WO2001031047A2 - Mikrobiologisches verfahren zur herstellung aromatischer aldehyde und/oder carbonsäuren - Google Patents
Mikrobiologisches verfahren zur herstellung aromatischer aldehyde und/oder carbonsäuren Download PDFInfo
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- WO2001031047A2 WO2001031047A2 PCT/EP2000/010552 EP0010552W WO0131047A2 WO 2001031047 A2 WO2001031047 A2 WO 2001031047A2 EP 0010552 W EP0010552 W EP 0010552W WO 0131047 A2 WO0131047 A2 WO 0131047A2
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- 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
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- the invention relates to an oxidative microbiological process for the preparation of aromatic aldehyde and / or carboxylic acid derivatives using recombinant microorganisms expressing xylene monooxygenase or alkane monooxygenase.
- Xylene monooxygenase e.g. encoded by the TOL plasmid pW O from Pseudomonas putida mt-2 is an enzyme system that plays a key role in the breakdown of toluene and xylenes.
- XMO belongs to the family of alkyl group hydroxylases and selectively hydroxylates a methyl group on the aromatic ring. This is the first step of a metabolic pathway (see FIG. 1 (A)) which leads to the formation of carboxylic acid derivatives, which are then converted into substrates for the Krebs cycle via the so-called meta-degradation pathway.
- XMO consists of two polypeptide subunits XylM and XylA, which are encoded by the genes xylM and xylA (xylMA GENBANK Accession No. M37480)
- XylA is a NADH acceptor reductase, i.e. an electron transport protein that transfers reduction equivalents of NADH to XylM, a hydroxylase located in the membrane.
- Alkane hydroxylase is the first enzyme in a medium chain alkane degradation pathway involving a set of enzymes encoded by two alk gene clusters on the catabolic OCT plasmid.
- the second enzyme in the pathway shown in Figure 1 (A) is benzyl alcohol dehydrogenase (BADH), a homodimeric member of a zinc-containing dehydrogenase family, the substrates of which are long-chain alcohols. This enzyme is encoded by the xylB gene.
- BADH benzyl alcohol dehydrogenase
- BZDH benzaldehyde dehydrogenase
- Escherichia coli which were genetically recombined in such a way that they express XMO, not only toluenes and xylenes, but also m- and p-ethyl-, methoxy-, nitro- and chlorine-substituted toluenes as well as m -Bromine-substituted toluene can oxidize to the corresponding benzyl alcohol derivatives (19, 20). Styrene is oxidized to styrene oxide (ee 95%).
- XMO also catalyzes the second step in the metabolic pathway shown in FIG.
- a simplified microbiological production method could surprisingly be provided.
- the invention is based in particular on the surprising finding that XMO or AMO are able to catalyze each individual step of the reaction path shown in FIG. 1 (A) or 1 (B).
- sieren ie the oxidation of alkyl-substituted aromatics to the carboxylic acid derivative via the corresponding alcohol derivative and the corresponding aldehyde derivative as intermediates.
- a first subject of the invention thus relates to a process for the preparation of aromatic aldehydes and / or carboxylic acids of the general formula I.
- Ar represents an optionally mono- or polysubstituted mononuclear aromatic ring
- R 1 represents an oxygen-containing group -CHO or -COOH
- n stands for an integer value from 0 to 15, such as O to 12, 1 to 6 or 6 to 12,
- R 3 represents H or OH; or, if R 1 is -COOH, R 2 is also
- R 4 can be in which n is as defined above and R 4 is -CHO;
- XMO xylene monoxygenase
- AMO alkane monooxygenase
- the reaction according to the invention can therefore be carried out in one or more stages using the same enzyme.
- the alkylated aromatic, the corresponding alcohol or the corresponding aldehyde can be used as the substrate.
- the degree of oxidation of the substrate used can be controlled in a simple manner, as described below.
- the aromatic ring system Ar in the compounds of the formulas I and II prepared according to the invention or used as a substrate can be mono- or polysubstituted.
- the position of the ring substituent (s) can be selected as desired. However, the meta and / or para position to the side chain to be oxidized is preferred.
- substrates of the formula II which can be oxidized by XMO by the process according to the invention are toluene, xylenes, styrene, m- and / or p-methyl, ethyl-, methoxy-, nitro- and chlorine-substituted toluenes and m-bromine substituted toluene and pseudocumene (ie trimethylbenzenes); as well as the corresponding alcohols or aldehydes of these compounds.
- substrates of the formula II which can be oxidized by AMO by the process according to the invention are toluene, ethylbenzene, n- and i-propylbenzene, n-butylbenzene, and the m- and / or p-methyl, ethyl, methoxy, nitro- and chlorine-substituted analogs of these compounds; and the corresponding alcohols and aldehydes of these compounds.
