AU782371B2 - Microbiological methods for the producing of aromatic aldehydes and/or carboxylic acids - Google Patents

Abstract

Production of aromatic aldehyde or carboxylic acid (I) by culturing a microorganism that expresses xylene mono-oxygenase (XMO) or alkane mono-oxygenase (AMO) in a medium containing an aromatic substrate (II). Production of aromatic aldehyde or carboxylic acid of formula (I) by culturing a microorganism that expresses xylene mono-oxygenase (XMO) or alkane mono-oxygenase (AMO) in a medium containing an aromatic substrate of formula (II). [Image] Ar : monocyclic aromatic ring, optionally with one or more substituents; R 1>formyl or carboxy; n : 0-15; R 2>vinyl or (CH 2) m-R 3>, also where R 1>= carboxy, R 2>may be (CH 2) n-CHO; m : n+1; and R 3>hydrogen or hydroxy. Independent claims are also included for the following: (a) recombinant microorganism harboring an expression vector that contains, under control of the alk-regulatory system of Pseudomonas oleovoransGpol, the XMO-encoding genes xylM and xylA, or the AMO-encoding genes alkB, alkG and alkT; and (b) expression construct containing the genes specified in (a), operably linked to the alk-regulatory system.

Description

0050/50842 Microbiological process for the preparation of aromatic aldehydes and/or carboxylic acids The invention relates to an oxidative microbiological process for the preparation of aromatic aldehydes and/or carboxylic acid derivatives using recombinant microorganisms which express xylene monooxygenase or alkane monooxygenase.

Xylene monooxygenase (XMO), as it is encoded, for example, by the TOL plasmid pWWO of Pseudomonas putida mt-2, is an enzyme system which plays a key role in the breakdown of toluene and xylenes.

XMO belongs to the family of the alkyl hydroxylases and selectively hydroxylates a methyl group on the aromatic ring.

This is the first step of a metabolic pathway (cf. Figure 1 which leads to the formation of carboxylic acid derivatives which are then transformed via the meta-catabolic pathway into substrates for the Krebs cycle.

XMO is composed of two polypeptide subunits XylM and XylA which are encoded by the genes xylM and xylA (xylMA GENBANK accession No. M37480). XylA is an NADH acceptor reductase, i.e. an electron transport protein which transfers reduction equivalents from NADH to XylM, a hydroxylase which is localized in the membrane.

XylM activity is dependent on the presence of phospholipids and iron (II) ions and has a pH optimum of 7. The amino acid sequence of XylM shows 25% homology with the amino acid sequence of the hydroxylase component AlkB of the P. oleovorans GPol alkane hydroxylase.

The alkane hydroxylase is the first enzyme of a catabolic pathway for alkanes of medium chain length in which a set of enzymes participates which is encoded by two alk gene clusters on the catabolic OCT plasmid.

The second enzyme in the metabolic pathway shown in Figure 1 (A) is benzyl alcohol dehydrogenase (BADH), a homodimeric member of a zinc-containing dehydrogenase family whose substrates are long-chain alcohols. This enzyme is encoded by the xylB gene. The third enzyme in the metabolic pathway shown in Figure 1 is benzaldehyde dehydrogenase (BZDH), again a homodimer, and encoded by the xylC gene.

0050/50842 2 More detailed information on the functions and properties of the abovementioned genes and enzymes are found in the references (1) to (18).

It was demonstrated that Escherichia coli which were genetically engineered in such a way that they express XMO are not only capable of oxidizing toluene and xylenes, but also m- and pethyl-, methoxy-, nitro- and chlorine-substituted toluenes and m-bromine-substituted toluene to give the corresponding benzyl alcohol derivatives (19, 20). Styrene is oxidized to give styrene oxide (ee In addition, XMO is also suspected to catalyze the second step in the metabolic pathway shown in Figure 1 viz. the oxidation in vivo in experiments on complete, live cells) of benzyl alcohols to give the corresponding aldehydes (2, 21). The transformation of benzaldehyde into benzoate after the third step of the metabolic pathway shown in Figure 1 has also already been observed, but was ascribed to unspecific dehydrogenases in E. coli Further experiments with partially purified XylMA (also used synonymously in the present description for XMO, i.e. for the functional enzyme composed of two polypeptide subunits, viz. XylM and XylA) which were carried out in vitro, in contrast, have shown that this enzyme has no activity with regard to benzyl alcohol The reasons for this discrepancy remain unclear.

Owing to the pronounced homology between xylene monooxygenase and alkane monooxygenase (AMO, also termed alkane hydroxylase; GENBANK Accession No.: AJ245436), the skilled worker would have expected similar restrictions for both systems with regard to nature and extent of the reactions catalyzed.

The prior-art biocatalytic processes for the preparation of aromatic aldehydes or carboxylic acids remain unsatisfactory in as far as a variety of enzymes seems to be required to carry out these processes. Also, the chemical synthesis proves to be difficult, owing to the regioselectivity and chemoselectivity required.

It is an object of the present invention to provide a simplified process for the preparation of aromatic aldehydes and/or carboxylic acids.

It has been found that this object is achieved in accordance with the invention, surprisingly, by a simplified microbiological preparation process. In particular, the invention is based on the surprising finding that XMO and AMO are capable of catalyzing each individual step of the reaction pathway shown in Figure 1 0050/50842 3 and 1 respectively, i.e. the oxidation of alkylsubstituted aromatics to give the carboxylic acid derivative via the corresponding alcohol derivative and corresponding aldehyde derivative as intermediates. Surprisingly, it has also been found in accordance with the invention that an activity which is 10 to times higher in comparison with earlier studies on XMO-expressing, recombinant E. coli (20, 21) can be achieved using a specific expression system for the XMO genes, i.e. xylM and xylA.

A first subject matter of the invention is thus a process for the preparation of aromatic aldehydes and/or carboxylic acids of the formula I Ar-(CH 2 )n-R 1

(I)

where Ar is a substituted or mono- or polysubstituted mononuclear aromatic ring,

R

1 is an oxygen-containing group -CHO or -COOH, and n is an integer from 0 to 15, for example from 0 to 12, from 1 to 6 or from 6 to 12, which comprises a) culturing, in particular aerobically, in a culture medium comprising an aromatic substrate of the formula II Ar-R 2

(II)

where Ar is as defined above and

R

2 is -CH=CH 2 or -(CH 2 )n+IR 3 where n is as defined above and

R

3 is H or OH; or, if R 1 is -COOH, R 2 can also be

-(CH

2 )nR 4 where n is defined above and R 4 is -CHO, a microorganism which expresses an enzyme selected from amongst xylene monooxygenase (XMO) and alkane monooxygenase (AMO), and b) isolating the compound(s) of the formula I from the culture medium.

r 0050/50842 4 Thus, the reaction according to the invention can be carried out in one or more steps using the same enzyme. Substrates which may be employed are the alkylated aromatic, the corresponding alcohol or the corresponding aldehyde. The degree of oxidation of the substrate employed can be controlled in a simple fashion, as described above.

