Abstract:
Method of modifying DNA by subjecting the DNA to a mutation-inducing treatment. The method includes the steps of bringing the DNA to be mutated and a gene encoding a mutation-inducing non-DNA polymerase protein together in cells, growing the cells in the presence of a stress factor, and selecting the mutant cells which have developed a desirable trait in the presence of the stress factor.

Description:
RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Application No. 60/001,381, filed Jul. 24, 1995, which is incorporated by reference herein. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a method of modifying the DNA of an organism, in particular of obtaining an organism capable of overproducing amino acids, antibiotics or proteins or able to degrade xenobiotics, wherein the DNA is subjected to a mutation-inducing treatment and a mutant organism with a desired trait is selected. 
     BACKGROUND OF THE INVENTION 
     Methods are known whereby an organism is treated with mutagens such as base analogues (for example 5-bromouracil and 2-aminopurine), substances that chemically modify bases (for example hydroxylamine) or intercalating agents (for example acridines). 
     This known method requires the addition of chemicals which are hazardous for both the technician handling them and, if not properly disposed of, to the environment. In addition, the modifications are not directed. That is, all the DNA of the organism is mutated and thus an organism with the desired trait will be selected carrying also many other mutations, which mutations may decrease its suitability for the intended use. 
     It is known that defective DNA polymerase results in an increase in mutations and may result in a desired mutated organism, but replication with defective DNA polymerase does not result in directed mutations. Cairns et al. (1988) describe the phenomenon of adaptive or selection-induced mutation, but this reference does not suggest the method of the present invention. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide a method of modifying the DNA, in particular of directed modification (also referred to as adaptive modification) of the DNA of an organism. In addition it is an object to provide an environmentally friendly method obviating the use of hazardous mutagens. 
     In accordance a method is provided characterized in that in an organism a gene encoding a mutation-inducing, non-DNA polymerase protein is brought together with the DNA to be mutated, and the organism is grown in the presence of a stress factor while the gene is expressed. 
     The applicants have found that genes exist which, upon expression, allow the introduction of mutations, in particular, in a way that suggests that the mutations are directed mutations. That is, in the presence of a stress factor those mutations appear to be favored which relieve the stress for the organism. Thus, for example, if an antibiotic is added as the stress factor, those mutations which allow the organism to grow in the presence of the antibiotic are favored. 
     According to a preferred embodiment the DNA to be mutated and the gene encoding a mutation-inducing protein are separated after the induction of mutations. 
     Thus, in case of accidental release of the mutated organism into the environment, the organism is no longer equipped with the means to rapidly adapt to the more hostile environment. This decreases its chances of survival, which increases the safety of the method for the environment. 
     The invention also relates to a method of improving the genetic stability of an organism comprising a gene encoding a mutation-inducing non-DNA polymerase protein, wherein said gene is disabled by methods known per se. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a DNA sequence of the present invention encoding a mutation inducing non-DNA polymerase protein, which protein is encoded from the complementary strand of the DNA sequence from nucleotide 1878 to 1339. Besides the nucleic acid sequence and amino acid sequence of the mut gene, FIG. 1 contains the nucleic acid sequence of a gene stimulating the transformation frequency, as well as its putative amino acid sequence. 
     FIG. 2 is a restriction map of plasmid pMEA300. 
     FIG. 3 shows the spontaneous resistance of wild-type, a pMEA300-free strain (WV1) and strain WV1 carrying pMEA300 against 1 mg/ml para-fluoro phenylalanine. 
     FIG. 4 shows the DNA and amino acid sequences of the mut and stf genes of the present invention. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The following terms are used throughout this description with the meanings defined below. 
     The term vector means a plasmid, phage or other such DNA molecule, as is well-known in the art, which may or may not be capable of being integrated in the chromosome of a host organism, and which is capable of being maintained in the host organism. 
     The term organism comprises any organism, prokaryotic or eucaryotic, single cell or multicellular, animal or vegetable, capable of maintaining and expressing said vector. 
     The term directed or adaptive mutation means the specific mutation of DNA relating to the stress factor. 
     The term stress factor refers to any chemical or physical factor, such as growth-inhibiting compounds and extreme temperature, as well as the absence of a suitable or essential substrate, which causes an organism to grow less well than without the stress factor. 
     The term xenobiotic refers to any compound which is not a compound naturally encountered by the organism, and which compound is desired to be degraded into compounds considered less harmful or harmless to man or the environment, or to be converted into a useful compound. 
     The term disabled in &#34;gene is disabled&#34; relates to a method as a result of which either the gene is no longer expressed, or, in the case the gene is expressed, the resulting protein product is not functionally active as a mutation-inducing non-DNA polymerase protein. As is known in the art, a gene can be disabled in many ways, for example by deleting it completely or partially, or by substituting at least one nucleotide by at least one other nucleotide or nucleotide sequence. 
     For many biotechnology applications, it is desired to have organisms with more desirable traits. For example, it is desirable to have organisms capable of degrading toxic substances. Even if such an organism is available, it may not be effective under all conceivable circumstances. For example, in land farming relatively low temperatures or the presence of heavy metals or other toxic compounds may render the organism ineffective. 
     Other biotechnology applications involve the production of antibiotics, (recombinant) proteins and amino acids. It is often desirable to increase the production of these products. The present invention is applicable to these and other areas, as will be evident to one of ordinary skill in the art, allowing production of desirable compounds in higher yields as well as allowing the development of organisms capable of degrading xenobiotics even under unfavorable conditions. 
     The actinomycete Amycolatopsis methanolica contains a 13.3 kb plasmid pMEA300 (Vrijbloed et al., 1994). FIG. 2 details the restriction map of pMEA300. This plasmid is capable of site-specific integration into the genome of the organism. As a result of the investigation of A. methanolica, it was surprisingly found that a gene which is carried by this plasmid is capable of introducing adaptive mutations into the DNA of an organism while it is under stress. This gene is referred to as the mut gene. Under conditions of stress, the mutation frequency increases over 100-fold in cells that contain pMEA300. 