- XMO encoded by the genes xylA and xylB according to xylMA GENBANK Accession No. M37480 and corresponding isoenzymes.
- XMO preferably originates from bacteria of the genus Pseudomonas, in particular of the species Pseudomonas putida, preferably strain mt-2 (ATCC 33015).
- AMO encoded by the genes alkB, alkG and alkT according to GENBANK Accession No. AJ245436 and corresponding isoenzymes (eg isoenzymes to alkB).
- AMO preferably originates from bacteria of the genus Pseudomonas, in particular of the species Pseudomonas oleovorans, preferably strain GPol (ATCC 29347).
- “functional equivalents” are understood to mean, in particular, enzyme mutants which, in at least the sequence position, have a different amino acid than the original one, but nevertheless catalyze one of the oxidation reactions mentioned above. “Functional equivalents” thus include those substituted by one or more amino acid additions or substituents , Deletions and / or inversions available mutants, the changes mentioned being able to occur in any sequence position as long as they lead to a mutant with the catalytic activity according to the invention. Functional equivalence is particularly given when the reactivity patterns between mutant and unchanged enzyme match qualitatively, i.e. for example, the same substrates can be implemented at different speeds.
- “Functional equivalents” naturally also include monooxygenases derived from other organisms, e.g. from bacteria other than those specifically mentioned here, as well as naturally occurring variants or isozymes. For example, regions of homologous sequence regions can be determined by sequence comparison and equivalent enzymes can be determined based on the specific requirements of the invention.
- nucleic acid sequences other than those specifically mentioned (single and double-stranded DNA and RNA sequences) which code for one of the above monooxygenases and their functional equivalents. Further nucleic acid sequences which can be used according to the invention thus differ from the specifically used sequences by addition, substitution, insertion or deletion of individual or more nucleotides, but continue to code for a monooxygenase with the desired property profile.
- the invention also includes the use of nucleic acid sequences which comprise so-called silent mutations or which have been changed in accordance with the codon usage of a specific source or host organism, in comparison to a specifically named sequence, as well as naturally occurring variants thereof, for example splice variants.
- the subject matter is likewise obtainable by conservative nucleotide substitutions (ie the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility).
- the invention also relates to expression constructs containing, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a monooxygenase enzyme which can be used according to the invention; and vectors comprising at least one of these expression constructs.
- Such constructs according to the invention preferably comprise a promoter 5 'upstream of the respective coding sequence and a terminator sequence 3' downstream and, if appropriate, further customary regulatory elements, in each case operatively linked to the coding sequence.
- An “operative linkage” is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence. Examples of sequences which can be linked operatively are targeting sequences and translation enhancers, enhancers, polyadenylation signals and the like. Other regulatory elements include selectable markers, amplification signals, origins of replication and the like.
- the natural regulatory sequence can still be present before the actual structural gene. This natural regulation can, if necessary, be switched off by genetic modification and the expression of the genes increased or decreased.
- the gene construct can, however, also have a simpler structure, that is to say no additional regulation signals are inserted in front of the structural gene and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that regulation no longer takes place and gene expression is increased or decreased.
- the nucleic acid sequences can be contained in one or more copies in the gene construct.
- Examples of useful promoters are: cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, tre , ara, SP6, 1-PR or in the 1-PL promoter, which are more advantageous find wise use in gram-negative bacteria; as well as the gram-positive promoters amy and SP02, the yeast promoters ADC1, MFa, AC, P-60, CYC1, GAPDH or the plant promoters CaMV / 35S, SSU, OCS, lib4, usp, STLS1, B33, not or the ubiquitin or phaseolin promoter.
- inducible promoters such as, for example, light- or temperature-inducible promoters, such as the P r P ⁇ promoter, is particularly preferred
- the regulatory sequences mentioned are intended to enable the targeted expression of the nucleic acid sequences and the 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 or decrease it.
- the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
- an increase in translation is also possible, for example, by improving the stability of the mRNA.
- An expression cassette according to the invention is produced by fusing a suitable promoter with a suitable monooxygenase nucleotide sequence and a terminator or polyadenylation signal. Common recombination and cloning techniques are used for this, as described, for example, in T. Maniatis et al (24) and in T.J. Silhavy et al. (32) and in Ausübel, F.M. et al. (33) are described.
- the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which enables optimal expression of the genes in the host.
- Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. et al. (34)).
- vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally.
- recombinant microorganisms can be produced which, for example, have been transformed with at least one vector according to the invention and can be used in the method according to the invention.
