JP2002523109A - Organisms for extracellular production of riboflavin - Google Patents

Abstract

(57) Abstract The present invention relates to unicellular or multicellular organisms, especially microorganisms, for producing riboflavin by biotechnology. This microorganism is characterized by altered intracellular mass transport such that a major amount of riboflavin accumulates extracellularly.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to unicellular or multicellular organisms for producing riboflavin by microorganisms.

[0002] The vitamin B _2, which is also referred to as riboflavin are essential for humans and animals. In vitamin B ₂ deficiency, inflammation of the oral mucosa and pharynx mucosa, tearing corner of the mouth, skin wrinkles, itching irritation and inflammation, conjunctivitis, opacities of blurred vision and corneal occurs particularly in skin damage. Growth arrest and weight loss may occur in infants and children. Therefore, vitamin B ₂ are as vitamin preparations in particular vitamin deficiency, as well as economic importance as a feed additive. Other,
As a food coloring agent, it is used, for example, in mayonnaise, ice cream, pudding and the like.

[0003] Riboflavin production is carried out chemically or using microorganisms. In chemical processes, riboflavin is generally obtained as a pure end product in multiple steps, but relatively expensive starting materials, for example D-ribose, must be used. Thus, the chemical synthesis of riboflavin is only considered for applications where pure riboflavin is essential, for example in human medicine.

[0004] An option for the chemical production of riboflavin provides for the microbial production of this substance. Microbial production of riboflavin is particularly suitable when high purity of this substance is not required. Such is the case, for example, when riboflavin is to be used as an additive in feed products. In such cases, microbial production of riboflavin has the advantage that the substance can be obtained in one step. Renewable raw materials, for example vegetable oils, can also be used as starting materials for the synthesis by microorganisms.

[0005] The production of riboflavin by fermentation of fungi, such as Acibia gossypii or Elimotecium abieii, is known (The Merck Index, Windholz et al., Eds.
Merck & Co., 1183, 1983; A. Bacher, F. Lingens, Angew. Chemie 1969, 3
93); however, yeasts, such as Candida or Saccharomyces, and bacteria, such as Clostridium, are also suitable for riboflavin production.

[0006] Further, a method using a yeast, Candida famata, is described in, for example, US Pat. No. 5,231,007.

[0007] Riboflavin overproducing strains are described, for example, in EP 405 370, wherein the strain is obtained by transformation of a riboflavin biosynthesis gene from Bacillus subtilis. Fermentative production of riboflavin by a special mutant of Bacillus subtilis is described in GB1434.
299, DE-A-3 420 310 and EP08210
No. 63 is also described.

[0008] However, this prokaryote-gene is not compatible with Saccharomyces cerevisiae (Sacc.
It is not suitable for the production of recombinant riboflavin in eukaryotes such as Hromyces cerevisiae or Acibia gossypii. Accordingly, WO 93/03183 describes a method for the isolation of a gene specific for riboflavin-biosynthesis from eukaryotes, ie Saccharomyces cerevisiae, and using this to produce a recombinant production process for riboflavin in eukaryotic production organisms. Offered. This type of recombinant production has no or very little effect on riboflavin production when the production of substrates for enzymes specifically involved in riboflavin biosynthesis is insufficient. It only brings.

DE 195 25 281 discloses a process for producing riboflavin, in which microorganisms which are resistant to substances which act in an inhibitory manner on isocitrate lyase are cultured.

DE-A-195 45 468.5-41 discloses a process for the production of riboflavin by microorganisms, in which the isocitrate lyase activity or the expression of isocitrate lyase gene of riboflavin-producing microorganisms is increased. ing.

However, all known methods have the disadvantage that most of the riboflavin accumulates in the cells. Therefore, cells must be lysed for this acquisition. Thus, continuous production of riboflavin has not heretofore been possible in biotechnological processes.

It is therefore an object of the present invention to provide a single-celled organism for the biotechnological production of riboflavin, which makes it possible to obtain riboflavin without disrupting the cells, in particular thereby enabling a continuous production of riboflavin. Or to provide a multicellular organism, advantageously a microorganism.

This problem is solved by a unicellular or multicellular organism with altered intracellular mass transport so that the major part of riboflavin accumulates extracellularly.

According to the present invention, the transport or the rate of transport of riboflavin into vacuoles is reduced,
Advantageously, a major part of the riboflavin is released through the cell membrane into the medium. It is particularly advantageous to at least partially block the transport of riboflavin into the vacuole. It is most advantageous to completely block the transport of riboflavin into the vacuolar membrane.

