WO1999050420A1 - Method for increasing the phenoxy propionic acid hydroxylation rate - Google Patents

Abstract

The invention relates to a method for producing 2-(4-hydroxyphenoxy)-propionic acid from 2-phenoxy propionic acid or the salts thereof by means of a micro-organism which is capable of converting 2-phenoxy propionic acid into 2-(4-hydroxyphenoxy)-propionic acid. The invention is characterized in that the micro-organism contains at least one of the genes contained in the sequences SEQ ID no. 1 or SEQ ID no. 4 or their functional equivalents. The invention also relates to isolated DNA sequences with the SEQ ID no. 1 or SEQ ID no. 4 and to a gene product containing these sequences. The invention further relates to a micro-organism containing the gene product and to the use of the sequences and the gene product for producing 2-(4-hydroxyphenoxy)-propionic acid.

Description

Process for increasing the rate of POPS hydroxylation

description

The invention relates to a for the production of 2- (4-hydroxyphenoxy) propionic acid from 2-phenoxypropionic acid or its salts with a microorganism which is able to convert 2-phenoxypropionic acid to 2- (4-hydroxyphenoxy) propionic acid, thereby characterized in that the microorganism contains at least one of the genes contained in the sequences SEQ ID No. 1 or SEQ ID No.4 or their functional equivalents. The invention also relates to isolated DNA sequences with SEQ ID No. 1 or SEQ ID No.4 and a gene construct containing the sequences mentioned. Furthermore, the invention relates to a microorganism containing the gene construct and the use of the sequences and the gene construct for the production of 2- (4-hydroxyphenoxy) propionic acid.

Filamentous fungi are known for their ability to perform a wide variety of biotransformations, e.g. the hydroxylation of aromatics, complex polyaromatics and steroids. Biotransformation is the selective chemical conversion of defined pure substances (starting materials) to defined end products, this conversion being catalyzed by microorganisms, animal or plant cell cultures or isolated enzymes.

The biotransformacion of carboxylic acids is carried out in order to achieve, for example, regio- or enantioselective reactions such as hydroxylations and epoxidations, which cannot be carried out or can only be carried out with great difficulty by means of chemical synthesis.

A sought-after hydroxylated carboxylic acid is (R) -2- (4-hydroxyphenoxy) propionic acid (= HPOPS), which can be prepared via complex chemical syntheses or via a biotransformation of (R) -phenoxypropionic acid (= POPS) to ( R) -2- (4-hydroxyphenoxy) propionic acid (see EP-B-0 465 494). (R) -2- (4-Hydroxyphenoxy) propionic acid is an important intermediate, especially for the production of grass herbicides. This biotransformation is carried out by a large number of microorganisms, in particular fungi and bacteria are capable of this reaction. EP-B-0 465 494 describes the regioselective hydroxylation of 2-phenoxypropionic acid to 2- (4-hydroxyphenoxy) propionic acid by means of microorganisms . Both race ate and enantiomerically pure compounds can be hydroxylated. It is advantageous in this process that no hydroxy-ionized by-products are formed. Suitable mushrooms are, for example 2 wise fungi of the genera Aspergillus, Beauveria, Paecilomyces, Sclerotium or Coprinus, bacteria of the genera Pseudomonas, Rhodococcus, Nocardia or Streptomyces are suitable as bacteria to name just a few of the suitable microorganisms. Suitable microorganisms can easily be isolated, for example, from soil samples. A method for this is described in EP-B-0 465 494. After screening a number of microorganisms, it was found that Beauveria bassiana is a particularly suitable filamentous fungus for the above-mentioned biotransformation and hydroxylates POPS with a conversion> 98% to HPOPS regioselectively (Dingler et al. Pestic. Sei. 1996, 46, 33-35; EP-A-0 758 398)

In order to further improve the method described in EP-B-0 465 494, a method was sought which made it possible to carry out the biotransformation of POPS to HPOPS with higher yields in a shorter time. The task was therefore to increase the productivity of the microorganisms.

The object was achieved by the process according to the invention for the preparation of 2- (4-hydroxyphenoxy) propionic acid from 2-phenoxypropionic acid or its salts with a microorganism which is capable of 2-phenoxypropionic acid to give 2- (4-hydroxyphenoxy) - Implementing propionic acid, characterized in that the microorganism contains at least one of the genes contained in the sequences SEQ ID No. 1 or SEQ ID No. 4 or their functional equivalents.

The microorganism preferably contains the two genes contained in the sequence SEQ ID No. 1 alone or in combination with the gene of the sequence SEQ ID No. 4.

The HPOPS produced after biotransformation in the process according to the invention is then worked up from the fermentation broth by methods known to the person skilled in the art (see, for example, the workup described in EP-B-0 465 494) and used for the chemical synthesis of the herbicides.

