WO2000012748A1

WO2000012748A1 - Organisms for the extracellular production of riboflavin

Info

Publication number: WO2000012748A1
Application number: PCT/EP1999/006328
Authority: WO
Inventors: Oskar Zelder; Reinhard Krämer; Carola FÖRSTER; Jose L. Revuelta Doval; Maria Angeles Santos Garcia
Original assignee: Basf Aktiengesellschaft
Priority date: 1998-08-31
Filing date: 1999-08-27
Publication date: 2000-03-09
Also published as: CN1316009A; JP2002523109A; EP1133570A1; DE19839567A1; CA2341715A1

Abstract

The invention relates to a monocellular or multicellular organism, especially 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 extracellular production of riboflavin

The present invention relates to a unicellular or multicellular organism for producing riboflavin by means of microorganisms.

Vitamin B ₂ , also called riboflavin, is essential for humans and animals. If there is a lack of Nitamine B ₂ , inflammation of the mucous membranes of the mouth and throat, cracks in the corners of the mouth, itching and inflammation in the skin folds, among other things, skin damage, conjunctivitis, decreased visual acuity and clouding of the cornea. In babies and children growth stagnation and weight loss can occur. Vitamin B ₂ is therefore of economic importance, particularly as a vitamin preparation for vitamin deficiency and as a feed additive. In addition, it is also used as a food coloring, for example in mayonnaise, ice cream, pudding, etc.

Riboflavin is produced either chemically or microbially. In the chemical production process, riboflavin is usually obtained as a pure end product in multi-stage processes, although relatively expensive starting products - such as D-ribose - must also be used. Therefore, the chemical synthesis of riboflavin is only considered for those applications for which pure riboflavin is necessary, such as. b. in human medicine.

An alternative to the chemical production of riboflavin is the production of this substance by microorganisms. The microbial production of riboflavin is particularly suitable for those cases in which high purity of this substance is not required. This is the case, for example, when the riboflavin is to be used as an additive to feed products. In such cases, the microbial production of riboflavin has the advantage that this substance can be obtained in a one-step process. Renewable raw materials such as vegetable oils can also be used as starting products for microbial synthesis.

The production of riboflavin by fermentation of fungi such as Ashbya gossypii or Eremothecium ashbyi is known (The Merck Index, Windholz et al., eds. Merck & Co., page 1183, 1983; A. Bacher, F. Lingens, Angew. Chemistry 1969, 393); but also yeasts, such as. B. Candida or Saccharomyces, and bacteria such as Clostridium are suitable for riboflavin production.

In addition, methods using the yeast Candida famata are described, for example, in US 05231007.

Riboflavin-overproducing bacterial strains are described, for example, in EP 405370, the strains being obtained by transforming the riboflavin biosynthesis genes from Bacillus subtilis. The fermentative production of riboflavin by means of special mutants of Bacillus subtilis is also described in GB 1434299, in German Offenlegungsschrift 3420310 and EP 0821063.

However, these prokaryotic genes were unsuitable for a recombinant riboflavin production process using eukaryotes such as Saccharomyces cerevisiae or Ashbya gossypii. Therefore, according to WO 93/03183, specific genes for riboflavin biosynthesis were isolated from a eukaryote, namely from Saccharomyces cerevisiae, in order to provide a recombinant production process for riboflavin in a eukaryotic production organism. Such recombinant

Manufacturing processes have no or only limited success for riboflavin production if the provision of substrate for the enzymes specifically involved in riboflavin biosynthesis is insufficient.

From German Patent 195 25 281 a process for the production of riboflavin is known, in which microorganisms are cultivated which are resistant to substances that inhibit isocitrate lyase.

A process for the microbial production of riboflavin is known from German published patent application 19545468.5-41, in which the isocitrate lyase activity or the isocitrate lyase gene expression of a riboflavin-producing microorganism is increased. However, all previously known processes have the disadvantage that a large part of the riboflavin is obtained intracellularly. Accordingly, the cell must be unlocked to obtain it. A continuous production of the riboflavin has therefore not been possible using biotechnology.

The object of the present invention is accordingly to provide a single-cell or multi-cell organism, preferably a microorganism, for the biotechnical production of riboflavin, which enables the riboflavin to be obtained without disruption of the cells and thus a u. a. enables continuous production of riboflavin.

