CA2477849A1 - Method for producing riboflavin - Google Patents

Abstract

The invention relates to transcription terminators, to an organism containing at least one of these transcription terminators, and to a method for producing riboflavin. According to the invention, an organism is grown, which is capable of producing riboflavin and has at least one of these transcription terminators, whereby the respective transcription terminator is operatively linked to at least one rib gene.

Description

METHOD FOR PRODUCING RIBOFLAVIN
The present invention relates to transcription terminators, to an organism which is able to produce riboflavin and comprises at least one of these transcription terminators, and to an improved process for producing riboflavin, in which an organism able to produce riboflavin and having at least one of said transcription terminators is cultured.
Vitamin B2, also called riboflavin, is produced by all plants and a large number of microorganisms. It is essential for humans and animals because they are unable to synthesize it. Riboflavin plays an important part in metabolism. Thus, for example, it is involved in the utilization of carbohydrates. Vitamin B2 deficiencies are associated with inflammations of the mucous membranes of the mouth and throat, pruritus and inflammations in the skinfolds and similar skin lesions, inflammations of the conjunctiva, diminished visual acuity and clouding of the cornea. In infants and children there may be cessation of growth and loss of weight. Vitamin B2 therefore has great economic importance, for example as vitamin product for vitamin deficiencies, and as animal feed additive. It is added to a wide variety of foodstuffs. It is also used as food coloring, for example in mayonnaise, ice cream, blancmange etc.
Vitamin B2 is produced either chemically or microbially (see, for example, Kurth et al., 1996, Riboflavin, in:
Ullmann's Encyclopedia of industrial chemistry, VCH
Weinheim). In the chemical production process, riboflavin is usually obtained as pure final product in multistage processes, it being necessary to employ relatively costly starting materials such as, for example, D-ribose.

An alternative to the chemical synthesis of riboflavin is the production of vitamin B2 by fermentation of microorganisms. The starting materials in this case are renewable raw materials such as sugar or vegetable oils. The production of riboflavin by fermentation of fungi such as Eremothecium ashbyii or Ashbya gossypii is known (The Merck Index, Windholz et al., eds. Merck & Co., page 1183, 1983), but yeasts such as, for example, Candida, Pichia and Saccharomyces or bacteria, such as, for example, Bacillus, Clostridia or Corynebacteria have also been described as riboflavin producers. EP-A-0 405 370 and EP-A-0 821 063 describe the production of riboflavin using recombinant strains of bacteria, the strains having been obtained from Bacillus subtilis by transformation with riboflavin biosynthesis genes.
The patent WO 95/26406 or WO 93/03183 and DE 44 20 785 describe the cloning of the genes specific for riboflavin biosynthesis from the eukaryotic organisms Ashbya gossypii and Saccharomyces cerevisiae, and microorganisms which have been transformed with these genes, and the use of such microorganisms for riboflavin synthesis.
In both organisms, 6 enzymes catalyze the formation of riboflavin starting from guanosine triphosphate (GTP) and from ribulose 5-phosphate. This involves conversion of GTP into 2,5-diamino-6-(ribosylamino)-4(3H)-pyrimidinone 5-phosphate by GTP cyclohydrolase-II (ribl gene product). The latter compound is then reduced by 2,5-diamino-6-(ribosylamino)-4(3H)-pyrimidinone-5-phosphate reductase (rib? gene product) to 2,5-diamino-6- ribitylamino-2,4(1H,3H)-pyrimidine 5-phosphate and subsequently deaminated by a specific deaminase (rib2 gene product) to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5-phosphate. The phosphate is then eliminated by a nonspecific phosphatase.
Val Gly Asn Lys Gly Asp Leu Leu Hi Ribulose 5-phosphate, the second starting material, besides GTP, in the last enzymatic steps of riboflavin biosynthesis, is converted by 3,4-dihydroxy-2-butanone-4-phosphate synthase (rib3 gene product) into 3,4-dihydroxy-2-butanone 4-phosphate (DBP).
Both DBP and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione are the precursors for the enzymatic synthesis of 6,7-dimethyl-8-ribityllumazine. This reaction is catalyzed by the rib4 gene product (DMRL
synthase). DMRL is subsequently converted by riboflavin synthase (ribs gene product) into riboflavin (Backer et al. (1993), Bioorg. Chem. Front. Vol. 3, Springer Verlag) .
Despite these advances in the production of riboflavin, there is still a need for vitamin B2 productivity to be improved and increased in order to meet the increasing demand and make the production of riboflavin more efficient.
It is an object of the present invention to improve vitamin B2 productivity further.
We have found that this object is achieved by a process for producing riboflavin, where an organism which is able to produce riboflavin and which has at least one transcription terminator as shown in SEQ ID No. 1, SEQ
ID No. 2 or SEQ ID No. 3, where the particular transcription terminator is operatively linked to at least one rib gene (gene of riboflavin biosynthesis), is cultured, and the riboflavin which is formed to an increased extent is recovered from the culture medium.
In the process for riboflavin production it is advantageous according to the invention for there to be operative linkage to at least one gene from the group of ribl, rib2, rib3, rib4, ribs or rib7.

