CA2255284A1 - Unicellular or multicellular organisms for preparing riboflavin - Google Patents

Abstract

The present invention relates to a unicellular or multicellular organism, in particular a micro-organism, for biotechnologically preparing riboflavin.
This organism is distinguished by the fact that it exhibits a glycine metabolism which is altered such that its synthetic output of riboflavin without any external supply of glycine is at least equal to that of a wild type of the species Ashbya gossypii, i.e.
ATCC10895, which is cultured under standard conditions with the addition of 6 g of external glycine/l.

Description

CA 022~284 1998-12-17 Unicellular or multicellular organisms for preparing riboflavin The present invention relates to a unicellular or multicellular organism for preparing riboflavin uslng mlcroorganlsms.
Vitamin B2, also termed riboflavin, is essential for humans and animals. Inflammations of the oral and pharyngeal mucous membranes, cracks in the corners of the mouth and pruritus and inflammations in the skin folds, among other damage to the skin, conjunctival inflammations, diminished visual acuity and clouding of the cornea appear in association with vitamin B2 deficiency. Cessation of growth and decrease in weight can occur in infants and children. Vitamin B2 therefore is of importance economically, in particular as a vitamin preparation in association with vitamin deficiency and as a feed'additive. In addition to this, it is also employed as a foodstuff colorant, for example in mayonnaise, ice cream, blancmange, etc.
Riboflavin is prepared either chemically or microbially. In the chemical methods of preparation, the riboflavin is as a rule isolated in multi-step processes as a pure end product, with, however, relatively expensive starting compounds, such as D-ribose, having to be employed. For this reason, the chemical synthesis of riboflavin is only suitable for those applications for which pure riboflavin is required, for example in human medicine.
Using microorganisms to prepare riboflavin offers an ~alternative to preparing this substance chemically. Preparing riboflavin microbially is particularly suitable in those instances in which this substance is not required to be of high purity. This is the case, for example, when the riboflavin is to be employed as an additive to feed products. In such CA 022~284 1998-12-17 cases, the microbial preparation of riboflavin has the advantage that the riboflavin can be obtained in a one-step process. In addition, renewable raw materials, such as vegetable oils, can be employed as starting compounds for the microbial synthesis.
It is known to prepare riboflavin by fermenting fungi such as Ashbya gossypii or Eremothecium ashbyi (The Merck Index, Windholz et al., eds. Merck & Co.), page 1183, 1983, A. Bacher, F. Lingens, Augen. Chem.
1969, p. 393); however, yeasts, such as Candida or Saccharomyces, and bacteria, such as Clostridium, are also suitable for producing riboflavin.
Methods using the yeast Candida famata are also described, for example in US 05231007.
Bacterial strains which overproduce riboflavin are described, for example, in EP 405370, GB 1434299, DE 3420310 and EP 0821063, where the strains were obtained by transforming the riboflavin biosynthesis genes from Bacillus subtilis. However, these prokaryotic genes were unsuitable for a method of preparing riboflavin recombinantly which used eukaryotes such as Saccharomyces cerevisiae or Ashbya gossypii. For this reason, the specific genes for riboflavin biosynthesis were, as described in WO 93/03183, isolated from a eukaryote, namely from Saccharomyces cerevisiae, in order thereby to provide a recombinant method for preparing riboflavin in a eukaryotic production organism. However, recombinant preparation methods of this nature are either unsuccessful, or only enjoy limited success, in producing riboflavin if there is inadequate provision of substrate for the enzymes which are specifically involved in the riboflavin biosynthesis.
In 1967, Hanson (Hanson AM, 1967, in Microbial Technology, Peppler, HJ, pp. 222-250, New York) found that adding the amino acid glycine increases the formation of riboflavin in Ashbya gossypii. However, such a method is disadvantageous because glycine is a CA 022~284 1998-12-17 very expensive raw material. For this reason, efforts were made to optimize riboflavin production by preparing mutants.
German Patent Specification 19525281 discloses a method for preparing riboflavin which involves culturing microorganisms which are resistant to sub-stances which have an inhibitory effect on isocitrate lyase.
German Laid-Open Specification 19545468.5-41 discloses another method for preparing riboflavin microbially in which the isocitrate lyase activity or the expression of the isocitrate lyase gene of a riboflavin-producing microorganism is increased.
However, even in comparison with these methods, there is still a need for a further optimization of the riboflavin preparation.
The object of the present invention is consequently that of making available a unicellular or multicellular organism, preferably a microorganism, for the biotechnological preparation of riboflavin, which microorganism enables formation of the riboflavin to be further optimized. In particular, an organism should be made available which is suitable for preparing riboflavin while economizing on raw materials and which consequently makes possible a production which is more economical than that of the previous state of the art.
In particular, the organism should permit an increased formation of riboflavin, without any addition of glycine, as compared with the previous organisms.
This object is achieved by means of a uni-cellular or multicellular organism which exhibits a glycine metabolism which is altered such that its synthetic output of riboflavin without any external supply of glycine is at least equal to that of a wild type of the Ashbya gossypii species ATCC10895, which is cultured under standard conditions with the addition of 6 g of external glycine/l.

