WO2009133096A2 - Souches produisant des b-lactames - Google Patents

Souches produisant des b-lactames Download PDF

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WO2009133096A2
WO2009133096A2 PCT/EP2009/055116 EP2009055116W WO2009133096A2 WO 2009133096 A2 WO2009133096 A2 WO 2009133096A2 EP 2009055116 W EP2009055116 W EP 2009055116W WO 2009133096 A2 WO2009133096 A2 WO 2009133096A2
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lactam
microbial strain
lactam compound
gene
mutant
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PCT/EP2009/055116
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WO2009133096A3 (fr
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Van Den Marco Alexander Berg
Hesselien Touw-Riel
Bianca Gielesen
Van Den Linda Hoogen
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Dsm Ip Assets B.V.
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Publication of WO2009133096A2 publication Critical patent/WO2009133096A2/fr
Publication of WO2009133096A3 publication Critical patent/WO2009133096A3/fr

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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
    • 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
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C12P17/184—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system containing a beta-lactam ring, e.g. thienamycin

Definitions

  • SSA's Semi-synthetic ⁇ -lactam antibiotics
  • ⁇ -lactam compounds such as penicillinG (PenG), penicillinV (PenV), 6- aminopenicillanic acid (6-APA), 7-amino-desacetoxy-cephalosporanic acid (7-ADCA), 7- aminocephalosporanic acid (7-ACA) and 7-amino-3-chloro-3-cephem-4-carboxylate (7- ACCA), 7-amino-3-[(Z/E)-1-propen-1-yl]-3-cephem-4-carboxylate (7-PACA), 7- aminodeacetylcephalosporanic acid (7-ADAC), 7-amino-3-carbamoyloxymethyl-3- cephem-4-carboxylic acid (7-ACCCA) and others.
  • ⁇ -lactam compounds such as penicillinG (PenG), penicillinV (PenV), 6- aminopenicillanic acid (6-APA),
  • PenicillinG can be used as a starting point to make semi-synthetic penicillins (SSP's) as amoxicillin and ampicillin, but it can also be used to make semi-synthetic cephalopsorins (SSCs).
  • SSP's semi-synthetic penicillins
  • SSCs semi-synthetic cephalopsorins
  • the first generation 7-ADCA product was derived from PenG whereby both the expansion of the 5-membered penem ring to the 6-membered cephem ring and the subsequent cleavage of the phenylacetic acid side chain of the phenylacetyl-7-ADCA were carried out using chemical reactions.
  • the next generation 7-ADCA product was still obtained from PenG but after the chemical ring expansion, the phenylacetic acid side chain of the phenylacetyl-7-ADCA was cleaved off enzymatically using a suitable (penicillin) acylase.
  • a suitable (penicillin) acylase Other processes have been developed wherein also the ring expansion of PenG to phenylacetyl-7-ADCA is carried out in vitro using a suitable expandase enzyme, but these processes are of little industrial importance.
  • the most recent and most elegant production process for 7-ADCA comprises the culturing of a Penicillium chrysogenum, transformed with and expressing a gene encoding a suitable expandase.
  • This engineered Penicillium chrysogenum strain when grown in the presence of adipic acid as the side chain precursor in the fermentation vessel, produces and excretes adipyl-7-ADCA - see WO93/05158.
  • the adipyl-7-ADCA is recovered from the fermentation broth, subjected to a suitable acylase to cleave off the adipic acid side chain after which the 7- ADCA thus obtained is further purified, crystallized and dried.