Without wanting to be bound by theory, 180 incorporation experiments in conjunction with fragmentation patterns obtained by mass spectrometry suggest that the most possible mechanism of the XMO-catalyzed alcohol oxidation proceeds via the formation of a germinal diol as intermediate which is then dehydrogenated, probably in a non-stereospecific manner, giving rise to the aldehyde (cf. Fig. 6).

The aromatic ring system Ar in the compounds of the formulae I and II which are prepared in accordance with the invention, or employed as substrate, can be mono- or polysubstituted. The position of the ring substituent(s) may be selected as desired.

However, the meta- and/or para-position for the side chain to be oxidized is preferred.

Specific nonlimiting examples of substrates of the formula II which can be oxidized by XMO in accordance with the process according to the invention are toluene, xylenes, styrene, nand/or p-methyl-, ethyl-, methoxy-, nitro- and chlorine-substituted toluenes, and m-bromo-substituted toluene and pseudocumene trimethylbenzenes); and the corresponding alcohols and aldehydes of these compounds. Specific nonlimiting examples of substrates of the formula II which can be oxidized by AMO in accordance with 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.

The processes according to the invention are preferably carried out employing the following enzymes: XMO, encoded by genes xylA and xylB in accordance with xylMA GENBANK Accession No. M37480 and corresponding isoenzymes. XMO is preferably derived from bacteria of the genus Pseudomonas, in particular the species Pseudomonas putida, preferably strain mt-2 (ATCC 33015).

0050/50842 AMO, encoded by genes alkB, alkG and alkT in accordance with GENBANK Accession No. AJ245436 and corresponding isoenzymes (for example isoenzymes to alkB). AMO is preferably derived from bacteria of the genus Pseudomonas, in particular the species Pseudomonas oleovorans, preferably strain GPol (ATCC 29347).

Also encompassed in accordance with the invention is the use of "functional equivalents" of the XMOs and AMOs which have been disclosed specifically.

"Functional equivalents" or analogs of the monooxygenases which have been disclosed specifically are for the purposes of the present invention enzymes which differ therefrom, which continue to show the desired reaction, and which are useful for the preparation of aldehydes and/or carboxylic acids of the above formula I.

In accordance with the invention, "functional equivalents" are to be understood as meaning, in particular, enzyme mutants which have an amino acid other than the original amino acid in at least [lacuna] sequence position, but nevertheless catalyze one of the abovementioned oxidation reactions. Thus, "functional equivalents" encompass the mutants obtainable by one or more amino acid additions, substituents [sic], deletions and/or inversions, it being possible for the modifications mentioned to occur in any sequence position as long as they lead to a mutant with the catalytic activity in accordance with the invention.

Functional equivalents exist in particular also when the reactivity patterns between mutant and unmodified enzyme agree in terms of quality, i.e. for example when identical substrates are converted at different rates.

Naturally, "functional equivalents" also encompass monooxygenases which can be obtained from other organisms, for example from bacteria other than those mentioned specifically herein, and naturally occurring variants or isoenzymes. For example, regions of homologous sequence regions can be established by means of sequence alignment, and equivalent enzymes can be determined with reference to the specific specifications of the invention.

The use of nucleic acid sequences other than those mentioned specifically (single- and double-stranded DNA and RNA sequences) which encode one of the above monooxygenases and their functional equivalents is also encompassed in accordance with the invention.

Further nucleic acid sequences which are useful in accordance with the invention thus differ from the sequences employed specifically by the addition, substitution, insertion or deletion 0050/50842 6 of one or more nucleotides, but continue to encode a monooxygenase with the desired property profile.

The use of such nucleic acid sequences which encompass so-called silent mutations or which have been modified in accordance with the codon usage of a specific organism of origin, or host organism, in comparison with a specifically mentioned sequence is also encompassed in accordance with the invention, as are naturally occurring variants, for example splice variants thereof. Another subject matter are sequences which can be obtained by conservative nucleotide substitutions the amino acid in question is replaced by an amino acid of identical charge, size, polarity and/or solubility).

Subject matter of the invention are furthermore expression constructs comprising, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence encoding a monooxygenase enzyme which is useful in accordance with the invention; and vectors encompassing at least one of these expression constructs. Such constructs according to the invention preferably encompass, 5'-upstream of the coding sequence in question, a promoter and, 3'-downstream, a terminator sequence and, if appropriate, further customary regulatory elements, in each case operatively linked to the coding sequence. "Operative linkage" is to be understood as meaning 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 intended function when the coding sequence is expressed. Examples of sequences which can be linked operatively are targeting sequences and translation enhancers, other enhancers, polyadenylation signals and the like.

Other regulatory elements encompass selectable markers, amplification signals, replication origins and the like.

In addition to the artificial regulatory sequences, the natural regulatory sequence may still be present before the actual structural gene. This natural regulation can, if appropriate, be switched off by means of genetic modification, and the expression of the genes can be increased or reduced. However, the gene construct may also have a simpler structure, that is to say no additional regulatory signals are inserted before the structural gene and the natural promoter with its regulation is not removed.

Instead, the natural regulatory sequence is mutated in such a way the regulation no longer takes place and gene expression is increased or reduced. One or more copies of the nucleic acid sequences may be present in the gene construct.

0050/50842 7 Examples of useful promoters are: cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, 1-PR or in the [sic] 1-PL promoter, all of which are advantageously used in Gram-negative bacteria; and 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 promoters. The use of inducible promoters, such as, for example, light- or temperature-inducible promoters, such as the PrPI promoter, is especially preferred.

In principle, all natural promoters with their regulatory sequences may be used. In addition, synthetic promoters may also advantageously be used.

The abovementioned regulatory sequences are intended to make possible the targeted expression of the nucleic acid sequences and protein expression. For example, depending on the host organism, this may mean that the gene is expressed or overexpressed only after induction, or that it is expressed and/or overexpressed immediately.

The regulatory sequences or factors can preferably have a positive effect on an expression, thus increasing or reducing it.

Thus, the regulatory elements may be enhanced advantageously at the transcriptional level by using strong transcription signals such as promoters and/or enhancers. However, enhanced translation is also possible, for example by improving mRNA stability.

An expression cassette according to the invention is generated by fusing a suitable promoter with a suitable monooxygenase nucleotide sequence and a terminator signal or polyadenylation signal. Customary recombination and cloning techniques, as are described, for example, in T. Maniatis et al and also in T.J. Silhavy et al. (32) and in Ausubel, F.M. et al. (33) are used for this purpose.