The operative linkage of a transcription terminator of the invention selected from the group as shown in SEQ
ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 to at least one gene of riboflavin biosynthesis (rib gene), when such a combination is present in bacteria, fungi, yeasts and plants, increases the expression of the corresponding gene or of the corresponding genes and/or exerts a stabilizing effect on the formed transcripts) and consequently increases riboflavin production (compared with the wild type of the genus Ashbya ATCC 10895) in these organisms. In this connection, operative linkage of a terminator of the invention from said group is possible to one or more rib genes, each gene being operatively linked individually to this terminator, or a gene group (e. g. cluster or operon) being operatively linked to such a terminator. The presence of different terminators among those mentioned in organisms suitable for riboflavin production is also included. In this case, the different terminators may each be operatively linked individually to a rib gene, or the different , terminators are present in an operative linkage to rib genes arranged in groups. It is thus possible for the purposes of the present invention for each of said transcription terminators individually, or two thereof or all three in every conceivable combination, to be present operatively linked to rib genes in an organism suitable for riboflavin production. Operative linkage of the transcription terminators of the invention to one or more other genes involved in riboflavin production is also conceivable.
An operative linkage means sequential arrangement of nucleotide sequences having regulatory and coding function, such as, for example, of promoter, coding sequence, terminator and, where appropriate, further regulatory elements in such a way that each of the regulatory elements is able to carry out its proper function in the expression of the coding sequence.
These regulatory nucleotide sequences may be of natural origin or be obtained by chemical synthesis. Genetic engineering procedures for the operative linkage of nucleotide sequences form part of conventional laboratory practice and can be referred to for example in D.M. Glover et al., DNA Cloning Vol.l, (1995), IRL
Press (ISBN 019-963476-9). Methods for the synthesis and for the exchange of bases in a nucleotide sequence, which are known to the skilled worker, can also be referred to here.
The operative linkage of at least one of the rib genes to one of the transcription terminators of the invention as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ
ID No. 3 advantageously leads to increased termination of transcription. This in turn indirectly influences the transcription rate of the gene which is upstream of the terminator, because the transcription apparatus, i.e. RNA polymerise and assisting transcription factors, is released more efficiently after reading of the terminator of the invention, and is thus available more quickly for a new round of transcription (Alberts et al., 3rd edition, Molekularbiologie der Zelle, VCH
Verlag). Consequently, an increased transcription rate of the corresponding genes is made possible in this way. This means that there is an increase in gene expression combined with an increase in the activity of the corresponding encoded gene products, which culminates in a distinctly increased riboflavin productivity. In addition, each of the transcription terminators of the invention may exert a stabilizing effect on the transcript which is formed. In this case, for example, degradation (e. g. by an exonuclease) from the 3' end of the transcript may be slowed down or virtually prevented. The result of this is that the transcripts, because they are degraded less rapidly, can be translated to an increased extent, in turn leading to an increased activity of the correspondingly encoded enzymes.

In a process which is advantageous according to the invention there is use of a transcription terminator as shown in SEQ ID No. 1, which is modified compared with the sequence of the transcription terminator of the rib2 gene of the wild type ATCC 10895 at position 13 by replacement of guanine by adenine. In an equally advantageous process there is use of a transcription terminator as shown in SEQ ID No. 2, which is modified compared with the sequence of the transcription terminator of the rib2 gene of the wild type ATCC 10895 at position 25 by replacement of guanine by adenine. In a likewise advantageous process there is use of a transcription terminator as shown in SEQ ID No. 3, which is modified compared with the sequence of the transcription terminator of the rib2 gene of the wild type ATCC 10895 at positions 13 and 25 by replacement of guanine by adenine. A process in which a transcription terminator shown in SEQ ID No. 3 is employed is preferred.
It is possible where appropriate for each of the transcription terminators of the invention to be present numerically once or else in multiple copies in the genome of the organism employed for riboflavin production. This depends on whether the terminator is operatively linked to one or more genes. Thus, for example, operative linkage of the terminator as shown in SEQ ID No. 1 to only one rib gene or to a plurality of, preferably separately organized, rib genes (or other genes involved in riboflavin synthesis) is conceivable . The same applies to a terminator as shown in SEQ ID No. 2 or equally to a terminator as shown in SEQ ID No. 3.
The invention thus includes the use of each individual transcription terminator or the combination of all three transcription terminators of the invention, in particular each in a single copy or multiple copies in the genome of an organism suitable for riboflavin production.