  • Group 1 is defined herein as the group with gDNA sequences with SEQ ID No 1,
  • Group 2 is defined herein as the group with gDNA sequences with SEQ ID No.2,
  • Group 3 is defined herein as the group with gDNA sequences with SEQ ID No.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
  • Group 5 is defined herein as the group with gDNA sequences with SEQ ID No.1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 15, 16, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 67, 68, 69, 70, 71, 72, 73, 75, 76, 78, 79, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
  • Group 6 is defined herein as the group with gDNA sequences with SEQ ID No.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104
  • mutant microbial strains which possess one or more of the desired properties selected from the group consisting of (i) an at least 10% higher concentration of the total ⁇ -lactam compounds in the culture medium compared to the parent microbial strain; and/or (ii) an at least 10% higher concentration of the one or more desired ⁇ -lactam compound(s) in the culture medium compared to the parent microbial strain; and/or (iii) an at least 10% improved yield ( ⁇ -lactam / consumed sugar); and/or
  • step a an at least 10% improved yield in the one or more desired ⁇ -lactam compound(s) on precursor ( ⁇ -lactam / consumed precursor).
  • step a identifying the gene(s) in the selected mutant microbial strain
  • step d optionally repeating step a. - d. whereby a selected mutant strain obtained in step d. is used as the parent strain in step a.
  • the genes identified by the method of the first aspect of the invention are referred to as 'negative genes'.
  • “Functionally inactivating” or “functionally inactive” is defined herein as the inactivation of a gene which results in a residual activity of the encoded enzyme as compared to the activity in the parent strain of preferably less than 50%, more preferably less than 40%, more preferably less than 30%, more preferably less than 20%, more preferably less than 10%, more preferably less than 5%, more preferably less than 2%.
  • a "parent microbial strain” may be defined as a micro-organism capable of producing ⁇ -lactam compounds, preferably N-adipylated ⁇ -lactam compounds.
  • a “mutant microbial strain” may be defined as a strain derived from the parent microbial strain by genetic engineering or classical mutagenesis.
  • the microbial strain capable of producing a ⁇ -lactam compound may be selected from the group consisting of a fungus, bacterium and yeast.
  • the microbial strain of the present invention is a fungus, more preferably a filamentous fungus.
  • a preferred filamentous fungus may be selected from the group consisting of Aspergillus, Acremonium, Trichoderma and Penicillium.
  • the microbial strain of the present invention belongs to the species Penicillium, most preferably Penicillium chrysogenum.
  • a preferred bacterium may be selected from the group consisting of Streptomyces, Nocardia, or Flavobacterium.
  • the microbial strain of the present invention capable of producing an N-adipylated ⁇ -lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum, which has been transformed with a gene encoding an expandase, preferably the Streptomyces clavuligerus cefE gene, which enables the strain to produce adipyl-7-ADCA when cultured in the presence of the precursor adipic acid.
  • the microbial strain of the present invention capable of producing an N-adipylated ⁇ -lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum and which, in addition to an expandase gene, preferably the Streptomyces clavuligerus cefE gene, has been transformed with a hydroxylase gene, preferably the Streptomyces clavuligerus cefF gene, whose expression product converts the 3-methyl side chain of adipyl-7-ADCA to 3-hydroxymethyl to give adipyl-7- aminodeacetylcephalosporanic acid (adipyl-7-ADAC).
  • Penicillium most preferably is Penicillium chrysogenum and which, in addition to an expandase gene, preferably the Streptomyces clavuligerus cefE gene, has been transformed with a hydroxylase gene, preferably the Streptomyces clavuligerus cefF gene, whose expression product converts the
  • ⁇ - lactam compounds are phenylacetyl- and adipyl-derivates of the intermediates listed before: 6-aminopenicillanic acid (6-APA), 7-amino-desacetoxy-cephalosporanic acid (7- ADCA), 7-aminocephalosporanic acid (7-ACA) and 7-amino-3-chloro-3-cephem-4- carboxylate (7-ACCA), 7-amino-3-[(Z/E)-1-propen-1-yl]-3-cephem-4-carboxylate (7- PACA), 7-aminodeacetylcephalosporanic acid (7-ADAC), 7-amino-3- carbamoyloxymethyl-3-cephem-4-carboxylic acid (7-ACCCA) and others.
  • Most preferred are N-adipylated cephalosporins, most preferred is adipyl-7-ADCA.
  • inactivation of the gene is defined as the modification of the gene in such a way so as to obtain a functionally inactive gene as defined hereinbefore.