By expressing the sequences SEQ ID No. 1 and / or SEQ ID No. 4 in a prokaryotic or eukaryotic host organism, the productivity of the biotransformation from POPS to HPOPS can be significantly increased. Expression of the sequences in a eukaryotic host organism is preferred. The expression of the genes increases the specific hydroxylation rate by at least a factor 1.3 compared to the control without these genes. The sequence SEQ ID No. 1 or the combination of the sequences SEQ ID No. 1 and SEQ ID No. 4 is preferred 3

Increasing the hydroxylation rate used, the sequence SEQ ID No. 1 is particularly preferably used.

After isolation and sequencing, the genes used in the method according to the invention can be obtained with the nucleotide sequences SEQ ID No. 1 and SEQ ID No.4 or their functional equivalents which are necessary for those in SEQ ID NO.2, SEQ ID No.3 and SEQ ID No. .5 specified amino acid sequences according to the invention or their functional equivalents such as. B. encode allele variations. Allel variants are understood to mean SEQ ID No. 1 or SEQ ID No. 4 variants which have 40 to 100% homology at the amino acid level, preferably 50 to 100%, very particularly preferably 80 to 100%. Allelic variants include in particular those functional variants which can be obtained by deleting, inserting or substituting nucleotides from the sequence shown in SEQ ID NO.1 or SEQ ID NO.4, but the enzymatic activity is retained.

Functional equivalents are also understood to mean analogs of SEQ ID NO.1 or SEQ ID NO.4, for example their bacterial, fungal, plant or yeast homologues, shortened sequences, single-stranded DNA or RNA of the coding and non-coding DNA sequence. These equivalents hybridize with the sequences SEQ ID No. 1 or SEQ ID No.4 under conditions known to the person skilled in the art, as described, for example, in Sambrook, Fritsch and Maniatis Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, Laboratory Press, 1989. Advantageous hybridization conditions are, for example, 42 ° C, 5 x SSC, 50% formamide.

In addition, functional equivalents are also to be understood to mean derivatives, for example promoter variants. The promoters which precede the specified nucleotide sequences can be changed by one or more nucleotide exchanges, by insertion (s) and / or deletion (s), but without the functionality or effectiveness of the promoters being impaired. Furthermore, the effectiveness of the promoters can be increased by changing their sequence, or completely replaced by more effective promoters, including organisms of other species.

Derivatives also mean variants whose nucleotide tidsequenz in the range from -1 to -100 in front of the start codon of the respective ^'genes or before all genes have been modified so that gene expression and / or protein expression is increased. This is advantageously done by means of a modified Shine-Dalgarno sequence. 4

The isolated gene sequences according to the invention with SEQ ID No. 1 or SEQ ID No. 4 are expressed alone or in combination in suitable eukaryotic or prokaryotic microorganisms for the method according to the invention. These organisms containing SEQ ID No. 1 and / or SEQ ID No. 4 are used in the process according to the invention.

In principle, all gram-negative or gram-positive bacteria which are able to convert POPS to HPOPS are possible as prokaryotic host organisms of the method according to the invention. Like all suitable microorganisms, these bacteria can easily be isolated from soil or water samples. A method for this is described in EP-B-0 4656 494. Examples of Gram-negative bacteria are Pseudomonadaceae and the genus Pseudomonas. Examples of Gram-positive bacteria are the genera Rhodococcus, Nocardia or Actinomycetes such as Streptoyces.

Bacteria of the genus Streptomyces are preferably used in the method according to the invention.

In principle, all POPS to organisms which transform HPOPS, such as fungi or yeasts, are suitable as eukaryotic host organisms of the method according to the invention. The fungi Aspergillus, Beauveria, Paecilomyces, Sclerotium or Coprinus are mentioned as examples.

Mushrooms of the genera Beauveria and Aspergillus are preferred as fungi for the process according to the invention, particularly preferably fungi of the genus Beauveria.

Advantageously, microorganisms such as bacteria, fungi or yeasts are used in the process according to the invention, which already have increased productivity compared to the wild-type isolates. Such microorganisms are expediently obtained by mutating wild strains which have the ability to hydroxylate POPS.

Known microbiological techniques can be used to generate such mutants. All common methods can be used to trigger mutations, such as the use of mutagenic substances, for example nitrosoguanidine, ethyl methanesulfonate, sodium nitrite, or the action of electromagnetic radiation, such as UV, gamma or X-rays. Furthermore, transposable genetic elements such as transposons or IS elements can also be used for mutagenesis. For the isolation of the mutants, for example, their property POPS to HPOPS 5 to be implemented to an increased extent (measurement by GC analysis, see EP-B-0 465 494).

