CA2341715A1 - Organisms for the extracellular production of riboflavin - Google Patents

Abstract

The invention relates to a monocellular or multicellular organism, especiall y a microorganism for biotechnically producing riboflavin. The organism is characterized in that the intracellular processes thereof for transporting material are modified in such a way that the predominant part of riboflavin accumulates in an extracellular manner.

Description

ORGANISMS FOR THE EXTRACELLULAR PRODUCTION OF RIBOFLAVIN
The present invention relates to a unicellular or multicellular organism for preparing riboflavin using microorganisms.
Vitamin B2, also termed riboflavin, is essential for humans and animals.
Vitamin BZ deficiency results in inflammation of the oral and pharyngeal mucosae, tears in the oral commissures, pruritus and inflammation in the skin folds, inter alia, skin damage, conjunctivitis, diminished visual acuity and keratitis. Cessation of growth and loss of weight may occur in infants and children. Vitamin B2 is therefore of economic importance, in particular as a vitamin preparation for use in vitamin deficiency and as a feed additive. In addition, it is also employed as a foodstuff colorant, for example in mayonnaise, ice cream, blancmange, etc.
Riboflavin is prepared either chemically of microbially. In the chemical methods of preparation, riboflavin is as a rule obtained in multistep processes as a pure end product, although it is also necessary to employ relatively expensive starting materials, such as D-ribose. For this reason, the chemical synthesis of riboflavin is only suitable for those applications which require pure riboflavin, as is the case, for example, in human medicine.
The use of microorganisms to prepare riboflavin provides an alternative to preparing the substance chemically. The microbial preparation of riboflavin is particularly suitable for those instances when it is not necessary for this substance to be of high purity. This is the case, for example, when the riboflavin is to be employed as an additive in feed products. In such cases, the microbial preparation of riboflavin has the advantage that this substance can be obtained in a single-step process. It is also possible to employ renewable raw materials, such as vegetable oils, as starting materials for the microbial synthesis.
The preparation of riboflavin by fermenting fungi such as Ashbya gossypii or Eremothecium ashbyi has been disclosed (The Merck Index, Windholz et al. eds.
Merck & Co., page 1183, 1983; A. Bacher, F. Lingens, Angew. Chemie 1969, 393); however, yeasts, such as Candida or Saccharomyces, and bacteria, such as Clostridium, are also suitable for producing riboflavin.

Moreover, methods using the yeast Candida famata have been described, for example in US 05231007.
Bacterial strains which overproduce riboflavin are described, for example, in EP
405370, in which document the strains were obtained by transforming the riboflavin biosynthesis genes from Bacillus subtilis. The use of special mutants of Bacillus subtilis to prepare riboflavin by means of fermentation is also described in GB 1434299, in German Laid-Open Specification 3420310 and in EP 0821063.
However, these prokaryotic genes were unsuitable for a recombinant method for preparing riboflavin using eukaryotes such as Saccharomyces cerevisiae or Ashbya gossypii. Genes which were specific for riboflavin biosynthesis were therefore isolated, as described in WO 93/03183, from a eukaryote, namely from 1 S Saccharomyces cerevisiae, in order thereby to provide a recombinant method for preparing riboflavin in a eukaryotic production organism. However, such recombinant preparation methods are of no value, or only of limited value, for producing riboflavin when the provision of substrate is insufficient for the enzymes which are specifically involved in the riboflavin biosynthesis.
German Patent Specification 195 25 281 discloses a method for preparing riboflavin which involves culturing microorganisms which are resistant to substances which have an inhibitory effect on isocitrate lyase.
German Laid-Open Specification 19545468.5-41 discloses a microbial method for preparing riboflavin in which the isocitrate lyase activity, or the isocitrate lyase gene expression, of a riboflavin-producing microorganism is increased.
However, all the previously known methods suffer from the disadvantage that a large part of the riboflavin accumulates intracellularly. Consequently, the cell has to be disrupted in order to isolate this intracellular riboflavin.
Accordingly, it has not so far been possible to produce riboflavin continuously in a biotechnological manner.
Consequently, it is an object of the present invention to make available a unicellular or multicellular organism, preferably a microorganism, for preparing riboflavin biotechnologically, which organism enables the riboflavin to be isolated without disrupting the cells and consequently makes it possible to produce the riboflavin continuously, inter alia.

