Jones, Jonathan Peter (University of Oxford Dept. of Chemistry South Parks Road Oxford OX1 3QR, GB)
Wong, Luet Lok (University of Oxford Dept. of Chemistry South Parks Road Oxford OX1 3QR, GB)
Jones, Jonathan Peter (University of Oxford Dept. of Chemistry South Parks Road Oxford OX1 3QR, GB)
| 1. | Process for oxidising a substrate which is a halo aromatic compound, which process comprises oxidising said substrate with a monooxygenase enzyme. |
| 2. | Process according to claim 1 in which the enzyme comprises a substitution of an amino acid in the active site by an amino acid with a less polar sidechain. |
| 3. | Process according to claim 2 in which the enzyme comprises one or more other amino acid substitutions in the active site. |
| 4. | Process according to any one of the preceding claims in which the enzyme is (i) P450cam7 or (ii) a naturally occurring homologue of (i), or (iii) a mutant of (i) or (ii). |
| 5. | Process according to claim 4 in which the enzyme is one in which amino acid 96 of P450Cam or the equivalent amino acid in a homologue, has been changed to an amino acid with a less polar sidechain. |
| 6. | Process according to any one of the preceding claims in which the halogen is chlorine. |
| 7. | Process according to any one of the preceding claims in which the aromatic compound is a benzene or biphenyl. |
| 8. | Process according to any one of the preceding claims in which the substrate has more than one halogen atom. |
| 9. | Process according to claim 8 in which the substrate is 1,2dichlorobenzene, 1,2,4trichlorobenzene, 3,3'dichlorobiphenyl or 2,2', 4,5,5'pentachlorobiphenyl. |
| 10. | Process according to claim 8 in which the substrate is pentachlorobenzene or hexachlorobenzene. |
| 11. | Process according to any one of the preceding claims which is carried out in a cell that expresses: (a) an enzyme as defined in any one of claims 1 to 5; (b) an electron transfer reductase; and (c) an electron transfer redoxin. |
| 12. | Process according to claim 11 in which: (b) is putidaretoxin reductase or a homologue; or a fragment thereof ; and/or (c) is putidaretoxin or a homologue; or a fragment thereof. |
| 13. | Process according to claim 11 or 12 wherein the cell is one in which the enzyme (a) does not naturally occur. |
| 14. | Process according to any one of claims 11 to 13 wherein the cell is one which in its naturally occurring form is able to oxidise a substrate as defined in any one of claims 6 to 10. |
| 15. | A cell as defined in claim 14. |
| 16. | A transgenic animal or plant whose cells are as defined in any one of claims 11 to 14. |
| 17. | Method of treating a locus contaminated with a substrate as defined in any one of claims 1 or 6 to 10 comprising contacting the locus with an enzyme as defined in any one of claims 1 to 5 or a cell as defined in any of claims 11 to 13, or an animal or plant as defined in claim 16. |
| 18. | Process for selecting a mutant of an enzyme as defined in claim 1,4 (i) or 4 (ii) for its ability to oxidise a substrate as defined in claim 1, or any one of the claims 6 to 10, which process comprises screening a library of said mutants for their oxidation effect on the substrate. |
| 19. | Process, cell, animal, plant or method according to any one of claims 1 to 17 wherein the enzyme is one that has been selected in a process according to claim 18. |
Chlorinated aromatic compounds such as the chlorobenzene and polychlorinated biphenyls (PCBs) are among the most wide-spread organic contaminants in the environment due to their common application as solvents, biocides, and in the heavy electrical industry. They are also some of the most problematic environmental pollutant, not only because of the health hazards (lipid solubility and hence accumulation in fatty tissues, toxicity and carcinogenicity) but also because of their slow degradation in the environment.
Whilst microorganisms have shown extraordinary abilities to adapt and evolve to degrade most of the organic chemicals released into the environment, the most chemically inert compounds such as PCBs do persist for two main reasons. First, these compounds have very low solubility in water and therefore their bioavailability is low. Research into this problem has focussed on the use of detergents and other surfactants to enhance their solubility and bioavailability. Second, these compounds require activation by enzymatic oxidation or reduction, and it can take a long time for the necessary genetic adaptations by microorganisms to occur, and even then the organisms may not be stable and viable.
We have now found, according to the present invention, that a monoxygenase, in particular P450Cam and its physiological electron transfer partners putidaretoxin and putidaretoxin reductase, can be used to oxidise halogenated aromatic compounds. Also mutants of the monoxygenase with substitutions in the active site have enhanced oxidation activity. Thus suitable monoxygenases can be expressed in microorganisms, animals and plants which are going to be used to oxidise the halogenated aromatic compounds.
Accordingly the present invention provides a process for oxidising a substrate which is a halo aromatic compound, which process comprises oxidising said substrate with a monooxygenase enzyme.
The process may be carried out in a cell that expresses: (a) the enzyme (b) an electron transfer reductase; and (c) an electron transfer redoxin.
The halo aromatic compound is typically a benzene or biphenyl compound. The benzene ring is optionally fused and can be substituted. The halogen is typically chlorine.
In many cases there is more than one halogen atom in the molecule, typically 2 to 5 or 6, for example 3. Generally 2 of the halogen atoms will be ortho or para to one another. The compound may or may not contain an oxygen atom such as a hydroxy group, an aryloxy group or a carboxy group. The compound may or may not be chlorophenol or a chlorophenoxyacetic compound.
Specific compounds which can be oxidised by the process of the present invention include 1,2; 1,3- and 1,4-dichlorobenzene, 1,2,4; 1,2,3-and 1,3,5- trichlorobenzene, 1,2,4,5-and 1,2,3,5-tetrachlorobenzene, pentachlorobenzene, hexachlorobenzene, 3,3'-dichlorobiphenyl and 2,3,4,5,6-and 2,2', 4,5,5'- pentachlorobiphenyl.
Other compounds which can be oxidised by the process include recalcitrant halo aromatic compounds, especially dioxins and halogenated dibenzofurans, and the corresponding compounds where one or both oxygen atoms is/are replaced by sulphur, in particular compounds of the formula: which possess at least one halo substituent, such as dioxin itself, 2,3,7,8-tetrachlorodibenzodioxin.