However, such organisms can also be selected in continuous culture from a population of less adapted individuals by appropriately adding increasing educt / product concentrations to the inflowing medium.

The genes according to the invention with the sequences SEQ ID No. 1 and / or SEQ ID No. 4 are introduced in one or more copies to increase productivity in wild-type organisms or in the mutants described above. They can be located on the same vector or have been integrated on separate vectors or else chromosomally or together or separately.

The gene construct according to the invention is to be understood as the gene sequences SEQ ID No. 1 and / or SEQ ID No. 4 and their functional equivalents such as variants, analogs or derivatives, which have been functionally linked to one or more regulation signals to increase gene expression. In addition to these new regulatory sequences, the natural regulation of these sequences may still be present before the actual structural genes and / or may have been genetically modified so that the natural regulation has been switched off and the expression of the genes increased. However, the gene construct can also have a simpler structure, that is to say no additional regulation signals have been inserted in front of the sequences SEQ ID No. 1 and / or SEQ ID No.4 and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence was mutated in such a way that regulation no longer takes place and gene expression is increased. Additional advantageous regulatory elements can also be inserted at the 3 'end of the DNA sequences. The genes with the sequences SEQ ID No. 1 and / or SEQ ID No. 4 can be contained in one or more copies in the gene construct.

Advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacis - T7, T5, T3 , gal, trc, ara, SP6, λ-P _R - or contained in the λ-P _L promoter, which are advantageously used in gram-negative bacteria. Further advantageous regulatory sequences are contained, for example, in the gram-positive promoters amy and SP02 or in the yeast promoters ADC1, MFα, AC, P-60, CYC1, GAPDH. 6

In principle, all natural promoters with their regulatory sequences such as those mentioned above can be used for the method according to the invention. In addition, synthetic promoters can also be used advantageously.

For expression in the above-mentioned host organism, the gene construct is advantageously inserted into a host-specific vector, which enables optimal expression of the genes in the host. Suitable vectors are, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUCl9, pKC30, pRep, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III ^{1: L} 3-Bl, λgtll or pBdces or in StreBomyces pIJlOl, pIJ364, pIJ702 or pIJ361, in yeasts YEp6, YEpl3 or pEMBLYe23. The vectors mentioned represent a small selection of the possible vectors. Further vectors are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018 ) can be removed. For fungi, for example, vectors such as pA04-13 (pyrG), PAB4-1, 2μ yeast plasmid, pBBH6, pFB9, pHY201, p3SR2, pAN7-l, pAN923-4lB, pALSl or pSL2 are suitable. Further advantageous vectors can also be found in the book Cloning Vectors.

Expression systems are to be understood as the combination of the host organisms mentioned by way of example above and the vectors which match the organisms, such as plasmids, viruses or phages such as the T7 RNA polymerase / promoter system or vectors with regulatory sequences for the phage λ.

The expression systems are preferably to be understood as the combination of pA04-13 (pyrG) or pAB4-1 and Beauveria bassiana or Aspergillus niger.

Further 3 'and / or 5' terminal regulatory sequences are also suitable for the advantageous expression according to the invention of SEQ ID No. 1 and SEQ ID No.4.

These regulatory sequences are intended to enable targeted expression of the genes and protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is immediately expressed and / or overexpressed.

The regulatory sequences or factors can preferably have a positive influence on the expression and thereby increase it. Thus, the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or enhancers 7 will be. In addition, an increase in translation is also possible, for example, by improving the stability of the mRNA.

“Enhancers” are understood to mean, for example, DNA sequences which bring about increased gene expression via an improved interaction between RNA polymerase and DNA.

An increase in the proteins derived from the sequences SEQ ID No. 1 or SEQ ID No.4 and their enzyme activity can be achieved, for example, compared to the starting enzymes by changing the corresponding gene sequences or the sequences of its homologues by classic mutagenesis such as UV radiation or treatment with chemical Mutagenic agents and / or by targeted mutagenesis such as site directed mutagenesis, deletion (s), insertion (s) and / or substitution (s). In addition to the described gene amplification, increased enzyme activity can also be achieved by eliminating factors that repress enzyme synthesis and / or by synthesizing active instead of inactive enzymes.

The process according to the invention advantageously increases the biotransformation of POPS to HPOPS and thus the overall productivity beyond the activity present in the organisms by expressing the sequences SEQ ID No. 1 and / or SEQ ID No.4.

In the method according to the invention, the microorganisms contained in SEQ ID No. 1 and / or SEQ ID No. 4 are grown in a medium which enables the growth of these organisms. This medium can be a synthetic or a natural medium. Depending on the organism, media known to the person skilled in the art are used. For the growth of the microorganisms, the media used contain a carbon source, a nitrogen source, inorganic salts and possibly small amounts of vitamins and trace elements.