_ 7 _ The process for increased production of riboflavin is advantageously carried out with an organism which is able to produce riboflavin. Suitable organisms or host organisms for the process according to the invention are in principle all organisms able to synthesize riboflavin. Organisms naturally able to synthesize riboflavin are preferred. However, organisms which, owing to the introduction of the complete vitamin B2 synthesis genes, are able to synthesize riboflavin are also suitable for the process according to the invention.
Organisms such as bacteria, yeasts, fungi or plants are suitable for the process according to the invention.
Examples which may be mentioned are 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, oilseed rape, barley, wheat, rye, rice, millet, cotton, legumes, sugar beet, sunflower, flax, hemp, canola, oats, tobacco, alfalfa, lettuce or the various tree, nut and vine species or prokaryotic organisms such as Gram-positive or Gram-negative bacteria such as Corynebacterium, Brevibacterium, Bacillus, Clostridium, Cyanobacter, Escherichia or Klebsiella.
Preferred organisms are selected from the group of genera Corynebacterium, Brevibacterium, Bacillus, Escherichia, Ashbya, Eremothecium, Candida or Saccharomyces or plants such as corn, soybean, oilseed rape, barley, wheat, potato or tomato.
Particularly preferred organisms are those of the genus and species Ashbya gossypii, Eremothecium ashbyii, Saccharomyces cerevisiae, Candida flaveri, Candida famata, Corynebacterium ammoniagenes or Bacillus subtilis. Particularly preferred plants are corn, soybean, oilseed rape, barley, wheat, potato and tomato. Ashbya gossypii or Eremothecium ashbyii are particularly preferred.
The invention also includes riboflavin producer strains. These can be produced for example starting from wild-type strains which are suitable for riboflavin production by classical (chemical or physical) or genetic engineering methods and have where appropriate further genetic modifications than those within the framework of the rib genes.
The aforementioned rib genes include according to the invention those derived from organisms which are able to produce riboflavin. Genes from organisms such as Bacillus subtilis, Saccharomyces cerevisiae or Ashbya gossypii are preferred. Ashbya gossypii rib genes are particularly preferred. Included herein are also functional analogs, functional equivalents or derivatives of these genes.
Functional analogs mean for example functional homologs of the rib genes or their enzymatic activities, i.e.
enzymes which catalyze the same reactions as the rib gene enzymes.
Functional equivalents mean, for example, allelic variants which have at least 35% homology at the derived amino acid level, preferably at least 400 homology, particularly preferably at least 450 homology, very particularly preferably 50o homology.
Allelic variants include in particular functional variants which are obtainable by deletion, insertion or substitution of nucleotides, with retention of the enzymatic activity of the derived synthesized proteins.
DNA sequences of this type can be isolated starting from the known DNA sequences of the rib genes, or parts of these sequences, for example using conventional _ g _ hybridization processes or the PCR technique, from other eukaryotes or prokaryotes than Ashbya gossypii as mentioned above. These DNA sequences hybridize under standard conditions with said sequences. It is advantageous to use for the hybridization short oligonucleotides of the conserved region which can be identified by comparisons with the corresponding genes from E. coli and B. subtilis in a manner known to the skilled worker.
Standard conditions mean, for example, temperatures between 42 and 58°C in an aqueous buffer solution with a concentration between 0.1 to 5 x SSC (1 X SSC - 0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50o formamide, such as, for example, 42°C in 5 x SSC and in the presence of 50o formamide.
The experimental conditions for the DNA hybridization are described in relevant textbooks of genetics, such as, for example, Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989. Homologs also mean truncated sequences or single-stranded DNA.
Derivatives mean variants whose nucleotide sequence in front of the start codon has been altered so that gene expression and/or protein expression is changed, preferably increased.
For optimal expression of heterologous genes in organisms, it is advantageous to modify the nucleic acid sequences to accord with the specific codon usage employed in the organism. The codon usage can easily be established by computer analyses of other known genes in the relevant organism.
Also advantageous for increasing vitamin B2 productivity is a combination of increasing the natural enzymic activity encoded by said rib genes and increasing the gene expression by additional introduction of at least one of the abovementioned genes or else a combination of a plurality of these - 1~ -genes into an organism which is able to produce riboflavin.
There is a large number of possibilities for increasing the enzymic activity of rib gene products in the cell.
One possibility consists of operative linkage of the genes to one of the terminators of the invention from the group as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ
ID No. 3, resulting in an increased transcription rate of the particular gene or plurality of said genes and/or a stabilization of the transcripts) formed.
A further possibility is to modify the endogenous rib genes in such a way that they code for enzymes having a rib activity which is increased compared with unmodified (initial or wild-type) enzymes. An increase in the enzymic activity can be achieved for example by modifying the catalytic centers to result in an increased substrate conversion or by abolishing the effect of enzyme inhibitors. This means that the enzymes have an increased specific activity or their activity is not inhibited.
It is also possible in a further advantageous embodiment to increase the enzymic activity by increasing enzyme synthesis in the cell, for example by eliminating factors which repress enzyme synthesis or by increasing the activity of factors or regulatory elements which promote enhanced synthesis. The introduction of further gene copies may also lead to an increased specific enzymic activity. These measures increase the total activity of the gene products in the cell without altering the specific activity. It is also possible to use a combination of these methods, i.e.
increasing the specific activity and increasing the total activity. These modifications can be introduced into the nucleic acid sequences of the genes, regulatory elements, terminators or promoters thereof in principle by all methods known to the skilled worker.

