WO2006123007A1 - Producteur de micro-organisme de 6hna, procede d'obtention, elements destines a sa realisation et leurs applications - Google Patents

Producteur de micro-organisme de 6hna, procede d'obtention, elements destines a sa realisation et leurs applications Download PDF

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WO2006123007A1
WO2006123007A1 PCT/ES2006/070063 ES2006070063W WO2006123007A1 WO 2006123007 A1 WO2006123007 A1 WO 2006123007A1 ES 2006070063 W ES2006070063 W ES 2006070063W WO 2006123007 A1 WO2006123007 A1 WO 2006123007A1
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microorganism
gene
nucleotide sequence
nicc
sequence
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José Ignacio JIMENEZ ZARCO
José Luis GARCÍA LOPEZ
Eduardo Diaz Fernandez
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Consejo Superior De Investigaciones Científicas
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring

Definitions

  • the invention falls within the area of biotechnology, and more specifically in a process of biotransformation by microorganisms. It is the production of 6-hydroxynicotinic acid (hereinafter referred to as 6HNA), an important precursor in the synthesis of modern insecticides.
  • 6HNA 6-hydroxynicotinic acid
  • Nicotinic acid (hereinafter referred to as NA) is a very abundant compound in nature and can be used by some bacteria as the sole source of carbon, nitrogen and energy.
  • the ability to use NA as a growth substrate is widely distributed in microorganisms of the genus Pseudomonas, and the different enzymatic stages of the catabolism of this compound are known (Behrman and Stanier J. Biol. Chem. 1957, 228, 923-45).
  • the first enzymatic reaction consists in the hydroxylation of NA to produce 6HNA (Hughes. Biochem. J. 1955, 60, 303). Although the biochemistry of hydroxylation was known, to date the genetic determinants responsible for this process were unknown.
  • the bacterium Pseudomonas putida KT2440 is one of the model organisms in the study of the degradation of aromatic compounds. It is a soil bacterium with great metabolic versatility and whose genome has been completely sequenced (Nelson et al. Environ .. Microbiol. 2002, 4, 799-808).
  • the genomic analysis of P. putida KT2440 has allowed to determine in its global catabolic map the location of different "gene clusters" ("clusters") already known in other bacteria for the degradation of aromatic compounds (Jiménez et al. Environ. Microbiol, 2002, 4, 824-841). In addition, new clusters have been identified within this map, such as those called pcm and nic, located at positions 2.8 Mb and 4.4 Mb of the genome, respectively, whose function is unknown.
  • An object of the present invention is a genetically transformed microorganism by means of genetic engineering techniques useful for the production of 6HNA from NA based on the fact that it presents a genetic modification of one or more of the genes of the nic route, whether of origin endogenous (from the genetically modified microorganism itself), or exogenous (from another microorganism other than the genetically modified one), belonging to the following nic group: nicA, nicB and nicC.
  • a particular embodiment of this microorganism is that developed in the present invention called P. putida KT2440dr ⁇ icC deposited in the Spanish Type Culture Collection (CECT) with the number CECT 7048 or the microorganism P. fluorescens R2f (pNicAB) deposited in the CECT with the number CECT 7049 or the so-called Pseudomonas sp. DSM 6412 (pNicAB).
  • Another object of the present invention is a method of obtaining a type of microorganism of the invention based on genetic alteration by endogenous or exogenous manipulation of the nic path genes.
  • a particular possibility of genetic alteration of the nicC gene is a homologous recombination mutagenesis procedure that causes the disruption of the nucleotide sequence of said gene and therefore the inability to degrade 6HNA.
  • Another particular object of the present invention is the complete nucleotide sequence of the nicC gene SEQ ID N05 and the fragment of the nicC gene with the sequence SEQ ID N07.
  • Another particular object of the present invention is a cloning vector comprising a nucleotide sequence of those described in the present invention that allows disruption of the nicC gene by homologous recombination.
  • Another particular object of the invention is the process of the invention which consists in a process of transformation of a microorganism that is initially incapable of degrading NA with the nucleotide sequences encoding NicA and NicB proteins from another microorganism.