Advantageous carbon sources are, for example, sugars such as mono-, di- or polysaccharides such as glucose, fructose, mannose, xylose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose, complex sugar sources such as molasses, sugar phosphates such as fructose -1, 6-bisphosphate, sugar alcohols such as mannitol, polyols such as glycerol, alcohols such as methanol or ethanol, carboxylic acids such as citric acid, lactic acid or acetic acid, fats such as soybean oil or rapeseed oil, amino acids such as glutamic acid or aspartic acid or amino sugar, which also act as nitrogen can be used. 8th

Advantageous nitrogen sources are organic or inorganic nitrogen compounds or materials that contain these compounds. Examples are ammonium salts such as NH ₄ C1 or (NH ₄ ) ₂ Sθ ₄ , nitrates, urea, or complex nitrogen sources such as corn steep liquor, brewer's yeast autolysa, soybean meal, wheat gluten, yeast extract, meat extract, casein hydrolyzate, yeast or potato protein, which are often also used simultaneously as a nitrogen source can serve.

Examples of inorganic salts are the salts of calcium, magnesium, sodium, manganese, potassium, zinc, copper, iron and other metals. The chlorine, sulfate and phosphate ions are particularly worth mentioning as the anion of these salts. An important factor for increasing productivity in the process according to the invention is the addition of Fe ^{2 + _} or Fe ³⁺ salts and / or potassium salts to the production medium.

If necessary, further growth factors are added to the nutrient medium, such as vitamins or growth promoters such as riboflavin, thiamine, folic acid, nicotinic acid, pantothenate or

Pyridoxine, amino acids such as alanine, cysteine, asparagine, aspartic acid, glutamine, serine, methonine or lysine, carboxylic acids such as citric acid, formic acid, pimelic acid or lactic acid, or substances such as dithiothreitol.

Antibiotics can optionally be added to the medium to stabilize the genes in the cells.

The mixing ratio of the nutrients mentioned depends on the type of fermentation and is determined in each individual case.

In general, POPS concentrations of about 1 to 150 g / 1, preferably 50-120 g / 1, are suitable for carrying out the process according to the invention.

The breeding conditions are determined so that the best possible yields are achieved. Depending on the microorganism, preferred cultivation temperatures are between 20 ° C and 40 ° C. Temperatures between 25 ° C to 30 ° C are particularly advantageous. The fermentation time is between 10 to 100 hours. The starting material can be added to the nutrient medium all at once, after growth has taken place or during cultivation in several portions or continuously. The educt is preferably added in the form of 2-phenoxypropionic acid, but it is also possible to use its salts, preferred salts are alkali and alkaline earth metal salts such as the Na, K or Li salts. The product is usually worked up after the reaction has ended. But it can also during 9 of the biotransformation take place discontinuously or continuously.

For the process according to the invention, in order to achieve the highest possible productivity, it is advantageous to control the pH during the fermentation in such a way that the optimum amount of freely dissociated 2-phenoxypropionic acid (= starting material) is contained in the medium in each phase of the biotransformation ( see EP-A-0 758 398). These values are as follows:

In this first phase of the biotransformation, the pH is therefore advantageously controlled so that the proportion of undissociated form of the educt is between 1 and 30, preferably between 3 and 20% of the growth-inhibiting concentration.

The growth-inhibiting concentration can easily be determined for a certain carboxylic acid and a given microorganism by simple preliminary tests familiar to the person skilled in the art. Furthermore, after knowing the pKa value of the carboxylic acid and using the buffer equation, the pH value to be set can easily be calculated.

In the second phase of the biotransformation, the majority of the starting material is implemented with high productivity. To ensure a high space-time yield, the concentration of the undissociated form of the educt is adjusted so that it is 5 to 65, preferably 15 to 40% of the concentration-inhibiting growth for a given organism.

The third phase of the biotransformation is characterized in that no more educt is metered in and the educt present in the medium is converted as completely as possible into product.

In this phase, the pH is generally reduced, with the result that the concentration of the undissociated form of the educt is kept as long as possible in the optimal range of the second phase even when the educt concentration drops. It is hereby achieved that the diffusion rate limiting productivity maintains a sufficiently high value.

This phase generally takes up 10 to 30% of the total biotransformation time.

The mixing ratio of the nutrients mentioned depends on the type of fermentation and is determined in each individual case. The medium components can all be placed at the beginning of the fermentation after being sterilized separately if necessary 10 or have been sterilized together, or given as needed during the fermentation.

Advantageous breeding conditions or biotransformation conditions are EP-B-0 465 494 (see examples 1 to 7) and EP-A-0 758 398 (see examples).

