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WO2003048367A1 - Optimisation de souche genetique pour la production amelioree de riboflavine - Google Patents

Optimisation de souche genetique pour la production amelioree de riboflavine Download PDF

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Publication number
WO2003048367A1
WO2003048367A1 PCT/EP2002/013660 EP0213660W WO03048367A1 WO 2003048367 A1 WO2003048367 A1 WO 2003048367A1 EP 0213660 W EP0213660 W EP 0213660W WO 03048367 A1 WO03048367 A1 WO 03048367A1
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gene
riboflavin
genes
rib
increased
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PCT/EP2002/013660
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German (de)
English (en)
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Henning ALTHÖFER
Jose L. Revuelta Doval
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Basf Aktiengesellschaft
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Priority to EP02791764A priority Critical patent/EP1456388A1/fr
Priority to AU2002358083A priority patent/AU2002358083A1/en
Priority to US10/497,526 priority patent/US20050239161A1/en
Priority to JP2003549544A priority patent/JP2005511053A/ja
Publication of WO2003048367A1 publication Critical patent/WO2003048367A1/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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C12P25/00—Preparation of compounds containing alloxazine or isoalloxazine nucleus, e.g. riboflavin
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor

Definitions

  • the present invention relates to a genetic process for the production of riboflavin.
  • the riboflavin production in these organisms is increased by the special selection of riboflavin biosynthesis genes or their combination in organisms of the genus Ashbya and their expression.
  • Vitamin B2 also called riboflavin, is produced by all plants and a variety of microorganisms. It is essential for humans and animals as they are unable to synthesize it. Riboflavin plays an important role in metabolism. For example, it is involved in the utilization of carbohydrates. With vitamin B2 deficiency, inflammation of the mucous membranes of the mouth and throat, itching and inflammation in the skin folds and similar skin damage, conjunctivitis, decreased visual acuity and clouding of the cornea occur. Growth and weight loss may occur in infants and children. Vitamin B2 is therefore of great economic importance, for example as a vitamin preparation for vitamin deficiency and as a feed additive. Various foods are added. It is also used as a food coloring, for example in mayonnaise, ice cream, pudding etc. used.
  • Vitamin B2 is produced either chemically or microbially (see e.g. Kurth et al., 1996, Riboflavin, in: Ulimann's Encyclopedia of industrial chemistry, VCH Weinheim). In the chemical production process, riboflavin is usually obtained as a pure end product in multi-stage processes, with relatively expensive starting products, e.g. D-Ribose, must be used.
  • riboflavin is the fermentative production of vitamin B2 by microorganisms. Renewable raw materials such as sugar or vegetable oils are used as starting materials.
  • the production of riboflavin by fermentation of fungi such as Eremothecium ashbyii or Ashbya gossypii is known (The Merck Index, Windholz et al., Eds. Merck & Co., page 1183, 1983), but also yeasts, e.g. Candida, Pichia and Saccharomyces or bacteria, e.g.
  • Bacillus, clostridia or corynebacteria are described as riboflavin producers.
  • EP-A-0 405 370 and EP-A-0 821 063 describes the production of riboflavin with recombinant bacterial strains, the strains being obtained by transformation with riboflavin biosynthetic genes from Bacillus subtilis.
  • WO 95/26406 and WO 94/11515 describe the cloning of the genes specific for riboflavin biosynthesis from the eukaryotic organisms Ashbya gossypii or Saccharomyces cerevisiae, as well as microorganisms which have been transformed with these genes, and the use of such microorganisms for riboflavin synthesis described.
  • WO 99/61623 describes the use of the selection of riboflavin biosynthesis genes (rib3, rib4, rib5) for increasing the riboflavin formation.
  • GTP guanosine triphosphate
  • ribulose-5-phosphate guanosine triphosphate
  • GTP guanosine triphosphate
  • the GTP cyclohydrolase II ribl gene product converts GTP to 2,5-diamino-6- (ribosylamino) -4- (3H) -pyrimidinone-5-phosphate. This compound is then replaced by the 2,5-diamino-6- (ribosylamino) -4-
  • 2,5-diamino-ribitylamino-2,4- (1H, 3H) -pyrimidine-5-phosphate reduced and then by a specific deaminase (rib2 gene product) to 5-amino-6-ribitylamino-2,4- (1H, 3H) -pyrimidinedione-5-phosphate deaminated.
  • the phosphate is then split off by an unspecific phosphatase.
  • Ribulose-5-phosphate in addition to GTP the second starting product of the last enzymatic steps in riboflavin biosynthesis, is converted to 3,4-dihydroxy by the 3,4-dihydroxy-2-butanone-4-phosphate synthase (rib3 gene product).