We have now found that this object is achieved by means of a unicellular or multicellular organism whose intracellular substance transport processes are altered such that the majority of the riboflavin accumulates extracellularly.
According to the invention, preference is given to the transport, or the rate of transport, of the riboflavin into the vacuole being decreased, such that the majority of the riboflavin is released through the cytoplasmic membrane into the fermentation medium. Particular preference is given to the transport of the riboflavin into the vacuole being at least partially blocked. The greatest preference is given to the transport of the riboflavin into the vacuole membrane being completely blocked.
The aim of this sought-after change in the intracellular transport of materials can be achieved using the known methods for improving organism strains; i.e., in the simplest case, appropriate strains can be obtained by means of screening following the selection which is customary in microbiology. It is also possible to use mutation with subsequent selection. In this connection, the mutation can be carned out either by means of chemical mutagenesis or by means of physical mutagenesis.
Another method comprises selection and mutation with subsequent recombination.
Finally, the organisms according to the invention can be prepared by means of genetic manipulation.
According to the invention, preference is given to the organism being changed in such a way that it almost exclusively produces riboflavin extracellularly.
According to the invention, this increase in the extracellular production of riboflavin can be achieved, for example, by preparing an organism in which the enzyme activity of the vacuolar H+-ATPase (V-ATPase) is decreased or completely blocked. This results in the transport of the riboflavin through the vacuole membrane being partially or completely inhibited.
This situation can be achieved, for example, by altering the catalytic center so as to decrease substrate turnover or by increasing the effect of enzyme inhibitors.
It is also possible to decrease the enzyme activity of the V-ATPase by decreasing synthesis of the enzyme, for example by eliminating factors which activate biosynthesis of the enzyme.
According to the invention, the ATPase activity can preferably be decreased or completely inhibited by mutating, inactivating or removing the ATPase gene.