The oxidation typically gives rise to 1,2 or more oxidation products. These oxidation products will generally comprise 1 or more hydroxyl groups. Generally, therefore, the oxidation products are phenols which can readily be degraded. It is particularly noteworthy that pentachlorobenzene and hexachlorobenzene can be oxidised in this way since they are very difficult to degrade. In contrast the corresponding phenols can be readily degraded by a variety of Pseudomonas and other bacteria. The atom which is oxidized is generally a ring carbon.
The enzyme is typically a natural monooxygenase or a mutant thereof. The
natural monooxygenase is generally a prokaryotic or eukaryotic enzyme. Typically it is a haem-containing enzyme and/or a P450 enzyme. The monooxygenase may or may not be a TfdA (2,4-dichlorophenoxy) acetate/a-KG dioxygenase. The monooxygenase is generally of microorganism (e. g. bacterial), fungal, yeast, plant or animal origin, typically of a bacterium of the genus Pseudomonas. These organisms are typically soil, fresh water or salt water dwelling. In the case of a mutant monooxygenase the non-mutant form may or may not be able to oxidize the substrate.
The monooxygenase typically has a coupling efficiency of at least 1%, such as at least 2%, 4%, 6% or more. The monooxygenase typically has a product formation rate of at least 5 min'', such as at least 8,10,15,20,25,50,100,150 min''or more. The coupling efficiency or product formation rate is typically measured using any of the substrates or conditions mentioned herein. Thus they are typically measured in the in vitro conditions described in Example 2, in which case the relevant monooxygenase, reductase and redoxin would be present instead of, but at the same concentration as, P450Cam, putidaretoxin reductase and putidaretoxin.
The mutant typically has at least one mutation in the active site. A preferred mutant comprises a substitution of an amino acid in the active site by an amino acid with a less polar side chain. Thus the amino acid is typically substituted with an amino acid which is above it in Table 1.
Table 1. HYDROPATHY SCALE FOR AMINO ACID SIDE CHAINS Side Chain Hydropathy Ile 4.5 Val 4.2 Leu 3.8 Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly-0.4 Thr-0.7 Ser-0.8 Trp-0.9 Tyr-1.3 Pro-1.6 His-3.2 Glu-3.5 Gln-3.5 Asp-3.5 Asn-3.5 Lys-3.9 Arg-4.5 An amino acid'in the active site'is one which lines or defines the site in which the substrate is bound during catalysis or one which lines or defines a site through which the substrate must pass before reaching the catalytic site. Therefore such an amino acid typically interacts with the substrate during entry to the catalytic site or during catalysis.
Such an interaction typically occurs through an electrostatic interaction (between charged or polar groups), hydrophobic interaction, hydrogen bonding or van der Waals forces.
The amino acids in the active site can be identified by routine methods to those skilled in the art. These methods include labelling studies in which the enzyme is allowed to bind a substrate which modifies ('labels') amino acids which contact the substrate.
Alternatively the crystal structure of the enzyme with bound substrate can be obtained in order to deduce the amino acids in the active site.
The monooxygenase typically has 1,2,3,4 or more other mutations, such as substitutions, insertions or deletions. The other mutations may be in the active site or outside the active site. Typically the mutations are in the'second sphere'residues which affect or contact the position or orientation of one or more of the amino acids in the active site. The insertion is typically at the N and/or C terminal and thus the enzyme may be part of a fusion protein. The deletion typically comprises the deletion of amino acids which are not involved in catalysis, such as those outside the active site. The monooxygenase may thus comprise only those amino acids which are required for oxidation activity.
The other mutations in the active site typically alter the position and/or conformation of the substrate when it is bound in the active site. The mutation may make the site on the substrate which is to be oxidized more accessible to the haem group. Thus the mutation may be a substitution to an amino acid which has a smaller or larger, or more or less polar, side chain.
The other mutations typically increase the stability of the protein, or make it easier to purify the protein. They typically prevent the dimerisation of the protein, typically by removing cysteine residues from the protein (e. g. by substitution of cysteine at position 334 of P450cam, or at an equivalent position in a homologue, preferably to alanine). They typically allow the protein to be prepared in soluble form, for example by the introduction of deletions or a poly-histidine tag, or by mutation of the N-terminal membrane anchoring sequence. The mutations typically inhibit protein oligomerisation, such as oligomerisation arising from contacts between hydrophobic patches on protein surfaces.
Typically the mutant monoxygenase is at least 70% homologous to a natural monooxygenase on the basis of amino acid identity.
Any of the homologous proteins mentioned herein are typically at least 70% homologous to a protein or at least 80 or 90% and more preferably at least 95%, 97% or 99% homologous thereto over at least 20, preferably at least 30, for instance at least 40,60 or 100 or more contiguous amino acids. The contiguous amino acids may include the active site. This homology may alternatively be measured not over contiguous amino acids or nucleotides but over only the amino acids in the active site.
The monoxygenase is preferably: (i) P450,.., (ii) a naturally occurring homologue of (i),
(iii) a mutant of (i) or (ii).
Typically (i) is any allelic variant of P450am of Pseudomonas putida (e. g. of the polypeptide sequence shown in SEQ ID No. 1). Typically (ii) is a species homologue of (i) which has sequence homology with (i), and is typically P450BM-3 of Bacillus megaterium (e. g. the polypeptide sequence shown in SEQ ID No. 2), P450, e"of Pseudomonas sp, P450eXyF of Saccharopollyspora erythraea, or P450 105 D l (CYP 15) of Streptomyces griseus strains.
The active site of (ii) or (iii) may be substantially the same as the active site of (i) or any of the mutants of (i) mentioned herein. Thus the site may comprise the same amino acids in substantially the same positions.
Typically in (iii) amino acid 96 of P450Cam, or the equivalent amino acid in a homologue, has been changed to an amino acid with a less polar side chain.
The'equivalent'side chain in the homologue is one at the homologous position.