- 1. I. -It is possible for this purpose to subject the sequences to, for example, mutagenesis such as site-directed mutagenesis as described in D.M. Glover et al., DNA Cloning Vol.l, (1995), IRL Press (ISBN 019-963476-9), chapter 6, pages 193 et seq. Spee et al.
(Nucleic Acids Research, Vol. 21, No. 3, 1993: 777-778) describe a PCR method using dITP for mutagenesis.
The use of an in vitro recombination technique for molecular evolution is described by Stemmer (Proc.
Natl. Acad. Sci. USA, Vol. 91, 1994: 10747 - 10751).
Moore et al. (Nature Biotechnology Vol. 14, 1996: 458 467) describe the combination of the PCR method and recombination method. The modified nucleic acid sequences are then returned to the organisms via vectors.
To increase the enzymic activities it is also possible to place altered promoter regions in front of the natural genes, so that expression of the genes is increased and thus finally the activity is raised.
Advantageous promoter sequences are present, for example, as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq~ T7, T5, T3, gal, trc, ara, SP6 or y-PR or in the y-PL promoter, which are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are, for example, present in the Gram-positve promoters amy and SP02, in the yeast or fungal promoters ADC1, MFa, 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.Mol.
Biol.22(1993)], SSU, OCS, lib4, usp, STLS1, B33, LEB4, nos or in the ubiquitin or phaseolin promoter. Also advantageous in this connection are the promoters of the pyruvate decarboxylase and the methanol oxidase from, for example, Hansenula. Further advantageous plant promoters are, for example, a benzenesulfonamide-inducible (EP 388186), a tetracycline-inducible (Gatz et al., (1992) Plant J. 2,397-404), an abscisic acid-inducible (EP335528) or an ethanol- or cyclohexanone-inducible (W09321334) promoter. Particularly advantageous plant promoters are those which ensure expression in tissues or parts of plants in which biosynthesis of purines or its precursors takes place.
Particular mention should be made of promoters which ensure leaf-specific expression. Mention should be made of the promoter of the cytosolic FBPase from potato or the ST-LSI promoter from potato (Stockhaus et al., EMBO
J. 8 (1989) 2445-245). It is also possible and advantageous to use the promoter of the phosphoribosyl-pyrophosphate amidotransferase from Glycine max (see also Genebank Accession Number U87999) or another node-specific promoter as in EP 249676.
It is possible in principle to use all natural promoters with their regulatory sequences like those mentioned above for the process according to the invention. It is also possible advantageously to use synthetic promoters.
It is also possible at the 3' end, besides the more efficient transcription termination according to the invention, through insertion of at least one of the transcription terminators from the group as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 for example to increase the stability of the mRNA and thus make increased translation possible. This likewise results in higher enzymic activity of the correspondingly expressed genes. Stabilization of the transcripts can be further enhanced by additionally introduced sequences at the 3' end.
It is advantageous additionally to introduce at least one of the abovementioned rib genes or a combination of a plurality of these genes, in operative linkage with at least one of the transcription terminators of the invention, in an organism which is able to produce riboflavin in order to increase gene expression by increasing the gene copy number. These gene copies may be subject to natural regulation or modified regulation, in which case the natural regulatory regions have been modified in such a way they make increased expression of the genes possible, or else regulatory sequences of foreign genes or even heterologous genes can be used. A combination of the abovementioned methods is particularly advantageous.
The present invention also relates to a gene construct comprising at least one transcription terminator as shown in 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 of the invention preferably comprises at least one gene from the group of ribl, rib2, rib3, rib4, ribs or rib7.
An advantageous variant of the present invention includes a vector comprising at least one transcription terminator as shown in 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 of the invention may also comprise other 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, further biosynthesis genes which make increased synthesis possible.
For expression, the gene construct is inserted into the abovementioned host organism advantageously into a vector such as, for example, a plasmid, a phage or other DNA, which makes optimal expression of the genes in the host possible. Examples of suitable plasmids are in E. coli pLG338, pACYC184, pBR322, pUCl8, pUCl9, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113_B1, ~,gtll or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALSl, pIL2 or pBB116, in yeasts 2~m, pAG-1, YEp6, YEpl3 or pEMBLYe23 or in plants pLGV23, pGHlac+, pBINl9, pAK2004 or pDH51 or derivatives of the abovementioned plasmids. Said plasmids represent a small selection of the possible plasmids. Further plasmids are well known to the skilled worker and can be found, for example, in the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Suitable plant vectors are described inter alia in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chapters 6/7, pages 71-119.
The gene construct advantageously contains, for expression of the other genes present, in addition 3' and/or 5' terminal regulatory sequences to increase expression, these being selected for optimal expression depending on the host organism and gene or genes selected.
These regulatory sequences are intended to make specific expression of these genes and of the protein expression possible. This may mean, for example depending on the host organism, that the gene is expressed and/or overexpressed only after induction, or that it is immediately expressed and/or overexpressed.
The regulatory sequences or factors may for this purpose preferably have a beneficial effect on expression of the introduced genes, and thus increase it. Thus, an enhancement of the regulatory elements can advantageously take place at the level of transcription, by using strong transcription signals such as promoters and/or enhancers. However, it is also possible to enhance translation by, for example, improving the stability of the mRNA.
In another embodiment of the vector the gene construct of the invention can also be advantageously introduced in the form of a linear DNA into the microorganisms, and be integrated by heterologous or homologous recombination into the genome of the host organism.
This linear DNA may consist of a linearized plasmid or only of the nucleic acid fragment as vector.
It is also possible to use as vector any suitable plasmid (but especially a plasmid which harbors the origin of replication of the 2E.tm plasmid from S. cerevisiae) which undergoes autonomous replication in the cell, but also, as described above, a linear DNA
fragment which integrates into the host's genome. This integration can take place by hetero- or homologous recombination. But preferably, as mentioned, by homologous recombination (Steiner et al., Genetics, Vol. 140, 1995: 973 - 987). It is moreover possible for the rib genes to be present singly in the genome at different sites or on different vectors, or to be present together in the genome or on one vector.
The transcription terminators of the invention, the aforementioned gene construct or the aforementioned vectors comprising according to the invention preferably at least one rib gene and regulatory sequences appropriately operatively linked thereto, such as, inter alia, at least one of the transcription terminators of the invention can in principle be introduced into the organisms used by all methods known to the skilled worker.
They are advantageously introduced into the organisms or cells thereof by transformation, transfection, electroporation, using the so-called particle gun or by microinjection. Methods appropriate for microorganisms can be found by the skilled worker in the textbooks by Sambrook, J. et al. (1989) Molecular cloning: A
laboratory manual, Cold Spring Harbor Laboratory Press, by F.M. Ausubel et al. (1994) Current protocols in molecular biology, John Wiley and Sons, by D.M. Glover et al., DNA Cloning Vol.l, (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 the introduction of the DNA by homologous or heterologous recombination, for example with the aid of the ura-3 gene, specifically the Ashbya ura-3 gene as described in the German Application DE 19801120.2 and/or by the REMI method (= restriction enzyme-mediated integration) which is described below.
The REMI technique is based on the cotransformation of a linear DNA construct which has been 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 has been introduced together with the restriction enzyme. This leads to activation of the cell's own repair mechanisms. These repair mechanisms repair the strand breaks caused by the endonuclease in the genomic DNA, and this is associated with a certain frequency of incorporation of the cotransformed DNA construct into the genome too. It is usually the case that the restriction cleavage sites are retained at both ends of the DNA.
This technique was described by Bolker et al. (Mol Gen Genet, 248, 1995: 547-552) for insertion mutagenesis of fungi. Schiestl and Petes (Proc. Natl. Acad. Sci. USA, 88, 1991: 7585-7589) used the method to discover whether heterologous recombination occurs in Saccharomyces. The method was described by Brown et al.
(Mol. Gen. Genet. 251, 1996: 75-80) for stable transformation and regulated expression of an inducible reporter gene. The system has not to date been used as genetic engineering tool for optimizing metabolic pathways or for commercial overexpression of proteins.