The sequences SEQ ID No. 1 and / or SEQ ID No.4 according to the invention or a gene construct containing these sequences are advantageously suitable for use in a biotransformation reaction in which POPS is converted to HPOPS.

The invention is further illustrated by the following examples.

Examples

Unless otherwise stated, media such as: Dingler et al. EP-A-0 758 398 and Pontecorvo et ai. The Genetics of Aspergillus nidulans. Adv. Genet. 5, 141-238 (1953).

Preculture medium:

Yeast extract 10 g / i

MgS04 7H ₂ 0 2 g / i CaCl ₂ -2H ₂ 0 1 g / i

Glucose 30 g / i

KH ₂ P0 ₄ 0.75 g / i

K2HPO4 1.8 g / i pH (NaOH) 7.0 POPS 30 g / i

Aqua Bidest. ad 11

Main culture medium:

Yeast extract 6 g / i MgS0 7H ₂ 0 2 g / i

CaCl ₂ .2H ₂ 0 1 g / i

Glucose 50 g / i

KH ₂ P0 0.75 g / i

K ₂ HP0 ₄ 1.8 g / i pH (NaOH) 7.0

Aqua Bidest . ad 11 11POPS 50-70 g / i

Aqua Bidest. ad 11 11

POPS agar

Yeast extract (Difco) 10.0 g / i

MgS0 ₄ • 7H ₂ 0 1.0 g / i

POPS 5 g / i

Agar 18.0 g / i

D-glucose 55 g / i

KH ₂ P0 ₄ 1.5 g / i

K2HPO4 3.6 g / i pH (NaOH) 7.0

Aqua bidest. ad 11

Tabelle I: Verwendete Beauveria ba

Table I: Beauveria ba used

Lu number genotype phenotype

700 wild type uridine positive / POPS positive

2793 pyrG uridine negative / POPS positive

2794 2793:: pA04-13 uridine positive / POPS negative

8979 2793:: pA04-13 uridine positive / POPS negative

8980 2793:: pA04-13 uridine positive / POPS negative

8987 Lu 8980:: 2Lambda2 uridine negative / POPS positive

8935 Lu 8980:: 2Lambda7 uridine negative / POPS positive

8988 Lu 8980:: 2Lambda20 uridine negative / POPS positive

8982 Lu 2793:: 2Lambda20 uridine negative / POPS positive

8985 Lu 8980:: 2Lambda7 uridine negative / POPS positive

Plasmids

A restriction map of plasmid pA04-13 is shown in Figure 1. The Lambda BlueSTAR cloning vectors are commercially available from Novagen (Madison, Wisconsin, U.S.A). The plasmids cloned in this work are shown in FIG. 3.

Based on the hypothesis that the gene of benzoate para-hydroxylase (bph A) is also involved in the hydroxylation of POPS, this gene was first isolated and characterized from B. bassiana. Deletion experiments, however, showed that this gene had no effect on POPS hydroxylation.

In order to identify and isolate the genes involved in the POPS hydroxylation, a REMI (restriction enzyme mediated integration) mutagenesis was carried out. Among the REMI transformants, 3 mutants were found that are defective for the POPS hydroxylation. It was not possible from two mutants 12 lent to isolate the defective gene. However, the defective gene was isolated from the third mutant. The corresponding "wild-type gene" (= WT gene) was isolated from the parent strain for the mutagenesis.

The transformation of the POPS-negative strain with the "wild-type gene" again led to a POPS-positive phenotype, i.e. the strain was again able to hydroxylate POPS to HPOPS. The introduction of the "WT gene" into a wild-type Beauveria bassiana led to an increased hydroxylation rate from POPS to HPOPS. The isolated gene is thus involved in the hydroxylation reaction.

Example 1: Isolation of a PyrG mutant

To isolate the genes involved in the POPS hydroxylation, genetically labeled HPOPS-negative mutants were produced using REMI mutagenesis. Since the Beauveria wild-type strains of Bialaphos are resistant, a different selection marker was used. Therefore 5-fluororotic acid (= 5-FOA) resistant, uridine negative mutant was isolated. After analysis of the mutants, it was ensured that it was a pyrG mutation. This 5-fluororotic acid-resistant, uridine-negative strain Lu 2793 was used for the REMI experiments. PA04-13 (pyrG, FIG. 1) was used as the insertion vector.