This can be deduced by lining up the P450 sequence and the sequence of the homologue based on the homology between the two sequences. The PILEUP, BLAST and BESTFIT algorithms can be used to line up the sequences (for example as described in Altschul S. F.
(1993) J Mol Evol 36: 290-300; Altschul, S, F et al (1990) J Mol Biol 215: 403-10 and (Devereux etal (1984) Nucleic Acids Research 12, p387-395)). These algorithms can also be used to calculate the levels of homology discussed herein (for example on their default settings). The equivalent amino acid will generally be in a similar place in the active site of the homologue as amino acid 96 in P450, The discussion below provides examples of the positions at which substitutions may The same substitutions may be made at equivalent positions in the homologues. Standard nomenclature is used to denote the mutations. The letter of the amino acid present in the natural form is followed by the position, followed by the amino acid in the mutant. To denote multiple mutations in the same protein each mutation is listed separated by hyphens. The mutations discussed below using this nomenclature specify the natural amino acid in P450cam, but it is to be understood that the mutation could be made to a homologue which has a different amino acid at the equivalent position. Note that the amino acid numbering shown in SEQ ID No. 1 for P450cm does not correspond to the numbering used in the description to denote mutations. The numbering in SEQ ID No.
1 is one more than the numbering in the description for a particular position.
An additional mutation is typically an amino acid substitution at amino acid 87, 98,101,185,244,247,248,296,395,396 or a combination of these, for example as shown in table 2.
The following combinations of substitutions are preferred: (i) Substitution at position 87 to amino acids of different side-chain volume, such as substitutions (typically of F) to A, L, I and W, combined with substitutions at position 96 to amino acids of different side-chain volume such as (typically Y to) A, L, F, and W. These combinations alter the space available in the upper part of the substrate pocket compared to the wild-type enzyme, for example, from Y96W-F87W (little space) to Y96A-F87A (more space), as well as the location of the space, for example from one side in Y96F-F87A to the other in Y96A-F87W.
(ii) Substitution at position 96 to F combined with substitutions at positions 185 and 395. Both T185 and I395 are at the upper part of the substrate pocket, and substitution with A creates more space while substitution with F will reduce the space available and push the substrate close to the haem.
(iii) Substitutions at position 96 to A, L, F, and W combined with substitutions at residues closer to the haem including at 101,244,247,295,296 and 396 to A, L, F, or W. These combinations will create or reduce space in the region of the different side- chains to offer different binding orientations to substrates of different sizes. For example, the combinations Y96W-L244A and Y96L-V247W will offer very different pockets for the binding of the substrate.
(iv) Triple substitutions at combinations of positions 87,96,244,247,295, 296,395 and 396 with combinations of A, L, F, and W. The aim is to vary the size and shape of the hydrophobic substrate binding pocket. For example, the Y96A-F87A-L244A combination creates more space compared to the Y96F-F87W-V396L combination, thus allowing larger substrates to bind to the former while restricting the available binding orientations of smaller substrates in the latter. The combinations Y96F-F87W-V247L and Y96F-F87W-V295I have comparable substrate pocket volumes, but the locations of the space available for substrate binding are very different. The combination Y96F-F87L- V247A has a slightly larger side-chain volume at the 96 position than the combination Y96L-F87L-V247A, but the L side-chain at the 96 position is much more flexible and the substrate binding orientations will be different for the two triple mutants.
(v) The mutants with four or five substitutions were designed with similar principles of manipulating the substrate volume, the different flexibility of various side- chains, and the location of the space available in the substrate pocket for substrate binding so as to effect changes in selectivity of substrate oxidation.
Mutations are generally introduced into the enzyme by using methods known in the art, such as site directed mutagenesis of the enzyme, PCR and gene shuffling methods or by the use of multiple mutagenic oligonucleotides in cycles of site-directed mutagenesis.
Thus the mutations may be introduced in a directed or random manner. Typically the mutagenesis method produces one or more polynucleotides encoding one or more different mutants. In one embodiment a library of mutant oligonucleotides is produced which can be used to produce a library of mutant enzymes.
The process is typically carried out in the presence of the natural cofactors of the monooxygenase. Thus typically in addition to the enzyme (a) and the substrate the process is carried out in the presence of an electron transfer reductase (b), an electron transfer redoxin (c), cofactor for the enzyme and an oxygen donor. In this system the flow of electrons is generally: cofactor-+ (b)-+ (c)--+ (a).
(b) is generally an electron transfer reductase which is able to mediate the transfer of electrons from the cofactor to (c), such as a naturally occurring reductase or a protein which has homology with a naturally occurring reductase, such as at least 70% homology; or a fragment of the reductase or homologue. (b) is typically a reductase of any of the organisms mentioned herein, and is typically a flavin dependent reductase, such as putidaredoxin reductase.
(c) is generally an electron transfer redoxin which is able to mediate the transfer of electrons from the cofactor to (a) via (b). (c) is typically a naturally occurring electron transfer redoxin or a protein which has homology with a naturally occurring electron transfer redoxin, such as at least 70% homology; or a fragment of the redoxin or homologue. (c) is typically a redoxin of any of the organisms mentioned herein. (c) is typically a two-iron/two sulphur redoxin, such as putidaredoxin.
The cofactor is any compound capable of donating an electron to (b), such as NADH. The oxygen donor is any compound capable of donating oxygen to (a), such as dioxygen.
Typically (a), (b) and (c) are present as separate proteins; however they may be
present in the same fusion protein. Typically only two of them, preferably (b) and (c), are present in the fusion protein. Typically these components are contiguous in the fusion protein and there is no linker peptide present.
Alternatively a linker may be present between the components. The linker generally comprises amino acids that do not have bulky side chains and therefore do not obstruct the folding of the protein subunits. Preferably the amino acids in the linker are uncharged. Preferred amino acids in the linker are glycine, serine, alanine or threonine. In one embodiment the linker comprises the sequence N-Thr-Asp-Gly-Gly-Ser-Ser-Ser-C.
The linker is typically from at least 5 amino acids long, such as at least 10,30 or 50 or more amino acids long.