[0015] The goal of efforts to alter intracellular mass transport is achieved by known methods of strain improvement of organisms. That is, the simplest method is to produce the corresponding strain by selection using routine screening in microbiology. Similarly, mutations and subsequent selections can be used. This mutation can be performed by chemical or physical mutagenesis. Other methods include selection and subsequent recombinational mutations. Finally, the organism according to the invention can be produced by genetic engineering.

Advantageously, according to the invention, the organism is modified to produce riboflavin almost completely extracellularly. Increased extracellular production of riboflavin can be achieved, for example, by producing an organism in which the enzymatic activity of vacuolar H ⁺ -ATPase (V-ATPase) is reduced or completely blocked according to the present invention. Achieved. As a result, the transport of riboflavin across the vacuolar membrane is partially or completely suppressed.

This can be achieved, for example, by changing the catalytic center, by achieving a reduced substrate conversion or by increasing the action of the enzyme inhibitor. A decrease in the enzymatic activity of V-ATPase can be caused by a decrease in enzyme synthesis, for example, by cleavage of a factor that activates enzyme biosynthesis.

ATPase activity can advantageously be reduced or completely inhibited by mutation, inactivation or removal of the ATPase gene according to the invention. Such mutations can be made untargeted by classical methods, such as, for example, by UV-irradiation or mutagenic chemicals, or by genetic engineering methods, such as structural genes or the regulatory elements, promoters and transgenes associated therewith. Targeting can be achieved by deletion, insertion, nucleotide exchange or substitution in the transcription factor.

ATPase-gene expression can be reduced or completely blocked, for example, by removing or inactivating ATPase and / or by altering a regulatory factor that affects gene expression. The reduction of the regulatory elements is thus advantageously effected at the transcription level, in particular the transcription signal is changed.
However, it is also possible to reduce the translation with it, so that, for example, m-RN
A changes.

According to the invention, the ATPase-gene altered in the manner described can be incorporated into a gene construct or into a vector. Finally, the riboflavin-producing microorganism is transformed with the genetic construct or vector.

According to the present invention, V-ATPase gene expression can be reduced or completely suppressed by exchanging a promoter. In this case, the reduction of the enzyme activity can be achieved by incorporation of the gene copy or by replacement of the promoter. Similarly, it is possible to achieve the desired change in enzyme activity by simultaneously exchanging the promoter and incorporating the gene copy.

For the isolation of the gene according to the invention, any organism, ie plant and animal cells, whose cells have sequences for the formation of V-ATPase, can be considered. The altered gene is advantageously isolated from a microorganism, particularly preferably from a fungus. Particularly advantageous are fungi of the genus Acibia. Most advantageous are those of the species Ashibia gossypii.

[0023] Isolation of the gene can be achieved through homologous or heterologous complementation of the ATPase-deficient mutant or ATPase-deleted mutant. The isolation of the gene is carried out on the basis of amino acid homology to the already known Vma-protein, by PCR-assay using degenerate primers and subsequently by screening gene libraries. Subsequently, the gene was completely sequenced by subcloning of the complementary clone or of the positive clone of the screened gene library (cutting with suitable restriction enzymes and cloning the resulting fragment into a suitable vector). It is determined. Disruption c
The onstruct (deleted / replaced allele) is completed by cutting a part of the gene sequence with a restriction enzyme and supplementing this fragment with an antibiotic resistance cassette plus a promoter on a plasmid. Such a disrupted construct can be used for transformation of a riboflavin producer.

After isolation and sequencing, a gene according to the invention having a nucleotide sequence encoding the amino acid sequence shown in FIG. 1 or an allelic variant thereof is obtained.
Allelic variants include derivatives which are obtained, in particular, by deletion, insertion and substitution of nucleotides from the corresponding sequence, while retaining V-ATPase activity. The corresponding sequence is the amino acid sequence in the range of nucleotides 1 to 3881 described above, wherein AGVMA1 itself has nucleotides 910 to 27 in FIG.
63 corresponds to the range.

[0025] The gene according to the invention is in particular a nucleotide from 1 to 3881 according to said amino acid sequence.
Or a DNA sequence having mainly the same effect. Thus, for example, one or more nucleotide exchanges with a promoter having said nucleotide sequence may be preceded by a promoter which differs by insertion and / or deletion. Further, the promoter may be altered in its action by altering its sequence, or may be completely replaced by a different promoter.

