WO2003074721A2 - Procede de fabrication de riboflavine - Google Patents

Procede de fabrication de riboflavine Download PDF

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Publication number
WO2003074721A2
WO2003074721A2 PCT/EP2003/002101 EP0302101W WO03074721A2 WO 2003074721 A2 WO2003074721 A2 WO 2003074721A2 EP 0302101 W EP0302101 W EP 0302101W WO 03074721 A2 WO03074721 A2 WO 03074721A2
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gene
transcription terminator
seq
riboflavin
organism
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PCT/EP2003/002101
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German (de)
English (en)
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WO2003074721A3 (fr
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Henning ALTHÖFER
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Basf Aktiengesellschaft
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Priority to JP2003573166A priority Critical patent/JP2005527200A/ja
Priority to EP03704701A priority patent/EP1483398A2/fr
Priority to AU2003206964A priority patent/AU2003206964A1/en
Priority to CA002477849A priority patent/CA2477849A1/fr
Priority to KR10-2004-7013634A priority patent/KR20040096643A/ko
Publication of WO2003074721A2 publication Critical patent/WO2003074721A2/fr
Publication of WO2003074721A3 publication Critical patent/WO2003074721A3/fr

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    • 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
    • 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
    • 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

Definitions

  • the present invention relates to transcription terminators, an organism capable of producing riboflavin, comprising at least one of these transcription terminators, and to an improved method for producing riboflavin, in which an organism capable of producing riboflavin is cultivated which comprises at least one of the transcription types mentioned. Has terminators.
  • 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, reduced 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.
  • Vitamin B2 is produced either chemically or microbially (see e.g. Kurth et al., 1996, Riboflavin, in: Ullmann's Encyclopedia of industrial chemistry, VCH Weinheim).
  • riboflavin is usually obtained as a pure end product in multi-stage processes, with relatively expensive starting products, such as D-ribose, having to be used.
  • An alternative to the chemical synthesis of riboflavin is the fermentative production of vitamin B2 by microorganisms. Renewable raw materials such as sugar or vegetable oils are used as starting materials.
  • 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, such as Candida, Pichia and Saccharomyces or bacteria such as Bacillus, Clostridia or Corynebacteria are described as riboflavin producers.
  • yeasts such as Candida, Pichia and Saccharomyces or bacteria such as Bacillus, Clostridia or Corynebacteria are described as riboflavin producers.
  • EP-A-0 405 370 and EP-A-0 821 063 describe the production of riboflavin with recombinant bacterial strains, the strains being obtained from Bacillus subtilis by transformation with riboflavin biosynthetic genes.
  • GTP guanosine triphosphate
  • ribulose-5-phosphate catalyze the formation of riboflavin.
  • GTP cyclohydrolase II ribl gene product converts GTP to 2,5-diamino-6- (ribosylamino) -4- (3H) -pyrimidinone-5-phosphate.
  • This compound is then converted to 2,5-diamino-ribitylamino-2,4- (2,5-diamino-6- (ribosylamino) -4- (3H) -pyrimidinone-5-phosphate reductase (rib7 gene product) 1H, 3H) -pyrimidine-5-phosphate reduced and then deaminated by a specific deaminase (rib2 gene product) to 5-amino-6-ribitylamino-2,4- (1H, 3H) -pyrimidinedione-5-phosphate. 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-2- by the 3,4-dihydroxy-2-butanone-4-phosphate synthase (rib3 gene product) butanone-4-phosphate (DBP) implemented.
  • DBP and 5-amino-6-ribitylamino-2,4- (1H.3H) -pyrimidinedione are the starting materials for the enzymatic synthesis of - 6,7-dimethyl- ⁇ -ribityllumazine.
  • This reaction is catalyzed by the rib4 gene product (DMRL synthase).
  • DMRL is then converted to riboflavin by riboflavin synthase (rib ⁇ gene product) (Bacher et al. (1993), Bioorg. Chem. Front. Vol. 3, Springer Verlag).
  • the task was therefore to further improve vitamin B2 productivity.
  • This object was achieved by a process for the production of riboflavin, in which an organism capable of producing riboflavin is cultivated which has at least one transcription terminator according to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3, wherein the respective transcription terminator is operatively linked to at least one rib gene (gene of riboflavin biosynthesis) and the riboflavin formed is processed from the culture medium.
  • an organism capable of producing riboflavin is cultivated which has at least one transcription terminator according to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3, wherein the respective transcription terminator is operatively linked to at least one rib gene (gene of riboflavin biosynthesis) and the riboflavin formed is processed from the culture medium.
  • the riboflavin production process advantageously involves an operative linkage with at least one gene from the group ribl, rib2, rib3, rib4, rib ⁇ or rib7.