In the process the concentration of (a), (b) or (c) is typically from 10-8 to 10-2M, preferably from 10-6 to 10-4M. Typically the ratio of concentrations of (a): (b) and/or (a): (c) is from 0.1: 01 to 1: 10, preferably from 1: 0.5 to 1: 2, or from 1: 0.8 to 1: 1.2.
Generally the process is carried out at a temperature and/or pH at which the enzyme is functional, such as when the enzyme has at least 20%, 50%, 80% or more of peak activity.
Typically the pH is from 3 to 11, such as 5 to 9 or 6 to 8, preferably 7 to 7.8 or 7.4.
Typically the temperature is 10 to 90°C, such as 25 to 75°C or 30 to 60°C.
In the process different monooxygenases may be present. Typically each of these will be able to oxidise different substrates, and thus using a mixture of monooxygenases will enable a wider range of substrates to be oxidised.
In one embodiment the process is carried out in the presence of a substance able to remove hydrogen peroxide by-product (e. g. a catalase).
In one embodiment the process is carried out in the presence of the enzyme, substrate and an oxygen atom donor, such as hydrogen peroxide or t-butylhydroperoxide, for example using the peroxide shunt.
In one embodiment in the process the (a), (b) and (c) together are typically in a substantially isolated form and/or a substantially purified form, in which case together they will generally comprise at least 90%, e. g., at least 95%, 98% or 99% of the protein in the preparation.
The process may be carried out inside or outside a cell. The cell is typically in culture, at a locus, in vivo or in planta (these aspects are discussed below).
The process is typically carried out at a locus such as in land (e. g. in soil) or in
water (e. g. fresh water or sea water). When it carried out in culture the culture typically comprises different types of cells of the invention, for example expressing different monooxygenases of the invention. Generally such cells are cultured in the presence of assimible carbon and nitrogen sources.
Typically the cell in which the process is carried out is one in which the monooxygenase does not naturally occur. In another embodiment the monooxygenase is expressed in a cell in which it does naturally occur, but at higher levels than naturally occurring levels. The cell may produce 1,2,3,4 or more different monooxygenases of the invention. These monoxygenases may be capable of oxidising different halo aromatic compounds. Typically the cell also expresses any of the reductases and/or redoxins discussed above.
The cell is typically produced by introducing into a cell (i. e. transforming the cell with) a vector comprising a polynucleotide that encodes the monooxygenase. The vector may integrate into the genome of the cell or remain extrachromosomal. The cell may develop into the animal or plant discussed below. Typically the coding sequence of the polynucleotide is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell. The control sequence is generally a promoter, typically of the cell in which the monooxygenase expressed.
The term"operably linked"refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence"operably linked"to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
The vector is typically a transposon, plasmid, virus or phage vector. It typically comprises an origin of replication. It typically comprises one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid. The vector is typically introduced into host cells using conventional techniques including calcium phosphate precipitation, DEAE-dextran transfection, or electroporation.
Components (b) and (c) may be expressed in the cell in a similar manner.
Typically (a), (b) and (c) are expressed from the same vector, or may be expressed from different vectors. They may be expressed as three different polypeptides. Alternatively they may be expressed in the form of fusion proteins. The cell typically expresses more
than one type of monooxygenase.
In one embodiment the three genes encoding the three protiens of the P450am system, i. e. camA, camB, and camC are placed in the mobile regions of standard transposon vectors and incorporated into the genome of Pseudomonas and flavobacteria.
Alternatively plasmid vectors for expressing these genes may used, in which case the P450cam gene cluster will be extra-chromosomal.
The cell may be prokaryotic or eukaryotic and is generally any of the cells or of any of the organisms mentioned herein. Preferred cells are Pseudomanas, flavobacteria or fungi cells (e. g. Aspergillus). In one embodiment the cell is one which in its naturally occurring form is able to oxidise any of the substrates mentioned herein. Typically the cell is in a substantially isolated form and/or substantially purified form, in which case it will generally comprise at least 90%, e. g. at least 95%, 98% or 99% of the cells or dry mass of the preparation.
The invention provides a transgenic animal or plant whose cells are any of the cells of the invention. The animal or plant is transgenic for the monooxygenase gene and typically also an appropriate electron transfer reductase and/or redoxin gene. They may be homozygote or heterozygote for such genes, which are typically transiently introduced into the cells, or stably integrated. (e. g. in the genome). The animal is typically a worm (e. g earthworm) or nematode. The plant or animal may be obtained by transforming an appropriate cell (e. g. embryo stem cell, callus or germ cell), fertilising the cell if required, allowing the cell to develop into the animal or plant and breeding the animal or plant true if required. The animal or plant may be obtained by sexual or asexual (e. g. cloning) propagation of an animal or plant of the invention or of the F 1 organism (or any generation removed from the F1, or the chimera that develops from the transformed cell).
As discussed above the process may be carried out at a locus. Thus the invention also provides a method of treating a locus contaminated with a halo aromatic compound comprising contacting the locus with a monooxygenase, cell, animal or plant of the invention. These organisms are then typically allowed to oxidise the halo aromatic compound. In one embodiment the organisms used to treat the locus are native to the locus. Thus they may be obtained from the locus (e. g. after contamination), transformed/transfected (as discussed above) to express the monooxygenase (and optionally an appropriate electron transfer reductase and/or redoxin.
In one embodiment the locus is treated with more than one type of organism of the invention, e. g. with 2,3,4, or more types which express different monooxygenases which oxidise different halo aromatic compounds. In one embodiment such a collection of organisms between them is able to oxidise all halobenzenes, e. g. all chlorobenzenes.
The organisms (e. g. in the form of the collection) may carry out the process of the invention in a bioreactor (e. g. in which they are present in immobilised form). Thus the water or soil to be treated may be passed through such a bioreactor. Soil may be washed with water augmented with surfactants or ethanol and then introduced into the bioreactor.
The invention also provides a process for selecting a mutant of a monooxygenase for its ability to oxidise any of the substrates mentioned herein, which process comprises screening a library of said mutants for their oxidation effect on the substrate. Thus typically the substrate is provided to the library and mutants are selected based on their ability to oxidise the substrate, for example at a particular rate or under particular conditions. The mutant may be selected based on its ability to oxidise the substrate to a particular oxidation product.