     It is expected that other such genes exist in other organisms, such as the closely related Amycolatopsis mediterranei (genus Nocardia) and Saccharopolyspora erythraea, as well as in more distantly related and unrelated organisms. Such genes may be found by using assays for activity, or hybridization techniques using part of the DNA sequence of the gene identified within pMEA300 as a probe, or a complementary strand thereof, because of the genetic resemblance with the gene of pMEA300. Alternatively, pMEA300 derivatives may be used. 
     Therefore, a method is provided which allows for the directed mutation of DNA using said mutation-introducing genes. The invention eliminates the use of hazardous mutagenic compounds, though it is recognized that xenobiotic compounds, which are to be broken down and are added as stress factors, may be mutagenic themselves. This elimination of mutagenic compounds increases the safety of the technician and obviates the disposal of the toxic waste resulting from an experiment with mutagenic compounds. 
     It is hypothesized that a stress factor results in an increased level of the mutation-inducing non-DNA polymerase protein. The DNA present in the organism is mutated randomly. If a mutation occurs that relieves the stress due to the stress factor, the expression of the mutation-inducing non-DNA polymerase protein is reduced to its ordinary level. Thus, in contrast to chemical mutagenesis or mutations due to a defective DNA polymerase, the DNA of the organism is not mutated more than necessary, i.e., after the desired mutation has been brought about. Consequently, the average number of mutations per organism for every desired mutant is lower, hence directed mutation. 
     From the above it will be clear that the method according to the invention is very versatile. A deliberate choice of stress factor will result in an organism with the desired trait. For example, adding a stress factor inhibiting the synthesis of a certain amino acid will result in a mutated organism capable of expressing the amino acid in sufficient quantities in the presence of the stress factor. If the stress factor is removed the organism may well overproduce the amino acid. In a similar way it is possible to increase the yield of proteins, antibiotics and other such compounds produced in the organism. 
     For example, if an organism is not or only barely capable of growing on a certain carbon source and that is the only carbon source available, mutant organisms may be selected for their increased ability to grow on that particular carbon source. The method according to the invention greatly increases the naturally occurring (i.e. spontaneous) mutation frequency. 
     The method according to the invention may also be used to obtain an organism capable of overproducing recombinant proteins. When an organism is induced to produce a recombinant protein, its resources are partly used to accomplish this task. If a gene encoding a mutation-inducing protein is introduced and expressed in the organism, the organism may end up better capable of dealing with the stress it is under while producing recombinant protein. Although a large proportion of the mutations may involve the elimination of the activity producing the recombinant protein, other mutations may involve beneficial mutations. 
     The presence of the gene encoding a mutation-inducing protein may offset the benefit of the elimination of the use of mutagens should the mutated organisms be accidentally released into the environment. As it is generally considered undesirable that organisms containing DNA that has been tampered with are released into the environment, the organisms chosen for use are usually incapable of maintaining themselves when released. If they had the ability of rapidly adapting to stress conditions, the released organism could overcome this situation. Therefore it is desirable to separate the gene and the mutated DNA. 
     According to one embodiment, the gene resides in a vector, the organism carrying the DNA to be mutated is transformed with said vector, a desired mutant organism is selected and cured from said vector. This provides a simple method of obtaining the desired mutant organism. 
     According to another embodiment the DNA to be mutated resides in a vector, an organism carrying the gene is transformed with said vector, a desired mutant organism is selected and the mutated vector is used to transform a second organism. 
     Thus, a second organism can be chosen which does not contain any undesired mutated DNA and which is optimal for the particular purpose. The second organism is not necessarily different from the original organism which was used to modify the DNA. On the other hand, the second organism may be entirely different, as long as the vector can be replicated and the gene expressed in the second organism. 
     The invention also relates to a DNA sequence encoding a mutation-inducing non-DNA polymerase protein, said DNA sequence having the complementary sequence of nucleotides 1917 to 1339 as shown in FIG. 1. FIG. 4 likewise shows the complementary DNA sequence (nucleotides 1 to 579) (SEQ ID NO. 3) and the protein sequence of the invention. In addition, the invention encompasses DNA sequences encoding proteins with activity similar to those encoded by the DNA sequences of FIG. 4. 
     The DNA sequence shown in FIG. 4 is from the open reading frame 179 (orf179) which region is responsible for the increased mutation frequency. In FIG. 2, orf179 corresponds to the region identified as the mutation frequency (mut) gene. 
     In addition the invention relates to a DNA sequence hybridizing to the DNA sequence having the sequence of nucleotides 1 to 579 (SEQ ID NO. 3) as shown in FIG. 4, said DNA sequence coding for a non-DNA polymerase protein having mutation-inducing activity. 
     The above DNA sequences may be used to develop probes capable of detecting and of isolating genes in organisms different from Amycolatolsis methanolica, said genes encoding a protein with an activity similar to that encoded by the sequence of nucleotides 1 to 579 (SEQ ID NO. 3) shown in FIG. 4. 
     Thus, the invention relates to the use of a nucleic acid probe comprising a nucleic acid sequence capable of hybridizing with the DNA sequence of nucleotides 1 to 579 (SEQ ID NO. 3) shown in FIG. 4 or the complementary strand thereof for the detection and/or isolation of a gene encoding a mutation-inducing non-DNA polymerase protein. 
     The development of probes and their use for detection and/or isolation of genes is well known to one of ordinary skill in the art. 
     Furthermore, the invention relates to an organism comprising DNA modified by the method according to the invention. 
     The invention also relates to a method of improving the genetical stability of an organism, wherein the organism comprises a gene encoding a mutation-inducing non-DNA polymerase protein and said gene is disabled by methods known per se. 
     It is well known that actinomycetes strains are often instable during large scale fermentations. Spontaneous occurring mutations result in reduced product yields at great cost. The method of stabilizing an organism according to the present invention allows for, for example, stabilizing organisms such as actinomycetes species and bacteria used for the production of, for example, metabolites such as amino acids and antibiotics. 
     Circumstantial evidence that the presence of mut genes are widespread in actinomycetes is that in natural, polluted environments they are among the first to evolve in the pollution degrading organisms. 