Lu 700 spores were plated with 5-FOA containing medium. Several resistant colonies were picked and tested for the correct phenotype. Of the colonies, clone # 6 had the phenotype sought (5-FOA ^R , uridine). The strain was cleaned twice, each time testing for the uridine negative phenotype. In subsequent transformation experiments it was shown that Lu 2793 is a pyrG ^" mutant. For this purpose the strain was grown for 32 hours in 500 ml of YPD medium, mycelium was harvested and incubated overnight with NOVOzym234 (Novo Nordisk Biotechnologie GmbH, Mainz, Germany) Protoplasts were washed and incubated without DNA (control), circular plasmid pAB4-I (pyrG gene from A. niger) and circular plasmid pA04-13 (pyrG gene from A.oryzae). Each transformation mixture was divided and each on a minimal medium plate with sorbitol and a plate with minimal medium sorbitol, casaminoacids (0.1%) and vitamins. After 12 days, transformants could be isolated from the transformation batches which contained one of the two plasmids. No colonies could be detected without DNA. From these results it was concluded that the 5-FOA resistant mutant Lu 2793 is a pyrG mutant. 13

Example 2: Isolation of plasmid and phage DNA from a B. bassiana gene library

B. bassiana created a gene library in Lambda BlueSTAR (Novagen). Plasmid and phage DNA were isolated from this library. Unless otherwise stated, the molecular biological work and the methods used were carried out as in Current Protocols in Molecular Biology, January 1998, John Wiley & Sons, Inc. New York.

Example 2: REMI transformation

The transformation of Lu 2793 with the plasmid pA04-13 (FIG. 1) led to a reduction in the transformation frequency in the presence of different amounts (2.5-25 U) of restriction enzyme Mbol. No or only minor effects on the transformation rate were observed when 0.5 or 1.25 units Mbol or 10 or 50 units BamHI were added to the transformation approach. After Southern analysis of randomly selected REMI transformants, it was found that the transformation with linear DNA or in the presence of Mbol led to the integration of more than two copies of the plasmid in approximately 50% of the transformants. No multiple copies were found in the presence of BamHI during transformation. In 50% of the cases the vector integrated into a BamHI site.

After transformation with pA04-13, 1192 REMI mutants were obtained. Three of these mutants Lu 2794, 8978 and 8980 were no longer able to hydroxylate POPS to HPOPS. Southern analyzes showed that the mutant Lu 2794 had integrated an intact copy and two deleted copies of the plasmid at one locus. With Mutante Lu 8979, several copies in tandem form are probably integrated in one place. In mutant Lu 8980 two copies of the vector and a deleted copy are integrated at one point (FIG. 2).

In order to isolate the genes which were marked by the REMI vector in these transformants, the chromosomal DNA sequence flanking the REMI vector had to be reisolated via E. coli. A variety of re-isolated plasmids were analyzed from the mutants Lu 2794 and Lu 8979, but no plasmids could be isolated which contained the flanking sequences of the integration site. The integration was mapped by mutant Lu 8980 and the sequences flanking on the right and left were isolated. These sequences were sequenced and two ORF (- open reading frames) could be found. The REMI vector integrated 1.6 kb downstream from ORF 1 and 1.0 b 14 upstream from ORF 2. Of both ORF's, the function was unknown (FIG. 2).

By screening a B. bassiana gene bank, 6 different Lambda BlueSTAR clones were found which contained the chromosomal fragment into which the REMI vector was integrated at mutant Lu 8980. Three of these clones contained both ORF 1 and ORF 2 (2 lambda 2, 7 and 20) (see Figure 3).

A transformation experiment with mutants 2793 and Lu8980 resulted in transformants that contained at least one copy of either 2Lambda2, 7 or 20. The Lu 8980 transformants were able to hydroxylate POPS again and some of the Lu 2793 transformants showed significantly higher POPS hydroxylation rates compared to the parent strain. The sequence of the isolated gene is given in SEQ ID No. 1. It is involved in the hydroxylation of POPS to HPOPS.

500 ml of YPD medium with uridine were inoculated with a culture of the Lu 2793 strain. After 32 hours the mycelium (approx. 4 g) was harvested and incubated with NOVOzym234 for 16 hours. The resulting 3xl0 ⁹ protoplasts were centrifuged off and washed. In order to determine the viability of the protoplasts, dilution series were plated out on: minimal medium; Casaminoacids; Vitamins with and without uridine (= without MMcv with Mmcvuri) and with and without 1.2 M sorbitol (= without Mmsorbcv with Mmsorbcvuri). 1.5xl0 ⁷ protoplasts were mixed without DNA, with circular plasmid PA04-13 or linearized plasmid pA04-13 (BamHI - digested) in the presence or absence of different amounts of Mbol and / or BamHI. These different approaches were made on plates

Minimal medium; 1.2 M sorbitol; Casaminoacids and vitamins plated. The vitality of the protoplasts was determined after 5 days. No colonies were found when the plates did not contain uridine (Mmcv and Mmsrobcv). The vitality of the protoplasts on stabilized medium (Mmsrobcvuri) was approx. 4% and on non-stabilized medium (Mmcvuri) 2%. After 8 days you could see the first transformants.