Mutations of this nature can either be generated in a random manner by means of classical methods, for example using LTV irradiation or mutation-inducing chemicals, or else generated specifically by means of recombinant methods, such as deletion, insertion, or nucleotide exchange or substitutions in the structural gene or the regulatory elements, promoters and transcription factors which are associated with this gene.
Accordingly, expression of the ATPase gene can be decreased or completely blocked by, for example, removing or inactivating the ATPase gene and/or altering regulatory factors which affect expression of the gene. Thus, it is possible to decrease regulatory elements, preferably at the transcriptional level, by, in particular, altering the transcription signals. However, in addition, it is also possible to decrease translation by, for example, altering the mRNA.
According to the invention, the ATPase gene, which has been altered in the above-described manner, can be incorporated into a gene construct or into a vector.
A
riboflavin-producing microorganism is then transformed with this gene construct or vector.
According to the invention, expression of the V-ATPase gene can also be decreased or completely inhibited by exchanging the promoter. In this connection, it is possible to achieve the decrease in enzymic activity either by incorporating gene copies or, alternatively, by exchanging the promoter. However, it is also equally possible to achieve the desired change in the enzyme activity by simultaneously exchanging the promoter and incorporating gene copies.
Any arbitrary organisms whose cells contain the sequence for forming the V-ATPase, that is plant and animal cells as well, are suitable for isolating the gene according to the invention. The altered gene is preferably isolated from microorganisms, particularly preferably from fungi. Particular preference is given to fungi of the genus Ashbya. The greatest preference is given to the species Ashbya gossypii.
The gene can be isolated by homologously or heterologously complementing a mutant in which the ATPase is defective or in which the ATPase is deleted. The gene can also be isolated by way of a PCR assay using degenerate primers based on amino acid homology with previously known Vma proteins and then screening a gene library. The gene can then be completely sequenced by subcloning (cutting with suitable restriction enzymes and cloning the resulting fragments into vectors) the complementing clone or the positive clone from the screened gene library.
Disruption constructs (deletion/substitution alleles) can be prepared by excising a part of the sequence of the gene with restriction enzymes and replacing this fragment with an antibiotic-resistance cassette plus a promoter on a plasmid.
Such S disruption constructs can then be employed for transforming a riboflavin producer.
Following isolation and sequencing, it is possible to obtain the genes according to the invention containing nucleotide sequences which preferably encode the amino acid sequence shown in Figure 1 or its allelic variant. Allelic variants comprise, in particular, derivatives which can be obtained by deleting, inserting and substituting nucleotides in corresponding sequences, with the V-ATPase activity being retained. In the abovementioned amino acid sequence, a corresponding sequence is the region from nucleotide 1 to nucleotide 3881, with AGVMAI itself corresponding to nucleotides 910-2763 in the sequenced region, as shown in Figure 2.
A promoter of the nucleotide sequence from nucleotide 1 to nucleotide 3881, in accordance with the abovementioned amino acid sequence, or a DNA sequence having essentially the same effect, can, in particular, be placed upstream of the genes according to the invention. Thus, a promoter which differs, by means of one or more nucleotide exchanges or by the insertion and/or deletion of nucleotides, from the promoter having the given nucleotide sequence can, for example, be placed upstream of the gene. Furthermore, the activity of the promoter can be altered by altering its sequence, or the promoter can be completely replaced with other promoters.
Furthermore, regulatory gene sequences or regulatory genes, which decrease the activity of the V-ATPase gene, in particular, can be assigned to the V-ATPase gene. Thus, expression of the V-ATPase gene can, for example, be decreased by altering the interaction between the RNA polymerase and the DNA.
One or more DNA sequences can be placed upstream and/or downstream of the V-ATPase gene, which has been altered or inactivated in accordance with the invention and which does or does not possess an upstream promoter and/or does or does not possess a regulatory gene, such that the gene is contained in a gene structure. By cloning the gene according to the invention, it is possible to obtain plasmids or vectors which contain the altered or inactivated V-ATPase gene, or do not contain V-ATPase gene, and are suitable for transforming a riboflavin producer. The cells which can be obtained by transformation contain the gene in replicatable form, i.e. in additional copies on the chromosome, with. the gene copies being integrated by homologous recombination at arbitrary sites on the genome and/or being on a plasmid or vector.
Another option for increasing the riboflavin which accumulates extracellularly consists in increasing the rate at which the riboflavin is transported through the cytoplasmic membrane into the fermentation medium. This thereby displaces the equilibrium between transport through the vacuole membrane and transport through the cytoplasmic membrane in such a way that the majority of the riboflavin accumulates extracellularly.
This can be achieved, for example, by expressing the gene which encodes the transport more powerfully or by increasing its activity.
The altered unicellular or multicellular organisms according to the invention can be any arbitrary cells which can be employed for biotechnological processes.
These cells include, for example, fungi, yeasts, bacteria and plant and animal cells.
According to the invention, the cells are preferably transformed fungal cells, particularly preferably fungal cells from the order of the Endomycetales. The Saccharomycetaceae family is particularly preferred. Fungi of the genus Ashbya and Eremothecium are very particularly preferred. In this connection, the species Ashbya gossipii and Eremothecium ashbyii are most highly preferred.
In principle, the described changes in the transport processes can also be achieved by altering the fermentation conditions. In particular, an at least partial blockage of the intracellular accumulation of riboflavin can be achieved by adding chemical substances to the fermentation medium. For example, according to the invention, an inhibitor which inactivates the V-ATPase can be added to the fermentation medium.
Examples of such substances are concanamycins, bafilomycins, N-ethylmaleimide and nitrate.
In that which follows, the invention is explained in more detail with the aid of examples, without this being associated with any limitation to the subject-matter of the examples:
1. Compartmentalization of riboflavin in A. ~ssypii under production conditions _7_ 1.1 Permeabilization technique The selective permeabilization technique was used, in four independent experiments, to determine the production and compartmentalization of the more strongly producing mutant ItaGS01 with or without the addition of concanamycin A. For this, the cells were grown on production medium (yeast extract: 10 g/l;
soybean oil: 10 g/1; glycine: 6 g/1) and harvested at defined times during the course of production. The technique of selectively permeabilizing the A. gossypii plasma membrane was used for the compartmentalization analysis. The aim of selectively permeabilizing the plasma membrane is to be able to analyze the cytosolic and vacuolar compartments separately. For this, the mycelium was harvested by filtration (circular glass fiber filter, diameter 4.5 cm), at the desired time, and washed with NaP buffer. The fungal cells (0.5 g moist mass) were resuspended in 10 ml of permeabilization solution, 0.003% (w/v) digitonin in 50 mM NaP
buffer, and incubated in this permeabilization solution, at 30°C for 10 min and at 120 rpm, in a laboratory shaker, in order to permeabilize the plasma membrane. At the end of the incubation, the cells were separated from the solution by filtration and the filtrate was used for analyzing cytosolic cell constituents. The cell suspension was incubated for a further 10 min with 0.02% (w/v) digitonin in order to destroy all the cellular membranes. It was then possible, by filtration, to isolate the vacuolar content of the cells for analyses (determination of riboflavin). The cell residues which remained on the filter were discarded.
1.2 Quantitative determination of dry biomass and riboflavin The riboflavin content of the permeabilization filtrates is determined by HPLC
under the following separation conditions:
Column: LiCrospher ~ 100 RP-18 (5 Vim) (Merck, Darmstadt) Eluent: 50 mM NaHZP04 1 mM Tetramethylammonium chloride 12% Acetonitrile Flow rate: 1 ml/min Elution: Isocratic Detection: 270 nm The riboflavin content of the individual compartments was then related to the dry biomass employed in order to calculate the compartmentalization between - g medium, cytosol and vacuole. In order to determine the dry fungal mass, 20 -50 ml of soybean oil culture were first of all transferred to SO ml Falcon tubes and centrifuged at 1500 x g for 10 min; the upper oily layer was then carefully removed with a pipette. After that, the oil-free suspension was then filtered off through tared circular glass fiber filters and washed with a 10-fold volume of NaP buffer.
The mycelium on the filter was dried to constant weight at 60° in a drying oven and then weighed.
1.3 Compartmentalization of riboflavin underproduction conditions The cytosolic and vacuolar contents of riboflavin, and the quantity of riboflavin secreted into the medium, were in each case determined for control and concanamycin-treated cells (5 pM) at the 3 time points of 24 h, 43 h and 67 h:
The diagrams clearly show (as can also readily be seen optically or using the light 1 S microscope) that the cells of the mutant ItaGS01 can no longer store any riboflavin in their vacuoles when the V-ATPase inhibitor concanamycin A is added to the medium at the beginning of the production phase. The total production is the same in control and concanamycin A assays, with the quantity of riboflavin which is otherwise stored within the vacuoles (30 - 60% of the total production) being secreted into the medium when concanamycin A is added.
2. Cloning, sequencing and disruption of the A. ~ossypii VMA1 -eene An experiment was undertaken to reproduce the diversion in the flow of riboflavin, which was achieved using the inhibitor concanamycin A, molecular biologically by constructing a strain having a dysfunctional V-ATPase. To do this, the catalytic subunit A of the V-ATPase complex was cloned and disrupted. The catalytic subunit A is encoded by the VMAl gene (v_acuolar membrane ATPase protein 1).
2.1 Cloning and seguencing A fragment of the AgVMAl gene (evolutionarily st:ongly conserved) was amplified by PCR (degenerate oligonucleotide primers designed from highly conserved sequences in the VMAIp alignment): for the PCR, degenerate oligonucleotide primers were developed from highly conserved amino acid segments in various VMA1 proteins: CFla (5-ATYCARGTBTAYGARAC-3) and GFIb (5-ATVACRGTYTTRCCRCA-3) were ordered from MWG Biotech (Ebersberg). The PCR (30 s 94°C, 60 s 52°C, 60 s 72°C, 35 cycles) was earned out in the Gene Amp~ PCR System 9700 (Applied Biosystems) using Taq polymerase (Boehringer Mannheim, Germany), buffer as recommended by the manufacturer, 8 uM primer CFIa and 4 p,M primer CFIb. The amplified PCR fragment was then used to screen a cosmid gene library (cosmid vector SuperCos 1, Stratagene) prepared from A. gossypii. In order to identify positive cosmid clones which contained homologous regions of DNA, the PCR fragment was radioactively labeled with (a-32P)dCTP using T7 polymerase. The cosmid DNA of the positive clones was then digested with BamHI and screened once again with the radioactive PCR fragment. The gene was then sequenced after having being subcloned in the Bluescript, Stratagene, plasmid vector. The nucleotide sequence can be obtained in the EMBL database, under accession number AJ009881, after the patent and the appurtenant publication have been filed. Aligning the AgVMAIp protein with the V-ATPase A subunits of various other organisms showed that a strongly conserved protein sequence is also present in Ashbya. In addition, the phylogenetic genealogical tree for the VMA1 proteins showed that, in AgVMAlp, that VMA1 protein had been found, among those so far known, which was most closely related to the corresponding protein in the yeast S. cerevisiae. Genomic Southern analysis confirmed that the gene is present as a single copy (as, incidentally, in yeast as well).
2.2 Disruption of the vmal~ne For the disruption, a vmal deletion/substitution allele, termed vmal: 6418, was constructed. For this, the 0.25 kb Barnes 1 /Pst 1 fragment in the VMA 1 PCR
fragment, which former fragment contains a part of the VMA1 ORF, was replaced with the geneticin-resistance cassette TEF-6418. The VMA1 PCR fragment was firstly cloned into a modified pGEM~T vector (Promega) without any Pstl cleavage site, resulting in the generation of the plasmid pJR1767. The sequence encoding VMA1 on the plasmid was then disrupted by replacing the 0.25 kb BamHl Pstl region with the TEF-6418 marker, resulting in the generation of the plasmid pJR1773. Finally, the linear 2.5 kb Ncol/Spel fragment from this plasmid was used to transform germinated spores of A. gossypii (starting strains: wild type and the more strongly producing mutant ItaGS01 ). Geneticin-resistant clones were selected and disruption of the vmal gene was confirmed by Southern blotting (Fig. 2).
2.3 Diversion of the flow of riboflavin by disrupting VMA-1 On plates, the disruptants exhibit characteristic colonies which grow thickly and in a crated manner, in contrast to the flat growth exhibited by the starting strains.