  • a transcription terminator selected from the group according to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 with at least one gene of riboflavin biosynthesis (rib gene)
  • rib gene riboflavin biosynthesis
  • an operational linkage of a terminator according to the invention from the group mentioned is possible with one or more rib genes, each gene being operatively linked individually to this term gate or a gene group (for example cluster or operon) being operatively linked to such a terminator.
  • the presence of various of the terminators mentioned is included in the organisms suitable for producing riboflavin.
  • the various terminators can each be operatively linked individually to a rib gene, or the various terminators are operatively linked to grouped rib genes.
  • each of the transcription terminators mentioned can be operatively linked in an imaginable combination with rib genes in any conceivable combination in an organism suitable for riboflavin production.
  • An operative linkage of the transcription terminators according to the invention with one or more other genes involved in riboflavin production is also conceivable.
  • An operative link is the sequential arrangement of nucleotide sequences with a regulatory and coding function, such as a promoter, coding sequence, terminator and possibly to understand further regulatory elements, such that each of the regulatory elements can fulfill its function in the expression of the coding sequence as intended.
  • a regulatory and coding function such as a promoter, coding sequence, terminator and possibly to understand further regulatory elements, such that each of the regulatory elements can fulfill its function in the expression of the coding sequence as intended.
  • These regulatory nucleotide sequences can be of natural origin or can be obtained by chemical synthesis. Genetic engineering procedures for the operative linking of nucleotide sequences are common laboratory practice and can be found, for example, in DM Glover et al., DNA Cloning Vol. 1, (1995), IRL Press (ISBN 019-963476-9). Also to be read here are methods for the synthesis and exchange of bases in a nucleotide sequence which are known to the person skilled in the art.
  • the operative linkage advantageously leads to at least one of the rib genes with one of the transcription terminators according to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 to an increased termination of the transcription.
  • This in turn has an indirect influence on the transcription rate of the gene upstream of the terminator, since the transcription apparatus, ie RNA polymerase and supporting transcription factors, is released more efficiently after reading the terminator according to the invention and is thus more quickly available for a new round of transcription (Alberts et al., 3rd edition, molecular biology of the cell, VCH Verlag). Consequently, an increased transcription rate of the corresponding genes is made possible in this way.
  • each of the transcription terminators according to the invention can have a stabilizing effect on the transcript formed.
  • the degradation for example by an exonuclease
  • the transcripts being less can be broken down quickly, can be increasingly translated, which in turn leads to increased activity of the correspondingly coded enzymes.
  • a method is advantageous in which a transcription terminator according to SEQ ID No. 1 is used, which is changed compared to the sequence of the transcription terminator of the rib2 gene of the wild type ATCC 10895 at position 13 by exchanging guanine for adenine.
  • each of the transcription terminators according to the invention can occur in number once or else in multiple copies in the genome of the organism used for riboflavin production. This depends on whether the terminator is operatively linked to one or more genes. For example, the operational linkage of the terminator according to SEQ ID No. 1 with only one rib gene or with several, preferably separately organized, rib genes (or other genes involved in riboflavin synthesis) is conceivable.
  • the process for the increased production of riboflavin is advantageously carried out with an organism capable of producing riboflavin.
  • all organisms which are able to synthesize riboflavin are suitable as organisms or host organisms for the process according to the invention.
  • Organisms that can synthesize riboflavin naturally are preferred.
  • organisms which are able to synthesize riboflavin due to the introduction of the complete vitamin B2 synthesis genes are also suitable for the process according to the invention.
  • Organisms such as bacteria, yeast, fungi or plants are suitable for the process according to the invention.
  • Examples include eukaryotic organisms such as fungi, which are described in Indian Chem Engr. Section B. Vol 37, No 1.2 (1995) on page 15, Table 6, such as Ashbya or Eremothecium, yeasts such as Candida, Saccharomyces or Pichia or plants such as Arabidopsis, tomato, potato, corn, soybean, rape, barley , Wheat, rye, rice, millet, cotton, legumes, sugar beet, sunflower, flax, hemp, canola, oats, tobacco, alfalfa, lettuce or the various tree, nut and wine species or prokaryotic organisms such as gram-positive or gram- negative bacteria such as Corynebacterium, Brevibacterium, Bacillus, Clostridium, Cyanobacter, Escherichia or Klebsiella.