- the recombinant constructs according to the invention described above are advantageously introduced and expressed in a suitable host system.
- Common cloning and transfection methods known to the person skilled in the art such as, for example, co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, are preferably used here in order to bring the nucleic acids mentioned into expression in the respective expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al. (35).
- all organisms which allow expression of the nucleic acids according to the invention, their allele variants, their functional equivalents or derivatives and which can be used to carry out the microbiological oxidation reaction according to the invention are suitable as host organisms.
- Host organisms are, for example, bacteria, fungi, yeasts, plant or animal cells.
- Preferred organisms are bacteria.
- microorganism expressing XMO is preferably used which has essentially no benzyl alcohol dehydrogenase (BADH) and / or no benzaldehyde dehydrogenase (BZDH) activity.
- BADH benzyl alcohol dehydrogenase
- BZDH benzaldehyde dehydrogenase
- microorganisms expressing AMO which have essentially no alkanol dehydrogenase (AODH) and / or alkanol dehydrogenase (AADH) activity which are encoded by the alkJ or alkH genes.
- AODH alkanol dehydrogenase
- AADH alkanol dehydrogenase
- a bacterium of the genus Escherichia such as. B. E. coli
- the strain W3110 and one of the K12 strains, such as JM101 and DH5 ⁇ , or one of the Pseudomonas putida strains, such as the strain KT 2440 are used.
- the characteristics of some preferred E. coli strains are given in Table I.
- microorganisms with a vector is carried out according to the invention using established standard techniques (24) and therefore does not require any detailed discussion.
- Successfully transformed organisms can be selected using marker genes which are also contained in the vector or in the expression cassette. Examples of such marker genes are genes for antibiotic resistance and for enzymes which catalyze a coloring reaction which stains the transformed cell. These can then be selected using automatic cell sorting.
- Microorganisms which have been successfully transformed with a vector and which carry an appropriate antibiotic resistance gene for example G418 or hygromycin
- Marker proteins that are presented on the cell surface can be used for selection by means of affinity chromatography.
- the combination of the host organisms and the vectors suitable for the organisms such as plasmids, viruses or phages, such as, for example, plasmids with the RNA polymerase / promoter system, the phages ⁇ or ⁇ or other temperate phages or transposons and / or further advantageous regulatory ones Sequences form an expression system.
- a recombinant microorganism which is transformed with an expression vector which, e.g. under the genetic control of the alk regulation system from Pseudomonas oleovorans GPol, which contains the genes xylM and xylA coding for XMO or the genes alkB, alkG and alkT coding for AMO in operative linkage.
- an expression vector which, e.g. under the genetic control of the alk regulation system from Pseudomonas oleovorans GPol, which contains the genes xylM and xylA coding for XMO or the genes alkB, alkG and alkT coding for AMO in operative linkage.
- the microorganism is particularly preferably transformed with the xylMA-encoding expression plasmid pSPZ3.
- the alk regulation system from Pseudomonas oleovorans GPol is known per se.
- the expression of the first of the two alk gene clusters mentioned above is under the control of alkBp, the alk promoter, and begins in the presence of the functional regulatory protein alkS, which is encoded by the second alk gene cluster, and in the presence of an inducer.
- an inducer such as B. an alkane, for example n-octane, or a little related to these compounds, such as.
- DCPK Dicyclopropyl ketone
- n-octane and DCPK are preferably used as inductors, particularly preferably in an amount of 0.001 to 0.5% (v / v) in the case of n-octane and in an amount of 0.005 to 0.05 % (V / V) in the case of DCPK. It can mixtures of n-octane and DCPK can of course also be used. When working in these concentration ranges, the induction is maximum.
- the invention also relates to a microbiological process for the oxidation of organic compounds of the above type with the aid of the recombinant microorganisms just described.
- the recombinant microorganism used according to the invention can be cultivated and fermented by known methods. Bacteria can be propagated, for example, in TB or LB medium and at a temperature of 20 to 40 ° C and a pH of 6 to 9. Suitable cultivation conditions are described in detail, for example, in T. Maniatis et al., Cited above.
- the microorganisms are preferably first cultivated in the presence of oxygen and in a complex medium, such as e.g. TB or LB medium, at a cultivation temperature of about 20 to 40oC or more, and a pH of about 6 to 9 until a sufficient cell density is reached.
- a complex medium such as e.g. TB or LB medium
- the use of an inducible promoter is preferred.
- the cultivation is carried out after induction of monooxygenase production in the presence of oxygen, e.g. 1 hour to 3 days, continued.
- the oxidation product or product mixture formed can then be processed in a conventional manner, e.g. by extraction or chromatography, separated from the medium and cleaned.