For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which makes possible optimal expression of the genes in the host. Vectors are well known to the skilled worker and can be found, for example, in "Cloning Vectors" (Pouwels P. H. et al. In addition to plasmids, vectors are also to be understood as meaning all vectors which are known to the skilled worker, such as, for example, phages, viruses such as SV40, CMV, baculovirus 0050/50842 8 and adenovirus, transposons, IS elements, phasmids, cosmids and linear or circular DNA.

These vectors can be replicated autonomously in the host organism or chromosomally.

It is possible, with the aid of such vectors according to the invention, to generate recombinant microorganisms which are transformed for example with at least one vector according to the invention and which can be employed in the process according to the invention. The above-described recombinant constructs according to the invention are advantageously introduced into, and expressed in, a suitable host system. To do this, it is preferred to use customary cloning transfection methods known to the skilled worker, such as, for example, coprecipitation, protoplast fusion, electroporation, retroviral transfection and the like, in order to express the abovementioned nucleic acids in the expression system in question. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al. Suitable host organisms are, in principle, all organisms which make possible an expression of the nucleic acids according to the invention, of their allelic variants, of their functional equivalents or their derivatives, and which can be employed for carrying out the microbiological oxidation reaction according to the invention. Host organisms are to be understood as meaning, for example, bacteria, fungi, yeasts, plant cells or animal cells. Preferred organisms are bacteria.

However, a preferred XMO-expressing microorganism which is used is one which has essentially no benzyl alcohol dehydrogenase (BADH) and/or no benzaldehyde dehydrogenase (BZDH) activity.

It is furthermore preferred to use those AMO-expressing microorganisms which have essentially no alkanol dehydrogenase (AODH) and/or alkanal dehydrogenase (AADH) activity and are encoded by genes alkJ and alkH, respectively. For example, a microorganism which can be used is a bacterium of the genus Escherichia, such as, for example, E. coli, for example strain W3110, and one of the K12 strains such as JM101 and DH5a, or one of the Pseudomonas putida strains such as the strain KT 2440. The characteristics of some preferred E. coli strains are shown in Table I.

0050/50842 9 The transformation of microorganisms with a vector is carried out in accordance with the invention using established standard techniques and a detailed description can therefore be dispensed with.

Successfully transformed organisms may be selected by means of marker genes, which are also present in the vector or in the expression cassette. Examples of such marker genes are genes for resistance to antibiotics and genes for enzymes which catalyze a coloring reaction, which causes the transformed cell to become stained. These cells can then be selected by means of automatic cell sorting. Microorganisms which have successfully been transformed with a vector and which carry a suitable gene for resistance to antibiotics (for example G418 or hygromycin) can be selected by suitable solid or liquid media containing antibiotics. Marker proteins presented at the cell surface can be used for selection by means of affinity chromatography.

The combination of the host organisms and the vectors matching the organisms, such as plasmids, viruses or phages, for example plasmids with the RNA polymerase/promoter system, phages X or R or other temperant phages or transposons and/or further advantageous regulatory sequences constitutes an expression system.

In an especially preferred embodiment, a recombinant microorganism is used which has been transformed with an expression vector which contains the XMO-encoding genes xylM and xylA or the AMO-encoding genes alkB, alkG and alkT, linked operatively and, for example under the genetic control of the alk regulatory system of Pseudomonas oleovorans GPol.

It is especially preferred for the microorganism to be transformed with the xylMA-encoding expression plasmid pSPZ3.

The alk regulatory system of Pseudomonas oleovorans GPol is known per se. The expression of the first of the two alk gene clusters stated above is under the control of alkBp, the alk promoter, and starts in the presence of the functional regulatory protein alkS which is encoded by the second alk gene cluster and in the presence of an inductor such as, for example, an alkane, for example n-octane, or a compound which shows little relation herewith, such as, for example, dicyclopropyl ketone (DCPK) (8, 22, 23). The use of the alk regulatory system in E. coli has the advantage that no catabolite repression takes place.

0050/50842 n-Octane and DCPK are preferably used as inductors in the process according to the invention, especially preferably in an amount of 0.001 to 0.5% in the case of n-octane and in an amount of 0.005 to 0.05% in the case of DCPK. Naturally, mixtures of n-octane and DCPK may also be used. If these concentration ranges are used, the induction maximum is achieved.

The invention furthermore relates to a microbiological process for the oxidation of organic compounds of the abovementioned type with the aid of the recombinant microorganisms which have just been described. The recombinant microorganism used in accordance with the invention can be cultured and fermented by known methods. For example, bacteria can be multiplied in TB or LB medium and at a temperature of from 20 to 40 0 C at a pH of 6 to 9.

Specific suitable cultivation conditions are described, for example, in T. Maniatis et al., loc. cit.

In the microbiological oxidation according to the invention using above-described recombinant microorganisms, the microorganisms are preferably first cultured in the presence of oxygen and in a complex medium, such as, for example, TB or LB medium, at a cultivation temperature of approximately 20 to 400C or more, and a pH of approximately 6 to 9, until a sufficient cell density has been reached. In order better to control the oxidation reaction, the use of an inducible promoter is preferred. After the production of monooxygenase has been induced, the cultivation is continued in the presence of oxygen, for example for 1 hour to 3 days. The oxidation product or oxygen product mixture formed can then be separated from the medium and purified in the customary manner, such as, for example, by extraction or chromatography.

The reactions according to the invention can advantageously also be carried out in bioreactors which contain the recombinant microorganism according to the invention for example in immobilized form.

The degree of oxidation of the substrates employed in accordance with the invention can be controlled in a simple manner. For example, samples are taken regularly from the culture medium, and they are examined for their content in the corresponding alcohol derivatives, aldehyde derivatives and/or carboxylic acid derivatives by gas chromatography, using a coupled gas chromatography and mass spectrometer system (GC-MS) or high-pressure liquid chromatography. Depending on which oxidized derivative is desired, or if a desired mixing ratio has established, the incubation is stopped. This can be done, for 0050/50842 11 example, by removing the microorganisms from the culture medium or by destroying them, for example by centrifugation and decanting and/or by treatment with acid, for example trichloroacetic acid, or by thermal treatment. Acid formation may also be inhibited by metering in an oxidized substrate (such as, for example, toluene or pseudocumene).

The oxidized aromatic can then be isolated from the culture medium with the aid of customary separation methods, for example by simple distillation, fractional distillation, rectification, if appropriate in vacuo, or by applying suitable chromatographic methods, preferably by distillation. The microorganism cells are expediently removed from the culture medium before these products are isolated.

The construction of the expression vector pSPZ3 which is preferred in accordance with the invention is described in (31) Panke, et al., Applied and Environmental Microbiology (1999) 2324-2332, which is expressly incorporated herein by reference.