CA 022~284 1998-12-17 Culturing under standard conditions means culturing, at 30~C and 120 rpm, in 500 ml shaker flasks possessing two baffles. 50 ml of a solution of 10 g of yeast extract/l containing either 10 g of glucose/l or 10 g of soybean oil/l are employed per flask as the medium. The media are inoculated with 1~ of a 16 h culture carried out under the same conditions.
The objective of this sought-after alteration of the intracellular metabolism of glycine can be achieved using the known methods for improving organism strains.
This means that, in the simplest case, appropriate strains can be prepared by means of screening after the selection which is customary in microbiology. It is also possible to use mutation in conjunction with subsequent selection. In this case, the mutation can be carried out either by means of chemical mutagenesis or by means of physical mutagenesis. A further method is that of selection and mutation together with subsequent recombination. Finally, the organisms according to the invention can be prepared by means of genetic manipulation.
According to the invention, the organism is altered such that it produces glycine intracellularly in a quantity which is greater than its requirement for maintaining its metabolism. According to the invention, this increase in intracellular glycine production can be achieved by preparing an organism in which the activity of the enzyme threonine aldolase is increased.
This can be achieved, for example, by increasing substrate turnover by means of altering the catalytic center or by abolishing the effect of enzyme inhibitors.
An increase in the activity of the threonine aldolase enzyme can also be elicited by increasing the synthesis of the enzyme, for example by means of gene amplification or by eliminating factors which repress the biosynthesis of the-enzyme.
According to the invention, the endogenous threonine aldolase activity can preferably be increased CA 022~284 1998-12-17 by mutating the endogenous threonine aldolase gene.
Such mutations can either be produced randomly by means of classical methods, such as using W irradiation or mutation-provoking chemicals, or in a targeted manner using genetic engineering methods such as deletion, insertion and/or nucleotide exchange.
Increased expression of the threonine aldolase gene can be achieved by incorporating copies of the threonine aldolase gene and/or by enhancing regulatory factors which exert a positive effect on threonine aldolase gene expression. For example, regulatory elements can preferentially be enhanced at the transcriptional level by, in particular, increasing the transcription signals. In addition to this, however, it is also possible to enhance translation by, for example, improving the stability of the mRNA.
In order to increase the gene copy number, the threonine aldolase gene can, for example, be incorporated into a gene construct or a vector which preferably contains regulatory gene sequences which are assigned to the threonine aldolase gene, in particular those sequences which enhance gene expression. A
riboflavin-producing microorganism is then transformed with the gene construct containing the threonine aldolase gene.
According to the invention, the threonine aldolase can also be overexpressed by exchanging the promoter. In this context, it is also possible to achieve the higher enzymic activity in an alternative manner by incorporating gene copies or by exchanging the promoter. However, it is equally also possible to achieve the desired alteration in the enzymic activity by simulta~eously exchanging the promoter and incor-porating gene copies.
Since threonine is limiting in an organism which has been altered in this way, it is necessary to feed in threonine when the cell according to the invention is employed. The improved uptake of the CA 022~284 1998-12-17 threonine and its virtually quantitative conversion into glycine lead to a surprisingly large increase in riboflavin formation such as was not previously achievable by feeding in glycine. Alternatively, the endogenous formation of threonine in the organism can be increased, for example, by eliminating the feedback resistance of the aspartate kinase.
The threonine aldolase gene is preferably isolated from microorganisms, particularly preferably from fungi. Fungi of the genus Ashbya are once again preferred in this context. The species Ashbya gossypii is highly preferred.
However, all other organisms whose cells contain the sequence for forming threonine aldolase, that is animal and plant cells as well, are also suitable for isolating the gene. The gene can be isolated by means of homologous or heterologous comple-mentation of a mutant which is defective in the threonine aldolase gene or by means of heterologous probing or PCR using heterologous primers. For subcloning, the size of the insert in the complementing plasmid can subsequently be reduced to a minimum by means of suitable restriction enzyme steps. After the putative gene has been sequenced and identified, subcloning which gives an accurate fit is effected by means of fusion PCR. Plasmids which carry the resulting fragments as inserts are introduced into the threonine aldolase gene-defective mutant, which is then tested for the functionality of the threonine aldolase gene.
Functional constructs are finally used to transform a riboflavin producer.
Following isolation and sequencing, the threonine aldolase genes can be obtained with nucleo-tide sequences which encode the given amino acid sequence or its allelic variation. Allelic variations include, in particular, derivatives which can be obtained by deleting, inserting or substituting nucleotides from appropriate sequences while at the CA 022~284 1998-12-17 same time retaining the threonine aldolase activity. A
corresponding sequence, from nucleotide 1 to nucleotide 1149, is shown in Figure 2b.
A promoter having the nucleotide sequence from nucleotide -1231 to nucleotide -1 as depicted in the abovementioned sequence, or a DNA sequence which has essentially the same effect, is, in particular, placed upstream of the threonine aldolase genes. Thus, a promoter which differs by one or more nucleotide substitutions, by insertion and/or by deletion from the promoter which possesses the nucleotide sequence shown without, however, the functionality or the activity of the promoter being impaired, can, for example, be placed upstream of the gene. In addition, the activity of the promoter can be increased by altering its sequence, or the promoter can be completely replaced by active promoters.
Moreover, regulatory gene sequences or regulatory genes which, in particular, increase the activity of the threonine aldolase gene can be assigned to the threonine aldolase gene. Thus, enhancers, which increase threonine aldolase gene expression by improving the interaction between the RNA polymerase and the DNA, can, for example, be assigned to the threonine aldolase gene.
One or more DNA sequences can be placed upstream and/or downstream of the threonine aldolase gene, which does or does not possess an upstream promoter or does or does not possess a regulatory gene, such that the threonine aldolase gene is contained in a gene structure. Plasmids or vectors which contain the threonine aldolase gene and are suitable for trans-forming a . riboflavin producer can be obtained by cloning the threonine aldolase gene. The cells which can be obtained by transformation contain the gene in replicatable form, i.e-. in additional copies in the chromosome, with the gene copies being integrated at - CA 022~284 1998-12-17 arbitrary sites in the genome by means of homologous recombination.
The objective, according to the invention, of partial or complete intracellular formation of glycine can also be achieved by preparing organisms in which the intra-cellular degradation of glycine is at least partially blocked. Mutations of this nature can, as already described above, either be generated in a random manner by means of classical methods using physical or chemical mutagenesis, for example using W irradiation or mutation-provoking chemicals, or in a targeted manner by means of genetic engineering methods.
According to the invention, the objective of the increased intracellular formation of glycine can preferably be achieved by altering the gene for serine hydroxymethyltransferase. Such alterations can, for example, be achieved by mutations, such as insertions, deletions or substitutions, in the structural gene or the regulatory elements, such as promoters and trans-cription factors, which are associated with this gene.
According to the invention, it was observed,surprisingly, that these mutants include mutants which are resistant to glycine antimetabolites. The glycine antimetabolite-resistant mutants which are preferred are those unicellular or multicellular organisms which are resistant to alpha-aminomethylphosphonic acid and/or alpha-aminosulfonic acid.
This can, for example, be achieved in exactly the same way by selecting mutants which are replaced by the threonine structural analog ~-hydroxynorvaline and/or which are substituted at the threonine and/or lysine analogs.
Consequently, mutants which can be employed in accordance with the invention can also be prepared by appropriate selection. Such resistant unicellular or multicellular organisms can therefore be prepared using the classical screening methods which are in general use in microbiology.