  • 2-butanone-4-phosphate (DBP) implemented.
  • DBP and 5-amino-6-ribitylamino-2,4- (1H, 3H) -pyrimidinedione are the educts of the enzymatic synthesis of 6, 7-dimethyl-8-ribityllumazine. This reaction is catalyzed by the rib4 gene product (DMRL synthase). DMRL is then converted to riboflavin by ribofavin synthase (rib5 gene product) (Bacher et al. (1993), Bioorg. Chem. Front. Vol. 3, Springer Verlag).
  • the process for the increased production of riboflavin is advantageously carried out with an organism which is capable of synthesizing riboflavin, in which, for example, the combination of the following rib gene products (the numbers indicate the corresponding rib gene product) have increased activity : 1 + 2, 1 + 4, 1 + 7, 2 + 4, 2 + 7, 4 + 7.
  • Organisms in which the combination of the following rib gene products (the numbers indicate the corresponding rib gene product) have an increased activity are particularly preferred: 1 + 2 + 4, 1 + 2 + 7, 1 + 4 + 7, 2 + 4 + 7.
  • the increased activity of the rib gene products is evaluated in comparison to the Ashbya gossypii strain ATCC 10895, which serves as a reference organism.
  • the corresponding methods for measuring the activity of the rib gene products, i.e. the enzyme activities are familiar to the person skilled in the art and are described in the literature.
  • Another advantage of increasing vitamin B2 productivity is the combination of increasing the natural enzyme activity and introducing the above-mentioned gene combination to increase gene expression.
  • Rib gene products are not only the polypeptide sequences described in the sequence listing in SEQ ID NO: 2, 4, 6, 8, but also those of these sequences by exchange, insertion or deletion of up to 5%, preferably up to 3%, particularly preferably up to 2% of the amino acid codons available polypeptide sequences. Such sequences occur, for example, as natural allele variations or can be treated by mutation of the parent strain, for example by mutagenic substances or electromagnetic radiation and subsequent selection for increased riboflavin productivity can be obtained.
  • the combination according to the invention of the rib genes ribl, rib2, rib4 and rib7 and / or the increase in activity of the genes and their gene products leads to a significantly increased riboflavin productivity.
  • the genes mentioned can in principle be introduced into the organisms used by all methods known to the person skilled in the art; they are advantageously introduced into the organisms or their cells via transformation, transfection, electroporation, with the so-called particle gun or via microinjection.
  • particle gun or via microinjection for microorganisms, the person skilled in the art can use the corresponding textbooks from Sambrook, J. et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, by F.M. Ausubel et al.
  • the REMI technique is based on the cotransformation of a linear DNA construct, which was cut at both ends with the same restriction endonuclease, together with the restriction endonuclease, which was used for this restriction of the DNA construct, into an organism.
  • the restriction endonuclease then cuts the genomic DNA of the organism into which the DNA construct was introduced together with the restriction enzyme. This leads to an activation of the cell's own repair mechanisms. These repair mechanisms repair caused by the endonuclease strand breaks' of the genomic DNA and build it at a certain frequency and the cotransformed DNA construct into the genome one. As a rule, the restriction sites at both ends of the DNA are preserved.
  • the REMI method can be used to integrate biosynthetic genes into the genome of the above-mentioned organisms and thus production processes for the production of metabolic products of primary or secondary metabolism, especially of biosynthetic pathways, for example of amino acids such as lysine, methionine, threonine or Tryptophan, vitamins such as vitamins A, B2, B6 B12, C, D, E, F, S-adenosylmethionine, biotin, pantothenic acid or folic acid,
  • Carotenoids such as ⁇ -carotene, lycopene, canthaxanthin, astaxanthin or zeaxanthin or proteins such as hydrolases such as lipases, esterases, amidases, nitrilases, proteases, mediators such as cytokines such as lymphokines such as MIF, MAF, TNF, interleukins such as interleukin 1, interferons such as interferons , tPA, hormones such as proteohormones, glycohormones, oligo or polypeptide hormones such as vassopressin, endorphins, endostatin, angiostatin, growth factors erythropoietin, transcription factors, integrins such as GPIIb / IIIa or oC v ßlll, receptors such as the various glutamate receptors are optimized can.
  • hydrolases such as lipases, esterases, amidases, nitrilases, proteases,
  • the nucleic acid fragments according to the invention or other of the genes mentioned above can be placed at transcriptionally active sites in the genome.