Typically the library will be in the form of cells which comprise the mutant enzymes. Generally each cell will express only one particular mutant enzyme. The library typically comprises at least 500 mutants, such as at least 1,000 or 5,000 mutants, preferably at least 10,000 different mutants.
The library typically comprises a random population of mutants. The library may undergo one or more rounds of selection whilst being produced and therefore may not comprise a random population.
The library is typically produced by contacting any of the cells discussed herein which expresses the monooxygenase with a mutagen and/or when the cell is a mutator cell culturing the cell in conditions in which mutants are produced. The mutagen may be contacted with the cell prior to or during culturing of the cell. Thus the mutagen may be present during replication of the cell or replication of the genome of the cell.
The mutagen generally causes random mutations in the polynucleotide sequence which encodes (a). The mutagen is typically a chemical mutagen, such as nitrosomethyguanidine, methyl-or ethylmethane sulphonic acid, nitrite, hydroxylamine, DNA base analogues, and acridine dyes, such as proflavin. It is typically electromagnetic radiation, such as ultra-violet radiation at 260 nm (absorption maximum of DNA) and X-
rays. It is typically ionising radiation.
A mutator cell is generally deficient in one or more of the primary DNA repair pathways (such as E. Coli pathways mutS, mutD or mutT, or their equivalents in another organism), and thus has a high mutation rate. Simply culturing such cell leads to the DNA encoding (a) to become mutated. The cell may be ofE. Coli XL1 Red mutator strain.
The mutant selected from the library may be used in any aspect of the invention, thus it may be used to oxidise a substrate in the process of the invention or may be expressed in the cell, animal or plant of the invention. It may be used in the method of treating a locus.
The invention is also illustrated by the Examples: Example 1 Expression of mutants for in vitro work.
The P450C"I, enzymes were expressed using the vector pRH1091 (Baldwin, J. E., Blackburn, J. M., Heath, R. J., and Sutherland, J. D. Bioorg. Med Chenu. Letts., 1992,2, 663-668.) which utilised the trc promoter (a fusion of the trp and lac promoters). This vector incorporates a strong ribosome binding site (RBS) and the gene to be expressed is cloned using an Nde I site on the 5'end of the gene. We used Hind III as the cloning site at the 3'end of the camC gene. The procedure for protein expression is as follows: Cells are grown at 30°C until the OD600 reaches 1.0-1.2, the temperature is increased to 37°C and camphor added as a 1 M stock in ethanol to a final concetration of 1 mM. The culture is allowed to incubate at 37°C for another 6 hours. The P450C^m protein is expressed to high levels in the cytoplasm and the cells take on a red to orange-red colour.
We have also prepared a variant of pRH1091 (by PCR) which has a extraXba I site between the RBS and the Nde I site. This is important because Nde I is not unique in M13, and this restriction site is also present in the reductase gene as well as the backbone of the pGLWl 1 vector used for the in vivo system. Xba I is unique in the polylinker region of M13, but absent in the genes of all three proteins in the P450 am system and in the expression vectors. It therefore allows the can ? c gene to be moved between the mutagenic and expression vectors.
How the mutants were made.
Oligonucleotide-directed site-specific mutagenesis was carried out by the Kunkel method (Kunkel, T. A. Proc. Natl. Acad. Sci. USA 1985,82,488-492) using the Bio-Rad Mutagen kit. The recommended procedure is summarised as follows. An M 13 mp 19 subclone of was propagated in the E. coli strain CJ236.
This strain has the dutWung-phenotype and thus will tolerate the inclusion of uracil in place of thymine in DNA molecules. After three cycles of infection, uracil-containing single stranded (USS) M13 DNA was readily isolated by phenol extraction of mature M13 phage particles excreted into the growth medium. The mutagenic oligonucloetide (or oligonucleotides) were phosphorylated with T4 polynucleotide kinase and then annealed to the USS template. The four nucleotides, DNA polymerase, DNA ligase, ATP and other chemical components were added and the second strand was synthesised in vitro. The double stranded form thus obtained was transformed into the dut+ long+ E. coli strain MV1190, which should degrade the uracil-containing template strand and propagate the mutant strand synthesised in vitro. Plaques were picked and phages of possible mutants grown in E. coli strains MV1190 or TG1. The single-stranded DNA from these were sequenced to determine whether the mutagenesis reaction was successful. The mutagenic efficiency was 50-80%.
The mutant camC gene is excised from the M 13 subclone by restriction digest with Nde I and Hind III, and the fragment of appropriate size is ligated to the backbone of the expression vector prepared by a similar Nde IlHind III digest.
Multiple mutants were prepared either by further mutagenesis, also by the Kunkel method, or where the location of the sites in the sequence permits, simple cloning steps.
There are two unique restriction sites within the camC gene which are absent from the expression vector. One is Sph I which spans residues 121-123, and the other is Sal I which spans residues 338 and 339. Therefore, all mutations at, for example, residues 87,96,98, and 101 are readily combined with mutations at higher number residues by ligating appropriate fragments from restriction digests of mutant camC genes with Nde IlSph I and Sph IlHind HI and the backbone fragment from a Nde IlSph I digest of the expression vector. Mutations at, for example, 395 and 396 can be similarly incorporated by digests in which Sph I is replaced with Sal I The rationale for introducing the unique Xba I site is now clear: many mutants with
multiple mutations were prepared by the cloning procedure above. Without the Xha I site it would be impossible to clone the gene for these multiple mutants from the expression vector back into M13 for further rounds of mutagenesis. Of course these problems could be overcome by doing mutagenesis by PCR, for example.
Example 2 Substrate oxidation protocol: in vitro reactions Component Final concentration P450,enzyme 1 IlM Putidaredoxin 10 pm Putidaredoxin reductase 1 IlM Bovine liver catalase 20 llg/ml KC1 200 mM Substrate Typically 1 mM NADH 250-400 tLM * 50 mM Tris-HCl buffer pH 7.4 is added to make up the volume.
* Temperature controlled at 30'C, optional.