  • Another particular object of the present invention is the nicA gene with the nucleotide sequence SEQ ID NO1 or the nicB gene with the nucleotide sequence SEQ ID N03.
  • another particular object of the present invention is a gene expression or cloning vector that carries the nucleotide sequences of nicA and nicB or a genetic construct containing the foregoing and allowing the expression of NicA and NicB proteins.
  • Another object of the present invention is the use of a microorganism of the invention in a process for obtaining 6HNA under suitable culture conditions.
  • amino acid sequence proteins SEQ ID N02 and SEQ ID N04 encoded by the nucleotide sequences SEQ ID NO1 and SEQ ID N03; as well as any other homologous form isolated or constructed by means of the information of the present invention.
  • the invention faces the need to create new tools and methods for enzymatic synthesis of chemical compounds of industrial value.
  • the present invention is based on the fact that the inventors have identified and isolated by, first, the analysis of the genome of P. putida KT2440 (accession number in GenBank AE015451) in search of ORFs involved in degradation processes of aromatic compounds (see Example 1), and, secondly, by experimental tests of the activity of the expressed or canceled enzymes (see Examples 2 and 3), the presence of a cluster of genes, hereinafter referred to as cluster nic, responsible for heterocyclic catabolism aromatic, and that specifically contains the genes directly involved in the biotransformation pathway of nicotinic acid (NA) both in the first enzymatic step of transforming NA into 6HNA - nicA and nicB genes (see SEQ ID NO1 and N03) -, as in the subsequent hydroxylation of 6HNA - nicC gene (see SEQ ID N05) -.
  • NA nicotin
  • pNicAB plasmid, pNicAB, was transferred by triparental conjugation to different bacteria unable to degrade NA such as Pseudomonas fluorescens R2f (van Elsas et al., 1988, FEMS Microbiol. Ecol., 53, 299-306) and Pseudomonas sp. DSM 6412 (Blaschke et al. Arch. Microbiol. 1991, 155, 164-169).
  • the generated recombinant cells accumulate 6HNA in the culture broth supernatant, and this, contrary to what happens in the procedures mentioned above in the literature, does not suffer further degradation.
  • the advantages of this procedure with respect to the previous ones are that the rational design of strains that express exclusively nicAB genes specifically accumulate 6HNA with a yield close to 100% regardless of the NA present, and are also unable to continue the degradation of 6HNA .
  • an object of the present invention is a genetically transformed microorganism by means of genetic engineering techniques useful for the production of 6HNA from NA, hereinafter microorganism of the present invention, based on the fact that it presents a genetic modification of one or more of the genes of the nic cluster, whether of endogenous or exogenous origin, belonging to the following nic group: nicA, nicB and nicC.
  • nicA nicA
  • nicB and nicC nicC
  • the term "genetically transformed microorganism” refers to any microorganism that has been genetically modified by genetic engineering techniques to transform NA into 6HNA, that is, any microorganism that is capable of expressing nicA genes.
  • the microorganism of the invention belongs to the genus Pseudomonas.
  • a particular object of the present invention is a microorganism that possesses the genes of the nic cluster but that has inactivated its nicC gene such that said nicC gene is not able to express the NicC enzyme or produces an inactive NicC enzyme and, therefore, Therefore, this microorganism is unable to modify 6HNA.
  • a particular embodiment of this microorganism is that developed in the present invention called P. putida KT2440dr ⁇ icC and deposited in the Spanish Type Culture Collection (CECT) with the number CECT 7048, in which the nicC gene has been inactivated by homologous recombination (Example 2.1).
  • Another particular object of the present invention is a microorganism to which the nicA and nicB genes of another microorganism have been transferred so that this new genetically transformed microorganism is capable of converting NA into 6HNA.
  • a particular embodiment of this microorganism is that developed in the present invention called Pseudomonas fluorescens R2f (pNicAB) and deposited in the CECT with the number CECT 7049 or the so-called Pseudomonas sp. DSM 6412 (pNicAB) (Example 3). Therefore, another object of the present invention is a method of obtaining the microorganism of the invention, hereinafter procedure of the invention, based on genetic alteration by endogenous or exogenous manipulation of the genes of the cluster nic.