  • the nucleic acids are advantageously cloned together with at least one reporter gene into a DNA construct which is introduced into the genome.
  • This reporter gene should allow easy detection via a growth, fluorescence, chemo- or bioluminescence assay or via a photometric measurement.
  • Examples include reporter genes, antibiotic resistance genes, hydrolase genes, fluorescence protein genes, bioluminescence genes, glucosidase genes, peroxidase genes or biosynthesis genes such as the riboflaving genes, the luciferase gene, ⁇ -galactosidase gene, gfp gene, lipase gene, esterase gene, acoxidase gene, peroxidase gene, peroxidase gene, peroxidase gene Called adenyltransferase gene. These genes enable the transcription activity and thus the expression of the genes to be measured and quantified easily. This enables genome sites to be identified which show a productivity difference of up to a factor of 2 (see FIG. 1).
  • an additional reporter gene can be dispensed with.
  • genes are to be introduced into the organism, all of them can be introduced into the organism together with a reporter gene in a single vector or each individual gene with a reporter gene can be introduced into the organism, the different vectors being able to be introduced simultaneously or successively.
  • Gene fragments that code for the respective activities can also be used in REMI technology.
  • restriction enzymes are suitable for the method according to the invention for integrating biosynthesis genes into the genome of organisms. Restriction enzymes that only recognize 4 base pairs as a restriction site are less preferred because they cut too frequently in the genome or in the vector to be integrated; preference is given to enzymes that recognize 6, 7, 8 or more base pairs as an interface, such as BamHI, EcoRI, Bglll, SphI , Spei, Xbal, Xhol, Ncol, Sall, Clal, Kpnl, Hindl l, Sacl, PstI, Bpnl, Notl, Srfl or Sfil to name just a few of the possible enzymes.
  • the enzymes used no longer have interfaces in the DNA to be introduced, this increases the efficiency of the integration.
  • 5 to 500 U, preferably 10 to 250, particularly preferably 10 to 100 U of the enzymes are used in the REMI approach.
  • the enzymes are advantageously used in an aqueous solution, the substances for osmotic stabilization such as sugar such as sucrose, trehalose or glucose, polyols such as glycerol or polyethylene glycol, a buffer with an advantageous buffering in the range from pH 5 to 9, preferably 6 to 8, particularly preferably 7 to 8 such as Tris, MOPS, HEPES, MES or PIPES and / or substances for stabilizing the
  • Nucleic acids contain such as inorganic or organic salts of Mg, Cu, Co, Fe, Mn or Mo. If appropriate, other substances may also be present, such as EDTA, EDDA, DTT, ⁇ -mercaptoethanol or nuclease inhibitors. However, it is also possible to carry out REMI technology without these additives.
  • the process according to the invention is carried out in a temperature range from 5 to 80 ° C., preferably from 10 to 60 ° C., particularly preferably from 20 to 40 ° C. All known methods for destabilizing cell membranes, such as electroporation, fusion with loaded vesicles or destabilization via various alkali or alkaline earth metal salts such as lithium, rubidium or calcium salts, the lithium salts are preferred.
  • nucleic acids can be used for the reaction according to the invention directly or after purification.
  • the combination of the rib genes according to the invention can be introduced into plants by all methods known to the person skilled in the art.
  • transformation The transfer of foreign genes into the genome of a plant is called transformation.
  • the methods described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the use of a gene cannon, electroporation, the incubation of dry embryos in DNA-containing solution, microinjection and the gene transfer mediated by Agrobacterium.
  • the methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, published by S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol.
  • the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium turne faciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
  • pBin19 Bevan et al., Nucl. Acids Res. 12 (1984) 8711.
  • the transformation of plants with Agrobacterium tumefaciens is described, for example, by Höfgen and Willuser in Nucl. Acid Res. (1988) 16, 9877.
  • Agrobacteria transformed with an expression vector according to the invention can also be used in a known manner to transform plants, in particular crop plants, such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa , Lettuce and the various tree, nut and wine species and legumes can be used, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • crop plants such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa , Lettuce and the various tree, nut and wine species and legumes can be used, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • the genetically modified plant cells can be regenerated using all methods known to the person skilled in the art. Appropriate methods can be found in the above-mentioned writings by SD Kung and R. Wu, Potrykus or Höfgen and Willmitzer. There are a number of ways to increase the enzyme activity of the rib gene products in the cell.