  • yeasts such as Candida, Saccharomyces or Pichia or plants
  • plants such as Arabidopsis, tomato, potato, corn, soybean, rape,
  • Organisms are preferably selected from the group of the genera Cop / nebacterium, Brevibacterium, Bacillus, Escherichia, Ashbya, Eremothecium, Candida or Saccharomyces or plants such as corn, soybeans, rapeseed, barley, wheat, potatoes or tomatoes.
  • Organisms of the genus and species Ashbya gossypii, Eremothecium ashbyii, Saccharomyces cerevisiae, Candida flaveri, Candida famata, Corynebacterium ammoniagenes or Bacillus subtilis are particularly preferred.
  • Maize, soybean, rapeseed, barley, wheat, potato and tomato are particularly preferred as plants.
  • Ashbya gossypii or Eremothecium ashbyii are preferred.
  • Riboflavin production strains are also included according to the invention. These can e.g. B. be produced from wild-type strains suitable for riboflavin production by classic (chemical or physical) or genetic engineering methods and possibly have other genetic changes than those in the context of the rib genes.
  • the aforementioned rib genes include those which come from organisms which are capable of producing riboflavin. Genes from organisms such as Bacillus subtilis, Saccharamyces cerevisiae or Ashbya gossypii are preferred. Rib genes from Ashbya gossypii are particularly preferred. Functional analogs, functional equivalents or derivatives of these genes are also included here.
  • Functional analogs are understood to mean, for example, functional homologs of the rib genes or their enzymatic activities, that is to say enzymes which catalyze the same enzymatic reactions as the rib genes.
  • Functional equivalents are understood to mean, for example, allele variants which have at least 35% homology at the derived amino acid level, preferably at least 40% homology, particularly preferably at least 45% homology, very particularly preferably 50% Show homology.
  • Allelic variants include, in particular, functional variants which can be obtained by deleting, inserting or substituting nucleotides, the enzymatic activity of the derived synthesized proteins being retained.
  • DNA sequences can be isolated from the known DNA sequences of the rib genes or parts of these sequences, for example using conventional hybridization methods or the PCR technique, from eukaryotes or prokaryotes other than Ashbya gossypii as mentioned above. These DNA sequences hybridize under
  • Derivatives are to be understood as variants whose nucleotide sequence has been changed before the start codon in such a way that the gene expression and / or the protein expression is changed, preferably increased.
  • the "codon usage” can easily be determined on the basis of computer evaluations of other known genes of the organism in question.
  • Another advantage for increasing vitamin B2 productivity is a combination of increasing the natural enzyme activity coded by the rib genes mentioned and increasing the gene expression by additionally introducing at least one of the above-mentioned genes or a combination of several of these genes into one organism capable of producing riboflavin.
  • One possibility is the operative linking of the genes with one of the terminators according to the invention from the group according to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3, whereby an increased transcription rate of the respective gene or more of the said genes is achieved and / or a stabilization of the transcript (s) formed.
  • Enzyme activity can be increased, for example, by changing the catalytic centers to increase substrate turnover or by canceling the action of enzyme inhibitors. This means that the enzymes have an increased specific activity or their activity is not inhibited.
  • increased enzyme activity can also be increased by increasing the enzyme synthesis in the cell take place, for example, by switching off factors that repress enzyme synthesis or by increasing the activity of factors or regulatory elements that promote increased synthesis. The introduction of additional gene copies can also lead to an increased specific enzyme activity. These measures increase the overall activity of the gene products in the cell without changing the specific activity.
  • sequences can be subjected, for example, to mutagenesis such as "site directed mutagenesis” as described in D.M. Glover et al., DNA Cloning Vol.1, (1995), IRL Press (ISBN 019-963476-9), chapter 6,
  • modified promoter areas can also be placed in front of the natural genes, so that the expression of the genes is increased and the activity is ultimately increased.
  • Advantageous promoter sequences are, for example, cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, lacl q " '77, T5, T3, gal, trc , ara-, SP6-, ⁇ -P R - or in the ⁇ -P L -promotor, which are advantageously used in gram-negative bacteria
  • Further advantageous regulation sequences are, for example, in the gram-positive promoters amy and SPO2, in the yeast - or fungal promoters ADC1, MF ⁇ , AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV / 35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Ward et al ., Plant.
  • promoters of pyruvate decarboxylase and Methanol oxidase from, for example, Hansenula is advantageous