For the purposes of the studies in accordance with the invention, another vector used for transforming the microorganisms was one which in addition to the XMO genes xylM and xylA additionally also contains the benzyl alcohol dehydrogenase(BADH) gene xylB of Pseudomonas putida mt-2 in expressible form. To introduce the benzyl alcohol dehydrogenase gene xylB into the above-described plasmid pSPZ3 directly downstream relative to the xylA gene, the 2.3 kb XhoI/FspI fragment of plasmid pCK04 which contains the xylB gene, was first introduced into the XhoI- and the SmaI-digested vector pGEM-7Zf(+) (Promega, Zurich, Switzerland) giving rise to pGEMAB. The 2.3 kb fragment was excised from this construct with XhoI and BamHI and ligated into the XhoI- and BamHI-digested plasmid pSPZ3. The resulting plasmid was termed pRMAB (Figure 2).

However, experiments on pRMAB have shown that not only is benzyl alcohol oxidized to benzaldehyde with a lower activity if BADH in addition to XMO is present, but indeed the reverse reaction takes place and benzyl alcohol is formed.

For the purposes of the studies in accordance with the invention, plasmid pRS, which did not contain xyl genes, was constructed as negative control. pRMAB was digested with BamHI and SmaI and treated with Klenow enzyme. The larger fragment was isolated and the vector was then relegated.

0050/50842 12 In the following text, the process according to the invention is illustrated with reference to specific nonlimiting examples and the figures.

Figure 1 shows the stepwise oxidation of toluene to benzyl alcohol, benzaldehyde and benzoic acid by the enzymes of the upper TOL metabolic pathway, and the organization of the xyl genes of the upper TOL operon. BADH and BZDH represent benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, respectively. Pu indicates the upper TOL operon promoter, xylW a gene with unknown function, xylC the BZDH encoding gene, xylM the gene which encodes the terminal hydroxylase component of XMO, xylA the gene which encodes the NADH:Acceptor reductase component of XMO, xylB the BADH-encoding gene and xylN a gene with unknown function; shows the stepwise oxidation of an aryl-substituted alkane via the corresponding alkanol and alkanal to give the alkanecarboxylic acid, catalyzed by the enzymes alkane hydroxylase (AMO), alkanol dehydrogenase (AODH) and alkanal dehydrogenase (AADH).

Figure 2 shows the construction schemes of the expression plasmids pSPZ3 and pRMAB with the genes xylMA and xylMAB under the control of the alk regulatory system. alkBp indicates the promoter of the alk operon, alkS is the gene for the positive regulator AlkS. The genes xylM* and xylA encode the xylene monooxygenase (the means that an NdEI site has been removed in the xylM gene. The xylB gene encodes BADH. Km indicates the gene for kanamycin resistance, and T4t is the transcription terminator of the T4-phage.

Figure 3 shows the oxidation of toluene by E. coli JM101 (pSPZ3) and E. coli JM101 (pRMAB) Toluene (1.37 mM) was added to a suspension of resting E. coli JMIOI (pSPZ3/pBRMAB) cells (2.07-2.14 g*l- 1 CDW) in potassium phosphate buffer (50mM) pH 7.4, 1% glucose. Circles: toluene, squares: benzyl alcohol, triangles: benzaldehyde, diamonds: benzoic acid, crosses: total of the four concentrations.

Figure 4 shows the oxidation of pseudocumene, the corresponding alcohol and corresponding aldehyde by E. coli JM101 (pSPZ3) and E. coli JM101 (pRMAB) The substrates (0.46 mM) were added to suspension of resting E. coli JM101 (pSPZ3/pBRMAB) cells (0.86-0.92 g*l- 1 CDW) in potassium phosphate buffer (50 mM) pH 7.4, 1% glucose. Graphs and show the oxidation of pseudocumene, graphs and the oxidation of 3,4-dimethylbenzyl alcohol, and graph shows the oxidation of 3,4-dimethylbenzaldehyde. Circles: pseudocumene, squares: 0050/50842 13 3,4-dimethylbenzyl alcohol, triangles: 3,4-dimethylbenzaldehyde, diamonds: 3,4-dimethylbenzoic acid, crosses: total of all concentrations.

Figure 5 shows the growth and induction kinetics of XMO in E.

coli JM101 (pSPZ3). Graph shows the xylene monooxygenase activity and the cell dry weight (CDW) at different points in time after induction by n-octane, while graphs and indicate the same values 3.5 hours after induction with different amounts of n-octane and dicyclopropyl ketone (DCPK), respectively. Each activity determination was carried out on an independent culture. Pseudocumene (1.37 mM) was added to a suspension of resting E. coli JM101 (pSPZ3) cells (2.04-2.44 g*l- 1 CDW) in potassium phosphate buffer (50mM) pH 7.4, 1% (w/v) glucose. The specific activities are based on the product formation during the first 5 minutes of the reaction. The arrow in graph indicates the timing of the addition of 0.1% (v/v) n-octane in order to induce XylMA synthesis. Filled circles: CDW of noninduced cultures, open circles: CDW of induced cultures, crosses: specific activities of induced cultures.

Figure 6 shows a possible mechanistic explanation for the XMO-catalytic formation of benzaldehyde from benzyl alcohol.

General methods, of materials used a) Bacteria and plasmids: TABLE I Bacterial strains and plasmids Strain or Properties Source or plasmid reference Strains E. coli supE44 AlacU169 (0801acZAM15) hsdR17 recAl endAl gyrA96 thi-1 relAl JM101 supE thi-1 A(lac-proAB) F'[traD26 pro AB (24) lacIq Plasmids pSPZ3 alkS Palk xylMA ori pMB1; Km f Invention pRMAB pBRMA; xylB; Km f Invention pRS pBRMA; AxylMA Invention pCK04 lacZa Pu xylWCMABN; pSC101 oriV; Cm f pGEM7Zf(+) ColEI fl ori lacZa; Ap f Promega pGEMAB pGEM7Zf(+); xylA*B; Ap f Invention 0050/50842 14 the plasmid only contains part of the xylA gene b) Chemicals and enzymes All chemicals and enzymes used are commercially available, for example from Boehringer Mannheim (Rotkreuz, Switzerland), NEB (Schwalbach, Germany), Gibco (Basle, Switzerland),

AGS

(Heidelberg, Germany), Promega (Zurich, Switzerland), Fluka (Buchs, Switzerland), Aldrich (Buchs, Switzerland) and Lancaster (MUhlheim, Germany). The QIAprep Spin Miniprep Kit from Qiagen (Basle), Switzerland) was used in accordance with the manufacturer's instructions for the small-scale preparation of plasmid DNA.

c) Recombinant methods To construct the vectors/plasmids used, and transfect and transform the bacteria, standard recombinant methods were used which are described in detail for example in the book by Sambrook, Fritsch and Maniatis Moreover, preference is made to the section Materials and Methods in the original papers listed in the reference section. A further discussion can therefore be dispensed with.

d) Bacterial cultures The bacteria were cultured either in Luria-Bertani(LB) broth (Difco, Detroit, Mich.) or in M9 minimal medium (24) containing three times the concentration of phosphate salts and 0.5% glucose as the sole carbon source. If appropriate, the cultures were supplemented with kanamycin (end concentration: 50 mg/liter), ampicillin (100 mg/liter), chloramphenicol (30 mg/liter), thiamine 1 mM indole and 0.5 mM IPTG (isopropyl-B-D-l-thiogalactopyranoside). Solid media contained 1.5% agar. Liquid cultures were grown routinely at 30 or 37 0 C on horizontal shakers at 200 rpm.

e) Determination of the enzyme activity For the sake of simplicity, enzyme activities were determined using intact cells.