* The NADH turnover rate could be determined by monitoring the absorbance at 340 nm with time.
* Catalase does not catalyse the substrate oxidation reactions but rather it is present to remove any hydrogen peroxide by-product which could otherwise denature the P450Cam.
The method can be increased in scale to, for example, 20 ml total incubation volume to allow purification of sufficient products by HPLC for spectroscopic characterisation. Fresh substrate (1 mM) and NADH (1-2 mM) are added periodically, such as every 20 minutes in a total reaction time of, typically, 3 hours.
Example 3 The in vivo svstem The in vivo systems were expressed using the vector pGLWl 1, a derivative of the plasmid pKK223 (Brosius, J. and Holy, A. Proc. Natl. Acad Sci. USA, 1984,81,6929- 6933). Expression is directed by the tac promoter and the vector incorporates a gene
conferring resistance to the antibiotic ampicillin.
Two systems were constructed. The first one expressed the electron transfer proteins putidaredoxin reductase (camA gene) andpputidaredoxin (camB gene) as a fusion protein with a seven amino acid peptide linker, and the P450e, enzyme (camC gene) was expressed by the same vector but it was not fused to the electron proteins. The second system expressed the three proteins as separate entities in the E. Coli host. Both systems were catalytically competent for substrate oxidation in vivo.
The general strategy was as follows. The genes for the three proteins were cloned using Eco RI and Hind III as flanking sites, with Eco RI at the 5'end. For both in vivo systems there are restriction sites between the genes, including between the reductase and redoxin genes in the fusion construct. These restriction sites were introduced by PCR, as detailed below. The first task, however, was to carry out a silent mutation to remove the Hind III site within the camA gene for the reductase. The AAGCTT Hind III recognition sequence in the camA gene was changed to AAGCCT, which is a silent mutation because GCT and GCC both encode alanine. The gene was completely sequenced to ensure that there were no spurious mutations.
1. The fusion protein system 1. a Manipulation of tSte camA gene by PCR For the camA gene the primer below was used at the 5'end of the gene to introduce the Eco RI cloning site and to change the first codon from GTG to the strong start codon ATG.
5'-GAG ATT AAG AAT TCA TAA ACA CAT GGG AGT GCG TGC CAT ATG AAC GCA AAC<BR> Eco RI RBS |-camA At the 3'end of camA the primer was designed such that 15 bases are complementary to nucleotide sequence of the last five amino acid residues of camA. The stop codon immediately after the GCC codon for the last amino acid was removed, and then part of a seven amino acid linker (Thr Asp Gly Gly Ser Ser Ser) which contained a Bam HI cloning site (GGATCC = Gly Ser) was introduced. The coding sequence was thus:
5'-GAA CTG AGT AGT GCC ACT GAC GGA GGA TCC TCA TCG-3' camA-Thr Asp Gly Gly Ser I Bani HI The primer sequence shown below is the reverse complement used for PCR: 5'-CGA TGA GGA TCC TCC GTC AGT GGC ACT ACT CAG TTC-3' 1.b Manipulations of tAte camB gene by PCR For the camB gene the primer at the 5'end incorporated the second half of the peptide linker between the reductase and redoxin proteins, and the restriction site Bam HI for joining the two amplified genes together.
5'-TCA TCG GGA TCC TCA TCG ATG TCT AAA GTA GTG TAT-3' Gly Ser Ser Ser l-camB lBam HI| Start At the 3'end of camB the primer incorporates 12 nucleotides complementary to the end of camB followed by the stop codon TAA, a 6 nucleotide spacer before the GGAG ribosome binding site. Xba I and Hitid sites were then added to allow cloning of the camC gene when required. The sequence of the coding strand was therefore: 5'-CCC GAT AGG CAA TGG TAA TCA TCG GGAG TCT AGA GCA TCG AAG CTT TCA TCG-3' Camp stop RBS Xba I Hind III The primer shown below is the reverse complement used for PCR: 5'-CGA TGA AAG CTT CGA TGC TCT AGA CTCC CGA TGA TTA CCA TTG CCT ATC GGG-3' I. c Preparation of theefullfusion construct The camA and camB genes were amplified by the PCR using the primers described above. The new camA was digested with Eco RI and Bam HI, while the new CamB was digested with Bam HI and Hind III. The pGLWl 1 expression vector was digested with Eco RI and Hind III. All three were purified by agarose gel electrophoresis and the three gel slices containing the separate fragments were excised from the gel and ligated together, and then transformed into E. Coli DH5a. Successful ligation of all the fragments were
confirme by a series of restriction digestion experiments, especially the presence of the new and unique Xba I site. The entire sequence of the insert from the Eco RI site to the Hind III site was determined to ensure that all the sequences were correct.
The new plasmid, named pSGBF was transformed into E. Coli and expression of the reductase and redoxin proteins was induced by IPTG. When a purified P450Cam enzyme was added to the cell-free extract, substrate oxidation was observed for a variety of substrates.
When the camC gene is cloned into the pSGBF plasmid using theXba I and Hind III restriction sites, the new recombinant plasmid thus generated expresses the reductase and redoxin as a fusion protein and the P450Cam enzyme as a operate entity both from the same mRNA molecule. This in vivo system is catalytically competent for terpene oxidation in whole cells.
2. The in vivo system with the protein expressed separately 2. a The basic strategy The starting point of the preparation of this in vivo system was the recombinant plasmid used to express the canA gene for putidaredoxin reductase. The camA gene was cloned into the pGLW 11 plasmid using the Eco RI and Bam HI restriction sites, with Eco RI being at the 5'end of the gene. Conveniently the polylinker region of the pGLWl 1 vector has a Hind III site downstream of the Bam HI site. The camB gene was therefore manipulated by PCR such that it can be cloned into pGLWl 1 using the Bam HI and Hind III sites. This new plasmid expresses the reductase and redoxin as separate proteins.