CA 022~284 1998-12-17 In the organisms described, riboflavin produc-tion can be further increased if the export into the medium of the glycine which is formed intracellularly is at least partially blocked. In the simplest case, it is sufficient to supplement with glycine in order to achieve this. As an alternative, the carrier which is responsible for the export can be switched off by disrupting the gene.
In addition, an increase in intracellular glycine concentration can be achieved by altering the glyoxylate metabolism, e.g. by increasing the activity of glyoxylate aminotransferase. Another option is to optimize the synthesis of intracellular glycine from carbon dioxide and ammonia.
In summary, it can be stated that the object according to the invention can preferably be solved by increasing intracellular synthesis of the glycine, at least partially blocking degradation of the glycine, at least partially inhibiting transport of the glycine out of the cell, altering the glyoxylate metabolism and optimizing glycine synthesis from ammonia and carbon dioxide. These solutions can be used as alternatives, or cumulatively or in any arbitrary combination.
An additional increase in riboflavin formation can be achieved by adding glycine to the nutrient medium.
The unicellular or multicellular organisms which are obtained in accordance with the invention may be any arbitrary cells which can be employed for biotechnological processes. Examples of these cells are fungi, yeasts, bacteria and plant and animal cells. In accordance with the invention, the cells are preferably transformed. fungal cells, particularly preferably fungal cells of the genus Ashbya. The species Ashbya gossypii is particularly preferred in this context.
In that which follows, the invention is explained in more detail with the aid of examples, CA 022~284 1998-12-17 without this being associated with any restriction of the invention to the subject matter of the examples:

Example 1 - Selecting a mutant which is resistant to alpha-amino-methylphosphonic acid (AMPS).

Ashbya gossypii spores were mutagenized with W
light. The spores were then added to plates treated with 70 mM alpha-aminomethylphosphonic acid. Inhibition of riboflavin formation can be recognized by the fungus forming yellow colonies without inhibition and white colonies with inhibition. Accordingly, the yellow organisms, i.e. those which were resistant to the inhibitor, were isolated. This method was used to obtain the resistant strain AMPS-NM-01, inter alia.
Experiments carried out on plates containing 200 mM AMPS demonstrated that this strain still exhibited a yellow colony color, in contrast to the starting strain, which remained completely white. In submerged culture, the mutant exhibited the same formation of riboflavin in the absence of glycine as did the wild type in the presence of glycine (cf.
Figure 1).
Investigations of the specific enzymic activities of the wild type and mutant showed that serine hydroxymethyltransferase activity was reduced by 50~
(~ig. 7). Since it was possible to demonstrate by feeding l3C-labeled threonine that formation of serine, which is presumably catalyzed by serine hydroxymethyltransferase, takes place from glycine (Table 1), the increase in riboflavin formation can be explained by a reduction in the quantity of glycine draining off to form serine.
The composition of the minimal medium used in Table 1 is as follows:

CA 022~284 1998-12-17 Solution A: KH2PO4200 g/l pH 6.7 with KOH
(100 times) Solution B: NH4Cl15 g/l 5 (10 times) Asparagine5 g/l NaCl 2 g/l MgSO4 x 7H2O 4 g/l MnSO4 x H2O 0.5 g/l ClCl2 x 2H2O 0.4 g/l Myoinositol 1.0 g/l Nicotinamide 2.5 g/l Yeast extract 2 g/l C source: Glucose or soybean oil 2.5 g/l In order to prepare the medium, the C source was added to one-times concentrated solution B and the mixture was sterilized by autoclaving. After the medium had cooled down, 1/100 of the volume of separately autoclaved solution A was added.