One unit is defined as the activity which results in 1 ptmol total products per minute. The specific activity is expressed here as the activity per g cell dry weight (CDW) (U g- 1 CDW), hereinbelow simply termed activity. Calculation 0050/50842 as mean activity based on the amount of products per g CDW formed during the first 5 minutes of the conversion. The experiments were replicated at least three times independently of each other.

The assay was carried out as follows. E. coli JM101 which had been recombined with the vectors in question were incubated in 40 or 100 ml of medium in the presence of kanamycin. When the optical density at 450 nm was approx. 0.3, the cells were induced by adding 0.05% DCPK or 0.1% n-octane, and incubation was continued for 3 to 3.5 hours until the

OD

450 had typically risen to 0.8-0.9. The cells were then harvested and resuspended up to a cell dry weight of 2.5 g/l in 50 mM potassium phosphate buffer pH 7.4 comprising 1% glucose. Portions of 1 or 2 ml were introduced into Pyrex tubes sealed with stoppers and incubated horizontally on an orbital shaker at 30 0 C and 250 rpm. After 5 minutes, the substrate in question was added to an end concentration of 1.5 mM in the form of a 20-fold concentrated stock solution in ethanol. In the experiments in which the formation of 3,4-dimethylbenzoic acid was determined, the cell dry weight was reduced to 1 g/l, and the substrates in question were added to an end concentration of 0.5 mM since this compound is sparingly soluble in water. The conversion was carried out for 5 minutes on the shaker and then terminated by placing the samples in ice and immediately treating them with 40 or 80 p1 of perchloric acid stock solution (10% v/v) so that the pH of the suspension was 2.

f) Determination of product formation as a function of time To examine product formation as a function of time, the cells were grown, induced, collected, resuspended and incubated for different periods, viz. 5, 10, 20, 30, 40 and 80 minutes with the substrate in question, as described above. Then, the conversions were terminated as described above, the cells were removed by centrifugation (7 800 g, 8 min), and the supernatants were analyzed.

High-performance liquid chromatography (HPLC) was used to separate benzyl alcohol, benzyl aldehyde and benzoic acid.

The column used was Nucleosil C18 (pore size 100 A, particle size 5 imu, length 25 cm, internal diameter 4 mm) (Macherey-Nagel, Oensingen, Switzerland), and the mobile phase 69.9% H 2 0/30% acetonitrile/0.1% H 3

PO

4 at a flow rate of 0.7 ml/min. The same column, but with 64.9% H 2 0/35% acetonitrile/0.1% H 3

PO

4 with the same flow rate, was used to 0050/50842 16 separate 3,4-dimethylbenzyl alcohol, 3,4-dimethylbenzaldehyde and 3,4-dimethylbenzoic acid. Detection was by UV absorption at 210 nm. The separated compounds were identified by comparing the retention times with those of commercially available standards.

In the case of toluene/pseudocumene and the corresponding alcohols, aldehydes and acids, separation was done by gas chromatography. The gas chromatograph (Fisons Instruments, England) was equipped with an OPTIMA 5 type capillary column (length 25 m, internal diameter 0.32 mm, film thickness 0.25 [im) by Macherey-Nagel (Oensingen, Switzerland). The carrier gas used was hydrogen, and injection was done without splitting. The following temperature profile was used: from 40 0 C to 70 0 C at 15 0 C/min, from 70 0 C to 105 0 C at 5 0 C/min, and from 105 0 C to 240 0 C at 20oC/min. The compounds were detected by a flame-ionization detector. The separated compounds were identified by comparing the retention times with those of commercially available standards. Alternatively, detection may also be carried out using a mass spectrometer (coupled GC-MS system). The latter has the advantage that not only the specific chromatographic retention time, but also the fragmentation pattern and the intensity distribution of the individual peaks can be used for the qualitative and quantitative determination of the reaction products.

The coupled GC-MS system was composed of a Fisons MD-800 type mass spectrometer and a gas chromatograph (Fisons Instruments, England) equipped with a CP-Sil-5CB column (Chrompack, the Netherlands). The carrier gas used was helium. The injection was split The temperature program was the same as in the separation by gas chromatography which has been described above.

An equal volume of ice-cold ether comprising 0.1 mM dodecane as the internal standard was added to the samples, both in gas chromatography and in the coupled GC-MS system. Then, sodium chloride was added to saturation and the aqueous phase was extracted at 30 0 C by vigorous shaking for 5 minutes, whereupon the phases were separated by centrifugation. The organic phase was dried over anhydrous sodium sulfate and then analyzed.

0050/50842 17 Example 1: Oxidation of toluene and derivatives thereof with xylene monooxygenase In the experiments which follow, all of the cells were grown to a cell density of 0.09 g CDW/1 and induced routinely with 0.1% n-octane. Then, the cultures were incubated for a further 3 to 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, benzylalcohol to benzaldehyde and benzaldehyde to benzoic acid.

Activities of up to 95-100 U/g CDW were found in the case of the first two oxidation reactions, while benzaldehyde was only oxidized at the lower activity of 10 U/g CDW.

Similar results were obtained with pseudocumene. Pseudocumene was oxidized to 3,4-dimethylbenzyl alcohol, 3,4-dimethylbenzyl alcohol to 3,4-dimethylbenzaldehyde and 3,4-dimethylbenzaldehyde to 3,4-dimethylbenzoic acid. An activity of 100 U/g CDW was found for the oxidation of pseudocumene, while 3,4-dimethylbenzaldehyde was formed more slowly with an activity of 50 U/g CDW and oxidized to 3,4-dimethylbenzoic acid with a markedly higher activity (55 U/g CDW) than benzaldehyde.

When the acids were added as substrates, neither reaction products nor a decrease in the acids were detected.

Uninduced E. coli JM101, which contained the plasmid pSPZ3 and induced E. coli JM101, which did not contain any plasmid acted as controls. To exclude any effects on E. coli by the alk regulatory system, 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 demonstrates that no transformation products were detected in the control experiments when toluene, pseudocumene and the corresponding alcohols were used as substrates.