After 12 days, the transformants were counted. A total of 1192 REMI transformants were isolated.

These transformants were tested in microtiter plates to determine whether they could hydroxylate POPS to HPOS. Among 1192 REMI transformants tested, 3 mutants Lu 2794, 8979 and 8980 were found which showed no defect in growth and were no longer able to hydroxylate POPS to HPOPS. 15

The assay was carried out in microtiter plates. The holes in the microtiter plates were filled with POPS agar and inoculated with one REMI transformant each and incubated at 28 ° C. for three days. Whether the transformants could still hydroxylate POPS to HPOPS was checked by overlaying with Pauly's reagent according to Kutacek (staining reagents for thin-layer and paper chromatography, Merk, Darmstadt (1980) Reagent No.: 304). The presence of HPOPS is indicated by a brown-black color.

The REMI mutant Lu 8980 came from a group of 321 REMI mutants, of which only Lu 8980 could no longer hydroxylate POPS. The further transformant Lu 2793 came from an approach in which Lu 2793 was transformed with linearized plasmid pA04-13 in the presence of 1.25 U Mbol.

To isolate the flanking sequences of the mutant Lu 8980, the chromosomal DNA was isolated and digested with EcoRI, BamHI, Bgll and Hindlll, ligated and the ligation mixture used to transform E. coli. A map of the integration pattern and the flanking sequences on the right and left could be determined from the analysis of these plasmids. (See Figure 2).

The next step in the analysis of mutant Lu 8980 was the isolation of wild-type B. densa chromosomal DNA containing the mutant gene contained in the mutant Lu 8980.

A LambdaBlueSTAR gene bank from Beauveria was used for the isolation. The Lambda BlueSTAR clones contain chromosomal B. densa DNA fragments of 10-20 kb, which were obtained by partial digestion with Sau3A.

The gene library was screened for clones that hybridize to the flanking sequences. The "left" 0.8 kb EcoRI / HindIII fragment (0.8 E / H) was used as a sample. Filter replicas on Hybond-N were produced from 14 plates containing approximately 3.8 x 10 ⁴ plaques with inserts (approximately 19 times the chromosome). These filters were hybridized with the 0.8 E / H fragment (65 ° C, 0.2xSSC, 0.1% SDS).

20 hybridizing plaques (2Lambdal - 20) were isolated and after a few purification steps the plasmid DNA was isolated. After restriction analysis with Kpnl and Hindlll it could be shown that 6 different plasmids were isolated (see FIG. 3). 16

In order to isolate further Lambda BlueSTAR clones which contained further upstream and downstream sequences of the insertion site, the filters were again treated with a mixture of a 3.0 kb BamHI / Kpnl and 1.0 kb Kpnl fragment of the left flank and with a 2.5 kb Hindll / Kpnl fragment the right flank hybridized as a sample. Lambda clones 21 and 22 were isolated in this way.

Figure 3 gives an overview of the restriction pattern of the clones. The following sequence was derived from these clones.

Example 3: Sequence analysis

Two open reading frames could be identified on a DNA fragment of 11.3 kb (= SEQ ID No. 1) whose translated sequence with sequences in the database showed significant homology. These are the orfl: Bp: 2468-3871 (1464 bp) and the orf 2: Bp7760-8917 (1158 bp, see Figure 4). The nucleotides (= G) contained in sequence SEQ ID No. 1 at positions 45, 2642, 3120, 3135 and 7845 are unclear. The sequence could not be clearly determined at these positions, that is to say the G nucleotides contained therein can also mean A, T or C. Sequencing problems also arose around position 3217, which means that other nucleotides could also be present here.

Orf 1 codes for a hypothetical protein with 488 aa and a molecular weight of 51167.2 daltons. The isoelectric point of the protein is 5.52. With the help of the Blast program, databases (Swissprot and PIR) were searched for homology. This BLAST search identified a sequence homolog to orfl: an open reading frame with accession number 0 06598 from Mycobacterium tuberculosis. This orf with a length of 446 aa was examined in more detail with the help of the MegAlign program. A pairwise alignment of both aa sequences resulted in an identity of 30.8% over a range of 411 aa. A psfi analysis of orf 1 showed no homology to known motifs.

