KR20040096643A - Method for Producing Riboflavin - Google Patents

Abstract

The present invention relates to a transcription terminator, an individual containing at least one transcription terminator, and a method of making riboflavin. In accordance with the present invention, riboflavin can be prepared and grows with one or more such transcription terminators, each transcription terminator operably linked to one or more rib genes.

Description

Method for Producing Riboflavin {Method for Producing Riboflavin}

Vitamin B2, also referred to as riboflavin, is produced by all plants and many microorganisms. Humans and animals are essential to them because they cannot synthesize them. Riboflavin plays an important role in metabolism. Thus, for example, this involves the use of carbohydrates. Vitamin B2 deficiency is associated with inflammation of the mucous membranes of the mouth and throat, itching and inflammation of the skin wrinkles, and similar skin damage, inflammation of the conjunctiva, loss of vision, and cloudiness of the cornea. Infants and children may have growth stoppages and weight loss. Thus, vitamin B2 has enormous economic importance, for example, as a vitamin product and animal feed additive for vitamin deficiency. It is added to a wide variety of foodstuffs. It is also used for coloring foods such as mayonnaise, ice cream, blaze and the like.

Vitamin B2 is prepared chemically or microbiologically (see, eg, Kurth et al., 1996, Riboflavin, in: Ullmann's Encyclopedia of industrial chemistry, VCH Weinheim). In chemical preparation methods, riboflavin is usually obtained as a pure final product in a multistage process where the use of relatively expensive starting materials, such as D-ribose, is required.

An alternative to the chemical synthesis of riboflavin is the production of vitamin B2 by microbial fermentation. In this case, the starting material is a renewable raw material such as sugar or vegetable oil. The production of riboflavin by fermentation of fungi such as Eremothecium ashbyii or Ashbya gossypii is known, but the Merck Index, Windholz et al., Eds. Merck & Co., page 1183, 1983], yeast, for example Candida, Pichia and Saccharomyces or bacteria, for example Bacillus, Clostridia Clostridia or Corynebacteria are also described as manufacturers of riboflavin. EP-A-0 405 370 and EP-A-0 821 063 describe the preparation of riboflavin using recombinant strains of bacteria, which strain is obtained from Bacillus subtilis transformed with a riboflavin biosynthesis gene. do.

Patents WO 95/26406 or WO 93/03183 and DE 44 20 785 disclose the cloning of genes specific for riboflavin biosynthesis from the eukaryotes Ashvija Gosipyi and Saccharomyces cerevisiae, transgenic with such genes. The use of such microorganisms for the synthesis of converted microorganisms and riboflavin is described.

In both individuals, six enzymes promote the formation of riboflavin from guanosine triphosphate (GTP) and ribulose5-phosphate. This includes converting GTP to 2,5-diamino-6- (ribosylamino) -4 (3H) -pyrimidinone 5-phosphate by GTP cyclohydrolase-II (rib1 gene product). . Subsequently, 2,5-diamino-6- (ribosylamino) -4 (3H) -pyrimidinone 5-phosphate was added to 2,5-diamino-6- (ribosylamino) -4 (3H) -pyridine. Specific reduction after reduction with 2,5-diamino-6-ribitylamino-2,4 (1H, 3H) -pyrimidine 5-phosphate by midinone-5-phosphate reductase (rib7 gene product) Deamination with 5-amino-6-ribitylamino-2,4 (1H, 3H) -pyrimidinedione 5-phosphate by amination enzyme (rib2 gene product). The phosphate is then removed with nonspecific phosphatase. In the final enzymatic step of riboflavin biosynthesis, the second starting material other than GTP, ribulose 5-phosphate, was synthesized by 3,4-dihydroxy-2-butanone-4-phosphate synthase (rib3 gene product). Dihydroxy-2-butanone 4-phosphate (DBP).

DBP and 5-amino-6-ribitylamino-2,4 (1H, 3H) -pyrimidinedione are both precursors for the enzymatic synthesis of 6,7-dimethyl-8-ribityllumazine. This reaction is facilitated by the rib4 gene product (DMRL synthase). DMRL is then converted to riboflavin by riboflavin synthase (rib5 gene product) [Bacher et al. (1993), Bioorg. Chem. Front. Vol. 3, Springer Verlag].

Despite these advances in the production of riboflavin, there is still a need for increased and increased productivity of vitamin B2 for the production of more efficient riboflavin.

The present invention relates to an improved method for producing riboflavin, which is capable of producing a transcription terminator, riboflavin and an individual comprising one or more such transcription terminators, and cultures an individual that can produce riboflavin and comprises one or more such transcription terminators. will be.

It is an object of the present invention to further improve the productivity of vitamin B2.

The inventors have found that an object for this purpose is to produce riboflavin and has one or more transcription terminators as set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, wherein the specific transcription terminator is operably linked to one or more rib genes (riboflavin biosynthesis genes). It was found that this is achieved by a method of preparing riboflavin, which is incubated and recovers riboflavin formed in increased amounts from the culture medium.

In the method of preparing riboflavin, it is advantageous according to the invention if it is operably linked to one or more genes from the group of rib1, rib2, rib3, rib4, rib5 or rib7.