Table III below shows that aldehydes added as substrates in this experiment were nevertheless reduced to the alcohols with constant activity. The formation of 3,4-dimethylbenzoic acid was also identified, but with a very low activity of 2 to 2.5 U/g CDW. This permits the conclusion that most of the acid formed by induced E. coli JM101 (pSPZ3) (this abbreviation means in this context that the microorganisms contain the plasmid stated, can be attributed to the presence of XMO.

0050/50842 18 It should be mentioned at this point that no significant differences were found with regard to the products formed and the activities, or formation rates, between the routinely used inductor 0.1% n-octane and the inductor 0.05% DCPK, which was used as an alternative.

TABLE II Oxidation of toluene and derivatives by XMO Substrate Specific activity [U g- 1

CDW]

E. coli JM101 E. coli JM101 E. coli JM101 (pSPZ3) (pSPZ3) (pRS) inducedb uninduced inducedb toluene 100 0 0 Benzyl alcohol 95 0 0 Benzaldehyde 10 s.b.c s.b.c Pseudocumene 100 0 0 3,4-Dimethylbenzyl 50 0 0 alcohol 3,4-Dimethyl- 55 s.b.c s.b.c benzaldehyde a The assay for determining the activity was carried out as described above. The unit is as defined above, and the specific activity was calculated as described above.

b The cells were induced by addition of 0.1% n-octane.

c see below (Table III) TABLE III Transformation of aldehydes in the control experiments Specific activitya [U g- 1

CDW]

Benzaldehyde as substrate 3,4-Dimethylbenzaldehyde as substrate Type of E. coli JM101 E. coli JM101 E. coli JM101 E. coli product (pSPZ3) (pRS) (pSPZ3) JM101 formed uninduced inducedb uninduced (pRS) inducedb Alcohol 19 15 9.4 8.3 formation Acid 0.5 0 2.2 2.4 formation a, b cf. Table II 0050/50842 19 The reduction of the aldehydes to the alcohols which was found in these control experiments using uniduced E. coli JM101 (pSPZ3) and induced E. coli JM101 (pRS) can be explained by the action of E-coli alcohol dehydrogenases which catalyze a reaction whose equilibrium is on the alcohol side, for thermodynamic reasons.

The reformation of benzyl alcohol at the end of the biotransformation of toluene by E. coli JM101 (pSPZ3) (cf. Fig.

can also be assigned to the E. coli dehydrogenases, whose activity becomes significant since the XMO activity decreases over time.

Example 2: Determination of the course of the oxidation of various substrates over time The course of the oxidation of toluene, pseudocumene, 3,4-dimethylbenzyl alcohol and 3,4-dimethylbenzaldehyde over time is shown in Figures 3 and 4. The assays were carried out as described above. The substrate in question was added in each case to a suspension of the respective resting cells in 50 mM potassium phosphate buffer, pH 7.4, comprising 1% glucose.

When toluene or pseudocumene were added as substrates, a subsequent formation of the corresponding alcohols, aldehydes and acids was established (Figures 3A and 4A). As also established in the activity assays, the activities with which benzyl alcohol, 3,4-dimethylbenzyl alcohol and benzaldehyde were formed were high. The activities with which 3,4-dimethylbenzaldehyde and 3,4-dimethylbenzoic acid were formed were medium, and benzoic acid was formed with low activity. During the first 5 minutes, the products of the two substrates pseudocumene and toluene were formed at a specific activity of 100 U/g CDW (cf. see also Table II). In the interval between minutes 5 and 10, benzaldehyde was formed with an activity of 80 U/g CDW, while 3,4-dimethylbenzaldehyde was formed more slowly (37 U/g CDW).

Acid formation started when the consumption of toluene or pseudocumene was complete, with an activity of 3.2 U/g and 21 U/g CDW in the case of benzoic acid and 3,4-dimethylbenzoic acid, respectively, during the interval between 10 and 30 minutes. A low benzyl alcohol concentration always remained. Between 40 and 80 minutes, the benzyl alcohol concentration increased again, while the benzaldehyde concentration dropped (Figure 3A).

Pseudocumene, 3,4-dimethylbenzyl alcohol and 3,4-dimethylbenzaldehyde were consumed completely, with 3,4-dimethylbenzoic acid being formed (Figure 4A).

0050/50842 When 3,4-dimethylbenzyl alcohol was added as substrate, the activity with which 3,4-dimethylbenzaldehyde was formed was U/g CDW (Table II), while the activity with which 3,4-dimethylbenzoic acid was formed was 23 U/g CDW, all during the period between 10 and 30 minutes (Figure 4C). When benzyl alcohol was used as substrate, the activity with which benzaldehyde was formed was 95 U/g CDW and the activity with which benzoic acid was formed was a constant 2.9 U/g CDW (results not shown). Within the test period, all of the 3,4-dimethylbenzyl alcohol was converted completely into the acid, which is in contrast to benzyl alcohol.

When 3,4-dimethylbenzaldehyde was added as substrate (Figure 4E), 3,4-dimethylbenzoic acid was formed with an activity of 55 U/g CDW and constituted the predominant species after as little as minutes. When benzaldehyde was used as substrate, the slow and constant formation of benzoic acid (3 U/g CDW) was established (results not shown). Here, the initial activity during the first minutes was 10 U/g CDW (Table II).

Example 3: Transformation of toluene and pseudocumene by E. coli JM101 (pRMAB).

These experiments were intended to clarify whether the activity with which the aldehyde is formed from toluene and pseudocumene increases or decreases with the simultaneous presence of BADH and XMO. Since some alcohol dehydrogenases catalyze a reaction whose equilibrium is on the alcohol side at the physiological pH, the last-mentioned possibility might indeed exist and BADH would thus reduce the rate at which aldehyde is formed.

Indeed, clear differences with regard to biotransformation activity were observed in comparison with E. coli JM101 (pSPZ3).

Toluene was transformed into its corresponding oxidation products with an initial specific activity of 87 U/g CDW. The corresponding activities for benzyl alcohol, pseudocumene and 3,4-dimethylbenzyl alcohol were 66, 73 and 42 U/g CDW. The presence of BADH also seems to cause a reduction in the biotransformation activity of recombinant E. coli MJ101 as biocatalysts.

The addition of toluene resulted in the consecutive formation of benzyl alcohol, benzaldehyde and benzoic acid (Figure 3B). During the period between minutes 5 and 10, benzyl alcohol was formed with an activity of 34 U/g CDW, and the acid was formed with an activity of 1 U/g CDW over the period between 10 and 40 minutes.

After approximately 20 minutes, the benzyl alcohol concentration 0050/50842 21 started to rise again (probably owing to the reverse reaction starting). Virtually no benzaldehyde was left after 80 minutes, and only a very small amount of benzoic acid was formed.