     Thus according to a preferred embodiment the method of improving the genetical stability of an organism is characterized in that the organism is an actinomycetes species, the actinomycetes species comprising a gene encoding a mutation-inducing non-DNA polymerase protein and said gene is disabled by methods known per se. 
     For organisms harboring pMEA300 or derivatives of pMEA300 the gene disabled by methods known per se comprises a functional nucleotide sequence chosen from pMEA300 or derivatives of pMEA300. To increase the viability of the organism it may be advantageous to disable the stf gene as well. 
     EXAMPLES 
     Amycolatopsis methanolica carrying the plasmid pMEA300 (wild type, wt) is available from the National Collection of Industrial Bacteria as NCIB11946 (Torry Research Station, Aberdeen, Scotland). Amycolatopsis methanolica strain WV1 is derived from NCIB11946 and differs from NCIB11946 in that the plasmid pMEA300 is not present. Amycolatopsis methanolica WV1 can be obtained by curing the plasmid from NCIB11946 using methods well known in the art. NCIB11946 was cured from pMEA300 by protoplast formation and regeneration (Moretti et al. 1985) and subsequently screening by Southern hybridization (Sambrook et al. 1989) for derivative strains of NCIB11946 that had lost pMEA300. The nucleic acid sequence of pMEA300 is available from the GenBank database accession no. L36679. 
     The mutation-inducing protein has a molecular weight of 21,721 and comprises 192 amino acids plus the &#34;STP&#34; codon (SEQ ID NO. 4). A restriction map of pMEA300 showing the location of the gene (mut) encoding this protein is shown in FIG. 2. The stimulating transformation frequency (stf) protein has a molecular weight of 40,233 and comprises 373 amino acids (SEQ ID NO. 2). 
     A. methanolica grows as chains of several cells, thus in the examples below the term colony forming unit is used (1 colony forming unit corresponding to about 10 cells). 
     Example I 
     Shikimate as a xenobiotic. Amycolatopsis methanolica (wild type; NCIB11946) and Amycolatopsis methanolica WV1 are not capable of growing on shikimate. The xenobiotic shikimate is neither available as a carbon source nor as an energy source (Euverink et al. 1992). 
     1-5×10 8  colony forming units of Amycolatopsis methanolica NCIB11946 and WV1 respectively were incubated on agar plates (de Boer et al., 1988) and incubated at 37° C. in the presence of shikimate without any other carbon source present. After 10 days Shikimate-utilizing mutants of the wild type appeared at a frequency of 5.0×10 -7 . Mutants of WV1 appeared at a 10 times lower frequency. 
     Example II 
     Overproduction of phenylalanine. 
     1-5×10 8  colony forming units of A. methanolica NCIB11946 and WV1 respectively were grown on glucose minimal medium agar plates (de Boer et al. 1988) in the presence of 1 mg/ml para-fluorophenylalanine (pFPhe) at 37° C. pFPhe is a growth inhibiting analogue of phenylalanine. After prolonged incubation, pFPhe-resistant mutants of the wild-type gradually appeared, reaching a final frequency of 5.7×10 -4  after 6-8 days. Strain WV1 showed a markedly lower frequency (2.0×10 -6 ), this latter frequency corresponding to the upper limit of the frequency of spontaneous mutation normally observed for other organisms. 
     Transformation of WV1 with pMEA300 resulted in a transformant exhibiting a similar frequency (5.5×10 -4 ) as the wild-type. 
     Using the method described by Euverink et al. (1995), it was shown that the activity of the prephenate dehydratase and chorismate mutase enzymes was markedly increased. The enzymes were no longer sensitive for inhibition by phenylalanine. However, as wild-type A. methanolica possesses the enzymes to degrade any excess phenylalanine, the wild-type mutants obtained according to this example do not overproduce phenylalanine in the absence of pFPhe. 
     Example III 
     Plasmid pMEA300 Deletion Derivatives. 
     Deletion derivatives of pMEA300 were tested for the ability to restore in strain WV1 the high frequency of spontaneous resistance against pFPhe. Deletion derivatives pWV129, pWV136, pWV113 and pWV375 lack, respectively, the int and xis genes, the genes indicated by black arrows in FIG. 2 as a result of which conjugation is no longer possible, the stf and mut genes, and the stf gene. The table below shows the frequency of spontaneous resistance of the various strains. Orf179 was shown to be responsible for the increased mutation frequency. 
     
         ______________________________________Strain     Frequency Rel. Mutation Freq.                             Plasmid Char.______________________________________wt         5.9 × 10.sup.-4                143          wt  WV1 4.1 × 10.sup.-6 1 plasmid-free  WV1/pMEA300 5.6 × 10.sup.-4 136 wt  WV1/pWV129 13.0 × 10.sup.-4  317 nointegration  WV1/pWV136 4.3 × 10.sup.-4 104 noconjugation  WV1/pWV113 1.3 × 10.sup.-6 0.3 no stf &amp;orf179  WV1/pWV375 6.0 × 10.sup.-4 146 no stf______________________________________ 
    
     As can be seen, absence of integration of the gene in the chromosome results in higher mutation frequencies. It is thought that if there is no integration a higher number of plasmid copies is present and thus a higher level of expression of the mutation-inducing protein. Accordingly, a preferred embodiment of the present invention is characterized in that the vector comprises at least one gene for integrating the vector into the genome of the hostcell and that at least one of the genes for integrating into the genome is disabled. Thus for pMEA300 preferably at least one of the genes chosen from the group consisting of int, xis and att is disabled. 