  • One possibility is to change the endogenous rib genes 1, 2, 4 and 7 in such a way that they code for enzymes with increased rib 1,2,4 or 7 activity compared to the starting enzymes.
  • Another increase in enzyme activity can be achieved, for example, by changing the catalytic centers to increase substrate conversion or by canceling the action of enzyme inhibitors, ie they have an increased specific activity or their activity is not inhibited.
  • an increased enzyme activity can also take place by increasing the enzyme synthesis in the cell, for example by switching off factors which repress the enzyme synthesis or by increasing the activity of factors or regulatory elements which promote increased synthesis, or preferably by introducing further ones gene copies. These measures increase the overall activity of the gene products in the cell without changing the specific activity.
  • a combination of these methods can also be used, ie increasing the specific activity and increasing the overall activity.
  • these changes can be introduced into the nucleic acid sequences of the genes, regulatory elements or their promoters by all methods known to the person skilled in the art.
  • the sequences can be subjected, for example, to a mutagenesis such as a "site directed mutagenesis" as described in D.M. Glover et al., DNA Cloning Vol. 1, (1995), IRL Press (ISBN 019-963476-9), Chapter 6, page 193 ff.
  • modified nucleic acid sequences are then brought back into the organisms via vectors.
  • modified promoter regions can also be placed in front of the natural genes, so that the expression of the genes is increased and thus the activity is ultimately increased.
  • Sequences can also be introduced at the 3 'end which, for example, increase the stability of the mRNA and thereby enable increased translation. This also leads to higher enzyme activity.
  • rib genes 1, 2, 7 and 4 are preferably introduced into the cell together. These gene copies can be subject to natural regulation, a changed regulation, the natural regulatory regions being changed in such a way that they enable increased expression of the genes, or else regulatory sequences of foreign genes or even genes of other species can be used.
  • the gene expression of the rib genes 1, 2, 7 and 4 can advantageously be increased by increasing the ribl, 2, 7, 4 gene copy number and / or by strengthening regulatory factors which have a positive effect on the ribl, 2,7 and 4 gene expression become. So one can
  • Regulatory elements are strengthened preferably at the transcription level by using stronger transcription signals such as promoters and enhancers.
  • an increase in translation is also possible, for example, by improving the stability of the ribl, 2, 7 and 4 mRNA, or by increasing the reading efficiency of this mRNA on the ribosomes.
  • the rib genes 1, 2, 7 and 4, or homologous genes can be incorporated, for example, into a nucleic acid fragment or into a vector which preferably contains the regulatory gene sequences assigned to the respective rib genes or promoter activity acting in an analogous manner , In particular, those regulatory sequences are used which increase gene expression.
  • each of the genes described can be brought into a single vector and transformed into the respective production organism.
  • the rib gene sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 or to understand their functional equivalents which have been functionally linked with one or more regulation signals advantageously to increase gene expression.
  • these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid.
  • the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically modified so that the natural regulation has been switched off and the expression of the genes increased.
  • the gene construct can also have a simpler structure, which means that no additional regulatory signals have been placed in front of the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7 or their functional equivalents were inserted and the natural promoter with its regulation was not removed. Instead, the natural regulatory sequence was mutated in such a way that regulation no longer takes place and gene expression is increased. These modified promoters can also be placed in front of the natural genes to increase activity.
  • the gene construct may advantageously also comprise one or more "enhancer sequences" functionally linked to the promoter contain 0, which allow increased expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the DNA sequences.
  • the rib genes can be contained in one or more copies in the gene construct. 5
  • Advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacl ⁇ - T7, T5, T3 -, gal-, trc-, ara-, SP6-, ⁇ -P R - or contain in the ⁇ -P L promoter, which are advantageously used in gram-negative bacteria.
  • promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacl ⁇ - T7, T5, T3 -, gal-, trc-, ara-, SP6-, ⁇ -P R - or contain in the ⁇ -P L promoter, which are advantageously used in gram-negative bacteria.
  • Further advantageous regulatory sequences are, for example, in the gram-positive promoters ay and SP02, in the yeast or fungal promoters ADC1, MF ⁇ , AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV / 35S [Franck 5 et al., Cell 21 (1980) 285-294], PRPl [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33, LEB4, nos or in the ubiquitin or phaseolin promoter.
  • promoters of pyruvate decarboxylase and methanol oxidase from, for example, Hansenula are also advantageous.
  • Further advantageous plant promoters are, for example, one that can be induced by benzenesulfonamide (EP 388186), one that can be induced by tetracycline (Gatz et al., (1992) Plant J. 2, 397-404), one that can be induced by abscisic acid (EP335528) or an ethanol or cyclohexanone inducible (W09321334) promoter.
  • Plant promoters which express in tissues or parts of plants are particularly advantageous
  • the promoter of the phosphoribosyl pyrophosphate amidotransferase from Glycine max can also be used advantageously.
  • the nucleic acid fragment may also contain further genes which are to be introduced into the organisms. These genes can be under separate regulation or under the same regulatory region as the rib genes. These genes are for example
  • plasmid 35 for example a plasmid, a phage or other DNA, which enables an optimal expression of the genes in the host.
  • Suitable plasmids are, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 113 -Bl, ⁇ gtll or pBdCI,
  • plasmids The plasmids mentioned represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Suitable plant vectors are described in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, p.71-119.
  • the nucleic acid fragment for the expression of the other genes contained additionally contains 3 'and / or 5' terminal regulatory sequences for increasing expression, which are selected for optimal expression depending on the host organism selected and gene or genes.
  • regulatory sequences are intended to enable targeted expression of the genes and protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed and / or overexpressed after induction, or that it is immediately expressed and / or overexpressed.
  • the regulatory sequences or factors can preferably influence the gene expression of the introduced genes positively and thereby increase.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • the gene construct according to the invention can also advantageously be introduced into the microorganisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination.
  • This linear DNA can consist of a linearized plasmid or only of the nucleic acid fragment as a vector.
  • Any plasmid in particular a plasmid which carries the origin of replication of the 2 ° em plasmid from S. cerevisiae
  • the vector which replicates autonomously in the cell, but also a linear DNA fragment as described above, that integrates into the genome of the host.
  • This integration can take place via hetero- or homologous recombination.
  • preferred via homologous recombination Stepiner et al., Genetics, Vol. 140, 1995: 973-987.
  • the genes ribl, rib2, rib4 and rib7 can be present individually in the genome at different locations or on different vectors or together in the genome or on one vector.
  • the organisms used in the process according to the invention which contain the combination of the rib genes 1, 2, 7 and 4 or their functional equivalents show increased riboflavin production.
  • the organisms used for the production of riboflavin are grown in a medium which enables these organisms to grow.
  • This medium can be a synthetic or a natural medium.
  • media known to the person skilled in the art are used.
  • the media used contain a carbon source, a nitrogen source, inorganic salts and possibly small amounts of vitamins and trace elements.
  • Advantageous carbon sources are, for example, sugars such as mono-, di- or polysaccharides such as glucose, fructose, mannose, xylose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose, complex sugar sources such as molasses, sugar phosphates such as fructose -1, 6-bis-phosphate, sugar alcohols such as mannitol, polyols such as glycerol, alcohols such as methanol or ethanol, carboxylic acids such as citric acid, lactic acid or acetic acid, fats such as soybean oil or rapeseed oil, amino acids such as an amino acid mixture, for example so-called casamino acids (Difco ) or individual amino acids such as glycine or aspartic acid or aminosugar, the latter can also be used simultaneously as a nitrogen source.