The operable linkage of the transcription terminator of the invention selected from the group shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 to one or more riboflavin biosynthesis genes (rib genes), when such combinations are present in bacteria, fungi, yeast and plants, Increasing the expression of the gene (s) and / or stabilizing the formed transcript (s) and consequently increasing the production of riboflavin in the subjects (as compared to the wild type of genus AshCC 10895). In such linkages, operative linkages of the terminators of the invention from this group are possible in one or more rib genes, each gene being operably linked to this terminator, or in one gene group (e.g., a cluster or Operon) can be operably linked to this terminator. Also included are the presence of different terminators of those mentioned in the subject suitable for the preparation of riboflavin. In this case, different terminators may each be operably linked to the rib gene individually, or may be present in an operative linkage to the rib gene with different terminators arranged in a group. Thus, for the purposes of the present invention, each of these transcription terminators may be operably linked to rib genes in individuals suitable for the production of riboflavin, either individually or in any combination of two or all three possible. Operable linkage of the transcription terminator of the invention to one or more other genes involved in the preparation of riboflavin is also possible.

Operable linkage means that each regulatory element, such as a nucleotide sequence with regulatory and coding functions, such as a promoter, coding sequence, terminator, and, where appropriate, other regulatory elements, will perform its proper function in the expression of the coding sequence. It is arranged in order to be able to. Such regulatory nucleotide sequences may be natural or obtained by chemical synthesis. Genetic procedures of operable linkage of nucleotide sequences form part of conventional experimental practice and are described, for example, in D.M. Glover et al., DNA Cloning Vol. 1, 1995, IRL Press (ISBN 019-963476-9). Methods of synthesis and base exchange of nucleotide sequences known to those skilled in the art are also cited herein.

Operable linkage of one or more rib genes to one of the transcription terminators of the invention shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 advantageously results in an increase in transcription termination. This is because, after translation of the terminator of the present invention, the transcriptional device, i.e., the RNA polymerase and the co-transcription factor, is more efficiently released, it becomes available to new transcription cycles more quickly, thus increasing the rate of transcription of the gene upstream of the terminator. Indirectly affects [Alberts et al., 3rd edition, Molekularbiologie der Zelle, VCH Verlag]. As a result, the rate of transcription of the corresponding gene can be increased by this approach. This means that gene expression increases with increasing activity of the corresponding encoded gene product, apparently maximizing the increase in riboflavin productivity. In addition, each transcription terminator of the present invention may stabilize the formed transcript. In this case, for example, degradation from the 3 'end of the transcript (eg by nucleic acid protease) can be slowed down or substantially prevented. The result is that the transcript is degraded less quickly, leading to higher amounts of translation, thus increasing the activity of the correspondingly encoded enzyme.

In an advantageous method according to the invention, the transcription terminator of SEQ ID NO: 1 is used, which is modified by replacing guanine with adenine at position 13 compared to the sequence of the transcription terminator of the rib2 gene of wild type ATCC 10895. In an equally advantageous method, the transcription terminator of SEQ ID NO: 2 is used, which is modified by replacing guanine with adenine at position 25 as compared to the sequence of transcription terminator of the rib2 gene of wild type ATCC 10895. In a similarly advantageous method, the transcription terminator of SEQ ID NO: 3 is used, which is modified by replacing guanine with adenine at positions 13 and 25 as compared to the sequence of the transcription terminator of the rib2 gene of wild type ATCC 10895. Preference is given to using the transcription terminator of SEQ ID NO: 3. Where appropriate, each transcription terminator of the present invention may exist in numerical one or multiple copies in the genome of an individual used for the preparation of riboflavin. This depends on whether the terminator is operably linked to one or more genes. Thus, for example, operable linkages are possible to the sole rib gene of the terminator shown in SEQ ID NO: 1 or to a plurality of, preferably separately organized, rib genes (or other genes involved in riboflavin synthesis). The same applies to the terminator shown in SEQ ID NO: 2 or the terminator shown in SEQ ID NO: 3. Accordingly, the present invention encompasses the use of each individual transcription terminator or all three transcription terminators of the invention, within each of a single copy or multiple copies within the genome of an individual, particularly suitable for the preparation of riboflavin.

The method for the production of increased riboflavin is advantageously carried out with the individual capable of producing riboflavin. Individuals or host individuals suitable for the method according to the invention are in principle all individuals capable of synthesizing riboflavin. Preferred are individuals that can innately synthesize riboflavin. However, individuals which have been able to synthesize riboflavin thanks to the introduction of the complete vitamin B2 synthesis gene are also suitable for the method according to the invention.

Individuals such as bacteria, yeasts, fungi or plants are suitable for the method according to the invention.

Examples that may be mentioned include eukaryotic individuals, such as Indian Chem Engr. Section B, Vol 37, No. 1, 2, 1995, on page 15, Table 6] fungi, such as Ashvija or Herremoteum, yeasts such as Candida, Saccharomyces or Pichia, or plants such as Arabidopsis, tomatoes, potatoes, Corn, soybean, rapeseed, barley, wheat, rye, rice, millet, cotton, legumes, sugar beet, sunflower, flax, hemp, canola, oats, tobacco, alfalfa, lettuce or various woody, nuts and vine species or prokaryotic objects Gram-positive or gram-negative bacteria such as Corynebacterium, Brevibacterium, Bacillus, Clostridium, Cyanobacter, Escherichia or Klebsi There is Klebsiella.