The V-ATPase gene may be provided with a regulatory gene sequence or a regulatory gene that reduces V-ATPase-gene activity, in particular. Thus, reduced V-ATPase-gene-expression can be achieved, for example, due to a change in the interaction between RNA-polymerase and DNA.

V-ATP modified or inactivated according to the present invention, with or without a pre-linked promoter, or with or without a regulatory gene
The ase-gene may be linked before and / or after one or more DNA-sequences, such that the gene is contained in a genetic construct. By cloning the gene according to the present invention, a plasmid or vector suitable for the transformation of a riboflavin producer having an altered or inactivated V-ATPase gene or no V-ATPase gene Is obtained. The cells obtained by transformation have the gene in a replicable form, ie in additional copies on the chromosome, wherein the gene copy is integrated in any position in the genome in a homologous combination and / or Integrated on a plasmid or vector.

Another possibility for increasing riboflavin which accumulates extracellularly consists in increasing the transport rate of riboflavin through the cell membrane into the fermentation medium. This allows
The equilibrium between transport across the vacuolar membrane and across the cell membrane moves such that a major portion of riboflavin accumulates outside the cell.

This can be achieved, for example, by causing a strong expression of the gene encoding transport or an increase in its activity.

The unicellular or multicellular organisms altered according to the present invention may be any cells that can be used in biochemical operations. This includes, for example, fungi, yeast, bacteria and plant and animal cells. In the context of the present invention, it is preferably a fungal transformed cell, particularly preferably from the class of Endmycetal. In particular, those belonging to the family Saccharomyces are advantageous. Particularly advantageous are fungi of the genus Ashibia and the genus Eremothecium. In this case, the species of Ashibia gossypii and Eremothium ashivii are most advantageous.

In principle, the described changes in the transport process can also be achieved by changing the fermentation conditions. In particular, the addition of chemicals to the fermentation medium at least partially blocks the intracellular accumulation of riboflavin. For example, according to the invention, an inhibitor acting on the inactivation of V-ATPase can be added to the fermentation medium.

Examples of such substances are concanamycin, bafilomycin, N-ethylmaleimide and nitrate.

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples: Compartmentation of Riboflavin in Acibia gossypii under Production Conditions 1.1 Permeabilization Method The selective permeabilization method was used to produce and compartmentalize the strongly productive mutant ItaGS01 with the addition of conkanamycin. Measured in four independent experiments, with or without additions. For this, the production medium (yeast extract: 10 g / l; soybean oil: 1)
(0 g / l; glycine: 6 g / l) and harvested at defined time points during production. For compartmental analysis, a method of selective permeabilization of the cell membrane of Acibia gossypi was applied. The purpose of selective permeabilization of cell membranes is the separate analysis of cytosolic and vacuolar compartments. To this end, the mycelium up to the desired time point was harvested by filtration (glass fiber-round filter, 4.5 cm diameter) and washed with NaP-buffer. Fungal cells (wet mass 0.5 g) are permeabilized with 50 mM N
Digitonin 0.003% (w / v) in aP-buffer, resuspended in 10 ml, 10%
Incubate for minutes for cell membrane permeabilization. At the end of the incubation, the cells were separated from the solution by filtration and the filtrate was used for analysis of the cytosolic cell component. The cell suspension was further incubated with 0.02% (w / v) digitonin for an additional 10 minutes for disassembly of all cell membranes. The vacuolar content of the cells could then be obtained for analysis (riboflavin measurement) by filtration.
The cell residue remaining on the filter paper was discarded.

[0034] 1.2 riboflavin content of quantitative analysis permeabilization filtrate dried biomass and riboflavin by HPLC, and then examined in isolated conditions described: ^{Column: LiCrospher (R) 100RP-18} (5μm) (Merck, Darmstadt Eluent: NaH ₂ PO ₄ 50 mM tetramethylammonium chloride 1 mM acetonitrile 12% flow rate: 1 ml / min elution: isocratic detection: 270 nm The riboflavin content of each compartment is determined by the media, cytosol, dry biomass used And used to calculate compartmentalization between vacuoles. For the determination of the dry fungal mass, 20-50 ml of soybean oil medium was first added to a 50 ml Falcon tube (Falcon tube).
tube) and centrifuged at 1500 xg for 10 minutes, then carefully pipetted off the upper oil layer. Thereafter, the oil-free suspension was filtered through a tared glass fiber-round filter and washed with 10 volumes of NaP-buffer. The mycelium on the filter was dried in a drying cabinet at 60 ° C. to constant weight and subsequently weighed.