It has been shown by the example of riboflavin synthesis that biosynthesis genes can be integrated by the REMI method into the genome of the abovementioned organisms, and thus it is possible to optimize production processes for producing metabolic products of primary or secondary metabolism, specifically of biosynthetic pathways, for example of amino acids such as lysine, methionine, threonine or tryptophan, vitamins such as vitamin A, B2, B6, B12, C, D, E or F, S-adenosylmethionine, biotin, pantothenic acid or folic acid, carotenoids such as (3-carotene, lycopene, canthaxanthin, astaxanthin or zeaxanthin or proteins such as hydrolases such as lipases, esterases, amidases, nitrilases, proteases, mediators such as cytokines, e.g. lymphokines such as MIF, MAF, TNF, interleukins such as interleukin 1, interferons such as y-interferon, tPA, hormones such as proteohormones, glycohormones, oligo- or polypeptide hormones such as vasopressin, endorphins, endostatin, angiostatin, growth factors, erythropoietin, transcription factors, integrins such as GPIIb/IIIa or a~(3III, receptors such as the various glutamate receptors, angiogenesis factors such as angiotensin.
The REMI method can also be used to site the transcription terminators of the invention, the abovementioned gene constructs or the aforementioned vectors comprising at least one of the transcription terminators of the invention in operative linkage to at least one rib gene at transcription-active sites in the genome.
The transcription terminators and/or said gene constructs are advantageously cloned together with at least one reporter gene into a further DNA construct which is introduced into the genome. This reporter gene ought to make detectability easy by a growth, fluorescence, chemo- or bioluminescence assay or by a photometric measurement. Examples of reporter genes which may be mentioned are antibiotic-resistance genes, hydrolase genes, fluorescent protein genes, bioluminescence genes, glucosidase genes, peroxidase gene or biosynthesis genes such as the riboflavin genes, the luciferase gene, (3-galactosidase gene, gfp gene, lipase gene, esterase gene, peroxidase gene, (3-lactamase gene, acetyl-, phospho- or adenyltransferase gene. These genes make it possible easily to measure and quantify the transcription activity and thus the expression of the genes. In the case where the biosynthesis genes themselves make detectability easy, it is possible to dispense with an additional reporter gene as, for example, in the case of riboflavin.
If a plurality of genes are to be introduced into the organism, it is possible to introduce them all together with one reporter gene in a single vector or each individual gene with one reporter gene in one vector into the organism, it being possible to introduce the various vectors simultaneously or successively. Gene fragments which code for the particular activities can also be employed in the REMI technique.
In principle all known restriction enzymes are suitable for the process for integrating regulatory sequences or gene constructs into the genome of organisms.
Restriction enzymes which recognize only 4 base pairs as restriction cleavage site are less preferred because they cut too frequently in the genome or in the vector to be integrated; preferred enzymes recognize 6, 7, 8 or more base pairs as cleavage site, such as BamHI, EcoRI, BglII, SphI, SpeI, XbaI, XhoI, NcoI, SalI, ClaI, KpnI, HindIII, SacI, PstI, Bpnl, NotI, SrfI or SfiI, to mention only a few of the possible enzymes. It is advantageous if the enzymes used have no further cleavage sites in the DNA to be introduced, which increases the integration efficiency. As a rule, from 5 to 500 U, preferably 10 to 250, particularly preferably to 100 U of the enzymes are used in the REMI
mixture. The enzymes are advantageously employed in an aqueous solution which contains substances for osmotic stabilization, such as sugars such as sucrose, 5 trehalose or glucose, polyols such as glycerol or polyethylene glycol, a buffer advantageously buffering in the range 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 10 acids, such as inorganic or organic salts of Mg, Cu, Co, Fe, Mn or Mo. It is also possible where appropriate for other substances to be present, such as EDTA, EDDA, DTT, (3-mercaptoethanol or nuclease inhibitors. However, it is also possible to carry out the REMI technique without these additions . 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 are suitable for the process, such as, for example, electroporation, fusion with loaded vesicles or destabilization using various alkali metal or alkaline earth metal salts such as lithium, rubidium or calcium salts, preferably lithium salts.
The transfer of foreign genes into the genome of a plant is referred to as transformation. In this case, the methods described for the 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-induced DNA uptake, the use of a gene gun, electroporation, incubation of dry embryos in DNA-containing solution, microinjection and agrobacterium-mediated gene transfer. Said processes are 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 cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). The transformation of plants with Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877.