While the cells secrete riboflavin (agar plates colored yellow), the hyphae themselves are completely white and no longer store any riboflavin in their vacuoles (first demonstrated by means of fluorescence microscopy). On the other hand, the hyphae of the starting strains are colored yellow by the riboflavin which S is stored in the vacuoles (often even in the form of crystals).
The compartmentalization of the riboflavin was once again examined quantitatively using the selective membrane permeabilization technique (see 1.1.).
By disrupting the V-ATPase A subunit (as above when adding the V-ATPase inhibitor concanamycin), it was possible to divert the flows of riboflavin. 3 new strains which do not accumulate riboflavin in their vacuoles (product retention) are therefore now available.
Comments on the figures:
Fig.l: Compartmentalization of riboflavin in Ashbya under standard production conditions (left-hand column) and when production is taking place in the added presence of the V-ATPase inhibitor concanamycin A
Fig. 2: Disruption of AgVMAI with the TEF-6418 marker, (A) construction, (B) Southern analysis T1: Wild strain disruptant T2: ItaGS01 disruptant Fig. 3: Compartmentalization of riboflavin in the starting strain and in the ItaGS01 mutant disruptant - S -SEQUENCE LISTING
<110> BASF Aktiengesellschaft <120> Organisms for preparing riboflavin extracellularly <130> 1 <140> 198 39 567.1 <141> 1998-08-31 < 160> 2 <170> PatentIn Ver. 2.0 <210> 1 <211> 3699 <212> DNA
<213> Ashbya gossypii <220>
<221 > CDS
<222> (910)..(2763) <400> 1 ggatccgaca gctagtcata gataagcaga accagcgcgt cctccccaca tataaccgcc 60 tactcgaccg catggaggac gcgctcgt;c- gctggcggcc gcccgccagc tcccacgtcg 120 gctcctcgct agcaatccac ggcacgcacc cgtaccaaaa agagctgccc gagcccaagg i80 cgctcggccg cgacctctca gagtcgatcg aacaaatgaa gcagatcttg cagaactgtt 240 ggcccacgcg cgacctgcat accgatgcca ccgacgacag cgagaccctc agcgactatt 300 cccttccgcg cgccggctcg gccaccgcga acagcgccaa gcccaacggg ccctcgttcg 360 aaacgctgtc cctgagcagg cgcacgccgg ttgcattccc catcaaggcg gccggcgccc 420 gcgcggaccc gcctccggac gacctccaga gcacgctcaa cacctacaag ttccccgttc 480 cctcttacta ccttccgctg tcgctccagc gtcagtgcca acagccaagt acacgtgcgc 540 ccgcaagcga cgccgaacaa agcacaccat gggaggcctg gtgccaattc tggaaggagt 600 tctgggttga tctccacagc ttactcgtct gtcagatcga cgaagagctg tattagatag 660 gcttcattaa cgtatatatg ggaaatga=t tttacatctc tacaccacag ccaaacgcaa ~z0 ccagcaagcg ctctgcgaaa cagggggtca actggtgaac accgcacgcc ggtggttcgc 730 acggtaaaca cacaagcagt tcaaacgcag catacgtgca cagacagaga gcccgaatct 640 cgcggaccgt ccagagcaca gcccaaagca ggtggagttc atagatagca gaaaatca~c 9v0 attgcagag atg gca gga get ttg gaa aac geg egc aag gas ate aag ega 951 Met A2a Gly A_La Leu Glu Asn Ala Arg :.ys Glu Ile Lys Atg atc tct ttg gag gac csc aat gag aac gas tac ggg cag ata tac tcc 999 Iie Ser Leu Glu Rsp His Asn Glu Asn Glu Tyr Gly Gln Ile Tyr Ser 15 2~ ~5 30 gtg tcc ggc ccg gtg gtc gtg gcg gag aac atg gtt gga tgc gcg atg 1047 Va'_ Ser Gly pro val Va. Val Aia Giu As.~. Met Val ~-ly Cys Ala Met tac gag ctt gtg aag gtg ggc cac cac aac ctg gtt ggg gag gtg atc 1095 Tyr Glu Leu Val Lys Vai Gly His E~s As.~. Leu Val Glv_ Glu Val Ile cgg ctg gac ggc gac aag gcg acg acc cag gtg :ac Sag gag acg gcg 1193 Arg Leu F.sp Gly Fsp Lys F?a 2hr Ile Gln Vai Tyr Glu Glu Thr Aia 65 70 7~
ggc gtg acg gtc ggc gat cct gtg ctg cgg acg ggc aag ccg ctg tcg 1191 GIy Val Thr Val Gly Asp Pro Val i,eu Arg The G!_; Lys Prc Leu Se, 80 8~ 90 gtg gag ttg ggt ccg ggg ttg atc gag acc atc tac aac ggg atc cac 1239 Val Glu Leu Gly Pro Gly Leu Met Glu T!:r Ile :'y= Asp Giy I1e Gln cgg ccg ctg aag gcg atc aaa gag cag ~cg cag ~cc atc tac atc ccg 1287 Arg Pro Leu Lys P1a Ile Lys Giu Gin Ser G'~.~. Se. =ie Tyr Ile pro 115 1?'? i.5 cgc ggc gtc gac gca cca gcg ttg agc cgc gag gtg aag tgg gcg ttc '335 Arg Gly Vai Asp Ala Prc Ala Leu Ser P.rg Glu Val Lys Trp i-1a Phe aag ccg ggc aag ctg ggc gtt ggc gac cac ata tcc ggc ugc gat att 1323 Lys Pro Gly Lys Leu Gly Val Giy Asp :l~s Ile Sea Gly Gly Asp Ile I95 150 15g ttt ggg tcg atc ttt gag aac tct ctc tta gag gac cac aag atc ctg _431 Phe Gly Ser Iie Phe G'_u Asn Ser Leu Leu Glu Asp H~~s Lys Ile Leu ctt cca ccg cgg get cgg ggg zcc att acg tgg att gca cct gca ggc 1::79 Leu Pro Pro Arg Ala Arg Gly Thr Iie Thr Trp I1e Ala Pro Ala Gly 175 i80 I85 i90 gaa tac act gtc gac gaa acg gta ctg gag gtg gag ttc gac ggc aaa 1527 Glu Ty~ Thr Va'_ Asp Glu Thr Val Leu vlu Va'_ Flu Phe Asp Gly ~ys aag tac tca tac tcc atg ttc cac aca tgg ccc gtg cgt gtg cca agg '575 Lys Tyr Ser Tyr Ser Met Phe His Thr Trp Pro Val Arg Vai Pro Arg cct gta acc gaa aag ttg tct gcc gac tac ccg cta ttg acg ggg caa 1023 Prc Val Thr Glu Lys Leu Ser AIa Asp Tyr Prc Leu Leu Thr G1y Gln 23~~