  • a particular object of the invention is the process of the invention consisting in the inactivation of the endogenous nicC gene of a microorganism that possesses the nic cluster.
  • Any technique or method of mutagenesis known to an expert can be used to induce an inactivation of the nicC gene, such as chemical mutagenesis, irradiation mutagenesis, transposon mutagenesis, minitransposon mutagenesis, and other random mutagenesis techniques. .
  • mutagenesis must be performed by homologous recombination with part of the nicC gene or its boundary regions.
  • Another way to inactivate the function of the nicC gene would be through the use of antisense RNA systems.
  • the expression of the nicC gene could also be inactivated by the use of regulatory proteins that prevent its transcription.
  • a particular possibility of disruption of the nicC gene is a homologous recombination mutagenesis procedure that causes the disruption of the nucleotide sequence of said gene and therefore the inability to degrade 6HNA.
  • a particular embodiment is a homologous recombination process comprising the following steps: a) construction of a cloning vector, preferably a plasmid, containing a fragment of the nucleotide sequence of the nicC gene bound or not bound to a border region, and which is capable of replicating in a microorganism that acts as a host for its construction, but that is not able to replicate in the original microorganism that is to be mutagenized by genetic transformation, and b) genetic transformation of the original microorganism by homologous recombination with the vector of a) under suitable conditions.
  • An exemplary case of the process of the invention by disruption of the nicC gene with a plasmid is the method of obtaining the microorganism P. putida KT2440d.nicC by homologous recombination through a transformation procedure by triparental conjugation in which the fragment of the nucleotide sequence of the nicC gene is constituted by the nucleotide sequence SEQ ID N07, the cloning vector is plasmid pKnicC and the original microorganism is strain P. putida KT2440 (see Example 2.1).
  • sequence SEQ ID N05 the complete coding sequence of the nicC gene identified and isolated in the present invention is indicated as well as a part of the promoter of said gene (see sequence between nucleotide 1 and 321), so that they can be selected by Any person skilled in the art has multiple alternatives of an effective sequence for the disruption of the nicC gene by homologous recombination.
  • nucleotide sequence of the genes comprising said cluster nic - nicA, nicB and nicC - allows the use of said nucleotide sequences, fragments or analogous forms in genetic engineering procedures and in the transformation of microorganisms that produce 6HNA by a person skilled in the art who can isolate or produce said sequences of nucleotides
  • nucleotide sequence of the nicC gene refers to a nucleotide sequence belonging to the following group: a) the nucleotide sequence of the nicC gene of P. putida KT2440 constituted by SEQ ID N05, b) a nucleotide sequence analogous to the sequence of a), c) a fragment of any one of the sequences of a) and b), and d) a nucleotide sequence comprising any sequence belonging to a), b) and e).
  • Another particular object of the present invention is the complete nucleotide sequence of the nicC SEQ ID N05 gene and the nicC gene fragment with the nucleotide sequence SEQ ID N07.
  • analog is intended to include any nucleotide sequence that can be isolated or constructed based on the sequences shown herein, for example, by introducing conservative or non-conservative nucleotide substitutions. , including the insertion of one or more nucleotides, the addition of one or more nucleotides at any of the ends of the molecule or the deletion of one or more nucleotides at any end or within the sequence, and that allows the coding of a peptide or protein capable of mimicking the action of the NicA and NicB protein together, and in the case of the nicC gene any nucleotide sequence that allows inactivation of said gene by insertional mutagenesis by homologous recombination.
  • an analogous nucleotide sequence is substantially homologous to the nucleotide sequence discussed above.
  • the term "substantially homologous” means that the nucleotide sequences in question have a degree of identity of at least 30%, preferably of at least 85%, or more preferably of At least 95%.
  • the nucleotide sequence of the invention may also comprise (sequence d) above), if necessary and to allow a better isolation, detection or expression of the protein, to a nucleotide sequence encoding a peptide capable of being used for purposes for isolation, detection or secretion of said peptide.