Agrobacteria transformed with an expression vector according to the invention can likewise be used in a known manner for transforming plants, in particular crop plants such as cereals, corn, soybean, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and vine species, and legumes, e.g. by bathing wounded leaves or pieces of leaves in a solution of agrobacteria and then cultivating in suitable media.
The genetically modified plant cells can be regenerated by all methods known to the skilled worker. Appropriate methods can be found in the abovementioned publications by S.D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
The present invention also includes an organism which is able to produce riboflavin and comprises at least one transcription terminator as shown in 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.
The organism is advantageously one from the group of genera Corynebacterium, Brevibacterium, Bacillus, Clostridium, Escherichia, Cyanobacter, Ashbya, Eremothecium, Pichia, Candida or Saccharomyces or plants such as arabidopsis, corn, soybean, oilseed rape, barley, wheat, rye, millet, oats, sugar beet, potato, sunflower, legumes or tomato. A preferred organism is selected from the group of genera Bacillus, Corynebacterium, Brevibacterium, Escherichia, Candida, Eremothecium or Ashbya or corn, soybean, oilseed rape, barley, wheat, potato or tomato. It is particularly preferably Ashbya gossypii, Eremothecium ashbyii, Saccharomyces cerevisiae, Candida flaveri, Candida famata, Corynebacterium ammoniagenes or Bacillus subtilis. It is in particular Ashbya gossypii or Eremothecium ashbyii.
The present invention additionally includes organisms which are distinguished as riboflavin-producing mutants or producer strains. These can be prepared for example starting from wild-type strains by classical (chemical or physical) or genetic engineering methods and, where appropriate, have further genetic modifications than those in the framework of the rib genes.
The organism of the invention is further distinguished by showing, compared with the wild type of the genus Ashbya ATCC 10895, an increased transcription rate of at least one rib gene which is operatively linked to one of the transcription terminators as shown in SEQ ID
No. 1, SEQ ID No. 2 or SEQ ID No. 3. The organism of the invention is also capable of improved riboflavin production compared with the wild type of the genus Ashbya ATCC 10895.
The organisms used for producing riboflavin are cultured in the process of the invention in a medium which allows these organisms to grow. This medium may be a synthetic or a natural medium. The media used depending on the organism are known to the skilled worker. For growth of the microorganisms, the media used contain a carbon source, a nitrogen source, inorganic salts 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 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 amino saccharides, and the latter can also be used as nitrogen source.
Advantageous nitrogen sources are organic or inorganic nitrogen compounds or materials which contain these compounds. Examples are ammonium salts such as NH4C1 or (NH9)2504, nitrates, urea, or complex nitrogen sources such as corn steep liquor, brewer's yeast autolysate, soybean meal, wheat gluten, yeast extract, meat extract, casein hydrolysate, yeast or potato protein, which can often also serve as carbon source.
Examples of inorganic salts are the salts of calcium, magnesium, sodium, cobalt, molybdenum, manganese, potassium, zinc, copper and iron. The anion in these salts to be particularly mentioned is the chloride, sulfate or phosphate ion. An important factor for increasing the productivity in the process according to the invention is the control of the Fe2+ or Fe3+ ion concentration in the production medium.
Further growth factors are added to the nutrient medium where appropriate, such as, for example, 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 said nutrients depends on the type of fermentation and will be established in the individual case. The medium components may all be present at the start of the fermentation after they have been, if necessary, sterilized separately or sterilized together, or else be subsequently added continuously or discontinuously during the fermentation as required.
The cultivation conditions are established so that the organisms grow optimally and that the best possible yields are obtained. Preferred cultivation temperatures are from 15°C to 40°C. Temperatures between 25°C and 37°C are particularly advantageous. The pH is preferably kept in the range from 3 to 9. pH values between 5 and 8 are particularly advantageous. An incubation time of from a few hours to some days, preferably from 8 hours to 21 days, particularly preferably from 4 hours to 14 days, is generally sufficient. During this time, the maximum amount of product accumulates in the medium.
The possibilities for advantageous optimization of media can be found by the skilled worker for example in the textbook Applied Microbiol Physiology, "A Practical Approach (Eds. P.M. Rhodes, P.F. Stanbury, IRL-Press, 1997, pages 53-73, ISBN 0 19 963577 3}. Advantageous media and cultivation conditions are to be found for Bacillus and other organisms for example in the publication EP-A-0 405 370, specifically Example 9, for Candida in the publication WO 88/09822, specifically Table 3, and for Ashbya in the publication by Schmidt et al. (Microbiology, 142, 1996: 419-426).