aga gtg ctt gat tca ctg ttc cca tgt gtc cag ggc ggt acc acc tgt 1671 Arg Vai :~eu Asp Ser Le1 ?he Pro Cys Val Gln Gly Gly Thr Thr Cys 2~5 att ccc ggt gcg ttt ggg tgc gga aag act gtc att tcc cag tcc cta '_719 Ile Prc Gly Ala Phe G_y Cys Gly T_ys T!-:r 'vaI Ile Ser Gln Ser Leu 255 2c'0 26~ 270 tcc aaa tac tcc aac tcg gac gcc att atc tat gtc ggg tgt ggt gag 1767 Ser Lys Tyr Ser Asn Ser Asp Ala _'le Iie Tyr Val Gly Cys Gly Glu cgt ggt aat gag atg gcg gaa gtg ttg atg gag ttc ccc gag cta ttc 1815 Arg Gly Asn Giu Met Ala Glu Val Leu Met Giu Phe Prc Glu Leu Phe act gag gtc aat ggt aga aag gag cct att atg aaa cgt acc act ctg 1803 Thr Glu Val Asn Gly Arg Lys Glu Fro ile Met L..ls Arg Thr Thr Leu 305 310 3.5 gtg get aae act tet aac atg cct gtg get gec aga gag get teg att 1911 Val Ala Asn Thr Ser Asa Met Pro Val Ala Ala Arg Giu Aia Ser Ile tac act ggt att act ctg gcc gag tat ttc aca gac caa gqt aag aac 1959 Tyr Thr Gly IIe Thr Leu Aia Glu Tyr Pne Arg Asp Gln Gly Lys Asn 335 gq~i 3 =5 35G