     
         __________________________________________________________________________#             SEQUENCE LISTING   - -  - - (1) GENERAL INFORMATION:   - -    (iii) NUMBER OF SEQUENCES: 4   - -  - - (2) INFORMATION FOR SEQ ID NO:1:   - -      (i) SEQUENCE CHARACTERISTICS:       (A) LENGTH: 1950 base - #pairs       (B) TYPE: nucleic acid       (C) STRANDEDNESS: single - #stranded       (D) TOPOLOGY: linear   - -      (x) PUBLICATION INFORMATION:       (A) AUTHORS: L. de B - #oer, W. Harder, L. Dijkhuizen      (B) TITLE: Phenylalanine - #and tyrosine metabolism in the           facultative - #methylotroph Nocardia sp. 239      (C) JOURNAL: Arch. Micr - #obiol.      (D) VOLUME: 149      (F) PAGES: 459-465      (G) DATE: 1988  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J. Cairns, - # J. Overbaugh, S. Miller      (B) TITLE:      (C) JOURNAL: Nature      (D) VOLUME: 335      (F) PAGES: 142-145      (G) DATE: 1988  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: G.J.W. Euv - #erink, G.I. Hessels, J.W.           Vrijbloed, - #J.R. Coggins, L. Dijkhuizen      (B) TITLE: Purification - #and characterization of a dual           function - #3-hydroquinate dehydratase from Amycolatopsis           methanolica      (C) JOURNAL: J. Gen. - #Microbiology      (D) VOLUME: 138      (F) PAGES: 2449-2457      (G) DATE: 1992  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: G.J.W. Euv - #erink, D.J. Wolters, L.           Dijkhuizen      (B) TITLE: Prephenate d - #ehydratase of the actinomycete           Amycolatopsi - #s methanolica: purification and           characteriza - #tion of the wild-type and deregulatedmutant            enzymes       (C) JOURNAL: Biochem. J - #.       (D) VOLUME: 308       (F) PAGES: 313-320       (G) DATE: 1995   - -      (x) PUBLICATION INFORMATION:       (A) AUTHORS: P. Moretti - #, G. Hintermann, R. H tter      (B) TITLE: Isolation an - #d characterization of an           extrachromos - #omal element from Nocardia mediterranei      (C) JOURNAL: Plasmid      (D) VOLUME: 14      (F) PAGES: 126-133      (G) DATE: 1985  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J. Sambroo - #k, E.F. Frisch, T. Maniatis      (B) TITLE: Molecular cl - #oning: a laboratory manual      (C) JOURNAL: Cold Sprin - #g Harbor Laboratory Press      (G) DATE: 1989  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J.W. Vrijb - #loed, J. Madon, L. Dijkhuizen      (B) TITLE: A plasmid - #from the methylotrophic           actinomycete - # Amycolatopsis methanolica capable ofsite-            specific - #integration       (C) JOURNAL: J. Bacteri - #ol.       (D) VOLUME: 176       (F) PAGES: 7087-7090       (G) DATE: 1994   - -          (xi) SEQUENCE DESCRIPTION: - #SEQ ID NO: 1:   - - GGCGCCCAGC ACCTTCGGCA GTAGCCCGCT CCCACCAGTC GGGACCGGAA TC -#ATGTTGCC     60   - - CTTCTTGCCC ATGGCTACCT CCCAGGTTCT GCGGCTCGGC GGTTTCCGTT GC -#CATGCCTC    120   - - AAGATTCGTC TGGTTCCGGC TTCTTGGGGA GATGAGTCCC TGGTGAGTCG TT -#GGTGATTC    180   - - GCTCGACTCA CCAGGGGCTG ACCAGGCACA ATGCCCGCAA GGGAGGTGAC C - #GTGGGG GAC 240               - #                  - #                  - #   MetGly Asp               - #                  - #                  - #   1  - - AGC GGG GCA ACC GAG AGC AAG CTT CGC CGT GC - #T CGC CTT GCC GCC GGG288 Ser Gly Ala Thr Glu Ser Lys Leu Arg Arg Al - #a Arg Leu Ala Ala Gly5              - #     10             - #     15  - - ATG ACG CAA GGC GAG GTC CGG GCC AAG CTC AC - #G CAG GCT CGC CGT CGC336 Met Thr Gln Gly Glu Val Arg Ala Lys Leu Th - #r Gln Ala Arg Arg Arg 20                  - #25                  - #30                  - #35  - - CGG GGC AAG ATG CCC CCG AAG GAA GCC AGC TT - #G AAG CGG ATG TAC ACG384 Arg Gly Lys Met Pro Pro Lys Glu Ala Ser Le - #u Lys Arg Met Tyr Thr            40  - #                45  - #                50  - - TCG TGG GAG ACC GGC GCG GTG ATC CCG ACG GA - #C TGG CGG GAC GAA CTG432 Ser Trp Glu Thr Gly Ala Val Ile Pro Thr As - #p Trp Arg Asp Glu Leu        55      - #            60      - #            65  - - TGC GAG GTA TTC GAG CTT CCA CCG GCC GCG CT - #C GGG TTG GTC GAG ACC480 Cys Glu Val Phe Glu Leu Pro Pro Ala Ala Le - #u Gly Leu Val Glu Thr    70          - #        75          - #        80  - - ACA CCG CCA CCT GCG CTC GAC CTC CCG AGC AC - #A TTC GAG GTG GTC CGG528 Thr Pro Pro Pro Ala Leu Asp Leu Pro Ser Th - #r Phe Glu Val Val Arg85              - #    90              - #    95  - - CTC GAT CCA GCG GTG ATT TCG CTG CTA GAC CA - #G CAG ACG AAC TTC TAC576 Leu Asp Pro Ala Val Ile Ser Leu Leu Asp Gl - #n Gln Thr Asn Phe Tyr 100                 1 - #05                 1 - #10                 1 -#15   - - CGG CTG CAG GAC CGG CTG CTG GGG GCG GCG AT - #C ATT CCG CAG ACCGAA      624  Arg Leu Gln Asp Arg Leu Leu Gly Ala Ala Il - #e Ile Pro Gln Thr Glu            120  - #               125  - #               130  - - GCC CAC GTC CGC AAC