The process of the invention can be carried out continuously or batchwise in a batch or fed-batch procedure.
Depending on the initial level of productivity of the organism used, the riboflavin productivity can be increased to varying extents by the process according to the invention. The productivity can usually be increased advantageously by at least 50, preferably by at least 100, particularly preferably by 200, very particularly preferably by at least 1000, in each case compared with the initial organism.
The present invention also includes the use of an organism of the type according to the invention comprising at least one transcription terminator as shown in 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. The organism is preferably Ashbya gossypii.
The present invention likewise relates to the use of at least one transcription terminator as shown in SEQ ID
No. 1, SEQ ID No. 2 or SEQ ID No. 3 for preparing a producer organism for improved production of riboflavin. The invention likewise includes the use of at least one transcription terminator as shown in SEQ
ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 for increasing the transcription rate of genes involved in riboflavin production. The present invention thus also relates to the use of at least one transcription terminator as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 for the improved production of riboflavin.
The present invention is explained in more detail, but not limited, by the following examples.
General methods:

General nucleic acid methods such as, for example, cloning, restriction cleavages, agarose gel electrophoresis, linkage of DNA fragments, transformation of microorganisms, culturing of bacteria and sequence analysis of recombinant DNA were, unless described otherwise, carried out as described in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87959-309-6).
Recombinant DNA molecules were sequenced using a laser fluorescence DNA sequencer from ABI by 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.
Isolation of the rib genes:
The isolation of the rib genes 1,2,3,4,5 and 7 from Ashbya gossypii and Saccharomyces cerevisiae is described in the patents WO 95/26406 and WO 93/03183 and specifically in the examples, and was carried out correspondingly. Express reference is hereby made to these publications.
Terminator region sequence comparison Starting from the isolated rib genes, the terminator region of the rib2 gene was also identified and analyzed. This revealed the following sequence:
Terminator of the rib2 gene of Ashbya ATCC 10895 (SEQ
ID No. 4) Stop-TGATTTTGCTGCGAATTGTAGATGG
The terminators of the invention have the following sequences:
Terminator of the invention as shown in (SEQ ID No. 1):
Stop-TGATTTTGCTGCAAATTGTAGATGG

Terminator of the invention as shown in (SEQ ID No. 2):
Stop-TGATTTTGCTGCGAATTGTAGATGA
Terminator of the invention as shown in (SEQ ID No. 3):
Stop-TGATTTTGCTGCAAATTGTAGATGA
Different nucleotides are demonstrated by underlining.
The procedures for synthesizing nucleotide sequences or for replacing or mutagenizing single nucleotides were carried out in accordance with conventional laboratory practice and are described inter alia in D.M. Glover et al., DNA Cloning Vol.l, (1995), IRL Press (ISBN 019-963476-9).
The sequences of the terminators of the invention and of 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.

SEQUENCE LISTING
<110> BASF Aktiengesellschaft <120> Process for producing 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>
<223> Terminator 2 <220>
<221> terminator <222> (1)..(25) <223> Terminator 2 <400> 2 tgattttgct gcgaattgta gatga 25 <210> 3 <212> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Terminator 3 <220>
<221> terminator <222> (1)..125) <223> Terminator 3 <9C0> 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)

Claims:

1. A process for producing riboflavin, where a) an organism which is able to produce riboflavin and which has at least one transcription terminator as shown in SEQ ID No. 1, SEQ ID No.
2 or SEQ ID No. 3, where the particular transcription terminator is operatively linked to at least one rib gene, is cultured, and b) the formed riboflavin is recovered from the culture medium.