g gtc tcc atg att gca gac tcc tcg tcc aga tgg gcc gag gca ttg aga 2007 vai Ser Met Iie Ala Asp Se. Ser Ser Arg Trp Ala GIu Aia Leu Arg gaa att tcg ggg cgt ctt ggg gag atg cct gcc gac cag ggt ttc ccg 2055 Glu Ile Ser Gly Arg Leu Gly Glu Met Pro Ala Asp Gin G1y P::e Pro gcc tat ctc ggc gcc aag cta gcc tcc ttc tat gaa aga gca ggg aag 2103 Ala Tyr Leu Gly Ala Lys Leu Ala Ser Phe Tyr Giu Arg Pea Gly Lys gcg gtt gca ctg ggc tct ccc gac aga gtc ggt tcg gtt tct atc gtc 2I5i Ala Val Ala Leu GIy Ser Pro Asp Arg val Gly Ser Val Ser Ile val gca get gtc tcg eea get ggc ggt gac ttc teg gac ect gtc act act 2199 Rla Ala Vai Ser Pro Ala G1y Gly Asp Phe Ser Asp P=o Val Thr Thr tcg acc ttg ggt att act cag gtg ttt tgg ggt ttg gac aaa aag ctt 2247 Ser Thr Leu Gly Ile Thr ~~ln Val Phe Trp Gly Leu Asp Lys Lys Lei gcc cag aga aag cat ttc cct tcg atc aac acg tct gtc tcg tac tcg 2295 Psa Gln Arg Lys His Phe Pro Ser Ile Asn Thr Ser Val Ser Tyr Ser aag tac acc aac gtc cta aac aaa tac tat gat agc aat tac ccc gag 2343 Lys Tyr Thr Asn Val Leu Asn Lys Tyr Tyr Asp Ser Asz Tyr Pro Glu ttc cca gtc ctg aga gac cgt atc aag gag atc ctc tcc aac gcc gag 2391 Phe Pro Val Leu Arg Asp Arg Ile Lys Glu Ile Leu Ser Asn Ala Glu gaa ttg gag caa gtt gtt cag ctg gtc ggt aag tcc gcg ctc tct~gac 2439 G~~u Leu Glu Gln Val Val Gln Leu Val Gly Lys Ser Ala Leu Ser Asp aaa gac aag atc gta ctg gat gtc gcg acg cta atc aaa gaa gac ttc 2487 Lys Asp Lys Ile Val Leu Asp Vai Ala Thr Leu T_le Lys Glu Asp Phe ttg cag cag aac ggt tac tcg acc tac gac gca ttc tgc ccc atc tgg 2535 Leu Gin Gin Asn Gly Tyr Ser .Thr Tyr Asp A2a ?he Cys Pro ile Trp aag acc tac gat atg atg aaa gcg ttt gtg tcc X583 tac ttc gac gag gca Lys Thr Tyr Asp Met Met Lys Ala Phe Val Ser Tyr Phe Asp Glu Ala caa aag agc gtg tcc aac ggc gcc aac tgg gcc 2031 gtg ctg tcg gag gca Gln Lys Ser Val Ser Asn Gly Ala Asn Trp Ala Val Leu Ser Glu Ala acc ggc gac gtg aaa cac gcc gtt tcg tcc tcc 2679 aag ttc ttc gag ccc Thr Gly Asp Val Lys iiis A1a Vai Ser Ser Se_-Lys Phe Phe Glu Pro age egc ggc gag cgc gag gte cac gca gag ttc gaa aag ctc ttt get Ser Arg Gly Glu Arg Glu Val His Aia Glu Phe Glu Lys Leu Phe Ala 545 600 6u5 tcc atc cag gaa cgc ttc gcc gag tcc acc gac 2773 tga gccgttacat Ser Ile Gin Glu Arg Phe Ala Glu Ser Thr Asp agcctactac atacaaattg acgcaagcca ttttttaaaa 2833 agcggacatg aattagacct atagaagatg tatatattgg ctgccgtagc agcagcgcca tctccccggc gtcatcccag 2893 agcctggtgc gagtaatcat acgcgaccga gc~aagtagc ttcactcaac cgttcatttg 2953 tatggaccgc acgcttattt ggatctggct ctaaccttcc gtctctGtct cttcaatctt 3013 ctaactctct tctttctcca cttagctctc attgtgaaat gaaagttttc tggatatatg 30?3 gaagtaagaa ctaagaagga gtaataagtg gaagagagac gactgaaaag ctactcttgt 3133 acaagaggga tgtcggggaq atggggacgc gagggctccc cgccgcttgc ttggggtcag 3193 cctgtgtagg gtaactattt cacgtgaaca agggcgtcag ggctcggtgg cgtgcgagat .253 gcgacttcct ctgagtgggc acgtgattgg atgcgcgcga aatctacgcg gcaagcgtgt 3313 atgggtttcc tgtttataga tgtacggctg gagtagtccc gtcagcccac actgagagta 33- '?3 tgtagttcta tcacgacctc tgcagctact cgcccttgtg ctgcgcttgc tccttcgaga 3433 cgcatgcctc ctggcgcatt gccgtcttcg atgtgtcggc gtagttcgtg tatcttgtaa 3993 gttatctagt atgcgcggga gcaatgcctg gtcaagcaac atgcgctcag actcgccggg 3553 cgtggcgggc gccacattgt cggccatcaa ggcggagtac tcctggaggt ctgcgaagcg 3613 gagcggctgc atgtcgccct gcatcatgga ctcgaggtgc cgcgcgccgc tgtaggcctt 3673 gaagttgcag tacgcgcggt atgcca 3c99 <210> 2 <211> 617 <212> PRT
<213> Ashbya gossypii Met Aia Gly Ala Leu Glu Asn Aia Arg Lys Giu Ile Lys Arg Ile Ser Leu Glu Asp His Asn Glu Asn Glu Tyr Gly Gln Ile Tyr Ser Vai Ser Gly Pro Val Val Val Ala Glu Asn Met Vai Gly Cys A1a Met Tyr Glu Leu Val Lys Vai Gly His His Asn Leu Val G=y Glu Val Ile Arg Leu Asp Gly Asp Lys A1a Thr Ile Gln Val Tyr Giu Glu Thr Ala Gly val Thr Val Gly Asp Pro Val Leu Azg Thr Gly Lys Pro Leu Ser Val Glu Leu Gly Pro Gly Leu Met Glu Thr Ile Tyr Asp Giy Ile Gln Arg Pro 100 i05 110 Leu Lys Ala Ile Lys Glu Gln Ser Gln Ser Ile Tyr ile Pro Arg Gly lI5 120 125 Val Asp Ala Pro A1a Leu Ser Arg Glu Val Lys Trp Ala Phe Lys Pro GIy Lys Leu Gly Val Gly Asp His _'le Ser Gly Gly Asp Ile Phe Gly Ser Ile Phe Glu Asa Se_- Leu Lea Glu Asp f:is Lys Ii.e Leu Leu Pro Pro Arg Ala Arg Gly Trr Ile Thr mrp Ile A:a Pro Ala Gly Glu Tyr Thr Val Asp Glu Thr Val Leu Glu Va'_. Glu Phe Asp Gly Lys Lys Tyr 19s 2oc 2~5 Ser Tyr Se. Met Phe His Thr Trp Pro Va: Arg Val Pro Arg Pr~_ Val 210 215 - 2~0 Thr Glu Lys Leu Ser Ala Asp Tyr Pro Leu Leu Thr Gly Gln Arg Val Leu Asp Ser Leu Phe Pro Cys Val Gln Gly Gly Thr Thr Cys Ile Pro Gly Ala Phe Gly Cys Gly Lys Thr Val Ile Ser Gln Ser Leu Ser Lys Tyr Ser Asn Ser Asp Ala Ile Ile Tyr v'a'_ Gly Cys Gly Glu Arg Gly Asn Glu Met Ala Glu Val Leu Met Giu Phe Pro Glu Leu Phe Thr Glu Val Asn C-ly Arg Lys Glu Pro Ile Met Lys Arg Thr Thr Leu Val Ala 305 310 31~ 320 Asn Thr Ser Asn Met Pro Va- Aia A1a Arg Glu Ala Ser Ile Tyr Thr Giy Ile Thr Leu Ala Glu Tyr Phe Arg Asp Gln Giy Lys Asn Val Ser Met ile Rla Asp Ser Ser Ser Arg Trp Ala Glu AIa Leu Arg Glu Ile Ser Gly Arg Leu Gly Glu Met Pro Ala Asp Gln Gly Phe Pro Ala Tyr Leu Gly Ala Lys Leu Ala Ser Phe Tyr Glu Arg Ala Gly Lys F1 a Val Ala Leu Gly Ser Pro Rsp Arq Val Gly Ser Val Ser Ile Val Ala Ala Val Ser Pro P.la Gly Gly Asp Phe Ser Asp Pro Va1 Thr Thr Ser Thr Leu Gly iie Thr Gln Vai Phe Trp Gly Leu Asp Lys Lys Leu Rla Gln Arg Lys His Phe Pro Ser Ile Asn Thr Scr Val Ser Tyr Ser Lys Tyr 4so 455 460 Thr Asa Vai Leu Asn Lys Tyt Tyr Asp Ser Asn Tyt Pro G1~.: Phe Pro 465 970 - 9'S 490 Val Leu Arg Asp Arg Ile Lys Giu Ile Leu Ser Asn Ala Glu Glu Leu 485 490 qg5 Glu Gln Val Val Gln Leu Val Gly Lys Ser Ala Leu Ser Asp Lys Asp Lys Ile Val Leu Asp Vai Ala Thr Leu I1e Lys Giu Asp Phe Leu C-In Gln Asn Gly Tyr Ser Thr Tyr Asp Ala Phe Cys Pro Ile Trp Lys Thr Tyr Asp Met Met Lys Aia Phe Val Ser Tyr Phe Asp Glu Ala Gln Lys Ser Val Set Asn Gly Aia Asn Trp P.la Val Leu Ser Glu Ala Thr Gly Asp Val Lys His Al.a Val Ser Ser Ser Lys Phe the G_u Pro Ser Arg Gly Glu Arg Glu Val His Ala Glu Phe Glu Lys Leu Phe Ala Ser Ile Gln Glu Arg Phe Ala Gi=.; Ser Thr Asp 610 6'_5