Preferred subjects are of the genus Corynebacterium, Brevibacterium, Bacillus, Escherichia, Ashvija, Eremotesium, Candida or Saccharomyces or plants such as corn, soybean, rapeseed, barley, wheat, potato or Is selected from the group of tomatoes. Particularly preferred individuals are Ashvija Gosipyi, Eremoteium Ashvii, Saccharomyces cerevisiai, Candida flaberi, Candida famata, Corynebacterium ammoniagenes or Bacillus Individuals of the genus and species Subtilis. Particularly preferred plants are corn, soybeans, rapeseed, barley, wheat, potatoes and tomatoes. Particularly preferred are Ashviy Gosipyi or Eremoteium Ashviy.

The present invention also includes riboflavin manufacturer strains. They may be prepared by classical (chemical or physical) or genetic engineering methods, for example starting from wild type strains suitable for the preparation of riboflavin, and may have additional genetic modifications within the translational frame of the rib gene, as appropriate.

The rib genes mentioned above include those derived from individuals capable of producing riboflavin according to the present invention. Preferred are genes from individuals such as Bacillus subtilis, Saccharomyces cerevisiae or Ashvi Gosipi. Ashvija Gossip is particularly preferred for this rib gene. Also included herein are functional analogs, functional equivalents or derivatives of these genes.

Functional analogues refer to, for example, functional homologues of rib genes or their enzymatic activity, ie enzymes that promote the same reaction as rib gene enzymes.

By functional equivalent is meant an allelic variant that is at least 35% homologous, preferably at least 40% homologous, particularly preferably at least 45% homologous, more particularly preferably 50% homologous, for example at the derived amino acid level. . Allelic variants include functional variants obtainable by deletion, insertion or substitution of nucleotides, in particular retaining the enzymatic activity of the derived synthetic protein.

DNA sequences of this type may be derived from known rib gene DNA sequences, or portions of these sequences, e.g., eukaryotic or prokaryotic organisms other than the above-mentioned Ashvi Gosipi using conventional hybridization methods or PCR techniques, for example. It can be isolated from. Such DNA sequences hybridize with the sequences under standard conditions. It is advantageous to use short oligonucleotides in the conserved region for hybridization in a manner known to those skilled in the art in comparison with the corresponding genes from E. coli and B. subtilis. Standard conditions include, for example, a temperature of 42-58 ° C. and a concentration of 0.1-5 × SSC (1 × SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally 50% formamide present. Aqueous buffer, for example an aqueous buffer in which 50% formamide is present in 5 x SSC at 42 ° C. Experimental conditions for DNA hybridization are described in genetic literature, for example Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989. Homolog also refers to truncated sequence or single-stranded DNA.

Derivatives refer to variants in which the nucleotide sequence preceding the start codon is modified to alter gene expression and / or protein expression, and preferably increase.

For optimal expression of heterologous genes in an individual, it is advantageous to alter the nucleic acid sequence according to the specific codon usage used in the individual. Codon usage can be readily established by computer analysis of other known genes of the individual concerned.

It also increases the natural enzyme activity encoded by the rib gene to increase the productivity of vitamin B2 and further introduces one or more of the aforementioned genes or a combination of a plurality of these genes into an individual capable of producing riboflavin. Combinations that increase gene expression are also advantageous.

There are many possibilities for increasing the enzyme activity of rib gene products in cells. One possibility is to operably link the gene to one of the terminators of the invention from the group represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, thereby increasing the rate of transcription of a particular gene or plurality of said genes and / or Stabilizing the formed transfer body (s). Another possibility is to modify the endogenous rib gene to encode an enzyme with increased rib activity than an unmodified (early or wild type) enzyme. Increasing enzyme activity can be achieved, for example, by modifying the catalyst center to increase substrate conversion or to eliminate the effects of enzyme inhibitors. This means that the enzymes have increased specific activity or their activity is not inhibited. In another advantageous embodiment, for example, removing enzymes that inhibit enzymatic synthesis increases the enzymatic synthesis in the cell or increases the activity of a factor or regulatory element that enhances the synthesis to increase enzyme activity. It is also possible. Introduction of additional gene copies can also result in increased specific enzyme activity. This method increases the total activity of the gene product in cells without altering specific activity. It is also possible to use a combination of the above methods, ie a combination of an increase in specific activity and an increase in total activity. Such modifications can in principle be introduced into the nucleic acid sequence of a gene, its regulatory element, terminator or promoter by any method known to those skilled in the art. For this purpose, see D.M. Glover et al., DNA Cloning Vol. 1, 1995, IRL Press (ISBN 019-963476-9), chapter 6, pages 193 et seq.], Such sequences can be, for example, mutagenesis, such as site-directed mutagenesis. Spee et al., Nucleic Acids Research, Vol. 21, No. 3, 1993: 777-778, describes PCR using dITP for mutagenesis. The use of in vitro recombinant techniques for molecular evolution is described in Stemmer, Proc. Natl. Acad. Sci. USA, Vol. 91, 1994: 10747-10751. Moore et al., Nature Biotechnology Vol. 14, 1996: 458-467, describes a combination of PCR and recombination methods. The modified nucleic acid sequence is then returned to the individual via the vector.

In order to increase the enzyme activity, it is also possible to position the altered promoter region in front of the native gene to increase the expression of the gene, thereby increasing the final activity.