- the reactions according to the invention can advantageously also be carried out in bioreactors which contain the recombinant microorganism according to the invention, e.g. in immobilized form.
- the degree of oxidation of the substrates used according to the invention can be controlled in a simple manner. For example, samples are taken from the culture medium at regular intervals and checked for the content of the corresponding alcohol, aldehyde and / or carboxylic acid by gas chromatography or using gas chromatography-mass spectrometer coupling (GC-MS) or high-performance liquid chromatography. Derivatives examined. Depending on which oxidized derivative is desired, or when a desired mixing ratio has been established, the incubation is interrupted. This can be done, for example, by removing or killing the microorganisms from the culture medium, for example by centrifuging and decanting and / or by treatment with acid, for example trichloroacetic acid. or by treatment with heat. Acid formation can also be inhibited by metering in unoxidized substrate (such as toluene or pseudocumene).
- unoxidized substrate such as toluene or pseudocumene
- the oxidized aromatic can then be isolated from the culture medium using conventional separation processes, for example by simple distillation, fractional distillation, rectification, if appropriate in vacuo, or by using suitable chromatographic processes, preferably by distillation.
- the microorganism cells are expediently removed from the culture medium before these products are purified.
- a vector was also used to transform the microorganisms which, in addition to the XMO genes xylM and xylA, also contains the benzyl alcohol dehydrogenase (BADH) gene xylB from Pseudomonas putida mt-2 in an expressible form.
- BADH benzyl alcohol dehydrogenase
- plasmid pRS which contained no xyl genes, was constructed in the course of the studies according to the invention.
- pRMAB was digested with BamHI and Smal and treated with Klenow-Enzy. After isolation of the larger fragment, the vector was religated.
- Figure 1 shows (A) the gradual oxidation of toluene to benzyl alcohol, benzaldehyde and benzoic acid by the enzymes of the upper TOL pathway and the organization of the xyl genes of the upper TOL operon.
- BADH and BZDH stand for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase.
- P u denotes the top TOL operon promoter, xylW a gene with unknown function, xylC the gene encoding BZDH, xylM the gene encoding the terminal hydroxylase component of XMO, xylA the NADH: acceptor reductase Component of the gene encoding XMO, xylB the gene encoding BADH and xylN a gene with unknown function; (B) the stepwise oxidation of an aryl-substituted alkane via the corresponding alkanol and alkanal to the alkane carboxylic acid, catalyzed by the enzymes alkane hydroxylase (AMO), alkanol dehydrogenase (AODH) and alkane aldehyde (AADH).
- AMO alkane hydroxylase
- AODH alkanol dehydrogenase
- AADH alkane aldehyde
- FIG. 2 shows construction schemes of the expression plasmids pSPZ3 and pRMAB with the genes xylMA and xylMAB under the control of the alk regulation system.
- alkBp means the promoter of the alk operon
- alkS is the gene for the positive regulator AlkS.
- the xylM * and xylA genes encode the xylene monooxygenase (this * means that a Ndel site has been removed in the xylM gene).
- the xylB gene encodes BADH.
- Km is the gene for kanamycin resistance and T4t is the transcription terminator of phage T4.
- Figure 3 shows the oxidation of toluene by E. coli JM101
- FIG. 4 shows the oxidation of pseudocumene, the corresponding alcohol and the corresponding aldehyde by E. coli JM101 (pSPZ3) (A, C, E) and E. coli JM101 (pRMAB) (B, D).
- the substrates (0.46 mM) were converted into a suspension of resting E. coli JM101 (pSPZ3 / pBRMAB) cells (0.86-0.92 g *! " 1 CDW) in
- the arrow in graph (A) indicates when 0.1% (v / v) n-octane was added to induce XylMA synthesis.
- FIG. 6 shows a possible mechanistic explanation for the XMO-catalytic formation of benzaldehyde from benzyl alcohol.
- a * the plasmid only contains part of the xylA gene
- the bacteria were either in Luria-Bertani (LB) broth (Difco, Detroit, Mich.) Or in M9 minimal medium (24) containing three times the concentration of phosphate salts (M9 *) and 0.5% (w / v) Contained glucose as the only carbon source.
- the cultures were optionally with kanamycin (final concentration: 50 mg / liter), ampicillin (100 mg / liter), chloramphenicol (30 mg / liter), thiamine (10 _3 %, w / v ), 1 mM indole and 0.5 mM IPTG (isopropyl- ⁇ -D-1-thiogalactopyranoside).
- Solid media contained 1.5% (w / v) agar. Liquid cultures were routinely grown at 30 or 37 ° C on horizontal shakers at 200 rpm.