Example 2 Isolation of the GLY1 gene from Ashbya gossypii In order to isolate the gene for threonine aldolase, the glycine-auxotrophic Saccharomyces cerevisiae mutant YM 13F (SHM1 : : HIS3 shm2 : : LEU2 glyl : : URA3) was transformed, after selection for resistance to fluoroorotic acid, with an Ashbya gossypii gene library. The gene library consisted of genomic DNA which had been partially digested with Sau3A and from which fragments of 8 - 16 kb in size had been isolated by density gradient centrifugation and ligated into the BamHI-cut vector Yep352. The trans-formants were first of all selected for uracil proto-trophy. Selection for glycine prototrophy was carried out in a second step after replica plating. 25 glycine-prototrophic clones were isolated from about 70,000 .

CA 022~284 1998-12-17 uracil-prototrophic clones. Curing of the transformants and retransformation with the isolated plasmids demonstrated that the complementation was plasmid-encoded. Whereas there was no measurable threonine aldolase activity (~ 0.1 mU/mg of protein) in the glycine-auxotrophic Saccharomyces strain, it was possi-ble to measure significant enzyme activity (25 mU/mg of protein) in the strains which were transformed with the isolated gene library plasmids. A sub-cloned 3.7 kb Hind III fragment which exhibited complementation was sequenced (Figure 2). A threonine aldolase-encoding gene which was homologous to Saccharomyces cerevisiae GLYl was found.

Example 3 Overexpressing the GLYl gene in Ashbya gossypii In order to overexpress the GLYl gene, it was cloned into the expression vector pAG203 (cf.
W09200379). In this plasmid, the gene is under the control of the TEF promoter and the TEF terminator (Figure 3). A gene for resistance to G418 functions as a selective marker in Ashbya gossypii. After Ashbya gossypii had been transformed with this plasmid and single-spore clones had subsequently been isolated, because the spores are mononuclear and haploid, the threonine aldolase activity in the crude extract was then measured. Both when growing on glucose and when growing on soybean oil, at least ten-fold overexpression was measured in A.g.p.AG203GLYl as compared with a strain which had been transformed with the empty plasmid pAG203 (Figure 4).

CA 022~284 1998-12-17 Example 4 Increasing riboflavin formation by overexpressing GLY1 and feeding threonine Threonine was added to the medium in order to check whether the threonine which is formed in the cell limits the formation of glycine by the overexpressed threonine aldolase. When 6 grams of threonine were added per liter when A.g.pAG203GLYl was growing on glucose as the carbon source, the strain formed approximately twice as much riboflavin as it did when 6 grams of glycine were added per liter (Figure 5).
This effect was not observed when a wild type and a control strain which was transformed with the empty plasmid were tested. Analysis of the amino acids in the medium showed that only about 6 mM of the fed-in 52 mM
of threonine remained in the case of the GLY1 overexpresser and that, surprisingly, the concentration of glycine had increased from 2 mM to 42 mM. These results demonstrated that glycine formation was limited by threonine, that the overexpressed threonine aldolase was capable of functioning, that glycine which was formed intracellularly was more effective than glycine which was fed extracellularly, and that the fungal cells exported glycine massively.

Example 5 Inhibiting glycine export If the threonine aldolase-overexpressing strain A.g.pAG203GLY1 was cultured on soybean oil instead of glucose, as in Example 4, the increase in riboflavin formation Qbtained when feeding threonine did not exceed that when feeding glycine (Fig. 6). However, analysis of the medium showed that the threonine had been degraded down to about 13 mM. There cannot, there-fore, have been any limitation in the threonine. At the same time, it was found that the extracellular glycine CA 022~284 1998-12-17 had increased from 2 to about 44 mM. All the glycine which had been formed by the fungus had therefore been exported into the medium. It was possible to inhibit this export by introducing glycine into the medium, a measure which then resulted in the riboflavin formation being substantially increased in association with the same uptake of threonine (Table 2). In order to rule out the possibility that it was only the glycine which had been introduced which was responsible for the increased production, as much glycine was introduced, in a control, as was ultimately formed, as glycine, in the experiment using glycine and threonine. This finding underlines the fact that glycine which is formed intracellularly is much more effective than glycine which is added extracellularly.