  • another particular object of the present invention is a genetic construct comprising, in addition to the nucleotide sequence of one of the genes of the cluster nic, any other nucleotide sequence encoding a peptide or peptide sequence that allows isolation, the detection or expression, for example, by way of illustration and without limiting the scope of the invention, a polyhistidine sequence ( ⁇ xHis), a peptide sequence recognizable by a monoclonal antibody (for example, for identification, or any other that serve to purify the resulting fusion protein by immunoaffinity chromatography: tag peptides such as c-myc, HA, E-tag) (Using antibodies: a laboratory manual. Ed. Harlow and David La ⁇ e (1999). CoId Spring Harbor Laboratory Press. New York Chapter: Tagging proteins. Pp. 347-377).
  • nucleotide sequences of the cluster nic described and isolated in the present invention can be obtained by one skilled in the art by employing techniques widely known in the state of the art (Sambrook et al. "Molecular cloning, a Laboratory Manual 2 nd ed., CoId Sping Harbor Laoratory Press, NY, 1989 vol 1-3) Such nucleotide sequences can be integrated into a gene expression or cloning vector that allows the regulation of expression thereof in a host cell in proper conditions.
  • Another particular object of the present invention is a cloning vector, hereinafter a cloning vector, comprising a nucleotide sequence or a genetic construct of those described in the present invention that allows homologous recombination of an endogenous nicC gene .
  • This cloning vector must be able to replicate in a microorganism that acts as a host for its replication, but it must not be able to replicate in the original microorganism that is to be mutagenized by genetic transformation by homologous recombination.
  • a particular case of a cloning vector of the invention is a mobilizable plasmid capable of replicating in E. coli but not in P. putida KT2440 and conferring resistance to any antibiotic, such as plasmid pKnicC obtained from plasmid pKl8mob ( see Example 2.1).
  • an expression vector comprises, in addition to a nucleotide sequence or genetic construct described in the invention, a promoter that directs its transcription (for example, pT7, plac, ptrc, ptac, pBAD, etc.), to which it is operatively linked, and other necessary or appropriate sequences that control and regulate said transcription and, where appropriate, the translation of the product of interest, for example, transcription initiation and termination signals (tlt2, etc), origin of replication, ribosome binding sequences (RBS), coding sequences of transcriptional regulators, transcription enhancers (enhancers), transcriptional silencers (silencers), repressors, restriction enzyme cleavages, etc.
  • a promoter that directs its transcription for example, pT7, plac, ptrc, ptac, pBAD, etc.
  • other necessary or appropriate sequences that control and regulate said transcription and, where appropriate, the translation of the product of interest, for example, transcription initiation and termination signals
  • Examples of appropriate expression vectors can be selected according to the conditions and needs of each specific case among expression plasmids, viral vectors, cosmids, etc. which may also contain markers that can be used to select cells transfected or transformed with the gene or genes of interest.
  • the choice of the vector will depend on the host cell and the type of use to be performed. Therefore, according to a particular embodiment of the present invention said vector is a plasmid.
  • the obtaining of said vector can be carried out by conventional methods known to those skilled in the art, as well as for the transformation of microorganisms different widely known methods can be used - chemical transformation, electroporation, etc. - described in various manuals [Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2 nd ed. CoId Spring Harbor Laboratory, CoId Spring Harbor, NY].
  • a particular object of the invention is the process of the invention which consists of a transformation procedure with the nucleotide sequences encoding the NicA and NicB proteins of a microorganism that is initially unable to degrade NA.
  • a particular embodiment constitutes a method of genetic transformation of a microorganism comprising the following steps: a) construction of a cloning vector, preferably a plasmid, comprising the nucleotide sequences of the nicA and nicB genes,
  • the term "nicA gene” refers to a nucleotide sequence belonging to the following group: a) the nucleotide sequence of the P. putida nicot gene KT2440 constituted by SEQ ID NOl, b) a nucleotide sequence analogous to the sequence of a), c) a fragment of any one of the sequences of a) and b), and d) a nucleotide sequence comprising any sequence belonging to a), b) and c).
  • Another particular object of the present invention is the nicA gene with the sequence SEQ ID NOl.