The camB gene was cloned into pUC 118 by the Bam HI and Hind III cloning sites to express putidaredoxin for our general in vitro substrate oxidation work. Therefore, the PCR primer at the 3'end of the camB gene was designed to introduce a ribosome binding site and the Xba I restriction site upstream of the Hind III site so that the camC gene can be inserted downstream of camB using the Xba I and Hind HI sites. Therefore the three genes were cloned without fusion in the pGLWl 1 expression vector and arranged in the order 5'- camA-camB-camC-3', and each gene has its own RBS to initiate protein synthesis.
2. b Manipulations of tAte camB gene We used the internal and unique restriction site Mu I (recognition sequence ACGCGT) within the camB gene as the starting point so that the PCR product has a different size from the PCR template fragment. The primers were as follows: 5'-TCA TCG ACG CGT CGC GAA CTG CTG-3' where the Mu site site in bold.
The desired coding sequence at the 3'end of the camB gene was: 5'-CCC GAT AGG CAA TGG TAA GTA GGT GAA TAT CTA ATC CCC ATC camp stop TAT GCG CGA GTG GAG TCT AGA GTT CGA-3' RBS Xba I After the stop codon there is a 35 base spacer before the RBS which is used to initiate the synthesis of the P450am enzyme. The Xba I cloning site is located within the spacer between the RBS and the start codon (not in this primer) of the camC gene. The PCR primer used was the reverse complement of the sequence above. The PCR was carried out and the amplified fragment of the appropriate size was purified by agarose gel electrophoresis and the gel slice excised.
One extra step was necessary to complete the construction of the new plasmid. The plasmid for the fusion protein in vivo system was digested with Adlu I and Hind HI restriction enzymes, purified by agarose gel electrophoresis, and the gel slice for the small camB fragment excised. The pUC 118 plasmid for camB expression was similarly digested, and the gel slice for the backbone was excised. By ligating the two fragments together we prepared a new pUC 118-based plasmid which had an Xba I site followed by an Hind III site downstream of the stop codon of camB. This new plasmid was digested with the Mlu I and Xba I enzymes and the backbone was ligated with the new camB fragment described above to generate a plasmid with the following arrangement of the key components:
.. lac Promoter.. Bam HI.. camB gene.. spacer.. RBS. Xba I.. Hind IH..
2.c Preparation of the in vivo system plasmid Once the modified camB with the Xba I and Hind III restriction sites and appropriate spacers were prepared, the iri vivo system was constructed by cloning this into the pGLWI 1-based plasmid used to express the camA gene (reductase protein) using the Bam HI and Hind III sites. The new in vivo system vector has the following arrangement of the key components: .. tac Promoter.. Eco IRl.. RBS.. camA gene.. spacer.. Bam HI.. RBS.. camB gene.. spacer.. RBS. Xa I.. Hind III..
This new plasmid, named pSGB+, was transformed into E. Coli and expression of the reductase and redoxin proteins was induced by IPTG. When a purified P450Cam enzyme was added to the cell-free extract, substrate oxidation was observed for a variety of substrates.
When the camC gene is cloned into this pSGC+ plasmid using the Xba I and Hind III restriction sites, the new recombinant plasmid thus generated will express the three proteins separately, each under the direction of its own RBS but from the same mRNA molecule. Thus constitutes the in vivo system used in the vast majority of our terpene oxidation work.
3. Introduction of an Xba I site into pRH1091 This is the final step to enable the camC gene to be cloned into the in vivo systems by the two cloning sites XbaI and Hind III. The Xba I site was added by PCR of the entire pRH1091 plasmid using two primers. The presence of these two sites will also enable cloning of the camC gene into M 13 since both Xba I and Hind III are unique in camC and M13.
The primers shown below maintain the Hind III cloning site AAGCTT: 5'-TCA TCG AAG CTT GGC TGT TTT-3' Hind III vector
At the other end the coding sequence desired was: 5'-ACA ATT TCA CAC AGGA TCT AGA C CAT ATG TCA TCG AAG CTT TCA TCG-3' Vector RB S Xba I Nde I Hind III This sequence maintained the Nde I and Hitid III sites but the new Xba I site was introduced upstream of the Nde I site. The PCR primer used was the reverse complement of the desired sequence: 5'-CGA TGA AAG CTT CGA TGA CAT ATG GTC T AGA TCCT GTG TGA AAT TGT-3' The PCR product was then purified by agarose gel electrophoresis, digested with Hind III and circularised with T4 DNA ligase. Success of the PCR method was indicated by the presence of a new and unique Xba I site in plasmid DNA isolated from transformants.
4. Cloning of camC into the in vivo systems All existing camC mutants were cut out of pRH1091-based expression plastids with Nde I and Hind III. The new vector is similarly cut with the same restriction enzymes and the camC gene cloned into this plasmid with T4 DNA ligase. This DNA is transformed into E. Coli JM109 which then may be grown to express P450.
The camC gene is excised from the new vector using Xba I and Hind III restriction enzymes and cloned into either the in vivo vector systems or M13mpl9 for mutagenesis.
5. In vivo expression and substrate turnover For protein expression, cells are grown in LBamp medium (tryptone 10 g/litre, yeast extract 5 g/litre, NaCI 10 g/litre, 50 g/ml ampicillin) at 30°C until the OD6oom reaches 1.0-1.2. IPTG (isopropyl-p-D-thiogalactopyranoside) was added to a final concentration of 1 IlM (from a 1 M stock in H20) and the culture was incubated at 30°C overnight.
For simple screening the substrate can be added to culture and the incubation continued. However, due to impurities from the culture media the cells were generally washed twice with 0.5 vol. of buffer P, (KH2PO4 6.4 g, K2HPO4.3H2O 25.8 g, H20 to 4
litres, pH 7.4) and resuspended in 0.25 vol. oxygen saturated buffer P containing 24 mM glucose. Substrate was added to 1 mM and the incubation continued at 30°C. The reaction was allowed to run for 24 hours with periodic additions of substrate and glucose.