Example 6 Increasing the formation of riboflavin by selecting ~-hydroxynorvaline-resistant mutants Since it was not the conversion of threonine into glycine but the synthesis of threonine which first of all limited glycine formation, the threonine analog ~-hydroxynorvaline was used to search for resistant mutants. Radial growth was significantly inhibited on agar plates filled with minimum medium containing 2.5 mM ~-hydroxynorvaline. Mutants which grew more vigorously formed spontaneously at the edges of the colonies. Stable mutants which grew significantly more vigorously on the ~-hydroxynorvaline minimal medium than did the parental strains (Fig. 8) were produced by isolating spores and selecting once again. Investigation of riboflavin formation indicated a marked increase in productivity. First, in minimal medium containing soybean oil, the strain HNV-TB-25 formed 41 + 11 mg of riboflavin/l whereas its parental strain only produced 18 + 3 mg/l. The progeny strain HNV-TB-29 also exhibits a marked increase, with a formation of 116 + 4 mg/l, as CA 022~284 1998-12-17 compared with its strain of origin, i.e. Ita-GS-O1, which only formed 62 i 10 mg/l.

Table 1: 13C-enrichment in the C atoms of serine, threonine and glycine following growth of A. gossypii ATC10895 on the given media and subsequent total hydrolysis of the resulting biomass (MM: minimal medium; YE: yeast extract; YNB: yeast nitrogen base; n.d.: not determined) Medium MM + 0.2 g of YE/l MM + 0.2 g of YNB/l + 1 g of ethanol/l + 1 g of ethanol/l + 2.7 mg of 13C2-serine/l + 2.6-mg of 13C1-serine/l Serine C1 1.1 4.9 C2 5.9 1.1 C3 1.1 1.1 Threonine C1 n.d. 39.0 C2 1.1 C3 1.1 C4 1.1 Glycine C1 1.1 7.1 C2 4.3 1.1 CA 022~284 1998-12-17 Table 2: Effect of supplementation with threonine and glycine on riboflavin formation when GLY1 is simultaneously being overexpressed Carbon t = 0 t = 72 h t = 72 h t = 72 h Strain source Supple- Riboflavin Gly Thr ment [mg/l] [mM] [mM]
80 mM Gly 80 + 2 Soybean oil 50 mM Thr 22 i 1 42 + 0 WT 130 mM Gly 18 ~ 3 129 + 2 n.d.
80 mM Gly 80 + 0 Glucose 50 mM Thr 5 + 1 35 + 0 130 mM Gly 7 + 1 126 + 2 n.d.
80 mM Gly 117 + 2 Soybean oil 50 mM Thr 31 + 0 11 + 1 Ag pAG 130 mM Gly 20 + 3 129 + 1 n.d.
203 GLY1 80 mM Gly 113 + 2 Glucose 50 mM Thr 40 + 1 12 + 0.7 130 mM Gly 9 + 1 129 + 3 n.d.
n.d. = not detectable CA 022~284 1998-12-17 Comments on the figures Figure 1: Formation of riboflavin by the Ashbya gossypii strains ATCC 10895 (wild type, WT) and the AMPS-resistant mutant AMPS-MN-01 in the presence or absence of 6 g of glycine/l following growth on complete medium contain-ing 10 g of soybean oil/l as the carbon source. The measured values were obtained from three independent experiments.

Figure 2a: Gly 1 locus in the Ashbya gossypii genome.
The clones GB 7-1 and GB 26-9, and also the 3.7 kb Hind III subclone GB-26-9-6, comple-ment the S. cerevisiae mutant. GB-26-9-6 was sequenced entirely while GB 7-1 was sequenced in order to complete the C
terminus of GLYl.
~0 Figure 2b: Nucleotide sequence, and deduced amino acid sequence, of the A. gossypii GLYl gene together with the flanking nucleotide sequence.
~5 Figure 3: Diagrammatic depiction of the construction of the vector pAG203GLYl for overexpressing the GLYl gene in A. gossypii.