Advantageous promoter sequences are, for example, cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI ^q , T7, T5, T3, gal, trc, ara, SP6 or γ-P _R or present in the γ-P _L promoter, which is advantageously used in Gram-negative bacteria. Another advantageous regulatory sequence is, for example, in the gram-positive promoter amy and SPO2, in the yeast or fungal promoter ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, or the plant promoter CaMV / 35S [Franck et al., Cell 21, 1980, 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33, LEB4, nos or in the ubiquitin or phasolin promoters. Promoters of pyruvate decarboxylase and methanol oxidase, for example from Hansenula, are also advantageous in this context. Another advantageous plant promoter is, for example, benzenesulfonamide-induced (EP 388186), tetracycline-induced [Gatz et al., 1992, Plant J. 2, 397-404], absic acid-induced There is a sex (EP335528) or ethanol- or cyclohexanone-induced (WO9321334) promoter. Particularly advantageous plant promoters are those which ensure expression in the tissue or part of the plant in which the biosynthesis of purine and its precursors takes place. In particular, mention should be made of promoters which ensure leaf-specific expression. Mention should also be made of promoters of cytoplasmic FBP degrading enzymes obtained from potatoes or ST-LSI promoters obtained from potatoes (Stockhaus et al., EMBO J. 8 (1989) 2445-245). It is also possible and advantageous to use a promoter of phosphoribosyl-pyrophosphate amidotransferase obtained from glycine max (see also Genebank Accession No. U87999) or another nodule-specific promoter such as in EP 249676. In principle all natural promoters can be used with regulatory sequences such as those mentioned above in the method according to the invention. It is also advantageously possible to use synthetic promoters.

In addition to the more efficient transcription termination according to the invention, one or more transcription terminators from the group shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, for example, to increase the stability of the mRNA to enable increased translation Can be inserted into This likewise increases the enzyme activity of the correspondingly expressed genes. Stabilization of the transcript may be further enhanced by sequences introduced further at the 3 'end.

In order to increase gene expression by increasing the number of gene copies, one or more of the above-mentioned rib genes or combinations of a plurality of these genes that are operably linked to one or more transcription terminators of the invention may be capable of producing riboflavin. It is advantageous to introduce in addition. The gene copies may be subject to natural or modified regulation, but modified sequences of natural regulatory regions or other foreign or even heterologous genes may be used so that they can increase gene expression. Combinations of the above mentioned methods are particularly advantageous.

The invention also relates to a genetic construct comprising at least one transcription terminator set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and at least one rib gene operably linked to one of these terminators. The genetic construct of the present invention preferably comprises one or more genes selected from the group of rib1, rib2, rib3, rib4, rib5 or rib7. Advantageous variants of the invention include one or more transcription terminators set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or gene constructs of the aforementioned types and additional nucleotides for selection and replication in the host cell, or integration into the host cell genome. Included are vectors comprising the sequence.

Gene constructs of the invention may also include other genes to be introduced into an individual. Such genes may be separate from the rib gene or under the same regulatory region. These genes are, for example, biosynthetic genes that can increase synthesis.

For expression, the gene construct that optimally expresses the gene in the host is inserted into the above-mentioned host individual, advantageously a vector such as a plasmid, phage or other DNA. Examples of suitable plasmids include pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III ¹¹³ -B1, λgt11 or pBdCI, streptomyces (E. coli). Streptomyces) pIJ101, pIJ364, pIJ702 or pIJ361, pUB110, pC194 or pBD214 in Bacillus, pSA77 or pAJ667 in Corynebacterium, pALS1, pIL2 or pBB116 in fungi, 2 μm in yeast, pAG-1, YEp6, YEp13 or BYEe13 Or in plants are pLGV23, pGHlac ⁺ , pBIN19, pAK2004 or pDH51 or derivatives of the plasmids mentioned above. The plasmids are selected minority of possible plasmids. Other plasmids are well known to those skilled in the art and are described, for example, in Eds. Pouwels PH et al., Cloning Vectors, Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018. Suitable plant vectors are described in particular in Methods in Plant Molecular Biology and Biotechnology, CRC Press, Chapters 6/7, pages 71-119.

The gene construct may advantageously further contain 3 'and / or 5' terminal regulatory sequences selected for expression of other genes, for optimal expression according to the host individual and the gene or selected genes, thereby increasing expression. have. Such regulatory sequences are intended to enable specific expression of the genes and protein expression. This may mean, for example, that the gene is expressed and / or overexpressed only after induction, or immediately expressed and / or overexpressed, depending on the host individual.

Regulatory sequences or factors for this purpose may preferably increase expression because they have a beneficial effect on the expression of the introduced gene. Thus, regulatory elements can advantageously be enhanced at the level of transcription using strong transcriptional signals such as promoters and / or enhancers. However, translation can also be enhanced by, for example, improving the stability of the mRNA.

In another embodiment of the vector, the genetic constructs of the invention can also be advantageously introduced into microorganisms as linear DNA and integrated into the genome of a host individual by heterologous or homologous recombination. Such linear DNA may consist only of plasmids or nucleic acid fragments linearized as vectors.

In addition, any suitable plasmid that autonomously replicates intracellularly (but in particular a plasmid with a 2 μm plasmid replication initiation from S. cerevisiae) can be used as a vector, but also integrated into the genome of the host, as described above. Linear DNA fragments can also be used. Such integration can occur by hetero- or homologous recombination. Preferably, however, as mentioned, homologous recombination [Steiner et al., Genetics, Vol. 140, 1995: 973-987. The rib genes may also be present alone in the genome, at different locations or on different vectors, or together in the genome or on a single vector.