  • Methods for the modification of the gene in order to obtain the functionally inactive gene are known in the art and may include (but are not limited to): inactivation of the gene by base pair mutation resulting in a(n early) stop or frame shift; mutation of one or more codons which encode one or more a critical amino acids (such as the catalytic triad for hydrolases); mutations in the gene resulting in mutations in the amino acid sequence of the enzyme which lead to a decreased half-life of the enzyme; modifying the mRNA molecule in such away that the mRNA half-life is decreased; insertion of a second sequence (i.e.
  • RNAi RNAi
  • Another approach is a temporary one using an anti-sense molecule or RNAi molecule (Kamath et al. 2003. Nature 421 :231-237).
  • Another is using a regulatable promoter system, which can be switched off using external triggers like tetracycline (see Park and Morschhauser, 2005, Eukaryot Cell. 4:1328-1342).
  • Yet another one is to apply a chemical inhibitor or a protein inhibitor or a physical inhibitor (see Tour et al. 2003. Nat Biotech 21 :1505-1508).
  • the most preferred method is to remove part of or the complete gene(s) encoding the enzyme directly or indirectly mediating the incorporation efficiency of raw materials into the ⁇ -lactam compound.
  • the integrative cloning vector comprises a DNA fragment, which is homologous to a DNA sequence in a predetermined target locus in the genome of host cell for targeting the integration of the cloning vector to this predetermined locus.
  • the cloning vector is preferably linearized prior to transformation of the host cell.
  • Fungal cells may be transformed by protoplast formation, protoplast transformation, and regeneration of the cell wall. Suitable procedures for transformation of fungal host cells are described in EP 238023 and Yelton et at. (1984. Proc. Nat. Acad. Sci. USA 81 :1470-1474). Suitable procedures for transformation of filamentous fungal host cells using Agrobacterium tumefaciens are described by de Groot MJ. et al. (1998. Nat. Biotechnol. 16:839-842. Erratum in: Nat. Biotechnol. 1998. 16:1074).
  • Useful selectable markers include, but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC or sutB (sulfate adenyltransferase), trpC (anthranilate synthase), ble (phleomycin resistance protein), as well as equivalents thereof.
  • amdS acetamidase
  • argB ornithinecarbamoyltransferase
  • bar phosphinothricinacetyltransferase
  • hygB hygromycin phosphotransferase
  • niaD nitrate reduc
  • 5'-flank of target locus + selection marker gene + 3'-flank of target locus) and cells wherein the target sequence in the chromosomal DNA sequence is replaced by the desired replacement sequence can be selected by the presence of the selectable marker of the first DNA fragment and the absence of the second selection marker gene.
  • the 5'- and 3'-flanks of the target locus can be for example the promoter and terminator of a gene, or the 5'- and 3'-end of the gene or any combination of these.
  • the example provided in the present invention uses the promoter of the gene as 5'-flank and the gene as the 3'-flank to insert a selection marker between the promoter and gene, thereby disturbing (i.e.
  • the gene sequences given above can be used to make similar functionally inactivated genes.
  • the genes may be split in two, yielding a 5'-flank and a 3'-flank, but the gene may also be used to clone a larger piece of genomic DNA containing the promoter and terminator regions of the gene, which than can function as 5'-flank and a 3'-flank.
  • the parent and mutant microbial strains capable of producing a ⁇ -lactam compound may be cultured in a medium which allows the production of said ⁇ -lactam.
  • a medium which allows the production of said ⁇ -lactam.
  • the culture medium may comprise a side chain precursor, e.g. adipic acid or a suitable salt thereof for the production of adipylated ⁇ -lactam compounds (e.g.
  • the culture medium furthermore may comprise a suitable carbon source such as a sugar (glucose and others).
  • the mutant microbial strains capable of producing a ⁇ -lactam are selected for one or more of the desired properties cited. In one embodiment, mutant microbial strains are selected based on the concentration of the total ⁇ -lactam compounds produced in the culture medium under the conditions of the test.