Figure 4: Comparison of Ashbya gossypii wild type (solid symbols) and A.g.pAG203GLYl (open symbols) with regard to growth, riboflavin formation and specific threonine aldolase --activity when cultured on complete medium containing 10 g of soybean oil/l.
Figure 5: Growth and riboflavin formation of Ashbya gossypii strains ATCC 10895 (wild type), pAG203 and pAG203GLYl when cultured on YE

CA 022~284 1998-12-17 complete medium containing 10 g of glucose/l as the carbon source and in association with glycine or threonine supplementation.
The Table shows the glycine and threonine concentrations in the medium in each case before and after culture. The mean values and standard deviations shown represent the results from three independent experiments.

Figure 6: Growth and riboflavin formation of Ashbya gossypii strains ATCC 10895 (wild type), pAG203 and pAG203GLY1 when cultured on complete medium containing 10 g of glucose/l as the C source and in association with glycine or threonine supplementation. The Table shows the glycine and threonine con-centrations in the medium in each case before and after culture. The mean values and standard deviations shown represent the results from three independent experiments.

Figure 7: Comparison of Ashbya gossypii wild type (solid symbols) and the AMPS-resistant mutant AMPS-NM-01 with regard to growth, riboflavin formation and the specific activities of threonine aldolase, serine hydroxymethyltransferase and glutamate gly-oxylate aminotransferase when cultured on complete medium containing 10 g of soybean oil/l. The measured values were obtained from three independent experiments.

Figure 8: Effect of ~-hydroxynorvaline on Ashbya gossypii; growth of wild type (W) and HNV-TB-25 (H) on an agar plate which is filled with minimal medium containing 2.5 g of glucose/l and 2.5 mM ~-hydroxynorvaline.

2255284.seq SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANTS:
(A) NAME: Forschungszentrum Juelich GmbH
(B) STREET: Leo-Brandt-Str.
(C) CITY: Juelich (E) COUNTRY: Germany (F) POSTAL CODE (ZIP): D-52428 (G) TELEPHONE: 02461 61-2843 (H) TELEFAX: 02461 61-2710 (A) NAME: BASF Aktiengesellschaft (B) STREET: ZDX/A C6 (C) CITY: Ludwigshafen (E) COUNTRY: Germany (F) POSTAL CODE (ZIP): D-67056 (ii) TITLE OF INVENTION: UNICELLULAR OR MULTICELLULAR ORGANISMS
FOR PREPARING RIBOFLAVIN

(iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Robic (B) STREET: 55 St-Jacques (C) CITY: Montréal (D) STATE: QC
(E) COUNTRY: Canada (F) ZIP: H2Y 3X2 (G) TELEPHONE: 514-987-6242 (H) TELEFAX: 514-845-7874 (v) COM~Ul~ READABLE FORM:
(A) MEDIUM TYPE: Disk 3.5" / 1.44 MB
(B) COM~U'1'~K: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: TXT ASCII

(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,255,284 (B) FILING DATE: December 17, 1998 CA 022~284 1999-03-22 ....

2255284.seq (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: DE 197 57 180.8 (B) FILING DATE: December 22, 1997 (A) APPLICATION NUMBER: DE 198 40 709.2 (B) FILING DATE: September 09, 1998 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2744 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Ashbya gossypii (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1232..2377 (C) IDENTIFICATION METHOD: experimental (D) OTHER INFORMATION:/codon_start= 1232 /product= "Threonin-Aldolase"
/evidence- EXPERIMENTAL
/number= 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CA 022~284 1999-03-22 2255284.seq Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile Asp Thr Leu Lys Leu Glu Gln CA 022~284 1999-03-22 ,.. ~. ~,. .. .

2255284.seq His Val Ala Lys Leu Ala Gly Met Glu Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu Val Gly Ser His Lys Phe Ile CA 022~284 1999-03-22 2255284.seq Lys Lys Ala Asn His Phe Arg Lys Gln Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr CA 022~284 1999-03-22 . .

2255284.seq TCA~ATGGGA TTGTGGTGCG CAGTACATGC GCAGTGCTGC ACATTTGAGG ATCAATGGGT 2647 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 382 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn ~sp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu ~rg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg ~la His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly CA 022~284 1999-03-22 2255284. seq -Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu HiS Gly Ile Ile ~iS Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn ~sp Leu Arg Leu HiS Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu Val Gly Ser HiS Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln ~ln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met ~al Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys ~ys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu ~er Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr CA 022~284 1999-03-22 2255284 . seq

Claims (26)

1. A unicellular or multicellular organism, in particular a microorganism, for the biotechnological preparation of riboflavin, which exhibits a glycine metabolism which is altered such that its synthetic output of riboflavin without any external supply of glycine is at least equal to that of a wild type of the species Ashbya gossypii, i.e. ATCC10895, which is cultured under standard conditions with the addition of 6 g of external glycine/l.