2. The process according to claim 1, wherein the operative linkage is to at least one gene from the group of rib1, rib2, rib3, rib4, rib5 or rib7.

3. The process according to either of claims 1 or 2, wherein the transcription terminator as shown in SEQ ID No. 1 is used, the sequence thereof being modified compared with the sequence of the transcription terminator of the rib2 gene of the wild type ATCC 10895 at position 13 by replacement of guanine by adenine.

4. The process according to either of claims 1 or 2, wherein the transcription terminator as shown in SEQ ID No. 2 is used, the sequence thereof being modified compared with the sequence of the transcription terminator of the rib2 gene of the wild type ATCC 10895 at position 25 by replacement of guanine by adenine.

5. The process according to either of claims 1 or 2, wherein the transcription terminator as shown in SEQ ID No. 3 is used, the sequence thereof being modified compared with the sequence of the transcription terminator of the rib2 gene of the wild type ATCC 10895 at positions 13 and 25 by replacement of guanine by adenine.

6. The process according to any of claims 1 to 5, wherein a bacterium, a yeast, a fungus or a plant is employed as organism able to produce riboflavin.

7. The process according to any of claims 1 to 6, wherein organisms selected from the group of genera Corynebacterium, Brevibacterium, Bacillus, Clostridium, Escherichia, Cyanobacter, Ashbya, Eremothecium, Pichia, Candida or Saccharomyces or plants such as arabidopsis, corn, soybean, oilseed rape, barley, wheat, rye, millet, oats, sugar beet, potato, sunflower, legumes or tomato are employed.

8. The process according to any of claims 1 to 7, wherein Ashbya gossypii, Eremothecium ashbyii, Saccharomyces cerevisiae, Candida flaveri, Candida famata, Corynebacterium ammoniagenes or Bacillus subtilis are used as organisms.

9. The process according to any of claims 1 to 8, wherein Ashbya gossypii or Eremothecium ashbyii is used.

10. A transcription terminator having a sequence as shown in SEQ ID No. 1.

11. A transcription terminator having a sequence as shown in SEQ ID No. 2.

12. A transcription terminator having a sequence as shown in SEQ ID No. 3.

13. The transcription terminator according to claim 10, which is modified compared with the transcription terminator of the rib2 gene of Ashbya ATCC 10895 at position 13 by replacement of guanine by adenine.

14. The transcription terminator according to claim 11, which is modified compared with the transcription terminator of the rib2 gene of Ashbya ATCC 10895 at position 25 by replacement of guanine by adenine.

15. The transcription terminator according to claim 12, which is modified compared with the transcription terminator of the rib2 gene of Ashbya ATCC 10895 at positions 13 and 25 by replacement of guanine by adenine.

16. A gene construct comprising at least one transcription terminator according to any of claims 10 to 15 and at least one rib gene which is operatively linked to the particular terminator.

17. The gene construct according to claim 16, which comprises at least one gene from the group of rib1, rib2, rib3, rib4, ribs or rib7.

18. A vector comprising at least one transcription terminator according to any of claims 10 to 15 or a gene construct according to either of claims 16 or 17, and additional nucleotide sequences for selection and for replication in the host cell or for integration into the host cell genome.

19. An organism which is able to produce riboflavin, comprising at least one transcription terminator according to any of claims 10 to 15 or a gene construct according to either of claims 16 or 17 or a vector according to claim 18.

20. The organism according to claim 19, selected from the group of genera Corynebacterium, Brevibacterium, Bacillus, Clostridium, Escherichia, Cyanobacter, Ashbya, Eremothecium, Pichia, Candida or Saccharomyces or plants such as Arabidopsis, corn, soybean, oilseed rape, barley, wheat, rye, millet, oats, sugar beet, potato, sunflower, legumes or tomato.

21. The organism according to either of claims 19 or 20, which is Ashbya gossypii, Eremothecium ashbyii, Saccharomyces cerevisiae, Candida flaveri, Candida famata, Corynebacterium ammoniagenes or Bacillus subtilis.

22. The organism according to any of claims 19 to 21, which is Ashbya gossypii or Eremothecium ashbyii.

23. The organism according to any of claims 19 to 22, which, compared with the wild type of the genus Ashbya ATCC 10895, shows an increased transcription rate of a rib gene which is operatively linked to a transcription terminator from the group of SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

24. The organism according to any of claims 19 to 23, which is capable of improved riboflavin production compared with the wild type of the genus Ashbya ATCC 10895.

25. The use of an organism according to any of claims 19 to 24 for the improved production of riboflavin.

26. The use of at least one transcription terminator according to any of claims 10-15 for preparing a producer organism for the improved production of riboflavin.

27. The use of at least one transcription terminator according to any of claims 10 to 15 for increasing the transcription rate of genes involved in riboflavin production.