  • the culture medium of the mutant microbial strain has an at least 10% higher concentration, more preferably an at least 20% higher concentration, more preferably an at least 30% higher concentration, more preferably an at least 40% higher concentration, more preferably an at least 50% higher concentration, more preferably an at least 100% higher concentration, more preferably an at least 200% higher concentration, more preferably an at least 300% higher concentration of the total ⁇ - lactam compounds compared to the concentration of the total ⁇ -lactam compounds produced by the parent microbial strain.
  • mutant microbial strains are selected based on the concentration of the one or more desired ⁇ -lactam compound(s) produced in the culture medium under the conditions of the test.
  • the culture medium of the mutant microbial strain has an at least 10% higher concentration, more preferably an at least 20% higher concentration, more preferably an at least 30% higher concentration, more preferably an at least 40% higher concentration, more preferably an at least 50% higher concentration, more preferably an at least 100% higher concentration, an at least 200% higher concentration, more preferably an at least 300% higher concentration of the one or more desired ⁇ -lactam compound(s) compared to the concentration of the one or more desired ⁇ -lactam compound(s) produced by the parent microbial strain.
  • mutant microbial strains capable of producing a ⁇ - lactam are selected based on an at least 10% improved yield of total ⁇ -lactam on consumed sugar compared to the yield of the parent microbial strain (expressed as percentage - In Table 1 , this yield is referred to as Ybs).
  • This yield is defined herein as ratio of the amount or millimoles of total ⁇ -lactam compounds found in the culture medium and the amount or mole sugar consumed.
  • the amount of total ⁇ -lactam compounds found in the culture medium may be calculated as follows:
  • the amount of Cmol consumed sugar is calculated as follows:
  • the mutant microbial strain with an at least 5% improved yield produces the one or more desired ⁇ - lactam compound(s) with a yield of 84% (80 + 0.05 * 80).
  • the improved yield is higher than at least 5%, preferably at least 10%, more preferably at least 20%.
  • mutant microbial strains are selected based on an improved yield on precursor. It is well known in the art, that the precursors used for the production of the various ⁇ -lactam compounds (e.g. phenyl acetic acid, adipic acid etceteras), are not only incorporated in the desired ⁇ -lactam compounds, but may also be degraded via several metabolic routes for instance to serve as a carbon source to the microbial strain. Mutant microbial strains capable of producing a ⁇ -lactam are selected based on an at least 10% improved yield of the desired ⁇ -lactam compounds on consumed precursor compared to the yield of the parent microbial strain.
  • ⁇ -lactam compounds e.g. phenyl acetic acid, adipic acid etceteras
  • the yield is defined herein as ratio of the molar amount of the desired ⁇ -lactam compounds found in the culture medium and the molar amount of consumed precursor).
  • the amount of millimoles desired ⁇ -lactam compounds found in the culture medium is calculated as follows:
  • the invention provides a method for the construction of a mutant microbial strain capable of producing a ⁇ -lactam compound comprising the step of functionally inactivating preferably one or more genes selected from Group 1 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 1.
  • the invention provides a method for the construction of a mutant microbial strain capable of producing a ⁇ -lactam compound and whereby the mutant microbial strain has an at least 5% improved yield in desired beta- lactam compound compared to undesired/total beta-lactam compound which method comprises the step of functionally inactivating preferably one or more genes selected from Group 5 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 5.
  • the invention provides a method for the construction of a mutant microbial strain capable of producing a ⁇ -lactam compound and whereby the mutant microbial strain an has an at least 10% improved yield in desired ⁇ - lactam on precursor ( ⁇ -lactam / consumed precursor) which method comprises the step of functionally inactivating preferably one or more genes selected from Group 6 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 6
  • the functionally inactivation of the 'negative genes' as defined hereinbefore may be combined with an overexpression of one or more of the 'positive genes' as defined hereinbefore.
  • This combination may lead to a further increase in one or more of the desired properties.