A transcription terminator of the invention, according to the invention preferably the above-mentioned gene construct comprising at least one rib gene and a regulatory sequence operably linked thereto, such as in particular one or more transcription terminators of the invention, or The above-mentioned vectors can in principle be introduced into the subject by any method known to those skilled in the art. They can advantageously be introduced into the individual or cells thereof by transformation, transfection, so-called electroporation by means of particle guns or by microscopic injection. One skilled in the art can review methods suitable for microorganisms in Sambrook, J. et al., 1989, Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, by FM Ausubel et al., 1994, Current protocols in molecular biology, John Wiley and Sons, by DM Glover et al., DNA Cloning Vol. 1, 1995, IRL Press (ISBN 019-963476-9), by Kaiser et al., 1994, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press or Guthrie et al. Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, 1994, Academic Press.

Examples of advantageous methods which may be mentioned are homologous or heterologous recombination using the ura-3 gene, in particular the Ashvija ura-3 gene, as described, for example, in German application DE 19801120.2 and / or REMI as described below. Introduction of DNA by the method (= restriction enzyme-mediated integration).

The REMI technique is based on the technique of cotransforming a linear DNA construct cut at both ends with the same restriction endonuclease into a subject with a restriction endonuclease for restriction of the DNA construct. The restriction endonuclease then cleaves the genomic DNA of the individual into a DNA construct introduced with the restriction enzyme. This activates the repair mechanism of the cells themselves. This repair mechanism repairs strand breaks in genomic DNA by endonucleases, which incorporates cotransformed DNA constructs into the genome at a constant frequency. Typically, restriction cleavage sites are retained at both ends of the DNA. This technique is described by Boelker et al. In relation to fungal insertion mutagenesis. Mol Gen Genet, 248, 1995: 547-552. See Chistl and Petes, Proc. Natl. Acad. Sci. USA, 88, 1991: 7585-7589, used this method to determine if heterologous recombination occurs in Saccharomyces. The method is described in Brown et al., Mol. In terms of stable transformation and regulated expression of inducible reporter genes. Gen. Genet. 251, 1996: 75-80. The system has not been used as a genetic engineering tool for the optimization of metabolic pathways or commercial overexpression of proteins to date. As an example of riboflavin synthesis it has been shown that biosynthetic genes can be integrated into the genomes of the above-mentioned individuals by the REMI method, thus allowing for primary or secondary metabolism, especially for example amino acids such as lysine, methionine, threonine or tryptophan, vitamins, Such as vitamins A, B2, B6, B12, C, D, E or F, S-adenosylmethionine, biotin, pantothenic acid or folic acid, carotenoids such as β-carotene, lycopene, cantaxanthin, astaxanthin or zeaxanthin, Or proteins such as hydrolases such as lipolytic enzymes, esterases, amidases, nitrile enzymes, proteases, mediators such as cytokines such as lymphokines such as MIF, MAF, TNF, interleukins, Such as interleukin 1, interferon such as γ-interferon, tPA, hormones such as protein hormone, sugar hormone, oligo- or polypeptide hormones such as vasopress Of metabolism products of the biosynthetic pathway of leucine, endorphin, endostatin, angiostatin, growth factor, erythropoietin, transcription factor, integrins such as GPIIb / IIIa, or α _v βIII, receptors such as various glutamate receptors, angiogenic factors such as angiotensin The manufacturing method can be optimized.

The REMI method also provides for the positioning of the above-mentioned gene construct or the above-mentioned vector comprising the transcription terminator of the invention, the transcription terminator of the invention operably linked to one or more rib genes at a transcription-active site in the genome. Can be used.

The transcription terminator and / or the gene construct is advantageously introduced into the genome as a cloned DNA construct with one or more reporter genes. This reporter gene allows for easy detection by growth, fluorescence, chemo- or bioluminescence analysis or photometry. Examples of reporter genes that may be mentioned include antibiotic resistance genes, hydrolase genes, fluorescent protein genes, bioluminescence genes, glucosidase genes, peroxidase genes or biosynthetic genes such as riboflavin genes, luminase genes, β- Galactosidase, gfp, lipase, esterase, peroxidase, β-lactam hydrolase, acetyl-, phospho- or adenyltransferase genes. These genes allow for easy measurement and quantification of gene expression through transcriptional activity. If, for example, riboflavin, the biosynthetic gene itself is easily detected, no additional reporter gene is required.

When multiple genes are introduced into an individual, all of them can be put into a single vector with one reporter gene or each individual gene can be introduced into an individual with one reporter gene and introduced into the individual, and various vectors can be introduced simultaneously. Or may be introduced continuously. Gene fragments encoding specific activities can also be used in REMI techniques.

In principle, all known restriction enzymes are suitable for methods of incorporating regulatory sequences or genetic constructs into the genome of an individual. Restriction enzymes that recognize only four base pairs as restriction cleavage sites are less preferred because they cleave the genome or the vector to be integrated too frequently; Preferred enzymes recognize 6, 7, 8, or more base pairs as cleavage sites, and mention only a few of the possible enzymes such as BamHI, EcoRI, BglII, SphI, SpeI, XbaI, XhoI, NcoI, SalI, ClaI, KpnI, HindIII, SacI, PstI, Bpn1, NotI, SrfI or SfiI. It is advantageous because the integration efficiency increases if the enzyme used has no more cleavage sites in the DNA to be introduced. Typically, 5 to 500 U, preferably 10 to 250 U, particularly preferably 10 to 100 U, of enzyme is used in the REMI mixture.