1.3 Compartmentation of Riboflavin under Production Conditions For control and conkanamycin-treated cells (5 μM), respectively, 2
At three time points, 4, 43 and 67 hours, the cytosolic and vacuolar riboflavin content and the amount of riboflavin secreted into the medium were determined: the graph is clearly (visually / by light microscopy). Well visible)
If the V-ATPase-inhibitor concanamycin A is added to the medium at the beginning of the production phase, cells of the mutant ItaGS01 show that it can no longer store any riboflavin in the vacuole. The total production was the same for both the control and the concanamycin A-formulation, with concanamicin A
Under the addition, the amount of riboflavin (30-60% of the total production) that accumulates in the vacuoles when not added is secreted into the medium.

[0036] 2. Cloning, Sequencing and Disruption of the A. gossypii VMA1-Gene This experiment demonstrates the use of the inhibitor conkanamycin A to create a strain having a dysfunctional V-ATPase that diverts riboflavin flow of interest. It was performed in order to reproduce that it could be changed chemically. For this, the subunit A, which catalyzes the V-ATPase-complex, was cloned and destroyed. Subunit A having this catalysis is encoded by VMA1- gene (tonoplast ATP ase protein 1 (v acuolar m embrane A TPase Prote
in 1 )).

2.1. Cloning and Sequencing A fragment of the AgVMA1-gene (by degenerate oligonucleotide primers designed from sequences highly conserved in the VMAlp sequence) by PCR (
Evolutionary strong conservation): For PCR, various VMAs
1—Degenerate oligonucleotides were developed from strongly conserved amino acid fragments of the protein: CF1a (5-ATYCARGTTBTAYGARAC-3) and GF
1b (5-ATVACRGTYTTRCCRCA-3) was converted to MWG Biothe
ch (Ebersberg). PCR (94 ° C for 30 seconds, 52 ° C for 60 seconds, 72 ° C
60 seconds, 35 cycles) a Gene ^{Amp (R)} PCR System 9700 (App
lied Biosystems) in Taq-Polymerase (Boehringer Mannheim, Germany).
) Buffer was used as recommended by the manufacturer, primer CF1a 8μ
M, primer CF1b 4 μM was used. Using the amplified PCR fragment, a cosmid-gene library (cosmid vector Su
perCos1, Stratagene). Having homologous regions of DNA, in order to identify the positive cosmid clones were radioactively labeled by PCR- fragment with the T7- polymerase ^(a- 32 P) dCTP. The cosmid-DNA of the positive clone was digested with Baml and screened again with radioactive PCR-fragments. This gene was then transferred to the plasmid vector Bluesc.
The sequence was determined by subcloning in Rip (Stratagenen).
This nucleotide sequence has been assigned accession number A after the filing of the patent and the publication to which it belongs.
J009881 from the EMBL databank. VA of various organisms
Protein sequencing of AgVMA1p with the TPase-A-subunit indicated that a strongly conserved protein sequence was also present in Avia. Furthermore, VMA
The 1-protein phylogenetic tree showed that the previously known VMA1-protein of the strain closest to the corresponding protein of the yeast Saccharomyces cerevisiae was found in AgVMA1p. Southern analysis of the genome has demonstrated that this is a single copy gene (as is also common in yeast).

2.2 Disruption of the vmal gene For disruption, a Vmal-deletion / replacement-allele named vmal: G418 was constructed. For this purpose, a VMA1-P containing a part of the VMA1-ORF is used.
The BamHI-Pst1-fragment 0.25 kb in the CR-fragment is replaced with the Geneticin-resistance-cassette TEF-G418. First, VMA1-P
The CR-fragment was cloned into a modified pGEM ^(R) T-vector (Promega) without the Pst1-cleavage site, thereby producing plasmid pJR1767. Subsequently, the sequence encoding VMA1 was disrupted on the plasmid, whereby the 0.25 kb BamHI-Pst1-region was replaced by TEF-G418, resulting in plasmid pJR1773. The linear 2.5-kb-
The fragment Ncol-Spel was used to transform germinated spores of Acibia gossypii (starting strain wild-type, highly productive mutant ItaGS01). Geneticin-resistant clones were selected and disruption of the vma1-gene was confirmed by Southern blotting (FIG. 2).