- One unit (U) is defined as the activity that gives 1 ⁇ mol total products in 1 minute.
- the specific activity is expressed here as activity per g cell dry weight (CDW) (U g _1 CDW) (hereinafter also simply referred to as activity). Calculation as average activity, based on the amount of products per g CDW, which are formed in the first 5 minutes of implementation.
- the experiments were repeated independently at least three times.
- the assay was carried out as follows. E. coli JM101 recombined with the appropriate vectors were incubated in 40 or 100 ml of medium in the presence of kanamycin.
- the cells were induced by adding 0.05% (v / v) DCPK or 0.1% (v / v) n-octane and 3 to 3, Incubated 5 hours more until the OD 450 typically rose to 0.8-0.9. The cells were then harvested and resuspended to a dry cell weight of 2.5 g / l in 50 mM potassium phosphate buffer, pH 7.4, containing 1% (w / v) glucose. Aliquots of 1 or 2 ml were placed in stoppered Pyrex tubes and incubated horizontally on a rotary shaker at 30 ° C and 250 rpm.
- the respective substrate was added to a final concentration of 1.5 mM in the form of a 20-fold concentrated stock solution in ethanol.
- the dry cell weight was reduced to 1 g / 1 and the respective substrates were added to a final concentration of 0.5 mM, because this compound was low in water Has solubility.
- the reaction was carried out on the shaker for 5 minutes and then ended by placing the samples in ice and immediately adding 40 or 80 ⁇ l of perchloric acid stock solution (10% v / v) so that the pH of the suspension was 2.
- High performance liquid chromatography HPLC was used to separate benzyl alcohol, benzyl aldehyde and benzoic acid.
- Nucleosil C18 pore size 100 ⁇ , particle size 5 ⁇ m, length 25 cm, inner diameter 4 mm
- H 2 O-30% acetonitrile-0.1 served as the column and 69.9% H 2 O-30% acetonitrile-0.1 as the mobile phase % H 3 P0 4 at a flow rate of 0.7 ml / min.
- the gas chromatograph (Fisons Instruments, England) was equipped with an OPTIMA-5 quartz capillary column (length 25 m, inner diameter 0.32 mm, film thickness 0.25 ⁇ m) from Macherey-Nagel (Oensingen, Switzerland). Hydrogen was used as the carrier gas and the injection was splitless. The following temperature profile was used: from 40 ° C to 70 ° C at 15 ° C / min, from 70 ° C to 105 ° C at 5 ° C / min and from 105 ° C to 240 ° C at 20 ° C / minute The compounds were detected using a flame ionization detector. The separated compounds were identified by comparing their retention times with those of commercially available standards.
- the detection can also be carried out with a mass spectrometer (GC-MS coupling).
- GC-MS coupling a mass spectrometer
- the GC-MS coupling consisted of a Fisons type MD-800 mass spectrometer and a gas chromatograph (Fisons Instruments, England) equipped with a CP-Sil-5CB column (Chrompack, The Netherlands). Helium was used as the carrier gas. The injection was split (20: 1). The temperature program was the same as for the gas chromatographic separation described above.
- Example 1 Oxidation of toluene and derivatives thereof with xylene monooxygenase
- the cells were each grown to a cell density of 0.09 g CDW / 1 and routinely induced with 0.1% (v / v) n-octane. Then the cultures were a further 3 to Incubated for 3.5 hours and grown to a cell density of 0.23 to 0.27 g CDW / 1.
- Table II shows that XMO oxidizes toluene to benzyl alcohol, benzyl alcohol to benzaldehyde and benzyldehyde to benzoic acid. Activities of up to 95-100 U / g CDW were found for the first two oxidation reactions, whereas the oxidation of benzaldehyde was carried out with a low activity, namely only 10 U / g CDW.
- Controls were uninduced E. coli JM101, which contained the plasmid pSPZ3, and induced E. coli JM101, which contained no plasmid.
- E. coli JM101 which contained the plasmid pRS, were used as additional controls.
- the plasmid pRS still contains the alkS gene, but not the xyl genes. Table II shows that when toluene, pseudo-documol and the corresponding alcohols were used as substrates, no conversion products were detectable in the control experiments.
- the activity assay was performed as described above.
- the unit (U) is as defined above and the specific activity was calculated as described above.
- Example 2 Determination of the evolution of various substrates over time
- FIGS. 3 and 4 The time course of the oxidation of toluene, pseudocumene, 3,4-dimethylbenzyl alcohol and 3,4-dimethylbenzaldehyde is shown in FIGS. 3 and 4.
- the assays were carried out as described above.