The enzymes are advantageously osmotic stabilizing substances such as sugars such as sucrose, trehalose or glucose, polyols such as glycerol or polyethylene glycol, buffers, advantageously pH 5 to 9, preferably 6 to 8, particularly preferably 7 Buffers such as Tris, MOPS, HEPES, MES or PIPES and / or nucleic acid stabilizing agents such as inorganic or organic salts of Mg, Cu, Co, Fe, Mn or Mo. Also, where appropriate, other substances may also be present, such as EDTA, EDDA, DTT, β-mercaptoethanol or nuclease inhibitors. However, it is also possible to implement the REMI technique without the addition. The process is carried out in a temperature range of 5 to 80 ° C, preferably 10 to 60 ° C, particularly preferably 20 to 40 ° C. All known methods for destabilization of cell membranes, for example electroporation, fusion with loaded cells, or destabilization with various alkali metal or alkaline earth metal salts such as lithium, rubidium or calcium salts, preferably lithium salts Is suitable for this method.

Transferring foreign genes to the genome of plants is called transformation. In this case, the methods described for transformation and regeneration of plants from plant tissues or plant cells are used for transient or stable transformation. Suitable methods are protoplast transformation by polyethylene glycol-derived DNA uptake, use of gene guns, electroporation, incubation of dry embryos in DNA-containing solutions, microinjection and transfer of Agrobacterium-mediated genes. The method is described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press, 1993, 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42, 1991, 205-225. The construct to be expressed is preferably a vector suitable for transforming Agrobacterium tumefaciens, for example pBin19 [Bevan et al., Nucl. Acids Res. 12, 1984, 8711]. Plant transformation with Agrobacterium tumefaciens is described, for example, in Hoefgen and Willmitzer, Nucl. Acid Res., 1988, 16, 9877.

Agrobacteria transformed with an expression vector according to the invention are likewise known in plants, for example by soaking a wounded leaf or leaf piece in a solution of Agrobacteria and then cultivating in a suitable medium, thereby producing plants, in particular crops such as cereals, corn, It can be used to transform soybeans, rice, cotton, sugar beets, canola, sunflowers, flax, hemp, potatoes, tobacco, tomatoes, rapeseed, alfalfa, lettuce and various woods, nuts and vines, and legumes. Genetically modified plant cells can be regenerated by any method known to those skilled in the art. Suitable methods are described above in S.D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.

The present invention also encompasses individuals that can produce riboflavin and include one or more transcription terminators as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or a gene construct or vector of the type according to the present invention. The subject is advantageously of the genus or plant of Corynebacterium, Brevibacterium, Bacillus, Clostridium, Escherichia, Cyanobacter, Ashvilla, Eremotesium, Pichia, Candida or Saccharomyces. Such as the Arabidopsis, corn, soybean, rapeseed, barley, wheat, rye, millet, oats, sugar beets, potatoes, sunflowers, legumes or tomatoes. Preferred individuals are selected from the group of Bacillus, Corynebacterium, Brevibacterium, Escherichia, Candida, Eremotesium or Ashvija or Corn, Soybean, Rapeseed, Barley, Wheat, Potato or Tomato. Especially preferred are Ashvija Gosipyi, Eremoteium Ashvii, Saccharomyces cerevisiai, Candida flaberry, Candida palmata, Corynebacterium ammonia genes or Bacillus subtilis. In particular Ashvi Gosipi or Hermotesium Ashvii.

The invention further encompasses individuals that are distinguished as riboflavin-producing mutants or manufacturer strains. These can be prepared, for example, by classical (chemical or physical) or genetic engineering methods starting from wild type strains and, where appropriate, have genetic modifications beyond those of the rib gene translation frame.

Individuals of the invention may also be characterized by an increase in the rate of transcription of one or more rib genes operably linked to one of the transcription terminators as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, compared to wild type ATCC 10895 in the genus Ashville. Are distinguished. Subjects of the invention may also exhibit improved production of riboflavin when compared to wild type ATCC 10895 in the genus Ashvilla.

Individuals used to prepare riboflavin can be cultured according to the methods of the present invention in a medium that allows the growth of the individual. This medium may be a synthetic or natural medium. Medium used differently depending on the individual is known to those skilled in the art. The medium used for the growth of microorganisms contains a carbon source, a nitrogen source, an inorganic salt and, where appropriate, 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 sugars 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 amino acid mixtures, for example so-called casamino acids (Difco) or individual amino acids such as glycine or aspartic acid, or amino sugars, the latter also being a nitrogen source May also be used.

Advantageous nitrogen sources are organic or inorganic nitrogen compounds or materials containing such compounds. Examples include ammonium salts such as NH ₄ Cl or (NH ₄ ) ₂ SO ₄ , nitrate, urea, or complex nitrogen sources such as corn steep liquor, brewer's yeast autolysate, soy flour, wheat gluten, yeast Extracts, meat extracts, casein hydrolysates, yeast or potato proteins, which can also be frequently used as carbon sources.