This object is achieved by a single or multicellular organism whose intracellular mass transfer processes are modified in such a way that the majority of riboflavin is obtained extracellularly.

According to the invention, the transport or the transport speed of the riboflavin into the vacuole is preferably reduced, so that the predominant part of the riboflavin is released through the cytoplasmic membrane into the fermentation medium. It is particularly preferred that the transport of the riboflavin into the vacuole is at least partially blocked. Complete blocking of the transport of the riboflavin into the vacuole membrane is most preferred.

The goal of this desired change in intracellular mass transport can be achieved with the known methods of strain improvement of organisms. I.e. in the simplest case, corresponding strains can be produced by means of screening according to the selection which is customary in microbiology. The mutation with subsequent selection can also be used. The mutation can be carried out using chemical as well as physical mutagenesis. Another method is selection and mutation with subsequent recombination. Finally, the organisms according to the invention can be produced by means of genetic engineering.

According to the invention, the organism is preferably changed such that it produces riboflavin almost exclusively extracellularly. This increase in Extracellular production of riboflavin can be achieved according to the invention, for example, by producing an organism in which the enzyme activity of the vacuolar H ^ -ATPase (V-ATPase) is reduced or completely blocked. The consequence of this is that the transport of the riboflavin through the nacuole membrane is partially or completely inhibited.

This can be achieved, for example, by reducing the substrate conversion by changing the catalytic center or by increasing the action of enzyme inhibitors. A reduced enzyme activity of the V-ATPase can also be brought about by reducing the enzyme synthesis, for example by switching off factors which activate the enzyme biosynthesis.

According to the invention, the ATPase activity can preferably be reduced or completely inhibited by mutation, inactivation or removal of the ATPase gene. Such mutations can either be generated undirected by classic methods, such as by UV radiation or mutation-triggering chemicals, or specifically by means of genetic engineering methods, such as deletion, insertion, nucleotide exchange or substitutions in the structural gene or the associated regulatory elements, promoters and transcription factors.

The ATPase gene expression can accordingly z. b. be reduced or completely blocked by removing or inactivating the ATPase gene and / or by changing regulatory factors which influence gene expression. For example, regulatory elements can preferably be reduced at the transcription level, in particular by changing the transcription signals. In addition, a reduction in translation is also possible, for example, by changing the m-RΝA.

According to the invention, the ATPase gene modified in the manner described 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, the gene expression of the V-ATPase can also be reduced or completely inhibited by exchanging the promoter. It is possible to achieve the reduced enzymatic activity alternatively by incorporating gene copies or by exchanging the promoter. Equally, however, it is also possible to achieve the desired change in enzyme activity by simultaneously exchanging the promoter and incorporating gene copies.

Any organisms whose cells contain the sequence for the formation of the V-ATPase, that is to say also plant and animal cells, can be used for the isolation of the gene according to the invention. The modified gene is preferably isolated from microorganisms, particularly preferably from fungi. Mushrooms of the genus Ashbya are particularly preferred. The species Ashbya gossypii is most preferred.

The gene can be isolated by homologous or heterologous complementation of an ATPase defect mutant or an ATPase deletion mutant. The gene can also be isolated using a PCR approach with degenerate primers using amino acid homology to known Vma proteins and subsequent screening of a gene bank. The gene can then be completely sequenced by subcloning (cutting with suitable restriction enzymes and cloning the fragments obtained into suitable vectors) of the complementing clone or the positive clone of the screened gene bank. Disruption constructs (deletion / substitution alleles) can be prepared by cutting out part of the sequences of the gene with restriction enzymes and replacing this fragment with an antibiotic resistance cassette plus a promoter on a plasmid. Such disruption constructs can then be used to transform a riboflavin producer.

After isolation and sequencing, the genes according to the invention can be obtained with nucleotide sequences which are preferably according to the amino acid sequence. Figure 1 or encode allele variation. Allelic variations include, in particular, derivatives which can be obtained by deleting, inserting and substituting nucleotides from corresponding sequences, the V-ATPase Activity remains intact. A corresponding sequence in the amino acid sequence given above is the range from nucleotide 1 to 3881, AGVMA1 even in the sequenced range according to nucleotides 910-2763. Figure 2 corresponds.