CTT GAG CAG ATG CTG CG - #A AAT GCG CTG CCG AGC672 Ala His Val Arg Asn Leu Glu Gln Met Leu Ar - #g Asn Ala Leu Pro Ser        135      - #           140      - #           145  - - GGC CAC CTT CCG ACA GCG GCG GTG ACC CTC GC - #T GAG GCG GCT GCG CTC720 Gly His Leu Pro Thr Ala Ala Val Thr Leu Al - #a Glu Ala Ala Ala Leu    150          - #       155          - #       160  - - GCC GGT TGG CAA GCG CTC GAT GCG GGT GAT CT - #C CGG AAA GCG TGG GAC768 Ala Gly Trp Gln Ala Leu Asp Ala Gly Asp Le - #u Arg Lys Ala Trp Asp165              - #   170              - #   175  - - CTG CAC GAC ATC GCG AAG TCC GCA GCA CGG CA - #G GGG GAG AAC CCA GCC816 Leu His Asp Ile Ala Lys Ser Ala Ala Arg Gl - #n Gly Glu Asn Pro Ala 180                 1 - #85                 1 - #90                 1 -#95   - - GTG CTC GCG CAC GTC ACG GCG CAG CAG GCT TA - #C GTT CTG CTC GATGCC      864  Val Leu Ala His Val Thr Ala Gln Gln Ala Ty - #r Val Leu Leu Asp Ala            200  - #               205  - #               210  - - GGC CGG GCC GCC GAT GCG GTG GAG CTG GTC GA - #G TAT GCA AGC GAA CCC912 Gly Arg Ala Ala Asp Ala Val Glu Leu Val Gl - #u Tyr Ala Ser Glu Pro        215      - #           220      - #           225  - - AGG CTG CTC GGA CAG GTC CCC GCA CGC CTT CG - #G TCG TGG TTG GCC GCG960 Arg Leu Leu Gly Gln Val Pro Ala Arg Leu Ar - #g Ser Trp Leu Ala Ala    230          - #       235          - #       240  - - GCG CAC GCC GAG TTC CTG GCC GCG GCG GGG GA - #C CGA TCC GGC GCG ATG    1008 Ala His Ala Glu Phe Leu Ala Ala Ala Gly As - #p Arg Ser Gly Ala Met245              - #   250              - #   255  - - CGG CGG CTC GAT CAA GCG GCC GAC GTG CTG CC - #A GCT GGC GAC AAC GAC    1056 Arg Arg Leu Asp Gln Ala Ala Asp Val Leu Pr - #o Ala Gly Asp Asn Asp 260                 2 - #65                 2 - #70                 2 -#75   - - CCT GAG TTG CCG TAC CTG ATG CTG AAC GGC GC - #G CAC CTC GCC CGGTGG     1104  Pro Glu Leu Pro Tyr Leu Met Leu Asn Gly Al - #a His Leu Ala Arg Trp            280  - #               285  - #               290  - - CGG GGC AAC TGC TTG GCG CGA CTC GGC GAA GA - #C CAG GCG ATC GAG GAC    1152 Arg Gly Asn Cys Leu Ala Arg Leu Gly Glu As - #p Gln Ala Ile Glu Asp        295      - #           300      - #           305  - - CTG ACA GCG GCG CTC GAT GGG CTC ACC ACG CT - #C ACC TCA CGG CGA GCA    1200 Leu Thr Ala Ala Leu Asp Gly Leu Thr Thr Le - #u Thr Ser Arg Arg Ala    310          - #       315          - #       320  - - GAG GCG GGG CTT CGT GTA GAC CTC GCG CTT GC - #C CTG CGG AAG CGC GGC    1248 Glu Ala Gly Leu Arg Val Asp Leu Ala Leu Al - #a Leu Arg Lys Arg Gly325              - #   330              - #   335  - - GAC CTG GAC GAG TCG CGC GTG CAG GCC CGA CA - #A GCC GCC GAG CTG GCC    1296 Asp Leu Asp Glu Ser Arg Val Gln Ala Arg Gl - #n Ala Ala Glu Leu Ala 340                 3 - #45                 3 - #50                 3 -#55   - - GGC ACA ACA GGC TCA GCC CGG CAG CGA GCC CG - #G ATC GCG GAG CTACTT     1344  Gly Thr Thr Gly Ser Ala Arg Gln Arg Ala Ar - #g Ile Ala Glu Leu Leu            360  - #               365  - #               370  - - GCC GCC TAG CGCGAGAACG TGCAGCAGCC CGATCAGAGC GCCTGAGTT - #CCCGACCTGGC  1403  Ala Ala   - - CTGCTCGGAT GAGGTCGGGA ACGTCGCGGA AGGGCATCCA CTGGAAGGTG CC -#TTCGTTCT   1463   - - GCTCGGTCGG GTCGGCGACT TGCTCGACGC CTCGGACGAC AAAGAGGTGG TT -#CGGGTTGC   1523   - - GCAGCATGCC CACCGCGGGC TCGAACGTGA TCAGCGGCTC GATCGAGCGC GG -#CCGGTAGC   1583   - - CGGTCTCTTC CTCGATCTCG CGGACGACGG TCTCCTCAGG GGACTCGTCG CC -#GTCGATGA   1643   - - TGCCGCCGGG TACTTCCCAG CTCCAGATGT TTGGTGCGAA CCTGTGTCGC CA -#AGCCATGA   1703   - - GAACGTGATC CGCAGTGTCG TTGAAGACGA TCGCCATGGC GACGGGCGGA AA -#CCACACGG   1763   - - TGTGATGCTC GAAGCGCTCG CCCGATGGCT GCGAGATGTC GGCTAAGCCG AC -#TTTGACCC   1823   - - ACTCGGTCTC GTAGACGGGA CGCTCGCCGT GGACGATCCA TCGGTCCTTG TC -#CATGGGGA   1883   - - TAGGGTCTCC CGCTCTAGCG CTGTGTGGGG TCACGCCGAT GAACCTCCCG TG -#GGTCGTCT   1943   - - CGCATGT                 - #                  - #- #        1950   - -  - - (2) INFORMATION FOR SEQ ID NO:2:   - -      (i) SEQUENCE CHARACTERISTICS:       (A) LENGTH: 373 amino - #acids       (B) TYPE: amino acid       (D) TOPOLOGY: linear   - -     (ix) FEATURE:       (A) NAME/KEY: Modified - #amino acid, formylmethionine      (B) LOCATION: The first - # amino acid in the sequence,           described - #as methionine, is actually known as           formylmethio - #nine (&#34;fMet&#34;).  Polypeptide chains in           bacteria - #often start with formylmethionine.  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: L. de B - #oer, W. Harder, L. Dijkhuizen      (B) TITLE: Phenylalanine - #and tyrosine metabolism in the           facultative - #methylotroph Nocardia sp. 239      (C) JOURNAL: Arch. Micr - #obiol.      (D) VOLUME: 149      (F) PAGES: 459-465      (G) DATE: 1988  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J. Cairns, - # J. Overbaugh, S. Miller      (B) TITLE:      (C) JOURNAL: Nature      (D) VOLUME: 335      (F) PAGES: 142-145      (G) DATE: 1988  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: G.J.W. Euv - #erink, G.I. Hessels, J.W.           Vrijbloed, - #J.R. Coggins, L. Dijkhuizen      (B) TITLE: Purification - #and characterization of a dual           function - #3-hydroquinate dehydratase from Amycolatopsis           methanolica      (C) JOURNAL: J. Gen. - #Microbiology      (D) VOLUME: 138      (F) PAGES: 2449-2457      (G) DATE: 1992  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: G.J.W. Euv - #erink, D.J. Wolters, L.           Dijkhuizen      (B) TITLE: Prephenate d - #ehydratase of the actinomycete           Amycolatopsi - #s methanolica: purification and           characteriza - #tion of the wild-type and deregulatedmutant            enzymes       (C) JOURNAL: Biochem. J - #.       (D) VOLUME: 308       (F) PAGES: 313-320       (G) DATE: 1995   - -      (x) PUBLICATION INFORMATION:       (A) AUTHORS: P. Moretti - #, G. Hintermann, R. H tter      (B) TITLE: Isolation an - #d characterization of an           extrachromos - #omal element from Nocardia mediterranei      (C) JOURNAL: Plasmid      (D) VOLUME: 14      (F) PAGES: 126-133      (G) DATE: 1985  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J. Sambroo - #k, E.F. Frisch, T. Maniatis      (B) TITLE: Molecular cl - #oning: a laboratory manual      (C) JOURNAL: Cold Sprin - #g Harbor Laboratory Press      (G) DATE: 1989  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J.W. Vrijb - #loed, J. Madon, L. Dijkhuizen      (B) TITLE: A plasmid - #from the methylotrophic           actinomycete - # Amycolatopsis methanolica capable ofsite-            specific - #integration       (C) JOURNAL: J. Bacteri - #ol.       (D) VOLUME: 176       (F) PAGES: 7087-7090       (G) DATE: 1994   - -          (xi) SEQUENCE DESCRIPTION: - #SEQ ID NO: 2:   - - Met Gly Asp Ser Gly Ala Thr Glu Ser Lys Le - #u Arg Arg Ala ArgLeu  1               5   - #                10  - #                15  - - Ala Ala Gly Met Thr Gln Gly Glu Val Arg Al - #a Lys Leu Thr Gln Ala        20      - #            25      - #            30  - - Arg Arg Arg Arg Gly Lys Met Pro Pro Lys Gl - #u Ala Ser Leu Lys Arg    35          - #        40          - #        45  - - Met Tyr Thr Ser Trp Glu Thr Gly Ala Val Il - #e Pro Thr Asp Trp Arg50              - #    55              - #    60  - - Asp Glu Leu Cys Glu Val Phe Glu Leu Pro Pr - #o Ala Ala Leu Gly Leu 65                  - #70                  - #75                  - #80  - - Val Glu Thr Thr Pro Pro Pro Ala Leu Asp Le - #u Pro Ser Thr Phe Glu            85  - #                90  - #                95  - - Val Val Arg Leu Asp Pro Ala Val Ile Ser Le - #u Leu Asp Gln Gln Thr        100      - #           105      - #           110  - - Asn Phe Tyr Arg Leu Gln Asp Arg Leu Leu Gl - #y Ala Ala Ile Ile Pro    115          - #       120          - #       125  - - Gln Thr Glu Ala His Val Arg Asn Leu Glu Gl - #n Met Leu Arg Asn Ala130              - #   135              - #   140  - - Leu Pro Ser Gly His Leu Pro Thr Ala Ala Va - #l Thr Leu Ala Glu Ala 145                 1 - #50                 1 - #55                 1 -#60   - - Ala Ala Leu Ala Gly Trp Gln Ala Leu Asp Al - #a Gly Asp Leu ArgLys             165  - #               170  - #               175  - - Ala Trp Asp Leu His Asp Ile Ala Lys Ser Al - #a Ala Arg Gln Gly Glu        180      - #           185      - #           190  - - Asn Pro Ala Val Leu Ala His Val Thr Ala Gl - #n Gln Ala Tyr Val Leu    195          - #       200          - #       205  - - Leu Asp Ala Gly Arg Ala Ala Asp Ala Val Gl - #u Leu Val Glu Tyr Ala210              - #   215              - #   220  - - Ser Glu Pro Arg Leu Leu Gly Gln Val Pro Al - #a Arg Leu Arg Ser Trp 225                 2 - #30                 2 - #35                 2 -#40   - - Leu Ala Ala Ala His Ala Glu Phe Leu Ala Al - #a Ala Gly Asp ArgSer             245  - #               250  - #               255  - - Gly Ala Met Arg Arg Leu Asp Gln Ala Ala As - #p Val Leu Pro Ala Gly        260      - #           265      - #           270  - - Asp Asn Asp Pro Glu Leu Pro Tyr Leu Met Le - #u Asn Gly Ala His Leu    275          - #       280          - #       285  - - Ala Arg Trp Arg Gly Asn Cys Leu Ala Arg Le - #u Gly Glu Asp Gln Ala290              - #   295              - #   300  - - Ile Glu Asp Leu Thr Ala Ala Leu Asp Gly Le - #u Thr Thr Leu Thr Ser 305                 3 - #10                 3 - #15                 3 -#20   - - Arg Arg Ala Glu Ala Gly Leu Arg Val Asp Le - #u Ala Leu Ala LeuArg             325  - #               330  - #               335  - - Lys Arg Gly Asp Leu Asp Glu Ser Arg Val Gl - #n Ala Arg Gln Ala Ala        340      - #           345      - #           350  - - Glu Leu Ala Gly Thr Thr Gly Ser Ala Arg Gl - #n Arg Ala Arg Ile Ala    355          - #       360          - #       365  - - Glu Leu Leu Ala Ala370  - -  - - (2) INFORMATION FOR SEQ ID NO:3:  - -      (i) SEQUENCE CHARACTERISTICS:      (A) LENGTH: 612 base - #pairs      (B) TYPE: nucleic acid      (C) STRANDEDNESS: single - #stranded      (D) TOPOLOGY: linear  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: L. de B - #oer, W. Harder, L. Dijkhuizen      (B) TITLE: Phenylalanine - #and tyrosine metabolism in the           facultative - #methylotroph Nocardia sp. 239      (C) JOURNAL: Arch. Micr - #obiol.      (D) VOLUME: 149      (F) PAGES: 459-465      (G) DATE: 1988  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J. Cairns, - # J. Overbaugh, S. Miller      (B) TITLE:      (C) JOURNAL: Nature      (D) VOLUME: 335      (F) PAGES: 142-145      (G) DATE: 1988  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: G.J.W. Euv - #erink, G.I. Hessels, J.W.           Vrijbloed, - #J.R. Coggins, L. Dijkhuizen      (B) TITLE: Purification - #and characterization of a dual           function - #3-hydroquinate dehydratase from Amycolatopsis           methanolica      (C) JOURNAL: J. Gen. - #Microbiology      (D) VOLUME: 138      (F) PAGES: 2449-2457      (G) DATE: 1992  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: G.J.W. Euv - #erink, D.J. Wolters, L.           Dijkhuizen      (B) TITLE: Prephenate d - #ehydratase of the actinomycete           Amycolatopsi - #s methanolica: purification and           characteriza - #tion of the wild-type and deregulatedmutant            enzymes       (C) JOURNAL: Biochem. J - #.       (D) VOLUME: 308       (F) PAGES: 313-320       (G) DATE: 1995   - -      (x) PUBLICATION INFORMATION:       (A) AUTHORS: P. Moretti - #, G. Hintermann, R. H tter      (B) TITLE: Isolation an - #d characterization of an           extrachromos - #omal element from Nocardia mediterranei      (C) JOURNAL: Plasmid      (D) VOLUME: 14      (F) PAGES: 126-133      (G) DATE: 1985  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J. Sambroo - #k, E.F. Frisch, T. Maniatis      (B) TITLE: Molecular cl - #oning: a laboratory manual      (C) JOURNAL: Cold Sprin - #g Harbor Laboratory Press      (G) DATE: 1989  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J.W. Vrijb - #loed, J. Madon, L. Dijkhuizen      (B) TITLE: A plasmid - #from the methylotrophic           actinomycete - # Amycolatopsis methanolica capable ofsite-            specific - #integration       (C) JOURNAL: J. Bacteri - #ol.       (D) VOLUME: 176       (F) PAGES: 7087-7090       (G) DATE: 1994   - -          (xi) SEQUENCE DESCRIPTION: - #SEQ ID NO: 3:   - - ACATGCGAGA CGACCCACGG GAGGTTCATC GGC GTG ACC CCA CA - #C AGC GCTAGA      54               - #                  - # Met Thr Pro His Ser Ala Arg              - #                  - # 1               5  - - GCG GGA GAC CCT ATC CCC ATG GAC AAG GAC CG - #A TGG ATC GTC CAC GGC102 Ala Gly Asp Pro Ile Pro Met Asp Lys Asp Ar - #g Trp Ile Val His Gly    10          - #        15          - #        20  - - GAG CGT CCC GTC TAC GAG ACC GAG TGG GTC AA - #A GTC GGC TTA GCC GAC150 Glu Arg Pro Val Tyr Glu Thr Glu Trp Val Ly - #s Val Gly Leu Ala Asp25              - #    30              - #    35  - - ATC TCG CAG CCA TCG GGC GAG CGC TTC GAG CA - #T CAC ACC GTG TGG TTT198 Ile Ser Gln Pro Ser Gly Glu Arg Phe Glu Hi - #s His Thr Val Trp Phe 40                  - #45                  - #50                  - #55  - - CCG CCC GTC GCC ATG GCG ATC GTC TTC AAC GA - #C ACT GCG GAT CAC GTT246 Pro Pro Val Ala Met Ala Ile Val Phe Asn As - #p Thr Ala Asp His Val            60  - #                65  - #                70  - - CTC ATG GCT TGG CGA CAC AGG TTC GCA CCA AA - #C ATC TGG AGC TGG GAA294 Leu Met Ala Trp Arg His Arg Phe Ala Pro As - #n Ile Trp Ser Trp Glu        75      - #            80      - #            85  - - GTA CCC GGC GGC ATC ATC GAC GGC GAC GAG TC - #C CCT GAG GAG ACC GTC342 Val Pro Gly Gly Ile Ile Asp Gly Asp Glu Se - #r Pro Glu Glu Thr Val    90          - #        95          - #        100  - - GTC CGC GAG ATC GAG GAA GAG ACC GGC TAC CG - #G CCG CGC TCG ATC GAG390 Val Arg Glu Ile Glu Glu Glu Thr Gly Tyr Ar - #g Pro Arg Ser Ile Glu105              - #   110              - #   115  - - CCG CTG ATC ACG TTC GAG CCC GCG GTG GGC AT - #G CTG CGC AAC CCG AAC438 Pro Leu Ile Thr Phe Glu Pro Ala Val Gly Me - #t Leu Arg Asn Pro Asn 120                 1 - #25                 1 - #30                 1 -#35   - - CAC CTC TTT GTC GTC CGA GGC GTC GAG CAA GT - #C GCC GAC CCG ACCGAG      486  His Leu Phe Val Val Arg Gly Val Glu Gln Va - #l Ala Asp Pro Thr Glu            140  - #               145  - #               150  - - CAG AAC GAA GGC ACC TTC CAG TGG ATG CCC TT - #C CGC GAC GTT CCC GAC534 Gln Asn Glu Gly Thr Phe Gln Trp Met Pro Ph - #e Arg Asp Val Pro Asp        155      - #           160      - #           165  - - CTC ATC CGA GCA GGC CAG GTC GGG AAC TCA GG - #C GCT CTG ATC GGG CTG582 Leu Ile Arg Ala Gly Gln Val Gly Asn Ser Gl - #y Ala Leu Ile Gly Leu    170          - #       175          - #       180  - - CTG CAC GTT CTC GCG CTA GGC GGC AAG TAG  - #                  - #  612 Leu His Val Leu Ala Leu Gly Gly Lys185              - #   190  - -  - - (2) INFORMATION FOR SEQ ID NO:4:  - -      (i) SEQUENCE CHARACTERISTICS:      (A) LENGTH: 192 amino - #acids      (B) TYPE: amino acid      (D) TOPOLOGY: linear  - -     (ix) FEATURE:      (A) NAME/KEY: Modified - #amino acid, formylmethionine      (B) LOCATION: The first - # amino acid in the sequence,           described - #as methionine, is actually known as           formylmethio - #nine (&#34;fMet&#34;).  Polypeptide chains in           bacteria - #often start with formylmethionine.  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: L. de B - #oer, W. Harder, L. Dijkhuizen      (B) TITLE: Phenylalanine - #and tyrosine metabolism in the           facultative - #methylotroph Nocardia sp. 239      (C) JOURNAL: Arch. Micr - #obiol.      (D) VOLUME: 149      (F) PAGES: 459-465      (G) DATE: 1988  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J. Cairns, - # J. Overbaugh, S. Miller      (B) TITLE:      (C) JOURNAL: Nature      (D) VOLUME: 335      (F) PAGES: 142-145      (G) DATE: 1988  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: G.J.W. Euv - #erink, G.I. Hessels, J.W.           Vrijbloed, - #J.R. Coggins, L. Dijkhuizen      (B) TITLE: Purification - #and characterization of a dual           function - #3-hydroquinate dehydratase from Amycolatopsis           methanolica      (C) JOURNAL: J. Gen. - #Microbiology      (D) VOLUME: 138      (F) PAGES: 2449-2457      (G) DATE: 1992  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: G.J.W. Euv - #erink, D.J. Wolters, L.           Dijkhuizen      (B) TITLE: Prephenate d - #ehydratase of the actinomycete           Amycolatopsi - #s methanolica: purification and           characteriza - #tion of the wild-type and deregulatedmutant            enzymes       (C) JOURNAL: Biochem. J - #.       (D) VOLUME: 308       (F) PAGES: 313-320       (G) DATE: 1995   - -      (x) PUBLICATION INFORMATION:       (A) AUTHORS: P. Moretti - #, G. Hintermann, R. H tter      (B) TITLE: Isolation an - #d characterization of an           extrachromos - #omal element from Nocardia mediterranei      (C) JOURNAL: Plasmid      (D) VOLUME: 14      (F) PAGES: 126-133      (G) DATE: 1985  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J. Sambroo - #k, E.F. Frisch, T. Maniatis      (B) TITLE: Molecular cl - #oning: a laboratory manual      (C) JOURNAL: Cold Sprin - #g Harbor Laboratory Press      (G) DATE: 1989  - -      (x) PUBLICATION INFORMATION:      (A) AUTHORS: J.W. Vrijb - #loed, J. Madon, L. Dijkhuizen      (B) TITLE: A plasmid - #from the methylotrophic           actinomycete - # Amycolatopsis methanolica capable ofsite-            specific - #integration       (C) JOURNAL: J. Bacteri - #ol.       (D) VOLUME: 176       (F) PAGES: 7087-7090       (G) DATE: 1994   - -          (xi) SEQUENCE DESCRIPTION: - #SEQ ID NO: 4:   - - Met Thr Pro His Ser Ala Arg Ala Gly Asp Pr - #o Ile Pro Met AspLys  1               5   - #                10  - #                15  - - Asp Arg Trp Ile Val His Gly Glu Arg Pro Va - #l Tyr Glu Thr Glu Trp        20      - #            25      - #            30  - - Val Lys Val Gly Leu Ala Asp Ile Ser Gln Pr - #o Ser Gly Glu Arg Phe    35          - #        40          - #        45  - - Glu His His Thr Val Trp Phe Pro Pro Val Al - #a Met Ala Ile Val Phe50              - #    55              - #    60  - - Asn Asp Thr Ala Asp His Val Leu Met Ala Tr - #p Arg His Arg Phe Ala 65                  - #70                  - #75                  - #80  - - Pro Asn Ile Trp Ser Trp Glu Val Pro Gly Gl - #y Ile Ile Asp Gly Asp            85  - #                90  - #                95  - - Glu Ser Pro Glu Glu Thr Val Val Arg Glu Il - #e Glu Glu Glu Thr Gly        100      - #           105      - #           110  - - Tyr Arg Pro Arg Ser Ile Glu Pro Leu Ile Th - #r Phe Glu Pro Ala Val    115          - #       120          - #       125  - - Gly Met Leu Arg Asn Pro Asn His Leu Phe Va - #l Val Arg Gly Val Glu130              - #   135              - #   140  - - Gln Val Ala Asp Pro Thr Glu Gln Asn Glu Gl - #y Thr Phe Gln Trp Met 145                 1 - #50                 1 - #55                 1 -#60   - - Pro Phe Arg Asp Val Pro Asp Leu Ile Arg Al - #a Gly Gln Val GlyAsn             165  - #               170  - #               175  - - Ser Gly Ala Leu Ile Gly Leu Leu His Val Le - #u Ala Leu Gly Gly Lys        180      - #           185      - #           190__________________________________________________________________________