  • nicB gene refers to a nucleotide sequence belonging to the following group: a) the nucleotide sequence of the P. putida KT2440 nicB gene consisting of SEQ ID N03, b) a nucleotide sequence analogous to the sequence of a), c) a fragment of any one of the sequences of a) and b), and d) a nucleotide sequence comprising any sequence belonging to a), b) and e).
  • Another particular object of the present invention is the nicB gene with the nucleotide sequence SEQ ID N03.
  • An exemplary case of the process of the invention by genetic transformation with a plasmid is the method of obtaining the microorganism of the invention in which the nucleotide sequence of the nicA gene is SEQ ID NO1 and that of the nicB gene is SEQ ID N03 , the vector is the plasmid pNicAB, the transformation is performed by triparenteral conjugation and the original microorganism is a strain belonging to the following group: Pseudomonas fluorescens R2f and Pseudomonas sp. DSM 6412 (see Example 3).
  • another particular object of the present invention is a gene expression or cloning vector, which carries the nicA and nicB nucleotide sequences and allows the expression of the NicA and NicB proteins.
  • the term cloning vector also refers to an expression system consisting of two cloning vectors each with one of the nucleotide sequences of the nicA or nicB genes, respectively. The expression of these two genes generates a monooxygenase activity of NA inside the host cells that contain the nucleotide sequences of the nicA and nicB genes, producing 6HNA.
  • a particular case of a cloning vector of the invention is plasmid pNicAB, which allows the expression of NicA and NicB proteins.
  • nucleotide sequences of the nicA, nicB and nicC gene for the preparation of gene cloning vectors is part of the invention. as well as the use of sayings vectors for the transformation of 6HNA producing microorganisms.
  • 6HNA is an important precursor in the synthesis of modern insecticides.
  • the synthesis of 6HNA by living organisms is possible and has the advantages of process specificity, simplicity, low economic cost and being an environmentally friendly process (Example 2 and 3) . Therefore, another object of the present invention is the use of the NA / 6HNA microorganism of the invention in a method of obtaining 6HNA under suitable culture conditions.
  • Another particular object of the invention is the use of the NA / 6HNA microorganism in a method of obtaining 6HNA in which the microorganism is a strain that has the inactivated nicC gene.
  • a specific embodiment of the use of the invention is that in which the strain that is used is the mutant strain P. putida KT2440d nicC (CECT 7048) that is grown in a culture medium, for example, in a minimum M63 medium supplemented with a source of carbon, trace elements, antibiotics and NA (see Example 2.2).
  • the 6HNA production process can be carried out with both growing cells and cells in suspension.
  • Another particular object of the invention is the use of the NA / 6HNA microorganism in a method of obtaining 6HNA in which the microorganism is a microorganism transformed with the exogenous genes nicA and nicB.
  • a particular embodiment is the use of the microorganism of the invention in which the microorganism is strain P. fluorescens R2f (pNicAB) (CECT 7049) or strain Pseudomonas sp. DSM 6412 (pNicAB) that is grown in a culture medium, for example, minimum M63 medium, supplemented with a source of carbon, trace elements, antibiotics and NA (Example 3.3).
  • Rich media can also be used for the cultivation of these microorganisms.
  • amino acid sequence proteins SEQ ID N02 and SEQ ID N04 encoded by the nucleotide sequences SEQ ID NO1 and SEQ ID N03, corresponding to the NicA and NicB proteins of the P. putida strain. KT2440, respectively; as well as any other homologous form isolated or constructed by the information of the present invention; as well as its use in a 6HNA enzymatic synthesis procedure.
  • This cluster has allowed the identification and subsequent isolation of a series of nucleic acid sequences that after the activity studies of their expressed protein products (see subsequent examples) correspond to several genes involved in the degradation of NA: a first stage of the degradation pathway of NA, the conversion of this into 6HNA, which would be catalyzed by the protein products of the nicA (SEQ ID NO1) and nicB genes (SEQ ID N03) and, a second stage, the hydroxylation of 6HNA, which would be mediated by the protein product of the nicC gene (SEQ ID N05).
  • the aforementioned proteins NicA, NicB and NicC are identified by the sequences SEQ ID N02, SEQ ID N04 and SEQ ID N06, respectively.