Examples of inorganic salts include salts of calcium, magnesium, sodium, cobalt, molybdenum, manganese, potassium, zinc, copper and iron. Particularly mentioned anions of these salts are chloride, sulfate or phosphate ions. 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.

Where appropriate, additional growth factors such as vitamins or growth promoters such as biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid or pyridoxine, amino acids such as alanine, cysteine, proline, aspartic acid, glutamine, serine, phenylalanine, Substances such as ornithine or valine, carboxylic acids such as citric acid, formic acid, pimelic acid or lactic acid, or dithiothritol are added to the nutrient medium.

The mixing ratio of the nutrients depends on the type of fermentation and will be determined in each case. The components of the medium may, if necessary, be sterilized separately or together, either presented at the start of fermentation or else added continuously or discontinuously as needed during fermentation.

Culture conditions are set such that individuals grow optimally and are obtained in the highest yield. Preferred incubation temperatures are from 15 ° C to 40 ° C. A temperature of 25 ° C. to 37 ° C. is particularly advantageous. The pH is preferably maintained in the range of 3-9. PH values of 5 to 8 are particularly advantageous. Incubation times are generally sufficient for several hours to several days, preferably 8 hours to 21 days, particularly preferably 4 hours to 14 days. During this time the maximum amount of product accumulates in the medium.

Methods for advantageously optimizing the medium are described, for example, in the Applied Microbial Physiology textbook Eds. P.M. Rhodes, P.F. Stanbury, A Practical Approach, IRL-Press, 1997, pages 53-73, ISBN 0 19 963577 3. Advantageous media and culture conditions are described, for example, in Bacillus and other individuals, in particular in Example 8 of EP-A-0 405 370, in Candida in particular in Table 3 in WO 88/09822, and in Ashvilla in Schmidt et al. , Microbiology, 142, 1996: 419-426.

The method of the invention can be carried out continuously or by one batch or fed-batch procedure. Depending on the initial level of productivity of the individual used, the productivity of riboflavin can be increased to varying degrees by the method according to the invention. Productivity can typically be advantageously increased by at least 5%, preferably at least 10%, particularly preferably at least 20%, more particularly preferably at least 100% when compared to the initial subject.

The invention also includes the use of an individual of the type according to the invention comprising at least one transcription terminator set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a gene construct or vector of the type according to the invention for improved riboflavin production. do. The subject is preferably Ashvija Gossipi.

The invention likewise relates to the use of one or more transcription terminators set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 for preparing a manufacturer individual for improved riboflavin production. The invention likewise encompasses the use of one or more transcription terminators set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 to increase the transcription rate of genes involved in the preparation of riboflavin. Accordingly, the present invention also relates to the use of one or more transcription terminators set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 for improved riboflavin production.

The invention is illustrated in more detail by the following examples, but is not limited thereto.

Common way :

General nucleic acid related methods such as cloning, restriction cleavage, agarose gel electrophoresis, ligation of DNA fragments, transformation of microorganisms, bacterial cultures and recombinant DNA sequencing, unless otherwise stated, are described in Sambrook et al. , 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6.

Recombinant DNA molecules are described by Sanger method [Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467] were sequenced from ABI using a laser fluorescent DNA sequencer. Fragments generated by polymerase chain reaction were sequenced and checked to avoid errors of polymerase in the construct to be expressed.

Isolation of the rib gene :

Isolation of rib genes 1, 2, 3, 4, 5 and 7 from Ashvija Gosipyi and Saccharomyces cerevisiae are described in patents WO 95/26406 and WO 93/03183 and detailed examples, corresponding Can be done. Said documents are incorporated herein by reference.

Terminator Region Sequence Comparison

Starting from the isolated rib gene, the terminator region of the rib2 gene was also identified and analyzed. This represented the following sequence.

Terminator of the rib2 gene of the genus Ashville ATCC 10895 (SEQ ID NO: 4):

Stop-TGATTTTGCTGCGAATTGTAGATGG

Terminators of the present invention have the following sequence:

Terminator of the present invention shown in (SEQ ID NO: 1):

Stop-TGATTTTGCTGC A AATTGTAGATGG

Terminator of the present invention shown in (SEQ ID NO: 2):

Stop-TGATTTTGCTGCGAATTGTAGATG A

Terminator of the present invention shown in (SEQ ID NO: 3):

Stop-TGATTTTGCTGC A AATTGTAGATG A

Different nucleotides are underlined. Synthesis of nucleotide sequences and substitution or mutagenesis procedures of single nucleotides were performed according to routine experimental criteria, in particular D.M. Glover et al., DNA Cloning Vol. 1, 1995, IRL Press, ISBN 019-963476-9.

The sequence of the terminator of the present invention and the terminator of the rib2 gene of the genus ASCC 10895 is shown in SEQ ID NOs: 1-4.