  • Further advantageous plant promoters are, for example, one that can be induced by benzenesulfonamide (EP 388186) and one that can be induced by tetracycline (Gatz et al., (1992) Pla nt J.
  • all natural promoters with their regulatory sequences such as those mentioned above can be used for the method according to the invention.
  • synthetic promoters can also be used advantageously.
  • the stability of the mRNA can be increased, thereby enabling increased translation. This also leads to a higher enzyme activity of the correspondingly expressed genes.
  • the stabilization of the transcripts can also be strengthened by additional sequences at the 3 'end.
  • rib genes or also a combination of several of these genes, in operative linkage with at least one of the transcription terminators according to the invention, into an organism capable of producing riboflavin in order to increase gene expression by increasing the number of gene copies.
  • 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.
  • a combination of the above methods is particularly advantageous.
  • the present invention also relates to a gene construct containing at least one transcription terminator according to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 and at least one rib gene operatively linked to one of these terminators.
  • the gene construct according to the invention preferably contains at least one gene from the group ribl, rib2, rib3, rib4, rib ⁇ or rib7.
  • a vector is included, comprising at least one transcription terminator according to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 or a gene construct of the aforementioned type and additional nucleotide sequences for selection and for replication in the host cell or for integration into the host cell genome.
  • the gene construct according to the invention can 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, other biosynthetic genes that enable increased synthesis.
  • the gene construct is advantageously inserted into a vector such as, for example, a plasmid, a phage or other DNA, which enables optimal expression of the genes in the host.
  • a vector such as, for example, a plasmid, a phage or other DNA, which enables optimal expression of the genes in the host.
  • Suitable plasmids are, for example, in E.
  • 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 ) can be removed. Suitable plant vectors are described in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, p.71-119.
  • the gene construct for the expression of the further genes contained additionally contains 3'- and / or 5'-terminal regulatory sequences for increasing expression, which are selected depending on the selected host organism and gene or genes for optimal expression.
  • regulatory sequences are intended to enable the targeted expression of these 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 expressed and / or overexpressed immediately.
  • 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 that replicates the origin of replication of the 2 ⁇ m plasmid from S. cerevisiae), which replicates autonomously in the cell, but also, as described above, a linear DNA fragment 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 rib genes can be present individually in the genome at different locations or on different vectors or together in the genome or on one vector.
  • the transcription terminators according to the invention, the aforementioned gene construct or the aforementioned vectors containing according to the invention preferably at least one rib gene and corresponding regulatory sequences operatively linked thereto, such as i.a. at least one of the transcription terminators according to the invention can in principle be introduced into the organisms used by all methods known to the person skilled in the art.
  • the REMI technique is based on the cotransformation of a linear DNA construct that was cut at both ends with the same restriction endonuclease together with the restriction endonuclease 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 the strand breaks in the genomic DNA caused by the endonuclease and thereby also incorporate the co-transformed DNA construct into the genome at a certain frequency. As a rule, the restriction sites at both ends of the DNA are preserved.
  • Organisms can be integrated and thus production processes for 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 ß-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 Interferon, tPA, hormones such as proteohormones, glycohormones, oligo- or polypeptide hormones such
  • the transcription terminators according to the invention can be placed in operative linkage with at least one rib gene at transcriptionally active sites in the genome.
  • the transcription terminators and / or the gene constructs mentioned are advantageously cloned together with at least one reporter gene into a further 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.