  • Overexpression of the 'positive gene' is defined herein as the modification of the gene in such a way so as to obtain an activity of the enzyme by the positive gene as compared to the activity of the enzyme in the parent strain of preferably more than 100%, more preferably more than 110%, more preferably more than 120%, more preferably more than 150%, more preferably more than 200%, more preferably more than 300%, more preferably more than 400%.
  • the invention provides a mutant microbial strain capable of producing a ⁇ -lactam compound characterized in that preferably one or more genes selected from Group 1 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 1 have been functionally inactivated.
  • the invention provides a mutant microbial strain capable of producing a ⁇ -lactam compound and whereby the mutant microbial strain gives an at least 10% higher concentration of the total ⁇ -lactam compound in the culture medium compared to the parent microbial strain characterized in that preferably one or more genes selected from Group 2 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 2 have been functionally inactivated.
  • the invention provides a mutant microbial strain capable of producing one or more desired ⁇ -lactam compound(s) and whereby the mutant microbial strain gives an at least 10% higher concentration of the one or more desired ⁇ -lactam compound(s) in the culture medium compared to the parent microbial strain characterized in that preferably one or more genes selected from Group 3 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 3 have been functionally inactivated.
  • the invention provides a mutant microbial strain capable of producing a ⁇ -lactam compound and whereby the mutant microbial strain has an at least 10% improved yield of total ⁇ -lactam on consumed sugar compared to the yield of the parent microbial strain characterized in that preferably one or more genes selected from Group 4 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 4 have been functionally inactivated.
  • the invention provides a mutant microbial strain capable of producing a ⁇ -lactam compound and whereby the mutant microbial strain an has an at least 10% improved yield in desired ⁇ -lactam on precursor ( ⁇ -lactam / consumed precursor) characterized in that preferably one or more genes selected from Group 6 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 6 have been functionally inactivated.
  • the invention provides a mutant microbial strain capable of producing a ⁇ -lactam compound and whereby the mutant microbial strain has been improved in all 5 desired properties as defined hereinbefore in step d(i) - d(v) of the method of the first aspect characterized in that preferably one or more genes selected from Group 7 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 7 have been functionally inactivated.
  • mutant microbial strain of the third aspect of the invention as defined hereinbefore in addition to the one or more functionally inactivated genes i.e. 'negative gene(s)' further comprises one or more 'positive gene(s)' as defined before which is over expressed as defined before.
  • nucleotide sequences determined by sequencing a DNA molecule herein are determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, for any DNA sequence determined by this automated approach, any nucleotide sequence determined may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • the person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors.
  • the degree of identity between two amino acid sequences refers to the percentage of amino acids that are identical between the two sequences.
  • the degree of identity is determined using the BLAST algorithm, which is described in Altschul et al. (J. MoI. Biol. 215: 403-410 (1990)). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST program uses as defaults a word length (W) of 1 1 , the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
  • a substantially homologous polypeptide may encompass polymorphisms that may exist in cells from different populations or within a population due to natural allelic or intra-strain variation.
  • a substantially homologous polypeptide may further be derived from a fungus other than the fungus where the specified amino acid and/or DNA sequence originates from, or may be encoded by an artificially designed and synthesized DNA sequence. DNA sequences related to the specified DNA sequences and obtained by degeneration of the genetic code are also part of the invention. Homologues may also encompass biologically active fragments of the full-length sequence.
  • amino acids with basic side chains e.g. lysine, arginine and histidine
  • acidic side chains e.g. aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cystein
  • non-polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophane
  • ⁇ -branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine tryptophane, histidine
  • Figure legends Figure 1 is an overview of the various enzymes and intermediates in ⁇ -lactam biosynthesis routes (i.e. penicillins, cephalosporins and cephamycins).