Orf 2 encodes a hypothetical protein of 41326 daltons with an isoelectric point of 10.4. A blast search against Swissprot and PIR resulted in the following homologies:

d Y3E8 s. cerevisiae 17a YB9R S. cerevisiae b Y33K H. sapiens c Y38 C. elegans

d Y3E8 p. cerevisiae 17

A pairwise alignment of the orf 2 with the sequences found using the MegAlign program resulted in the following homologies:

orf2 - d 23 4 % 156 aaorf2 - a 21 6% 272 aa orf2 - b 21 5 277 aa orf2 - c 22 2 9 Ό- 337 aa

orf2 - d 23 4% 156 aa

Example 4; Implementation of POPS with Lambda transformants from Mutante Lu 8980 in shake culture

Transformants from strain Lu 8980 with lambda 2, 7 and 20 were isolated. It was checked whether these clones can hydroxylate POPS again. For this purpose, the strains were grown in 250 ml Erlenmeyer flasks with 30 ml medium with 50 g / 1 POPS (see POPS medium). After three days, the turnover was analyzed by GC. The inoculum of the cultures originated from precultures which were grown with 30 g / l POPS. The results are shown in Table II below:

Table II: HPOPS production

HPOPS strain after three days (%)

Lu 700 (wild type) 18.4

Lu 8980 0

Lu 8987 18.1

Lu 8935 15.5

Lu 8988 18.8

The results showed that the cloned fragments can complement the mutation generated in Lu 8980 and thus lead to a wild type phenotype.

Example 4 Implementation of POPS with Lambda Transformants from Lu 2793 in Shake Culture

Transformants from the Lu 2793 strain with lambda 7 and 20 were isolated as described. The transformants obtained were tested in shake culture to determine whether they had an increased POPS hydroxylation rate. For this purpose, the strains were grown in 250 ml Erlenmeyer flasks with 30 ml medium with 70 g / 1 POPS and after three days the conversion was analyzed by GC. The inoculum of the cultures came from precultures grown with 30 g / 1 POPS Turned 18. The results are shown in the following table In:

Table III: HPOPS productivity

Bio dry matter spec. Hydroxyl. -rate

Strain HPOPS g / 1 mg / g g HPOPS / mg BTM

Lu 2793 10.5 18.3 0.5

10 Lu 8982 15.5 14.8 1.04

Lu 8985 14.5 18.5 0.78

The results show that by additional copies of the characterized fragment, the hydroxylation rates of B. bassiana I ^{5 could be} significantly increased.

Example 5: Cloning of the reductase (= SEQ ID No.4)

Analogously to the 2 ° cloning strategy described for SEQ ID No. 1, the reductase, which is the reduction equivalents for the

Reaction delivers cloned. By combining the sequences SEQ ID No. 1 and SEQ ID No. 4, productivity could be further increased by a factor of 1.2.

25

30

35

40

5

Claims

19 patent claims 1. Process for the preparation of 2- (4-hydroxyphenoxy) propionic acid from 2-phenoxypropionic acid or its salts with a Microorganism which is able to convert 2-phenoxypropionic acid to 2- (4-hydroxyphenoxy) propionic acid, characterized in that the microorganism contains at least one of the genes contained in the sequences SEQ ID No. 1 or SEQ ID No.4 or their functional genes Equivalents contains. 2. The method according to claim 1, characterized in that the genes contained in SEQ ID No. 1 according to claim 1 or their functional equivalents in combination with the gene SEQ ID No.4 or its functional equivalents is used. 3. The method according to claim 1 or 2, characterized in that the functional equivalents are genes which have a homology of 40 to 100% on the amino acid level derived from the sequences SEQ ID No. 1 or SEQ ID No.4 . 4. Process according to claims 1 to 3, characterized in that a fungus or a bacterium is used as the microorganism. 5. Process according to claims 1 to 4, characterized in that fungi selected from the group Aspergillus, Beauveria, Paecilomyces, Sclerotium, Coprinus or bacteria selected from the group Pseudomonas, Rhodococcus, Nocardia or Streptomyces are used as the microorganism. 6. Isolated DNA sequence with the sequence SEQ ID N.l or SEQ ID No.4. 7. Proteins with the amino acid sequence derived from SEQ ID No. 1 or SEQ ID No. 4 and their functional equivalents with a homology of at least 80% at the amino acid level. 20th 8. Gene construct containing genes with SEQ ID No. 1 or SEQ ID No.3 or their combination, as well as their functional equivalents, which are functionally linked to one or more regulation signals to increase gene expression or protein expression or both and / or whose natural regulation has been switched off. 9. A gene construct according to claim 8, characterized in that it has been inserted in a vector which is suitable for the expression of the gene in a prokaryotic or eukaryotic host organism. 10. organisms containing a gene construct according to claim 8 or 9. 11. Use of sequences according to claim 1 for the preparation of 2- (4-hydroxyphenoxy) propionic acid from 2-phenoxypropionic acid. 12. Use of a gene construct according to claim 8 or 9 for the preparation of 2- (4-hydroxyphenoxy) propionic acid from 2-phenoxypropionic acid.