- the respective substrate was added to a suspension of the respective resting cells in 50 mM potassium phosphate buffer, pH 7.4, which contained 1% (w / v) glucose.
- Example 3 Conversion of toluene and pseudocumene by E. coli JMIOI (pRMAB).
- Example 4 Growth and induction kinetics of E. coli JM101 (pSPZ3).
- a single culture was grown for each activity point.
- the activity assay was carried out as described above.
- pseudocumene (1.37 mM) was made into a suspension of resting E. coli JM101 (pSPZ3) (2.04-2.26 g l- 1 CDW) in 50 M potassium phosphate buffer, pH 7.4. containing 1% (w / v) glucose.
- the specific activities were calculated using the gas-chromatographically determined amounts of the products formed in the first 5 minutes of the reaction.
- the arrow indicates the point in time at which 0.1% (v / v) n-octane was added to induce the xylMA synthesis.
- the filled circles indicate the dry cell weight (CDW) of the uninduced cultures, the open circles the dry cell weight of the induced cultures and the crosses the specific activities of the induced cultures.
- 5 (B) and (C) were obtained as in FIG. 5 (A), with the difference that the cell density was 2.26-2.44 gl " 1 CDW.
- FIG. 5 (B) shows the effects of different Amounts of n-octane and Figure 5 (C) that of DCPK
- the circles indicate the dry cell weight 3.5 hours after induction and the crosses indicate the specific activities of the induced cultures.
- XMO activity was monitored after induction with 0.1% (v / v) n-octane (Fig. 5 (A)) or 0.05% (v / v) DCPK.
- the XMO activity was quickly induced by both compounds and reached a constant strength of about 115 or 105 U / g CDW in the case of n-octane or DCPK after 3 to 3.5 hours of induction time. Compared to non-induced cells, the growth rates of the induced cells were significantly lower.
- the dependence of the XMO activity on the inductor concentrations [in the range 0.00001-1% (v / v)] was determined by attracting E. coli JM101 (pSPZ3) to a concentration of 0.09 g CDW per liter and the Cells with different amounts of n-octane and DCPK were induced. After a further 3.5 hours of culture, the cell dry weight and the XMO activity were determined for each inducer concentration (FIGS. 5 (B) and (C)). XMO activities were very low when less than 0.0001% (v / v) n-octane or 0.001% (v / v) DCPK was added to the culture medium.
- XMO clearly has a higher affinity for toluene and pseudocumene than for the corresponding aldehydes.
- BADH results in less activity for product formation and even the regression of benzyl alcohol.
- the cells containing BADH clearly accumulate the aldehydes more slowly.
- BADH appears to dramatically increase the effects of E. coli dehydrogenases, with the balance of this dehydrogenase reaction appearing to be on the alcohol side. This is confirmed by thermodynamic calculations according to methods known in the prior art (26 to 29) and by enzyme kinetic studies (16-18).
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CA002389138A CA2389138A1 (en) | 1999-10-27 | 2000-10-26 | Microbiological method for producing aromatic aldehydes and/or carboxylic acids |
AU13880/01A AU782371B2 (en) | 1999-10-27 | 2000-10-26 | Microbiological methods for the producing of aromatic aldehydes and/or carboxylic acids |
EP00975925A EP1224315B1 (de) | 1999-10-27 | 2000-10-26 | Mikrobiologisches verfahren zur herstellung aromatischer aldehyde und/oder carbonsauren |
JP2001533182A JP2003512078A (ja) | 1999-10-27 | 2000-10-26 | 芳香族アルデヒド及び/又はカルボン酸の微生物学的製造方法 |
IL14920400A IL149204A0 (en) | 1999-10-27 | 2000-10-26 | Microbiological method for producing aromatic aldehydes and/or carboxylic acids |
DE50012376T DE50012376D1 (de) | 1999-10-27 | 2000-10-26 | Mikrobiologisches verfahren zur herstellung aromatischer aldehyde und/oder carbonsauren |