SEQUENCE LISTING <110> BASF Aktiengesellschaft <120> Verfahren zur Herstellung von Riboflavin <130> 1 <160> 4 <170> PatentIn version 3.1 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Terminator 1 <220> <221> terminator (222) (1) .. (25) <223> Terminator 1 <400> 1 tgattttgct gcaaattgta gatgg 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> Terminator 2 <220> <221> terminator (222) (1) .. (25) Terminator 2 <400> 2 tgattttgct gcgaattgta gatga 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Terminator 3 <220> <221> terminator (222) (1) .. (25) <223> Terminator 3 <400> 3 tgattttgct gcaaattgta gatga 25 <210> 4 <211> 25 <212> DNA <213> Ashbya gossypii <220> <221> terminator (222) (1) .. (25) <223> Terminator rib2-Gen <400> 4 tgattttgct gcgaattgta gatgg 25

Claims (27)

a) a riboflavin may be prepared and cultured with an individual having one or more transcription terminators as set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, wherein a particular transcription terminator is operably linked to one or more rib genes, b) recovering the formed riboflavin from the culture medium, Method for preparing riboflavin. The method of claim 1, wherein one or more genes from the group of rib1, rib2, rib3, rib4, rib5 or rib7 are operably linked. The method according to claim 1 or 2, wherein the transcription terminator shown in SEQ ID NO: 1 is a sequence in which guanine at position 13 is modified by adenine to be modified as compared to the sequence of the transcription terminator of the rib2 gene of wild type ATCC 10895. The method according to claim 1 or 2, wherein the transcription terminator shown in SEQ ID NO: 2 is a sequence in which guanine at position 25 is modified by adenine to be modified as compared to the sequence of transcription terminator of the rib2 gene of wild type ATCC 10895. 3. The method according to claim 1 or 2, wherein the transcription terminator shown in SEQ ID NO: 3 is a sequence which is modified by replacing guanine at positions 13 and 25 with adenine as compared to the sequence of transcription terminator of the rib2 gene of wild type ATCC 10895. The method according to any one of claims 1 to 5, wherein bacteria, yeasts, fungi or plants are used as individuals capable of producing riboflavin. The method of claim 1, wherein Corynebacterium, Brevibacterium, Bacillus, Clostridium, Escherichia, Sia Novobter, Ashbya, Eremothecium, Pichia, Candida or Saccharomyces or plants, such as baby poles, corn, soybeans, How to use objects selected from the group of rapeseed, barley, wheat, rye, millet, oats, sugar beets, potatoes, sunflowers, legumes or tomatoes. The method according to any one of claims 1 to 7, wherein Ashbya gossypii, Eremothium ashbyii, Saccharomyces cerevisiae, Candida flaber ( Candida flaveri, Candida famata, Corynebacterium ammoniagenes or Bacillus subtilis as a subject. The method according to any one of claims 1 to 8, wherein Ashvi Gosipi or Eremoteium Ashvii are used. A transcription terminator having the sequence set forth in SEQ ID NO: 1. A transcription terminator having the sequence set forth in SEQ ID NO: 2. A transcription terminator having the sequence set forth in SEQ ID NO: 3. The transcriptional terminator of claim 10 wherein the guanine at position 13 is modified by adenine to be modified as compared to the sequence of the transcriptional terminator of the rib2 gene of genus AshCCilla ATCC 10895. 12. The transcriptional terminator of claim 11 wherein the guanine at position 25 is modified by adenine to be modified as compared to the sequence of the transcriptional terminator of the rib2 gene of genus Ashville 108 ATCC 10895. 13. The transcriptional terminator of claim 12 wherein the guanine at positions 13 and 25 is replaced with adenine and modified as compared to the sequence of the transcriptional terminator of the rib2 gene of the genus Ashville 108 ATCC 10895. A gene construct comprising at least one transcription terminator of any one of claims 10 to 15 and at least one rib gene operably linked to a particular terminator. The genetic construct of claim 16 comprising one or more genes from the group of rib1, rib2, rib3, rib4, rib5 or rib7. The transcriptional terminator of any one of claims 10-15 or the gene construct according to claim 16 or 17, and further selection of host cells and replication within the host cells and integration into the host cell genome. A vector comprising a nucleotide sequence for. An individual capable of producing riboflavin, comprising at least one transcription terminator of claim 10 or a gene construct according to claim 16 or 17 or a vector according to claim 18. 20. The genus or plant according to claim 19, wherein the genus Corynebacterium, Brevibacterium, Bacillus, Clostridium, Escherichia, Cyanobacter, Ashvija, Eremotesium, Pichia, Candida or Saccharomyces For example, an individual selected from the group of Arabidopsis, corn, soybean, rapeseed, barley, wheat, rye, millet, oats, sugar beets, potatoes, sunflowers, legumes or tomatoes. 21. The method according to claim 19 or 20, comprising: Ashvi Gosipi, Eremoteium Ashbi, Saccharomyces cerevisiai, Candida flaber, Candida Palmata, Corynebacterium ammonia genes or Bacillus subtilis. Object. 22. The subject of any one of claims 19-21, wherein the subject is Ashvi Gosipi or Eremoteium Ashvii. The transcription rate of any one of claims 19-22, wherein the rib gene is operably linked to a transcription terminator from the group of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 when compared to the wild type of genus ATCC 10895. An object representing the increase of. The individual of claim 19, wherein the production of riboflavin is improved when compared to the wild type of genus Ashville ATCC 10895. Use of an individual of any one of claims 19 to 24 for producing improved riboflavin. Use of one or more transcription terminators according to any one of claims 10 to 15 for the manufacture of a manufacturer individual for improved riboflavin production. Use of one or more transcription terminators according to any one of claims 10 to 15 for increasing the rate of transcription of a gene associated with the production of riboflavin.