  • sugars such as mono-,
  • Advantageous nitrogen sources are organic or inorganic nitrogen compounds or materials that contain these compounds.
  • ammonium salts such as NH 4 CI or (NH 4 ) S ⁇ 4 , nitrates , urea, or complex nitrogen sources such as corn steep liquor, brewer's yeast autolysate, soybean meal, wheat gluten, yeast extract, meat extract, casein hydrolyzate, yeast or potato protein, which can often also serve as a nitrogen source at the same time.
  • inorganic salts are the salts of calcium, magnesium, sodium, cobalt, molybdenum, manganese, potassium, zinc, copper and iron.
  • the chlorine, sulfate and phosphate ions are particularly worth mentioning as the anion of these salts.
  • An important factor in increasing the productivity in the process according to the invention is the control of the Fe 2 + _ or Fe 3+ ion concentration in the production medium.
  • growth factors are added to the nutrient medium, such as vitamins or growth promoters such as biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate or pyridoxine, amino acids such as alanine, cysteine, proline, Aspartic acid, glutamine, serine, phenylalanine, ornithine or valine, carboxylic acids such as citric acid, formic acid, pimelic acid or lactic acid, or substances such as dithiothreitol.
  • vitamins or growth promoters such as biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate or pyridoxine
  • amino acids such as alanine, cysteine, proline, Aspartic acid, glutamine, serine, phenylalanine, ornithine or valine
  • carboxylic acids such as citric acid, formic acid, pimelic acid or lactic
  • the mixing ratio of the nutrients mentioned depends on the type of fermentation and is determined in each individual case.
  • the medium components can all be introduced at the beginning of the fermentation, after they have been sterilized separately if necessary or sterilized together, or else they can be added continuously or discontinuously during the fermentation as required.
  • the breeding conditions are determined in such a way that the organisms grow optimally and that the best possible yields are achieved.
  • Preferred cultivation temperatures are 15 ° C to 40 ° C. Temperatures between 25 ° C and 37 ° C are particularly advantageous.
  • the pH is preferably held in a range from 3 to 9. PH values between 5 and 8 are particularly advantageous.
  • an incubation period of a few hours to a few days, preferably 8 hours to 21 days, particularly preferably 4 hours to 14 days, is sufficient. The maximum amount of product in the medium accumulates within this time.
  • the process according to the invention can be carried out continuously or batchwise in batch or fed-batch fashion.
  • the riboflavin productivity can be increased to different extents by the method according to the invention.
  • productivity can advantageously be increased by at least 5%, preferably by at least 10%, particularly preferably by 20%, very particularly preferably by at least 100% in each case compared to the starting organism.
  • Sequence 1 shows the DNA construct which, in addition to the selection marker required for the transformation, bears the gene fragments of ribl, rib2, rib4 and rib7.
  • the sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA74, 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed.
  • the vector TefG418Tefrib3, 4, 5 is described in WO 99/61623. This vector was cut with Kpnl, felled and released again and then partially digested with Nhel. The larger fragment, once cut with Nhel and Kpnl, was purified from an agarose gel.
  • the rib7 gene was amplified from vector pJR765 (described in WO 95/26406) with the aid of PCR (primer: TCGAGGTACCGGGCCCCCCCTCGA; TCGAACTAGTAGACCAGTCAT). The specific PCR product was cut with Kpnl / Spel and ligated with the Kpnl / Nhel cut vector described above.
  • TefG418Tefrib7 4.
  • the rib2 gene was amplified from the vector pJR758 (WO 95/26406) with PCR and the resulting product was cut with Spei and Nhel (primer: CCCAACTAGTCTGCAGGACAATTTAAA; AGTGCTAGCCTACAATTCGCAGCAAAAT). This DNA fragment is with the Cut and phosphatase-treated vector TefG418Tefrib7.4 was ligated.
  • the result was vector TefG418Tef-rib7,4,2.
  • the ribl gene was amplified from vector pJR765 (WO95 / 26406) by PCR (primer: GTAGTCTAGAACTAGCTCGAAACGTG;
  • the resulting DNA construct represents the vector Tef-G418-ribl, 2,7,4.
  • Example 2 Transformation of the DNA construct into the Ashbya gossypii fungus
  • the DNA construct described in Example 1 (vector Tef-G418-ribl, 2,4,7) was completely cut with the restriction enzyme Xbal and the insert which carries the rib gene sequences purified by agarose gel separation.
  • MA2 medium (10 g / 1 Bacto-Peptone, 1 g / 1 yeast extract, 0.3 g / 1 myo-inositol and 10 g / 1 D-glucose) was inoculated with Ashbya gossypii spores. The culture was incubated for 12 h at 4 C and then with shaking for 13 h at 28 ° C.
  • Figure 1 shows the riboflavin yields of the different clones.