NO20021906A NO20021906L (no) | 1999-10-27 | 2002-04-23 | Microbiologisk fremgangsmåte for fremstilling av aromatiske aldehyder og/eller karbosykliske syrer |
Applications Claiming Priority (2)
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DE19951768A DE19951768A1 (de) | 1999-10-27 | 1999-10-27 | Mikrobiologisches Verfahren zur Herstellung aromatischer Aldehyde und/oder Carbonsäuren |
DE19951768.1 | 1999-10-27 |
Publications (2)
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WO2001031047A2 true WO2001031047A2 (de) | 2001-05-03 |
WO2001031047A3 WO2001031047A3 (de) | 2002-02-07 |
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PCT/EP2000/010552 WO2001031047A2 (de) | 1999-10-27 | 2000-10-26 | Mikrobiologisches verfahren zur herstellung aromatischer aldehyde und/oder carbonsäuren |
Country Status (14)
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EP (2) | EP1645638B1 (de) |
JP (1) | JP2003512078A (de) |
KR (1) | KR100807899B1 (de) |
CN (1) | CN100354427C (de) |
AT (2) | ATE319846T1 (de) |
AU (1) | AU782371B2 (de) |
CA (1) | CA2389138A1 (de) |
DE (3) | DE19951768A1 (de) |
DK (1) | DK1224315T3 (de) |
ES (2) | ES2304738T3 (de) |
IL (1) | IL149204A0 (de) |
NO (1) | NO20021906L (de) |
PT (1) | PT1224315E (de) |
WO (1) | WO2001031047A2 (de) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003014368A2 (en) * | 2001-08-10 | 2003-02-20 | E.I. Du Pont De Nemours And Company | Use of xylene monooxygenase for the oxidation of substituted monocyclic aromatic compounds |
WO2005021766A1 (en) * | 2003-09-02 | 2005-03-10 | Martin Fussenegger | Regulatable gene expression in mammalian cells and mammals |
US7541168B2 (en) | 2000-07-18 | 2009-06-02 | National Research Council Of Canada | Recombinant cyclopentanone monooxygenase [cpmo] |
DE102009002811A1 (de) | 2009-05-05 | 2010-11-11 | Evonik Degussa Gmbh | Enzymatisches Verfahren zur Herstellung von Aldehyden |
EP2738260A3 (de) * | 2012-11-30 | 2014-09-10 | Samsung Electronics Co., Ltd | Verfahren zur enzymatischen Herstellung einer aromatischen Carbonsäure |
CN113897322A (zh) * | 2021-06-29 | 2022-01-07 | 迪嘉药业集团有限公司 | 一种3-甲基-4-硝基苯甲酸的工程菌及其制备方法 |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
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DE10117359A1 (de) | 2001-04-06 | 2002-10-10 | Basf Ag | Verfahren zur Oxidation aromatischer Verbindungen |
KR100659419B1 (ko) * | 2003-12-31 | 2006-12-18 | 주식회사 효성 | 슈도모나스 푸티다 유래의 벤즈알데히드 디히드로게나제유전자의 발현 벡터, 이 벡터로 형질전환된 미생물 및 이형질전환체를 이용한 고순도 2,6-나프탈렌 디카르복실산의제조 방법 |
KR100811385B1 (ko) * | 2004-11-16 | 2008-03-07 | 주식회사 효성 | 자일렌 모노옥시게나제를 발현하는 재조합 미생물의 제조방법과 이를 이용한 2,6-나프탈렌 디카르복실산의 제조방법 |
DE102010015807A1 (de) * | 2010-04-20 | 2011-10-20 | Evonik Degussa Gmbh | Biokatalytisches Oxidationsverfahren mit alkL-Genprodukt |
WO2015190632A1 (ko) * | 2014-06-12 | 2015-12-17 | 한국과학기술원 | 테레프탈산 생산능을 가지는 재조합 미생물 및 이를 이용한 테레프탈산의 제조방법 |
CN107974428A (zh) * | 2017-12-13 | 2018-05-01 | 迪沙药业集团有限公司 | 一种重组大肠杆菌及用于转化生产5-甲基吡嗪-2-羧酸的方法 |
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EP0442430A2 (de) * | 1990-02-13 | 1991-08-21 | Lonza Ag | Mikrobiologische Oxidation von Methylgruppen in Heterocyclen |
EP0466115A1 (de) * | 1990-07-10 | 1992-01-15 | Lonza Ag | Mikrobiologische Oxidation von Ethylgruppen in Heterocyclen |
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- 2000-10-26 WO PCT/EP2000/010552 patent/WO2001031047A2/de active IP Right Grant
- 2000-10-26 EP EP06000642A patent/EP1645638B1/de not_active Expired - Lifetime
- 2000-10-26 AU AU13880/01A patent/AU782371B2/en not_active Ceased
- 2000-10-26 JP JP2001533182A patent/JP2003512078A/ja not_active Withdrawn
- 2000-10-26 EP EP00975925A patent/EP1224315B1/de not_active Expired - Lifetime
- 2000-10-26 DK DK00975925T patent/DK1224315T3/da active