  • an inactivated P. putida KT2440 mutant was obtained in one of the postulated genes, the nicC gene, the first gene in one of the operons that make up the nic cluster.
  • Example 2 Construction of a mutant microorganism of the nicC gene and production of 6HNA with this microorganism
  • nih5 oligonucleotides GCAAGCTTCATCGGGTGGCAGGCG
  • oligonucleotides nih5 and nih3 include HindIII and EcoRI restriction targets respectively (underlined below the sequence), which were used to clone the amplification product in pKl ⁇ mojb, a mobilizable plasmid that replicates in E. coli but not in Pseudomonas and confers resistance to kanamycin (Schafer et al.
  • Plasmid pKnicC was transferred to P. putida KT2440 by triparental conjugation as described in the literature (Herrero et al. J. Bacteriol 1990, 172, 6557-6567). For this, LB medium (Sambrook, J., Fritsch, EF, and Maniatis, T. 1989, Molecular Cloning. CSHL Press, CoId Spring Harbor, New York) E. coli DHlOB cells (pKnicC) (Kanamycin 30) were cultured ⁇ g / ml), E.
  • coli HBlOl (pRK600) (chloramphenicol 34 ⁇ g / ml), a strain that provides the functions for the transfer of plasmid pKnicC (Herrero et al. J. Bacteriol 1990, 172, 6557-6567), and P. putida KT2440.
  • 1 ml of each culture is taken which, after three successive washes, is resuspended in 100 ⁇ l of LB medium. 100 ⁇ l of each culture are mixed and the mixture is deposited on a nitrocellulose filter (0.22 ⁇ m, Millipore) for 8 hours at 30 ° C.
  • the cells are resuspended in 1 ml of saline solution and grown in plates of M63-agar medium containing 0.2% citrate (w / v) and the antibiotic kanamycin (30 ⁇ g / ml) so that only those are selected P. putida KT2440 cells that have acquired the plasmid pKnicC. Since pKnicC is not able to replicate in Pseudomonas, the exconjugants that grow in M63-kanamycin are P. putida KT2440 cells that have homologously integrated plasmid pKnicC into the chromosomal copy of the nicC gene, that is, cells with a disruption of the nicC gene.
  • the mutant strain P. putida KT2440d.nicC was inoculated at a DO ⁇ oonm of 0.1 in minimal medium M63 supplemented with citrate
  • the mutant strain P. putida KT2440dr ⁇ icC is inoculated at a DO ⁇ oonm of 0.1 in a minimum medium M63 supplemented with 0.2% citrate (w / v) as a carbon source, trace elements, Kanamycin (30 ⁇ g / ml) and with NA (1 mM) as a conversion inducer. After incubation at 30 ° C with stirring (300 rpm) until reaching DO ⁇ oo of 0.8, the culture was centrifuged and the cells were washed with a volume of saline. The cell pellet was resuspended in 1/5 volume of M63 medium containing NA (5 mM).
  • the bacteria suspension (10 ml) was incubated at 30 ° C with stirring and samples (0.1 ml) were taken at different times.
  • the samples were centrifuged 2 minutes in a microcentrifuge (12000 rpm) and the cell-free supernatant was subjected to the analysis described in Example 2.1. It was observed that after 6 hours of incubation the NA had been transformed into 6HNA with a yield of 98% (see Table 1).
  • Example 3 Construction of microorganisms carrying the plasmid pNicAB and production of 6HNA with these microorganisms
  • plasmid pNicAB containing the nicAB genes under the control of the Plac promoter was performed in two stages.
  • cosmid 3n5 (kindly provided by Diana Stjepandic of the Krebsabastechnik of Heidelberg, Germany), which contains a cloned region of the genome of P. putida KT2440 spanning from position 4421 kb to 4464 kb, was digested with restriction enzymes EcoRI and Seal isolating a 4.1 kb fragment comprising the nicB gene and much of the nicA gene. Said fragment was subcloned into plasmid pUC19 (Yanisch-Perron et al.
  • nicA5 oligonucleotides GGGGATCCGTAACCGTTGCCGCCACCACCGTATTCG, contains the underlined BamHI restriction target and hybridized with the 5 'end of the nicA (SEQ ID NOlO) and Sgral (CGCTGTCATGGCCGGCATAGGG, hybridized in the nicB gene) (SEQ ID NOIl).
