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WO1999050420A1 - Procede pour l'augmentation du taux d'hydroxylation de l'acide phenoxypropionique - Google Patents

Procede pour l'augmentation du taux d'hydroxylation de l'acide phenoxypropionique Download PDF

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Publication number
WO1999050420A1
WO1999050420A1 PCT/EP1999/002205 EP9902205W WO9950420A1 WO 1999050420 A1 WO1999050420 A1 WO 1999050420A1 EP 9902205 W EP9902205 W EP 9902205W WO 9950420 A1 WO9950420 A1 WO 9950420A1
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seq
sequences
gene
propionic acid
pops
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PCT/EP1999/002205
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German (de)
English (en)
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Bernhard Hauer
Christoph Dingler
Robert Van Gorcom
Cora Van Zeijl
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Basf Aktiengesellschaft
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Priority to JP2000541308A priority Critical patent/JP2002509725A/ja
Priority to EP99914558A priority patent/EP1066384A1/fr
Priority to CA002324973A priority patent/CA2324973A1/fr
Publication of WO1999050420A1 publication Critical patent/WO1999050420A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/02—Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids

Definitions

  • 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.
  • 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.
  • (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)
  • 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.
  • 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.
  • sequences SEQ ID No. 1 and / or SEQ ID No. 4 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
  • sequence SEQ ID No. 1 is particularly preferably used.
  • 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.
  • promoter variants 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.
  • 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.
  • 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.
  • Gram-negative bacteria are Pseudomonadaceae and the genus Pseudomonas.
  • 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.
  • 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.
  • 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.
  • mutants can be generated using all common methods, 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.
  • transposable genetic elements such as transposons or IS elements can also be used for mutagenesis.
  • their property POPS to HPOPS 5 to be implemented to an increased extent (measurement by GC analysis, see EP-B-0 465 494).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, ⁇ -P R - or contained in the ⁇ -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, MF ⁇ , AC, P-60, CYC1, GAPDH. 6
  • 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, ⁇ gtll or pBdces or in StreBomyces pIJlOl, pIJ364, pIJ702 or pIJ361, in yeasts YEp6, YEpl3 or pEMBLYe23.
  • 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, 2 ⁇ 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 ⁇ .
  • the expression systems are preferably to be understood as the combination of pA04-13 (pyrG) or pAB4-1 and Beauveria bassiana or Aspergillus niger.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • media known to the person skilled in the art are used.
  • 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.
  • sugars such as mono-, di- or polysaccharides such as glucose, fructose, mannose, xylose, galactose, rib
  • Advantageous nitrogen sources are organic or inorganic nitrogen compounds or materials that contain these compounds.
  • ammonium salts such as NH 4 C1 or (NH 4 ) 2 S ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • EP-B-0 465 494 see examples 1 to 7
  • EP-A-0 758 398 see examples.
  • 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.
  • Lu 8980 2Lambda7 uridine negative / POPS positive
  • FIG. 3 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.
  • REMI restriction enzyme mediated integration
  • 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.
  • NOVOzym234 Novo Nordisk Biotechnologie GmbH, Mainz, Germany
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • flanking sequences of the mutant Lu 8980 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).
  • mutant Lu 8980 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.
  • 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).
  • Figure 3 gives an overview of the restriction pattern of the clones. The following sequence was derived from these clones.
  • 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.
  • 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:
  • 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:
  • Reaction delivers cloned.
  • productivity could be further increased by a factor of 1.2.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour la production d'acide (hydroxy-4 phénoxy)-2 propionique à partir d'acide phénoxy-2 propionique ou de ses sels, en présence d'un micro-organisme capable de transformer de l'acide phénoxy-2 propionique en acide (hydroxy-4 phénoxy)-2 propionique. Le procédé selon l'invention est caractérisé en ce que le micro-organisme contient au moins un des gènes contenus dans les séquences SEQ ID n° 1 ou SEQ ID n° 4 ou leurs équivalents fonctionnels. L'invention concerne également des séquences d'ADN isolées d'identification SEQ ID n° 1 ou SEQ ID n° 4, ainsi qu'un produit génique contenant les séquences nommées. L'invention concerne enfin un micro-organisme contenant le produit génique et l'utilisation des séquences et du produit génique pour la production d'acide (hydroxy-4 phénoxy)-2 propionique.
PCT/EP1999/002205 1998-04-01 1999-03-31 Procede pour l'augmentation du taux d'hydroxylation de l'acide phenoxypropionique WO1999050420A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2000541308A JP2002509725A (ja) 1998-04-01 1999-03-31 Ｐｏｐｓ−ヒドロキシル化率の増大方法
EP99914558A EP1066384A1 (fr) 1998-04-01 1999-03-31 Procede pour l'augmentation du taux d'hydroxylation de l'acide phenoxypropionique
CA002324973A CA2324973A1 (fr) 1998-04-01 1999-03-31 Procede pour l'augmentation du taux d'hydroxylation de l'acide phenoxypropionique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19814528A DE19814528A1 (de) 1998-04-01 1998-04-01 Verfahren zur Erhöhung der POPS-Hydroxylierungsrate
DE19814528.4 1998-04-01

Publications (1)

Publication Number Publication Date
WO1999050420A1 true WO1999050420A1 (fr) 1999-10-07

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Application Number Title Priority Date Filing Date
PCT/EP1999/002205 WO1999050420A1 (fr) 1998-04-01 1999-03-31 Procede pour l'augmentation du taux d'hydroxylation de l'acide phenoxypropionique

Country Status (5)

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EP (1) EP1066384A1 (fr)
JP (1) JP2002509725A (fr)
CA (1) CA2324973A1 (fr)
DE (1) DE19814528A1 (fr)
WO (1) WO1999050420A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113881715B (zh) * 2021-09-15 2023-06-06 湖北工业大学 一种r-（+）-2-（4-羟基苯氧基）丙酸的生物合成方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990011362A1 (fr) * 1989-03-28 1990-10-04 Basf Aktiengesellschaft Procede de production par fermentation d'un acide propionique de 2-(4-hydroxyphenoxy-)
WO1994029453A2 (fr) * 1993-06-11 1994-12-22 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Nouvelle oxydoreductase issue de champignons filamenteux, adn codant pour celle-ci et cellules transformees avec ledit adn
WO1995029249A1 (fr) * 1994-04-26 1995-11-02 Basf Aktiengesellschaft Procede de biotransformation d'acides carboxyliques en presence d'un micro-organisme

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990011362A1 (fr) * 1989-03-28 1990-10-04 Basf Aktiengesellschaft Procede de production par fermentation d'un acide propionique de 2-(4-hydroxyphenoxy-)
WO1994029453A2 (fr) * 1993-06-11 1994-12-22 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Nouvelle oxydoreductase issue de champignons filamenteux, adn codant pour celle-ci et cellules transformees avec ledit adn
WO1995029249A1 (fr) * 1994-04-26 1995-11-02 Basf Aktiengesellschaft Procede de biotransformation d'acides carboxyliques en presence d'un micro-organisme

Non-Patent Citations (1)

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Title
GRIFFITHS, D.A. ET AL.: "Metabolism of xenobiotics by Beauveria bassiana", XENOBIOTICA, vol. 23, no. 10, October 1993 (1993-10-01), pages 1085 - 1100, XP002111241 *

Also Published As

Publication number Publication date
CA2324973A1 (fr) 1999-10-07
EP1066384A1 (fr) 2001-01-10
DE19814528A1 (de) 1999-10-07
JP2002509725A (ja) 2002-04-02

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