- 2000-10-26 AT AT00975925T patent/ATE319846T1/de not_active IP Right Cessation
- 2000-10-26 PT PT00975925T patent/PT1224315E/pt unknown
- 2000-10-26 ES ES06000642T patent/ES2304738T3/es not_active Expired - Lifetime
- 2000-10-26 ES ES00975925T patent/ES2258982T3/es not_active Expired - Lifetime
- 2000-10-26 DE DE50012376T patent/DE50012376D1/de not_active Expired - Fee Related
- 2000-10-26 KR KR1020027005344A patent/KR100807899B1/ko not_active IP Right Cessation
- 2000-10-26 CN CNB008178666A patent/CN100354427C/zh not_active Expired - Fee Related
- 2000-10-26 IL IL14920400A patent/IL149204A0/xx unknown
- 2000-10-26 AT AT06000642T patent/ATE396276T1/de not_active IP Right Cessation
- 2000-10-26 CA CA002389138A patent/CA2389138A1/en not_active Abandoned
- 2000-10-26 DE DE50015174T patent/DE50015174D1/de not_active Expired - Fee Related
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2002
- 2002-04-23 NO NO20021906A patent/NO20021906L/no not_active Application Discontinuation
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EP0466115A1 (de) * | 1990-07-10 | 1992-01-15 | Lonza Ag | Mikrobiologische Oxidation von Ethylgruppen in Heterocyclen |
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B]HLER B ET AL.: "Xylene monooxygenase catalyzes the multistep oxygenation of toluene and pseudomucene to corresponding alcohols, aldehydes, and acids in Escherichia coli JM101" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 14, 7 April 2000 (2000-04-07), pages 10085-10092, XP002166422 * |
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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US7541168B2 (en) | 2000-07-18 | 2009-06-02 | National Research Council Of Canada | Recombinant cyclopentanone monooxygenase [cpmo] |
WO2003014368A2 (en) * | 2001-08-10 | 2003-02-20 | E.I. Du Pont De Nemours And Company | Use of xylene monooxygenase for the oxidation of substituted monocyclic aromatic compounds |
WO2003014368A3 (en) * | 2001-08-10 | 2004-09-16 | Du Pont | Use of xylene monooxygenase for the oxidation of substituted monocyclic aromatic compounds |
WO2005021766A1 (en) * | 2003-09-02 | 2005-03-10 | Martin Fussenegger | Regulatable gene expression in mammalian cells and mammals |
DE102009002811A1 (de) | 2009-05-05 | 2010-11-11 | Evonik Degussa Gmbh | Enzymatisches Verfahren zur Herstellung von Aldehyden |
EP2738260A3 (de) * | 2012-11-30 | 2014-09-10 | Samsung Electronics Co., Ltd | Verfahren zur enzymatischen Herstellung einer aromatischen Carbonsäure |
US9562240B2 (en) | 2012-11-30 | 2017-02-07 | Samsung Electronics Co., Ltd. | Process of biologically producing aromatic carboxylic acid and derivative thereof |
CN113897322A (zh) * | 2021-06-29 | 2022-01-07 | 迪嘉药业集团有限公司 | 一种3-甲基-4-硝基苯甲酸的工程菌及其制备方法 |
CN113897322B (zh) * | 2021-06-29 | 2023-01-17 | 迪嘉药业集团股份有限公司 | 一种3-甲基-4-硝基苯甲酸的工程菌及其制备方法 |
Also Published As
Publication number | Publication date |
---|---|
DE19951768A1 (de) | 2001-05-03 |
ATE319846T1 (de) | 2006-03-15 |
ES2304738T3 (es) | 2008-10-16 |
CN1415018A (zh) | 2003-04-30 |
NO20021906L (no) | 2002-04-26 |
CN100354427C (zh) | 2007-12-12 |
PT1224315E (pt) | 2006-07-31 |
NO20021906D0 (no) | 2002-04-23 |
DE50015174D1 (de) | 2008-07-03 |
EP1645638A1 (de) | 2006-04-12 |
ES2258982T3 (es) | 2006-09-16 |
KR20020060217A (ko) | 2002-07-16 |
KR100807899B1 (ko) | 2008-02-27 |
EP1224315A2 (de) | 2002-07-24 |
IL149204A0 (en) | 2002-11-10 |
AU782371B2 (en) | 2005-07-21 |
CA2389138A1 (en) | 2001-05-03 |
EP1645638B1 (de) | 2008-05-21 |
DE50012376D1 (de) | 2006-05-04 |
DK1224315T3 (da) | 2006-07-10 |
WO2001031047A3 (de) | 2002-02-07 |
JP2003512078A (ja) | 2003-04-02 |
ATE396276T1 (de) | 2008-06-15 |
AU1388001A (en) | 2001-05-08 |
EP1224315B1 (de) | 2006-03-08 |
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