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Abstract

L'invention concerne un procédé pour la production microbienne de riboflavine. Selon le procédé de l'invention, on cultive un micro-organisme du genre Ashbya, qui est capable de produire de la riboflavine et qui présente, dans au moins deux des produits géniques du groupe rib1, rib2, rib4 et rib7, des activités plus élevées que le type sauvage ATCC 10895. Puis on isole du milieu de culture la riboflavine produite.
PCT/EP2002/013660 2001-12-04 2002-12-03 Optimisation de souche genetique pour la production amelioree de riboflavine WO2003048367A1 (fr)

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EP02791764A EP1456388A1 (fr) 2001-12-04 2002-12-03 Optimisation de souche genetique pour la production amelioree de riboflavine
AU2002358083A AU2002358083A1 (en) 2001-12-04 2002-12-03 Genetic strain optimization for improving the production of riboflavin
US10/497,526 US20050239161A1 (en) 2001-12-04 2002-12-03 Genetic strain optimization for improving the production of riboflavin
JP2003549544A JP2005511053A (ja) 2001-12-04 2002-12-03 リボフラビンの産生改善のための遺伝的系統の最適化

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DE10159396A DE10159396A1 (de) 2001-12-04 2001-12-04 Genetische Stammoptimierung zur verbesserten Herstellung von Riboflavin

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Cited By (2)

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US7455438B2 (en) 2004-05-14 2008-11-25 C.R.F. Societa Consortile Per Azioni Module for projecting a light beam, an optical device for the module, and a vehicle front light assembly
EP3227430B1 (fr) * 2014-12-01 2021-05-19 Danisco US Inc. Souches d'hôtes fongiques, constructions d'adn, et méthodes d'utilisation

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CN100430471C (zh) * 2006-05-17 2008-11-05 天津大学 产核黄素的工程菌株及其生产核黄素的方法
US8138876B2 (en) * 2008-01-29 2012-03-20 International Business Machines Corporation On-chip integrated voltage-controlled variable inductor, methods of making and tuning such variable inductors, and design structures integrating such variable inductors
CN102809658A (zh) * 2012-09-03 2012-12-05 南开大学 一种维生素b2的酶联免疫检测试剂盒
KR102561864B1 (ko) * 2014-11-19 2023-08-01 바스프 에스이 Rib７ 프로모터를 사용하여 유전자 발현을 하향조절하기 위한 에레모테시움의 유전적 변형
CN114592000B (zh) * 2020-12-03 2023-07-21 上海市农业科学院 一种六基因组合在水稻种子中提高vb2含量的应用及方法
CN113817654B (zh) * 2021-11-08 2023-06-20 通辽梅花生物科技有限公司 一种生产核黄素的发酵培养基及发酵方法

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EP0405370A1 (fr) * 1989-06-22 1991-01-02 F. Hoffmann-La Roche Ag Souches de bactéries surproductrices de riboflavine
WO1995026406A2 (fr) * 1994-03-25 1995-10-05 Basf Aktiengesellschaft Biosynthese de la riboflavine dans des champignons
WO1999061623A2 (fr) * 1998-05-28 1999-12-02 Basf Aktiengesellschaft Procede genetique de production de riboflavine

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0405370A1 (fr) * 1989-06-22 1991-01-02 F. Hoffmann-La Roche Ag Souches de bactéries surproductrices de riboflavine
WO1995026406A2 (fr) * 1994-03-25 1995-10-05 Basf Aktiengesellschaft Biosynthese de la riboflavine dans des champignons
WO1999061623A2 (fr) * 1998-05-28 1999-12-02 Basf Aktiengesellschaft Procede genetique de production de riboflavine

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7455438B2 (en) 2004-05-14 2008-11-25 C.R.F. Societa Consortile Per Azioni Module for projecting a light beam, an optical device for the module, and a vehicle front light assembly
EP3227430B1 (fr) * 2014-12-01 2021-05-19 Danisco US Inc. Souches d'hôtes fongiques, constructions d'adn, et méthodes d'utilisation

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JP2005511053A (ja) 2005-04-28
EP1456388A1 (fr) 2004-09-15

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