  • reporter genes are antibiotic resistance genes, hydrolase genes, fluorescence protein genes, bioluminescence genes, glucosidase genes, peroxidase genes or biosynthesis genes such as the riboflavin genes Luciferase gene, ß-galactosidase gene, gfp gene, lipase gene, esterase gene, peroxidase gene, ß-lactamase gene, acetyl-, phospho- or adenyltransferase gene called. These genes enable the transcription activity and thus the expression of the genes to be measured and quantified easily. In the event that the biosynthesis genes themselves enable easy detection, as in the case of riboflavin, for example, 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 process of integrating regulatory sequences or gene constructs 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, Sphl , Spei, Xbal, Xhol, Ncol, Sall, Clal, Kpnl, Hindlll, Sacl, Pstl, Bpn1, Notl, Srfl or Sfil to name just a few of the possible enzymes.
  • 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, such as inorganic or organic salts of Mg, Cu, Co, Fe, Mn or Mo. Other substances such as EDTA, EDDA, DTT may also be present. ß-mercaptoethanol or nuclease inhibitors. However, it is also possible to carry out REMI technology without these additives.
  • the process 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 are preferred for the process.
  • the lithium salts are preferred.
  • 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, edited by SD 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 tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
  • Agrobacterium tumefaciens for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
  • the transformation of plants with Agrobacterium tumefaciens is described, for example, by Höfgen and Willmitzer 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.
  • 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 S.D. Kung and R. Wu, Potrykus or Höfgen and Willmitzer can be found.
  • the present invention further comprises an organism which is capable of producing riboflavin, comprising at least one transcription terminator according to SEQ ID No. 1, SEQ ID No. 2 or SEQ3 or a gene construct or a vector of the type according to the invention.
  • an organism from the group of the genera Corynebacterium, Brevibacterium, Bacillus, Clostridium, Escherichia, Cyanobacter, Ashbya, Eremothecium, Pichia, Candida or Saccharomyces or plants such as Arabidopsis , Corn, soybeans, rapeseed, barley, wheat, rye, millet, oats, sugar beet, potatoes, sunflowers, legumes or tomatoes.
  • An organism is preferably selected from the group of the genera Bacillus, Corynebacterium, Brevibacterium, Escherichia, Candida, Eremothecium or Ashbya or maize, soybean, rapeseed, Barley, wheat, potato or tomato.
  • Ashbya gossypii, Eremothecium ashbyii, Saccharomyces cerevisiae, Candida flaveri, Candida famata, Corynebacterium ammoniagenes or Bacillus subtilis are particularly preferred. In particular, it is Ashbya gossypii or Eremothecium ashbyii.
  • the present invention also includes organisms which are distinguished as riboflavin-producing mutants or production strains. These can e.g. B. based on wild-type strains by classic (chemical or physical) or genetic engineering methods and possibly have other genetic changes than those in the context of the rib genes.
  • the organism according to the invention is furthermore distinguished by the fact that it has an increased transcription rate of at least one rib gene compared to the wild type of the genus Ashbya ATCC 10895, which is associated with one of the transcription terminators according to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 is operatively linked. Furthermore, the organism according to the invention is capable of improved riboflavin production compared to the wild type of the genus Ashbya ATCC 10895.
  • 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-bisphosphate, 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-, di-
  • 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 ) 2 S0, 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 for increasing 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.
  • 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 riboflavin productivity can be increased by the method according to the invention to different extents.
  • the 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.
  • the present invention further comprises use of an organism of the type according to the invention containing at least one transcription terminator according to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 or a gene construct or a vector of the type according to the invention for the improved production of riboflavin. It is preferably the Ashbya gossypii organism.
  • the present invention also relates to the use of at least one transcription terminator according to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 for the production of a production organism for the improved production of riboflavin.
  • the present invention thus also relates to the use of at least one transcription terminator according to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 for improved production of riboflavin.
  • the present invention is explained in more detail by the following examples, but is not limited.
  • 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.
  • Terminator of the Ashbya ATCC 10895 rib2 gene (SEQ ID No. 4):
  • the terminators according to the invention have the following sequences:
  • Terminator according to the invention according to (SEQ ID No. 1):
  • Terminator according to the invention according to (SEQ ID No. 2):
  • sequences of the terminators according to the invention and the terminator of the rib2 gene from Ashbya ATCC 10895 are shown in the sequence listing as SEQ ID No. 1 to SEQ ID No.4.