A promoter of the nucleotide sequence of nucleotide 1 to 3881 according to. Above amino acid sequence or a DNA sequence having essentially the same effect. For example, the gene can be preceded by a promoter which differs from the promoter with the specified nucleotide sequence by one or more nucleotide exchanges, by insertion and / or deletion. Furthermore, the effectiveness of the promoter can be changed by changing its sequence or can be completely replaced by other promoters.

The V-ATPase gene can also be assigned regulatory gene sequences or regulatory genes which in particular reduce the V-ATPase gene activity. So z. B. a changed V-ATPase gene expression can be brought about via a modified interaction between RNA polymerase and DNA.

The V-ATPase gene modified or inactivated according to the invention with or without an upstream promoter or with or without a regulator gene can be preceded and / or followed by one or more DNA sequences so that the gene is contained in a gene structure. By cloning the gene according to the invention, plasmids or vectors are obtainable which contain the modified or inactivated V-ATPase gene or no V-ATPase gene and are suitable for transforming a riboflavin producer. The cells obtainable by transformation contain the gene in replicable form, i.e. H. in additional copies on the chromosome, the gene copies being integrated at any point in the genome by homologous recombination and / or on a plasmid or vector.

Another way to increase the extracellularly occurring riboflavin is to increase the transport speed of the riboflavin through the Increase cytoplasmic membrane in the fermentation medium. This shifts the equilibrium between the transport through the vacuole membrane and through the cytoplasmic membrane in such a way that the majority of the riboflavin is obtained extracellularly.

This can be done e.g. B. achieve that a stronger expression of the gene coding for transport or an increase in its activity is effected.

The single or multicellular organisms modified according to the invention can be any cells that can be used for biotechnical processes. These include, for example, fungi, yeasts, bacteria, and plant and animal cells. According to the invention, these are preferably transformed cells of fungi, particularly preferably those from the order of the Endomycetales. In particular, the family of Saccharomycetaceae is preferred. Fungi of the genus Ashbya and Eremothecium are very particularly preferred. The species Ashbya gossipii and Eremothecium ashbyii are most preferred.

In principle, the changes described in the transport processes can also be achieved by changing the fermentation conditions. In particular, the addition of chemical substances to the fermentation medium can at least partially block the intracellular accumulation of riboflavin. For example, according to the invention, an inhibitor can be added to the fermentation medium which inactivates the V-ATPase.

Examples of such substances are concanamycins, bafilomycins, N-ethylmaleimide, nitrate.

The invention is explained in more detail below with the aid of examples, without this being intended to limit the subject matter of the examples: L Compartmentalization of riboflavin in A. gossypii under production conditions

1.1 Permeabilization technology

With the help of the selective permeabilization technique, the production and compartmentalization of the stronger producing mutant ItaGSOl with / without the addition of concanamycin A was determined in 4 independent experiments. For this purpose, the cells were grown on production medium (yeast extract: 10 g / 1; soybean oil: 10 g / 1; glycine: 6 g / 1) and harvested at defined times in the course of production. The technique of selective permeabilization of the plasma membrane of A. gossypii was used for the compartmentalization analysis. The goal of the selective permeabilization of the piadma membrane is the separate analysis of cytosolic and vacuolar compartments. For this purpose, the mycelium was harvested by filtration at the desired point in time (glass fiber round filter, O 4.5 cm) and washed with NaP buffer. The fungal cells (0.5 g wet weight) were resuspended in 10 ml permeabilization solution 0.003% (w / v) digitonin in 50 mM NaP buffer and incubated in this permeabilization solution for 10 min at 30 ° C., 120 rpm in a laboratory shaker to permeabilize the plasma membrane. After incubation, the cells were separated from the solution by filtration and the filtrate was used for the analysis of cytosolic cell components. The cell suspension was incubated for a further 10 min with 0.02% (w / v) digitonin to destroy all cellular membranes. The vacuolar content of the cells for analysis (riboflavin determination) could then be obtained by filtration. The cell residues remaining 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 separation conditions described below:

Column: LiCrospher Q. D 100 RP-18 (5 µm) (Merck, Darmstac

I portion: 50 mM NaH ₂ PO ₄

I mM tramethylammonium chloride

12% acetonitrile

Flow: 1 ml / min

Elution: isocratic

Detection: 270 nm

The riboflavin content of the individual compartments was then related to the dry biomass used to calculate the compartmentalization between medium, cytosol and vacuole. To determine the dry mushroom mass, 20-50 ml soybean oil culture was first transferred to 50 ml Falcon tubes, 10 min. Centrifuged at 1500 x g and then carefully removed the oil layer above with a pipette. The oil-free suspension was then filtered through tared glass fiber round filters and washed with 10 times the volume of NaP buffer. The mycelium on the filter was dried to constant weight in a drying cabinet at 60 ° and then weighed.