When benzyl alcohol was added as substrate, the results were very similar (not shown). Again, the aldehyde concentration dropped during alcohol formation after approximately 20 minutes. Thus, the introduction of the xylB gene causes a considerable reduction of the aldehyde to give the alcohol, which suggests that the formation of aldehyde is not increased by addition of BADH.

When pseudocumene was added as substrate, again, a consecutive formation of 3,4-dimethylbenzyl alcohol, 3,4-dimethyl benzaldehyde and 3,4-dimethylbenzoic acid was established (Figure 4B). In the period during minutes 5 and 3,4-dimethylbenzaldehyde was formed with an activity of 15 U/g CDW. During the period between 30 and 40 minutes, the acid formed with approximately the same activity (13 U/g CDW). After the rapid alcohol formation, the aldehyde concentration remained relatively high for 20 minutes. Nevertheless, all of the alcohol and the aldehyde were transformed into the acid at the end of the reaction.

When 3,4-dimethylbenzyl alcohol was added as substrate, 3,4-dimethylbenzaldehyde and 3,4-dimethylbenzoic acid were formed (Figure 5B). In the period between 20 and 30 minutes, the acid was formed with a specific activity of 8 U/g CDW. The aldehyde concentration never exceeded the alcohol concentration. The longer presence of 3,4-dimethylbenzyl alcohol indicates that, besides the oxidation, aldehyde is again reduced to alcohol, for which BADH seems to be responsible.

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. In the case of Figure pseudocumene (1.37 mM) was added to a suspension of resting E. coli JM101 (pSPZ3) (2.04-2.26 g 1- 1 CDW) in 50 mM potassium phosphate buffer pH 7.4 comprising 1% glucose. The specific activities were calculated with the aid of the amounts which were formed during the first 5 minutes of the conversion, which were determined by gas chromatography. The arrow indicates the point in time at which 0.1% n-octane was added to induce xylMA synthesis. The filled circles indicate the cell dry weight (CDW) of the uninduced cultures, while the open circles indicate the 0050/50842 22 cell dry weight of the induced cultures and the crosses the specific activities of the induced cultures.

Figures 5(B) and were obtained as described for Fig. except that the cell density was 2.26-2.44 g 1- 1 CDW. Fig. shows the effect of different amounts of n-octane, and Fig. those of DCPK. The circles indicate the cell dry weight 3.5 hours after induction, and the crosses the specific activities of the induced cultures.

XMO activity was monitored after induction with 0.1% (v/v) n-octane (Fig. or 0.05% DCPK. XMO activity was induced rapidly by both compounds, and reached a constant value of approx. 115 and 105 U/g CDW in the case of n-octane and DCPK, respectively, after 3 to 3.5 hours. The growth rates of the induced cells were markedly lower in comparison with uninduced cells.

The dependence of XMO activity on the inductor concentrations [in the range 0.00001 1 was determined by growing E. coli JM101 (pSPZ3) up to a concentration of 0.09 g CDW per liter and inducing the cells with different amounts of n-octane and DCPK.

After a further 3.5 hours culturing, cell dry weight and XMO activity were determined for each inductor concentration (Fig. and When less than 0.0001% n-octane or 0.001% DCPK were added to the culture medium, the resulting XMO activities were very low. Induction maxima were found at DCPK concentrations of 0.005 to 0.01% and at n-octane concentrations of between 0.001 and 0.004% XMO activity remained constant at higher inductor concentrations. Owing to the high vapor pressure of n-octane, sealed shake flasks were used, but even then only some of the n-octane is dissolved in the aqueous medium. The solubility of n-octane in distilled water is very low and amounts to 0.7 mg/l, or 0.0001% The cell densities decreased in the inductor concentration ranges in which the enzyme activities increased to their maximum. The enzyme activity remained constant at higher n-octane concentrations. In contrast, higher DCPK concentrations caused a further decrease in cell density (Fig. 5 and In addition, the direct effect of various inductor concentrations on the growth of E. coli JM101 without plasmid was examined. High n-octane concentrations had no effect on cell growth. In contrast, DCPK concentrations of above 0.01% resulted in a decreased growth rate. At a DCPK concentration of 0.5% a cell dry weight of only 0.073 g/l was determined 3.5 hours after induction. Obviously, high DCPK concentrations have a toxic effect on the cells.

Thus, n-octane is the better inductor.

To summarize, the following can be said. There are two significant differences in the stepwise oxidation of toluene and pseudocumene. 3,4-dimethylbenzyl alcohol is oxidized more slowly than benzyl alcohol, and 3,4-dimethylbenzoic acid is formed with a markedly higher activity than benzoic acid. This suggests that the variation of substituents has different effects on the specific XMO activities for oxidized substrates (alcohols, aldehydes) than on the specific activity for unoxidized substrates such as toluene and pseudocumene, for which substrates XMO shows very similar activities. Another important result is that no acids are formed as long as more or less all of the toluene or pseudocumene has been consumed. In the event that this is a general fact, XMO clearly has a higher affinity for toluene and pseudocumene than for the corresponding aldehydes. The 15 presence of BADH results in lower activities for product formation and even in the reformation of benzyl alcohol. The BADH-containing cells clearly accumulate the aldehydes more slowly. BADH seems to increase the effect of the E. coli dehydrogenases drastically, and it seems that the equilibrium of this dehydrogenase reaction is on the alcohol side. This is confirmed by 20 thermodynamic calculations by known prior-art methods (26 to 29) and by studies into enzyme kinetics (16-18).

Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

N:\2\21272\au\00\20050317 Amended pages.doc\\ 0050/50842 24 References 1. Harayama, Kok, M. and Neidle, E.L. (1992), Annu. Rev.

Microbiol. 46, 565-601 2. Harayama, Rekik, Wubbolts, Rose, Leppik, R.A.

and Timmis, K.N. (1989), J. Bacteriol. 171(9), 5048-5055 3. Abril, Michan., Timmis, K.N. and Ramos, J.L.

(1989), J. Bacteriol. 171(12), 6782-6790 4. Williams, Shaw, Pitt, C.W. and Vrecl, M. (1997), microbiology 143, 101-107 Ramos, Marques, S. and Timmis, K.N. (1997), Ann. Rev.

Microbiol. 51, 341-373 6. Suzuki, Hayakawa, Shaw, Rekik, M. and Harayama, S. (1991), J. Bacteriol. 173(5), 1690-1695 7. Shaw, J.P. and Harayama, S. (1992), Eur. J. Biochem. 209, 1-61 8. Wubbolts, M. (1994),Dissertation, Rijksuniversiteit Groningen 9. Shaw, J.P. and Harayama, S. (1995), J. Ferm. Bioeng. 79(3), 195-199 Baptist, Gholson, R.K. and Coon, M.J. (1963), Bioch.