2. A unicellular or multicellular organism as claimed in claim 1, in which the intracellular synthesis of glycine is increased and/or the intracellular degradation of glycine and/or the transport of glycine out of the cell is at least partially inhibited.

3. A unicellular or multicellular organism as claimed in claim 1 or 2, which exhibits an increased threonine aldolase activity.

4. A unicellular or multicellular organism as claimed in one of claims 1 to 3, in which the intra-cellular formation of serine from glycine is at least partially blocked.

5. A unicellular or multicellular organism as claimed in claim 4, in which the activity of serine hydroxymethyltransferase is at least partially blocked.

6. A unicellular or multicellular organism as claimed in claim 4 or 5, which is resistant to glycine antimetabolites.

7. A unicellular or multicellular organism as claimed in claim 6, which is resistant to alpha-amino-methylphosphonic acid or alpha-aminosulfonic acid, .beta.-hydroxynorvaline and/or other threonine and/or lysine analogs.

8. A unicellular or multicellular organism as claimed in any one of claims 1 to 7, which is a fungus, preferably from the genus Ashbya.

9. A unicellular or multicellular organism as claimed in one of claims 1 to 8, which is a fungus of the species Ashbya gossypii.

10. A threonine aldolase gene having a nucleotide sequence which encodes the amino acid sequence shown in Figure 2b and its allelic variantion.

11. A threonine aldolase gene as claimed in claim 10 which has the nucleotide sequence of nucleotide 1 to 1149 as depicted in Fig. 2b or a DNA sequence which has essentially the same effect.

12. A threonine aldolase gene as claimed in claim 10 or 11 which possesses an upstream promoter having the nucleotide sequence from nucleotide -1231 to -1 as depicted in Fig. 2b or a DNA sequence which has essentially the same effect.

13. A threonine aldolase gene as claimed in one of claims 10 to 12 together with regulatory gene sequences which are assigned to this gene.

14. A gene structure which contains a threonine aldolase gene as claimed in one of claims 10 to 13.

15. A vector which contains a threonine aldolase gene as claimed in one of claims 10 to 13 or a gene structure as claimed in claim 14.

16. A transformed organism for preparing riboflavin which contains, in replicatable form, a threonine aldolase gene as claimed in one of claims 10 to 13 or a gene structure as claimed in claim 14.

17. A transformed organism as claimed in claim 16 which contains a vector as claimed in claim 15.

18. A process for preparing riboflavin, which comprises employing an organism as claimed in one of claims 1 to 9.

19. A process for preparing a riboflavin-producing unicellular or multicellular organism which comprises altering the organism such that it exhibits a glycine metabolism which is altered such that its synthetic output of riboflavin without any external supply of glycine is at least equal to that of a wild type of the species Ashbya gossypii, i.e. ATCC10895, which is cultured under standard conditions with the addition of 6 g of external glycine/l.

20. The process as claimed in claim 19, wherein the organism is altered using genetic engineering methods.

21. The process as claimed in claim 19 or 20, wherein the alteration of the organism is achieved by exchanging the promoter and/or increasing the gene copy number.

22. The process as claimed in one of claims 19 to 21, wherein an enzyme possessing increased activity is produced by altering the endogenous threonine aldolase gene.

23. The process as claimed in one of claims 19 to 22, wherein the activity of the serine hydroxymethyl-transferase is at least partially blocked by altering the endogenous serine hydroxymethyltransferase gene.

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

25. The use of the threonine aldolase gene as claimed in one of claims 10 to 13 and the gene structure as claimed in claim 14 for preparing an organism as claimed in one of claims 1 to 9 and 16 and 17.

26. The use of the vector as claimed in claim 15 for preparing an organism as claimed in one of claims 1 to 9 and 16 and 17.