1.3 Compartmentalization of riboflavin under production conditions

The cytosolic and vacuolar riboflavin contents and the amount of riboflavin secreted into the medium were determined for control and concanamycin-treated cells (5 μM) at the 3 points in time 24 h, 43 h and 67 h: The diagrams clearly show (can also be seen optically / highly microscopically) that the cells of the mutant ItaGSOl can no longer store riboflavin in their vacuoles if the V-ATPase inhibitor concanamycin A is added to the medium at the beginning of the production phase. The total production is the same for control and concanamycin A batches, with concanamycin A addition being used to secrete the otherwise intravacuously stored amount of riboflavin (30 - 60% of total production) into the medium.

2. Cloning. Sequencing and disruption of the A.gossvpii VMAl gene

An attempt was made to implement the riboflavin flow redirection achieved with the aid of the inhibitor concanamycin A by molecular biology by constructing a strain with dysfunctional V-ATPase. For this purpose, the catalytic subunit A of the V-ATPase complex was cloned and disrupted. The catalytic subunit A is encoded by the VMA1 gene (yacuolar membrane ATPase protein 1).

2.1. Cloning and sequencing

Using PCR (design of degenerate oligonucleotide primers from highly conserved sequences in the VMAlp alignment) it was possible to amplify a fragment of the Ag VMAl gene (highly conserved in evolution): for the PCR, degenerate oligonucleotide primers from highly conserved amino acid sections of various VMA1 proteins were used Developed: CFla (5- ATYCARGTBTAYGARAC-3) and GFlb (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 carried out in the Gene Amp® PCR System 9700 (Applied Biosystems) with Taq polymerase (Boehringer Mannheim, Germany), buffers like recommended by the manufacturer, 8 uM primer CFla, 4uM primer CFlb. The amplified PCR fragment was then used to screen a A. gossypii cosmid gene library (cosmid vector SuperCosl, Stratagene). To identify positive Cos id clones that contained homologous regions of DNA, the PCR fragment was analyzed with (a- ³² P) dCTP radioactively labeled by T7 polymerase. The cosmid-DANN of the positive clones was digested with Baml and screened again with the radioactive PCR fragment. The gene was then sequenced after subcloning into the plasmid vector Bluescript, Stratagene. The nucleotide sequence can be obtained in the EMBL database after filing of the patent and the associated publication under the accession number AJ009881. The protein alignment for AgVMAlp with the V-ATPase-A subunits of various other organisms showed that a strongly conserved protein sequence is also present in Ashbya. The phylogenetic pedigree of the VMA1 proteins also showed that the VMA1 protein most closely related to the corresponding protein of the yeast S. cerevisiae and known to date has been found with AgVMAlp. Genomic Southern analysis confirmed that it is a single-copy gene (like the rest of the yeast).

2.2 Disruption of the vmal gene

A vmal deletion / substitution allele, called vmal: G418, was constructed for the disruption. For this, the 0.25 kb BamHl-Pstl fragment in the VMA1-PCR fragment, which contains part of the VMA1-ORF, was replaced with the genetic resistance cassette TEF-G418. First, the VMA1 PCR fragment was cloned into a modified pGEM ^® T-vector (Promega) without PstI site was generated resulting in plasmid pJR1767. The coding sequence for VMA1 was finally disrupted on the plasmid by replacing the 0.25 kb BamHl-Pstl region with the TEF-G418 marker, thereby generating plasmid pJR1773. The linear 2.5 kb fragment Ncol-Spel of this plasmid was finally used to transform sprouted spores of A. gossypii (starting strains wild-type, stronger producing mutant ItaGSOl). Geneticin-resistant clones were selected and the disruption of the vma 1 gene was confirmed by Southern blot (Fig. 2).

