containing 2 l of one of the non-substrate inducers listed in Table 1 below in a 250ml Erlenmeyer flask adapted to take a non-porous septum seal. Growth was continued with shaking for 18 hours at 30"C. A similar culture was grown with no inducer as a control. Following growth, the cells were removed by centrifuging, washed once in 25 mM phosphate buffer pH 7.0 and resuspended in 50ml of the same buffer.

To each flask was added chlorobenzene (50,k1) and ethanol (50,t,1) as an energy source. The flasks were sealed with silicone rubber stoppers and the mixtures incubated at 30"C with shaking. Samples were removed at intervals and assayed by gas chromatography for the presence of chlorocatechol. The results are given after 1, 3 and 24 hours in Table 1 below in GC integrated peak area units (100,000 units is equivalent to lmg/ml).

Table 1 Inducing GC Integrated Units 3-chlorocatechol Compound 1 hr 3 hr 24 hr None (control) O 0 7750 Furan 0 trace 11569 Thiophene trace trace 11238 Pyridine (Note 1) 6149 9570 3-Picoline 5264 + 9252 11524 3932 diol Cyclohexane O 0 8193 Benzofuran O 0 10299 Cyclohexanol O 0 13273 c & -picoline o 0 13005 Cyclohexadiene o 0 8349 Mesitylene O 0 10195 Note 1. An additional 50 l of ethanol and 50 l chlorobenzene were added after 5 hours: the reaction mixture was black at 24 hours.

The results above indicate the presence of both enzymes necessary for production of chlorocatechol, i.e. the aromatic dioxygenase and the diol dehydrogenase.

Example 2 Oxidation of Fluorobenzene The procedure of Example 1 was repeated employing the microorganism NCIB 40034 instead of NCIB 40035 and, as substrate, fluorobenzene in place of chlorobenzene. The results are given in Table 2 below in GC integrated units (40,000 units is equivalent to lmg/ml).

Table 2 Inducing GC Integrated Units 3-fluorocatechol Compound 2 hr 5 hr 24 hr Cyclohexadiene 0 3916 613 3-Picoline (Note 1) 4627 10682 11761 (1178) (1425) Pyridine (Note 1) 4728 10558 10257 (836) (1634) None (control) 0 2573 2716 (Note 2) Note 1 The figures in brackets indicate the units of intermediate diene diol detected.

Note 2 The small amounts of fluorocatechol detected in the control were considered to be the result of carry over in the assay (GLC) from a previous injection because the actual reaction mixture remained colourless whereas those experiments using non-substrate inducers resulted in the development of purple coloration.

Claims

1. A method of modifying a strain of Pseudomonas putida to produce cells containing enzymes capable of converting benzene or a substituted benzene to the corresponding catechol comprising growing a wild type strain of Pseudomonas putida, which strain is non-constitutive of at least one of the enzymes necessary for converting benzene or a substituted benzene to the corresponding catechol but in which strain the missing enzyme or enzymes can be induced, in a culture medium in the presence of a non-substrate inducer compound capable of inducing said missing enzyme or enzymes.

2. A method according to claim 1 wherein the wild type strain is Pseudomonas putida NCIB 40034 or Pseudomonas putida NCIB 40035.

3. A method according to claim 1 or 2 wherein the inducer compound is a cyclic organic compound.

4. A method according to claim 3 wherein the inducer compound is selected from pyridine, 3-picoline, > -picoline, furan, benzofuran, thiophene, cyclohexane, cyclohexene, cyclohexadiene, cyclohexanol and mesitylene.

5. A method according to claim 4 wherein the inducer compound is pyridine or 3-picoline.

6. Modified cells of Pseudomonas putida when produced by the method of any one of the preceding claims.

7. A microbial process for the preparation of catechol or substituted catechol from as substrate, benzene or the corresponding substituted benzene comprising supplying the substrate to a culture comprising modified cells of Pseudomonas putida in accordance with claim 6 in a suitable medium and subsequently recovering the catechol or substituted catechol therefrom.

8. A microbial process according to claim 7 wherein the substrate is fluorobenzene or chlorobenzene.

9. Catechol or a substituted catechol when obtained by the process of claim 7 or 8.