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Abstract

L'invention concerne des terminateurs de transcription, un organisme contenant au moins un de ces terminateurs de transcription, ainsi qu'un procédé de fabrication de riboflavine. Ledit procédé consiste à cultiver un organisme capable de produire de la riboflavine, comportant au moins un de ces terminateurs de transcription, le terminateur de transcription correspondant étant combiné de façon fonctionnelle à au moins un gène </= rib >/= .
PCT/EP2003/002101 2002-03-02 2003-02-28 Procede de fabrication de riboflavine WO2003074721A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2003573166A JP2005527200A (ja) 2002-03-02 2003-02-28 リボフラビン製造法
EP03704701A EP1483398A2 (fr) 2002-03-02 2003-02-28 Procede de fabrication de riboflavine
AU2003206964A AU2003206964A1 (en) 2002-03-02 2003-02-28 Method for producing riboflavin
CA002477849A CA2477849A1 (fr) 2002-03-02 2003-02-28 Procede de fabrication de riboflavine
KR10-2004-7013634A KR20040096643A (ko) 2002-03-02 2003-02-28 리보플라빈의 제조 방법

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DE10209363.6 2002-03-02
DE10209363A DE10209363A1 (de) 2002-03-02 2002-03-02 Verfahren zur Herstellung von Riboflavin

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JP2005333907A (ja) * 2004-05-28 2005-12-08 Kyowa Hakko Kogyo Co Ltd ５−アミノレブリン酸の製造法

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KR101335853B1 (ko) * 2011-12-01 2013-12-02 씨제이제일제당 (주) Ｌ-아미노산 및 리보플라빈을 동시에 생산하는 미생물 및 이를 이용한 ｌ-아미노산 및 리보플라빈을 생산하는 방법
CN106434819A (zh) * 2016-12-22 2017-02-22 广济药业（孟州）有限公司 一种提高枯草芽孢杆菌发酵生产核黄素的产量的方法
CN110964769B (zh) * 2019-11-29 2022-11-11 河南巨龙生物工程股份有限公司 一种提高枯草芽孢杆菌发酵生产核黄素收率的方法
CN113801910A (zh) * 2021-08-30 2021-12-17 湖北广济药业股份有限公司 一种纯天然核黄素的制备方法
CN113755551A (zh) * 2021-09-30 2021-12-07 天津科技大学 一种提高核黄素产量的发酵方法
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
KR20230168477A (ko) * 2022-06-07 2023-12-14 씨제이제일제당 (주) 리보플라빈 생산능이 향상된 코리네박테리움 속 미생물 및 이를 이용한 리보플라빈을 생산하는 방법

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WO1995026406A2 (fr) * 1994-03-25 1995-10-05 Basf Aktiengesellschaft Biosynthese de la riboflavine dans des champignons
EP0821063A2 (fr) * 1996-07-24 1998-01-28 F. Hoffmann-La Roche Ag Production améliorée de riboflavine
WO1999061623A2 (fr) * 1998-05-28 1999-12-02 Basf Aktiengesellschaft Procede genetique de production de riboflavine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995026406A2 (fr) * 1994-03-25 1995-10-05 Basf Aktiengesellschaft Biosynthese de la riboflavine dans des champignons
EP0821063A2 (fr) * 1996-07-24 1998-01-28 F. Hoffmann-La Roche Ag Production améliorée de riboflavine
US6322995B1 (en) * 1996-07-24 2001-11-27 F. Hoffmann-La Roche Ag Riboflavin production
WO1999061623A2 (fr) * 1998-05-28 1999-12-02 Basf Aktiengesellschaft Procede genetique de production de riboflavine

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005333907A (ja) * 2004-05-28 2005-12-08 Kyowa Hakko Kogyo Co Ltd ５−アミノレブリン酸の製造法

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AU2003206964A1 (en) 2003-09-16
DE10209363A1 (de) 2003-09-11
JP2005527200A (ja) 2005-09-15
CA2477849A1 (fr) 2003-09-12
WO2003074721A3 (fr) 2003-12-24

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