2.2. Redirection of riboflavin by the disruption of VMA-1 The disruptants show characteristic, densely / crater-like colonies on plate in contrast to the flat growth of the original strains. The cells secrete riboflavia (yellow-colored agar plates), but the hyphae themselves are completely white and no longer store riboflavin in their vacuoles (first detection using fluorescence microscopy). In contrast, the hyphae of the parent strains are colored yellow by the intravacuously stored riboflavin (often even in crystals).

The riboflavin compartmentation was again quantitatively investigated using the selective membrane permeation technique (see 1.1.).

The disruption of the V-ATPase subunit A (as above with the addition of the V-ATPase inhibitor concanamycin) was able to redirect the riboflavin influences. So there are 3 new strains without vacuolar riboflavin accumulation (product retention) available.

Explanations to the figures:

Fig. 1: Compartmentalization of riboflavin in Ashbya under standard production conditions (left column) and during production with the addition of the V-ATPase inhibitor concanamycin A

Fig. 2: Disruption of AgVMAl with the TEF-G418 marker, (A)

Construction, (B) Southern analysis Tl: Disruptant from the wild strain

T2: Disruptant of ItaGSOl

Fig. 3: Riboflavin compartmentalization in parent strain and

Disruptant of the mutant ItaGSOl

Claims

Expectations

1. One or more organism, in particular microorganism, for the biotechnical production of riboflavin, characterized in that its intracellular

Mass transport processes are changed in such a way that the majority of riboflavin is obtained extracellularly.

2. Single- or multi-cell organism according to claim 1, characterized in that the

Transport of the riboflavin into the vacuole is reduced.

3. Single or multi-cell organism according to one of claims 1 or 2, characterized in that the transport of the riboflavin in the vacuole is at least partially inhibited.

4. single or multiple organism according to one of claims 1 to 3, characterized in that with it the V-

ATPase activity is at least partially blocked.

5. single or multi organism according to claim 4, characterized in that with him the transport speed of the riboflavin through the cytoplasmic membrane is higher than that through the vacuole membrane.

6. single or multiple organism according to one of claims 4 or 5, characterized in that it is a fungus.

7. single or multiple organism according to claim 6, characterized in that it is a filamentous

Is mushroom.

8. single or multi-cell organism according to one of claims 1 to 7, characterized in that it is a fungus from the family of the Saccharomycetaceae.

9. single or multiple organism according to one of claims 1 to 8, characterized in that it is a fungus from the

Genus of the Ashbya species, preferably Ashbya gossypii.

10. V-ATPase genes characterized in that they are at least partially inactivated by mutation.

11. V-ATPase genes according to claim 10 with a for the amino acid sequence acc. Figure 1 and their allele variation coding nucleotide sequence.

12. V-ATPase genes according to one of claims 10 or 11 with the nucleotide sequence of nucleotide 1 to 3881 acc. the amino acid sequence indicated in FIG. 2 or a DNA sequence which acts essentially in the same way.

13. V-ATPase genes according to one of claims 10 to 12 with this associated regulatory gene sequences.

14. Gene for the production of a riboflavin-producing single or multicellular organism, characterized in that it contains no V-ATPase gene.

15. Vector containing a gene according to one of claims 10 to 14.

16. Transformed organism for the production of riboflavin containing in a replicable form a gene according to one of claims 10 to 14.

17. A transformed organism containing a vector according to claim 15.

18. A process for the preparation of riboflavin, characterized in that an organism acc. one of claims 1 to 9 is used.

19. The method according to claim 18, characterized in that the

A chemical inhibitor is added to the fermentation medium.

20. The method according to claim 19, characterized in that the fermentation medium concanamycine, bafilomycine, N-ethylmaleimide,

Nitrate can be added as inhibitors.

21. A process for the production of a riboflavin-producing single or multicellular organism, characterized in that it is changed so that the predominant amount of the resulting riboflavin is obtained extracellularly.

22. The method according to claim 21, characterized in that the change in the organism takes place by means of genetic engineering methods.

23. The method according to any one of claims 20 to 22, characterized in that the activity of the ATPase is at least partially or completely blocked by changing or removing the V-ATPase gene.

24. Use of the organimus according to one of claims 1 to 9 and 16 and 17 for the production of riboflavin.

25. Use of the ATPase gene according to one of claims 10 to 13 for the production of an organism according to one of claims 1 to 9 and 16 and 17.

6. Use of the vector according to claim 14 for the production of an organism according to one of claims 1 to 9 and 15 and 16.