  • the arrows represent the following enzymes: 1 , L- ⁇ -aminoadipyl-L-cysteinyl-D-valine synthetase; 2, isopenicillin N synthase; 3, acyl-CoA:6-aminopenicillanic acid/isopenicillin N acyltransferase; 4, PenN epimerase; 5, deacetoxycephalosporin C synthase (expandase); 6, deacetylcephalosporin C hydroxylase; 7, 3'-hydroxymethylcephem-O- carbamoyltransferase; 8, O-carbamoyl-deacetylcephalosporin C hydroxylase; 9, methyltransferase; 10, acetyltransferase; 1 1 , phen
  • pSTamdSL was constructed by insertion of an inactive part of the amdS selectionmarker gene (see for example the PgpdA-amdS cassette of pHELY-A1 in WO04106347) by PCR amplification of the last 2/3 of the gene ⁇ amdS) and cloning it in the H/nc/lll-SamHI sites of pSTC1.3.
  • the promoter and ORF PCR fragments (SEQ ID No 2374 and SEQ ID No 2375) were ligated into the respective vectors (pSTamdSL and pSTamdSR, repectively) overnight using T4 ligase (Invitrogen) at 16 0 C, according to the STABY-protocol (Eurogentec) and transformed to chemically competent CYS21 cells (Eurogentec). Ampicillin resistant clones were isolated and used to PCR amplify the cloned fragments fused to the non-functional amdS fragments (see Fig. 2). This was done using the oligonucleotides SEQ ID No 2380 and SEQ ID No 2381.
  • Protoplastation was done for 2 hours at 37°C, using Glucanex at 10 mg/ml in the 24-well plates. Cells (protoplasts and remaining mycelium) are washed again and DNA was isolated using the Puragen DNA isolation kit (Gentra) according to the suppliers' instructions. The DNA was air-dried and dissolved in 100 ul water. PCR reactions were performed in a final 50 ⁇ l, with the following composition:
  • the first PCR reaction is to confirm the correct integration at left flanking, using for the locus of gene Pc12g12120 the specific forward oligonucleotide of SEQ ID NO 2382 and the reverse oligonucleotide of SEQ ID NO 2383; the former being specific for this gene locus and choosen just upfront of the fragment used for gene targeting and the latter annealing in the amdS selection marker, which can be used to verify all individual gene mutations.
  • Step 5 Repeat steps 2-4 for 35 cycles
  • Step 6 10min at 72°C

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Abstract

La présente invention concerne un procédé d’identification d’un ou de plusieurs gènes d’une souche microbienne parente capable de produire un ou plusieurs composés b-lactames souhaités, et des souches microbiennes mutantes dans lesquelles les gènes identifiés ont été fonctionnellement inactivés, ce qui aboutit à une concentration au moins 10 % supérieure d'un ou des composés b-lactames souhaités dans le milieu de culture, par rapport à la souche microbienne parente et/ou une concentration au moins 10 % supérieure d’un ou des composés b-lactames souhaités dans le milieu de culture, par rapport à la souche microbienne parente ; et/ou un rendement amélioré d’au moins 10 % (b-lactame / sucre consommé) ; et/ou un rendement amélioré d’au moins 5 % d’un ou des composés b-lactames souhaités par rapport au composés b-lactames totaux/non souhaités ; et/ou un rendement amélioré d’au moins 10 % d’un ou des composés b-lactames souhaités par rapport au précurseur (b-lactame / précurseur consommé).
PCT/EP2009/055116 2008-04-29 2009-04-28 Souches produisant des b-lactames WO2009133096A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CASQUEIRO JAVIER ET AL: "Gene targeting in Penicillium chrysogenum: Disruption of the lys2 gene leads to penicillin overproduction" JOURNAL OF BACTERIOLOGY, vol. 181, no. 4, February 1999 (1999-02), pages 1181-1188, XP002543928 ISSN: 0021-9193 *
THYKAER JETTE ET AL: "Metabolic engineering of beta-lactam production" METABOLIC ENGINEERING, ACADEMIC PRESS, US, vol. 5, no. 1, 1 January 2003 (2003-01-01), pages 56-69, XP002450423 ISSN: 1096-7176 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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