Biophys. Acta 69, 40-47 11. Chakrabarty, Chou, G. and Gunsalus, L.G. (1973), Proc.

Natl. Acad. Sci. USA 70(4), 1137-1140 12. Kok, Oldenhuis, v.d. Linden, Raatjes, P., Kingma, v. Lelyveld, P.H. and Witholt, B. (1989), J.

Biol. Chem. 264(10), 5435-5441 13. van Beilen, Wubbolts, M.G. and Witholt, B. (1994), Biodegradation 5, 161-174 14. Shanklin, Whittle, E. and Fox, B.G. (1994), Biochemistry 33, 12787-12794 Shanklin, J. Achim, Schmidt, Fox, B.G. and Miinck, E.

(1997), Proc. Natl. Acad. Sci. USA 94, 2981-2986 16. Shaw, J.P. and Harayama, S. (1990), Eur. J. Biochem. 191, 705-714 17. Shaw, Schwager, F. and Harayama, 5. (1992), Biochemical Journal 283, 789-794 18. Shaw, Rekik, Schwager, F. and Harayama, S. (1993), J. Biol. Chem. 268, 10842-10850 19. Kunz, D.A. and Chapman, P.J. (1981), J. Bacteriol. 146(1), 179-191 Wubbolts, Reuvekamp, P. and Witholt, B. (1994), Enzyme Microb. Technol. 16, 608-615 21. Harayama, Leppik, Rekik, Mermod, Lehrbach, Reineke, W. and Timinis, K.N. (1986), J. Bacteriol.

167(2), 455-461 0050/50842 22. Grund, Shapiro, Fennewald, Bacha, Leahy, J., Markbreiter, Nieder, M. and Toepfer, M. (1975), J.

Bacteriol. 123, 546-556 23. Yuste, Canosa, I. and Rojo, F. (1998), J. Bacteriol.

180(19), 5218-5226 24. Sambrook, Fritsch, E.F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

Panke, Witholt, Schmid, A. and Wubbolts, M.G. (1998), Appl. Environ. Microbiol. 64(6), 2032-2043 26. Mavrovouniotis, M.L. (1990), Biotechnol. Bioeng. 36, 1070-1082 27. Mavrovouniotis, M.L. (1991) The Journal of Biological Chemistry 266(22), 14440-14445 28. Dean, J.A. (1985), Lange's Handbook of Chemistry, 13th edition, McGraw-Hill Book Company 29. Leonardo, Dailly, Y. and Clark, D.P. (1996), J.

Bacteriol. 178(20), 6013-6018 Hanahan, D. (1983), J. Mol. Biol. 166, 557-580 31. Panke, et al., (1999) Applied and Environmental Microbiology 2324-2332 32. T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) 33. Ausubel, F.M. et al.,(1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience 34. Pouwels P. H. et al, (1985) "Cloning Vectors", ed., Elsevier, Amsterdam-New York-Oxford F. Ausubel et al.,(1997) Current Protocols in Molecular Biology, Wiley Interscience, New York

Claims (16)

1. A process for the preparation of aromatic aldehydes and/or carboxylic acids of the formula I Ar-(CH2)n-R 1 (I) where Ar is a substituted or mono- or polysubstituted mononuclear aromatic ring, R 1 is an oxygen-containing group -CHO or -COOH, and n is an integer from 0 to which comprises a) culturing, in a culture medium comprising an aromatic substrate of the formula II Ar-R 2 (II) where Ar is as defined above and R 2 is -CH=CH 2 or -(CH 2 )n+ 1 R 3 where n is as defined above and R 3 is H or OH; or, if R 1 is -COOH, R 2 can also be -(CH 2 )nR 4 where n is defined above and R 4 is -CHO, a microorganism which expresses an enzyme selected from amongst xylene monooxygenase (XMO) and alkane monooxygenase (AMO), and b) isolating the compound(s) of the formula I from the culture medium, wherein the XMO-expressing microorganism has essentially no benzyl alcohol dehydrogenase (BADH) and possibly no benzaldehyde dehydrogenase (BZDH) activity. AMENDED SHEET

2. A process as claimed in claim 1, wherein the AMO-expressing microorganism has essentially no alkanol dehydrogenase and/or alkanal dehydrogenase (AADH) activity.

3. A process as claimed in any one of the preceding claims, wherein a recombinant microorganism is used which has been transformed with an expression vector which contains the XMO-encoding genes xylM and xylA or the AMO-encoding genes alkB, alkG and alkT, linked operatively and under the genetic control of the alk regulatory system of Pseudomonas oleovorans GPol.

4. A process as claimed in claim 3, wherein the microorganism has been transformed with the expression plasmid pSPZ3. 15

5. A process as claimed in any one of the preceding claims, wherein the microorganism is a bacterium of the genus Escherichia.

6. A process as claimed in any one of claims 3 to 5, wherein the enzyme expression is started by adding an inductor to the culture medium.

7. A process as claimed in any one of the preceding claims, wherein a compound of the formula II, where R 2 is -CH=CH 2 -CH 3 or -CH 2 0H is converted using a microorganism which expresses XMO activity.

8. A process as claimed in any one of claims 1 to 6, wherein a compound of the formula II where R 2 is -(CH2)m-R 3 where R 3 is as defined above and m is an integer from 6 to 13 is converted using a microorganism which expresses AMO activity.

9. A process as claimed in claim 3, wherein xylene monooxygenase of Pseudomonas putida mt-2 is expressed.

N:\2\21272\au\00\20050317 Amended pages.doc\\ The use of a recombinant microorganism with an expression vector which contains the XMO-encoding genes xylM and xylA or the AMO-encoding genes alkB, alkG and alkT, linked operatively and under the genetic control of the alk regulatory system of Pseudomonas oleovorans GPol for the microbiological production of aromatic compounds of formula I.

11. The use as claimed in claim 10, wherein the microorganism is selected from the bacteria of the genus Escherichia and Pseudomonas.

12. The use as claimed in claim 10 or 11 wherein the microorganism has been transformed with the plasmid pSPZ3.

13. The use of an expression construct which contains the XMO-encoding 15 genes xylM and xylA or the AMO-encoding genes alkB, alkG and alkT, linked operatively and under the genetic control of the alk regulatory system of SPseudomonas oleovorans GPol for the microbiological production of aromatic compounds of formula I. 20

14. A process substantially as hereinbefore described with reference to the Examples.

15. The use of a recombinant microorganism substantially as hereinbefore described with reference to the Examples.

16. The use of an expression construct substantially as hereinbefore described with reference to the Examples. N:\2\21272\au\00\20050317 Amended pages.doc\\ DATED this 17th day of March, 2005 BASF AKTIENGESELLSCHAFT WATERMARK PATENT TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA P21 272AUOO N:\2\21 272\au\OO\2005031 7 Amended pages-doc\\