Claims (26)

Claims

1. A unicellular or multicellular organism, in particular a microorganism, for preparing riboflavin biotechnologically, characterized in that its intracellular substance transport processes are altered such that the majority of the riboflavin accumulates extracellularly.

2. A unicellular or multicellular organism as claimed in claim 1, characterized in that transport of the riboflavin into the vacuole is decreased in this organism.

3. A unicellular or multicellular organism as claimed in either claim 1 or 2, characterized in that transport of the riboflavin into the vacuole is at least partially inhibited.

4. A unicellular or multicellular organism as claimed in one of claims 1 to 3, characterized in that the V-ATPase activity is at least partially blocked in this organism.

5. A unicellular or multicellular organism as claimed in claim 4, characterized in that the rate of transport of the riboflavin through the cytoplasmic membrane is higher than that through the vacuolar membrane in this organism.

6. A unicellular or multicellular organism as claimed in claim 4 or 5, characterized in that it is a fungus.

7. A unicellular or multicellular organism as claimed in claim 6, characterized in that it is a filamentous fungus.

8. A unicellular or multicellular organism as claimed in one of claims 1 to 7, characterized in that it is a fungus from the Saccharomycetaceae family.

9. A unicellular or multicellular organism as claimed in one of claims 1 to 8, characterized in that it is a fungus from the genus of the species Ashbya, preferably Ashbya gossypii.

10. A V-ATPase gene, characterized in that it is at least partially inactivated by mutation.

11. A V-ATPase gene as claimed in claim 10 having a nucleotide sequence which encodes the amino acid sequence shown in Figure 1 and its allelic variant.

12. A V-ATPase gene as claimed in either claim 10 or 11 having the nucleotide sequence from nucleotide 1 to nucleotide 3881, in accordance with the amino acid sequence given in Figure 2, or a DNA sequence having essentially the same effect.

13. A V-ATPase gene as claimed in one of claims 10 to 12 having regulatory gene sequences which are assigned to it.

14. A gene for preparing a riboflavin-producing unicellular or multicellular organism, characterized in that it does not contain any V-ATPase gene.

15. A vector containing a gene as claimed in one of claims 10 to 14.

16. A transformed organism for preparing riboflavin, containing a gene as claimed in one of claims 10 to 14 in replicatable form.

17. A transformed organism containing a vector as claimed in claim 15.

18. A process for preparing riboflavin, characterized in that an organism as claimed in one of claims 1 to 9 is employed.

19. A process as claimed in claim 18, characterized in that a chemical inhibitor is added to the fermentation medium.

20. A process as claimed in claim 19, characterized in that concanamycins, bafilomycins, N-ethylmaleimide and nitrate are added to the fermentation medium as inhibitors.

21. A process for preparing a riboflavin-producing unicellular or multicellular organism, characterized in that the organism is altered such that the majority of the resulting riboflavin accumulates extracellularly.

22. A process as claimed in claim 21, characterized in that the alteration in the organism is effected by recombinant methods.

23. A process as claimed in one of claims 20 to 22, characterized in that the activity of the ATPase is at least partially or completely blocked by altering or removing the V-ATPase gene.

24. The use of the organism as claimed in one of claims 1 to 9 and also 16 and 17 for preparing riboflavin.

25. The use of the ATPase gene as claimed in one of claims 10 to 13 for preparing an organism as claimed in one of claims 1 to 9 and also 16 and 17.

26. The use of the vector as claimed in claim 14 for preparing an organism as claimed in one of claims 1 to 9 and also 15 and 16.