Example 4 The oxidation of halo aromatic compounds Mutant 6- Coupling Product Trichlorophenot Trichlorophenol Efficiency formation (%)rate(min Y96F 75 25 18 22 Y96A77 23 14 33 Y96H54 46 3 1 F87L-Y96F42 58 4 8 F87A-Y96F52 48 2 3 F87A-Y96-F-V247A43 57 4 7 Mutant2,3- 3, 4- Coupling Product Dichlorophenol Dichlorophenol Efficiency formation (%)rate(min- Y96A94 6 6 19 Y96F91 9 4 8 Y96A-V247L94 6 7 20 Y96L-V247A90 10 2 0.7 F87L-96F96 4 3 5 C334A95 5 2 0.5 All mutants have C334A. Coupling efficiency is the percentage of NADH consumed which was utilised for product formation, i. e. a percentage of the theoretical maximum efficiency. The product formation rates are given in (nmol product) (nmol P450)'' (min)''. The relative amount of product formed in each case is shown.
1,3- and 1,4-dichlorobenzene, 1,2,3-and 1,3,5-trichlorobenzene, 1,2,4,5-and 1,2,3,5- tetrachlorobenzene, and 2,3,4,5,6-and 2,2', 4,5,5'-pentachlorobiphenyl were also found to be oxidised.
Wild-type and mutant P450cnm enzymes were tested for their ability to oxidise 3,3'- dichlorobiphenyl and 2,2', 4,5,5'-pentachlorobiphenyl. Results are shown in terms of NADH turnover. Rates are given as nanomol NADH consumed per nanomol P450ni enzyme per minute. P450,,,,enzyme 4,5,5'- dichlorobiphenylpentachlorobiphenyl Wild-type0.4notdetected __ Y96F 15 1 F87A-Y96F 845 165 F87L-Y96F 174 13 F87W-Y96F 4 3 F87A-Y96F-V247A 112 12 Y96A-V247L 84 37 F87A-Y96F-L244A 669 321 F87A-Y97F-L244A-V247A173 214 The first product, 4-hydroxy-3,3'-dichlorobiphenyl was identified by the characteristic coupling patterns expected in the'H NMR spectrum and by mass spectroscopy. The further oxidation product, 4,4'-dihydroxy-3,3'-dichlorobiphenyl was identified by co-elution with an authentic sample, and by W-vis and mass spectroscopy.
This product did not constitute more than ca. 10% of the total products in any of the mutants tested.
For the second substrate product was established as 4'-hydroxy-2,2', 4,5,5'- pentachlorobiphenyl by the observation of the parent ion in the mass spectrum, and by comparison with literature'H NMR data.
P450,. mutants All mutants optionally contain the base mutation C334A.
Single mutants: Y96A, Y96F, Y96L, Y96W.
Double mutants: Y96A-F87A Y96F-F87A Y96F-V295A Y96L-F87A Y96L-A296L Y96A-F87L Y96F-F87I Y96F-V295L Y96L-F87L Y96L-A296F Y96A-F87W Y96F-F87L Y96F-V295I Y96L-F98W Y96L-V396A Y96A-F98W Y96F-F87W Y96F-A296L Y96L-TlOlL Y96L-V396L Y96A-L244A Y96F-F98W Y96F-A296F Y96L-TlOlF Y96L-V396F Y96A-V247A Y96F-TlOlL Y96F-I395F Y96L-L244A Y96L-V396W Y96A-V247L Y96F-T101F Y96F-I395G Y96L-L244F Y96A-I395F Y96F-T185A Y96F-V396A Y96L-V247A Y96A-I395G Y96F-T185F Y96F-V396L Y96L-V247L Y96W-F87W Y96F-T185L Y96F-V396F Y96L-V247F Y96W-F98W Y96F-L244A Y96F-V396W Y96L-V247W Y96W-L244A Y96F-V247A Y96L-G248L Y96W-V247A Y96F-V247L Y96L-V295L Y96W-V396A Y96F-G248L Y96L-V295F Triple Mutants: Y96A-F87A-L244A Y96L-V247A-V396L Y96F-F87W-V247A Y96A-F87A-V247AY96L-V247A-V396FY96F-F87W-V247L Y96A-F87L-L244A Y96L-V247A-V396W Y96F-F87W-V247F Y96A-F87L-V247A Y96L-V247F-V396A Y96F-F87W-V295L <BR> <BR> Y96A-L244A-V247AY96F-F87W-A296L<BR> Y96F-F87A-L244A Y96F-F87W-V396A Y96L-F87A-L244A Y96F-F87A-V247A Y96F-F87W-V396L Y96L-F87A-V247A Y96F-F87A-V247L Y96F-V247F-V396A Y96L-F87L-L244A Y96F-F87A-I395F Y96F-L244A-V396L<BR> Y96L-F87L-V247AY96F-F87A-I395GY96F-L244A-V396F<BR> Y96L-V247A-I395FY96F-F87L-V247AY96F-L244A-V396W<BR> Y96L-V247L-I395FY96F-F87L-V247LY96F-L244F-V396A Y96L-V247L-I395G Y96F-F87L-I395F Y96F-V247A-V396L Y96L-L244A-V396L Y96F-F87W-T185A Y96F-V247A-V396F i96L, L244A-V396F Y96F-F87W-T185F Y96F-V247A-V396W T96L-L244A-V396WY96F-F87W-T185L t96I-L244F-V396A Y96F-F87W-L244F Y96W-F87W-F98W Fivemutations:<BR> Y96F-F87W-T185L-V247L-V295L<BR> Y96F-F87W-T185L-V247L-V396<BR> Y96F-F87W-T185L-V247L-V396L Fourmutations:<BR> Y96A-F87A-L244A-V247A<BR> Y96A-F87L-L244A-V247A<BR> Y96L-F87A-L244A-V247A<BR> Y96L-F87L-L244A-V247A<BR> Y96F-F87W-L244A-V295L<BR> Y96F-F87W-L244F-V396A<BR> Y96F-F87W-L244A-A296L<BR> Y96F-F87W-V247A-V396L<BR> Y96F-F87W-V247A-V396F<BR> Y96F-F87W-V247L-V295A<BR> Y96F-F87W-V247L-V396A<BR> Y96F-F87W-V247F-V396A<BR> Y96F-F87W-V247A-I395F<BR> Y96F-F87W-V247L-I395G Table 2 (continued)