  • the amplified fragment was digested with the restriction enzymes BamEI and SgrAI and replaced the 1.2 kb BamHI-SgrAI fragment of plasmid pFUSAB generating a plasmid, pUCNAB (7.3 kb), which contains the complete nicA and nicB genes under the control of the Plac promoter . Since the plasmid pUCNAB only replicates in E. coli, the nicA and nicB genes were subcloned into a broad-spectrum host plasmid and conferring resistance to gentamicin, pBBRlMCS-5 (Kovach et al. Gene, 1995, 166, 175- 176).
  • the nicA and nicB genes were isolated from the pUCNAB plasmid after digesting it with the restriction enzymes EcoRI and Hindlll, and the resulting fragment was ligated with the plasmid pBBRlMCS-5 treated with the same enzymes.
  • the ligation mixture was transformed into strain E. coli DHlOB and gentamicin-resistant clones (10 ⁇ g / ml) and plasmid carriers, pNicAB were selected
  • DSM 6412 containing the control plasmid pBBRlMCS-5 incubated under conditions similar to those described above do not generate any conversion of NA, demonstrating that the nicA and nicB genes are necessary for the 6HNA biosynthesis from NA, and that the products of these genes are functional in bacteria other than the parental strain.

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

Abstract

L'invention concerne un micro-organisme génétiquement transformé utile dans la production de 6HNA (acide 6-hydroxynicotinique) à partir de NA (acide nicotinique) basé sur le fait qu'il présente une modification génétique d'un ou plusieurs gènes du processus nic, lesquels sont déjà d'origine endogène ou exogène, appartenant au groupe nic suivant: nicA, nicB et nicC; ainsi qu'un processus d'obtention de celui-ci par manipulation des gènes du processus nic. Un autre objet de l'invention consiste en l'utilisation de ce micro-organisme dans un processus d'obtention de 6HNA dans des conditions de culture adéquates. Le 6HNA est un important précurseur dans la synthèse d'insecticides modernes.
PCT/ES2006/070063 2005-05-18 2006-05-17 Producteur de micro-organisme de 6hna, procede d'obtention, elements destines a sa realisation et leurs applications WO2006123007A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP200501209 2005-05-18
ES200501209A ES2288070B1 (es) 2005-05-18 2005-05-18 Microorganismo productor de 6hna, procedimiento de obtencion, elementos para su realizacion y sus aplicaciones.

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WO2006123007A1 true WO2006123007A1 (fr) 2006-11-23

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ES (1) ES2288070B1 (fr)
WO (1) WO2006123007A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020137163A1 (en) * 2000-11-10 2002-09-26 Degussa-Huels Aktiengesellschaft Polynucleotides encoding the nadA gene and methods of producing nicotinic acid or nicotinic acid derivatives

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020137163A1 (en) * 2000-11-10 2002-09-26 Degussa-Huels Aktiengesellschaft Polynucleotides encoding the nadA gene and methods of producing nicotinic acid or nicotinic acid derivatives

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JIMENEZ J.I. ET AL.: "Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440", ENVIRONMENTAL MICROBIOLOGY, vol. 4, no. 12, December 2002 (2002-12-01), pages 824 - 841, XP003003933 *
NAKANO H. ET AL.: "Purification, characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5", EUROPEAN JOURNAL OF BIOCHEMISTRY/FEBS, vol. 260, February 1999 (1999-02-01), pages 120 - 126, XP003003934 *
NELSON K.E. ET AL.: "Complete genome sequence and comparative analysis of the metadolically versatile Pseudomonas putida KT2440", ENVIRONMENTAL MICROBIOLOGY, vol. 4, no. 12, December 2002 (2002-12-01), pages 799 - 808, XP002968819 *
THACKER R. ET AL.: "NIC, a conjugative nicotine-nicotinate degradative plasmid in Pseudomonas convexa", JOURNAL OF BACTERIOLOGY, vol. 135, no. 1, July 1978 (1978-07-01), pages 289 - 290, XP003003935 *

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