2.2 Reversal of Riboflavin Mass Flow by Disruption of VMA-1 This disruption differs from the flat growth of the starting strain and is a characteristic dense / crater-growing colony on the plate Is shown. These cells secrete riboflavin (yellow colored agar plates), the mycelium itself is completely white and does not accumulate any riboflavin in its vacuoles (first detection by fluorescence microscopy). In contrast, the mycelium of the starting strain is colored yellow (often crystallizes) by riboflavin accumulated in the vacuole.

[0040] Riboflavin compartmentalization was quantitatively examined by the selective membrane permeabilization method (1.1).

By disruption of the V-ATPase-subunit A (with the addition of the V-ATPase-inhibitor conkanamycin as described above), a redirection of the riboflavin stream could be achieved. 3 without accumulation of vacuolar riboflavin (product retention)
Two new strains were offered.

Description of the Figures FIG. 1: Compartmentation of riboflavin in Acibia in production under standard production conditions (left column) and with the addition of the V-ATPase-inhibitor conkanamycin A, FIG. Disruption of AgVMA1 at TEF-G418-marker, (A) construction, (
B) Southern analysis, T1: disrupted strain of wild type strain T2: disrupted strain of ItaGS FIG. 3: Riboflavin compartmentalization in starting strain and mutant ItaGS disrupted strain.

[Sequence list]

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the compartmentalization of riboflavin in Ashibia during production under standard production conditions (left column) and with the addition of the V-ATPase-inhibitor conkanamycin A.

FIG. 2 is a schematic diagram showing a configuration in which AgVMA1 is disrupted by a TEF-G418-marker.

FIG. 3 is a schematic diagram showing the results of Southern analysis when AgVMA1 was disrupted with the TEF-G418-marker.

FIG. 4 is a graph showing riboflavin compartmentalization in the starting strain and the mutant ItaGS-disrupted strain.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 (C12P 25/00 15/01 C12R 1: 645) C12P 25/00 C12N 15/00 ZNAA / / (C12P 25/00 5/00 A C12R 1: 645) X (72) Inventor Corolla Förster Germany, Germany Frankfurt Zach Henweiser Landwehrweg 218 (72) Inventor Jose El Reverta Dvar Spain Salamanca Grillyo 11- 4D (72) Inventor Maria Angeles Santos Garcia Spain Salamanco Versaljes 7 F term (reference) 4B024 AA01 AA05 BA80 CA01 FA17 GA19 GA25 4B064 AH02 CA05 CA19 CD30 DA01 DA10 4B065 AA57Y AB01 AC14 AC15 BA02 CA16 CA60

Claims (26)

[Claims] 1. In a unicellular or multicellular organism, especially a microorganism, for biotechnological production of riboflavin, its intracellular mass transport is altered such that a major amount of riboflavin accumulates extracellularly. A unicellular or multicellular organism for the biotechnological production of riboflavin, characterized in that: 2. The unicellular or multicellular organism according to claim 1, wherein the transport of riboflavin into vacuoles is reduced. 3. The unicellular or multicellular organism according to claim 1, wherein the transport of riboflavin into the vacuole is at least partially suppressed. 4. The unicellular or multicellular organism according to claim 1, wherein V-ATPase activity is at least partially blocked. 5. The unicellular or multicellular organism according to claim 4, wherein the transport rate of riboflavin across the cell membrane is higher than the transport rate across the vacuolar membrane. 6. The unicellular or multicellular organism according to claim 4, which is a fungus. 7. The unicellular or multicellular organism according to claim 6, which is a filamentous fungus. 8. A unicellular or multicellular organism according to claim 1, which is a fungus from the family Saccharomyces. 9. The unicellular or multicellular organism according to claim 1, which is a fungus from the genus Acibia, preferably Acibia gossypii. 10. A V-ATPase gene which is at least partially inactivated by mutation. 11. The V-ATPase gene according to claim 10, which has a nucleotide sequence encoding the amino acid sequence according to FIG. 1 and an allelic variant thereof. 12. Nucleotides 1 to 3 according to the amino acid sequence shown in FIG.
12. The V-ATPase gene according to claim 10 or 11, which has a nucleotide sequence of 881 or a DNA sequence which acts almost similarly. 13. The gene having a regulatory gene sequence related to this gene.
13. The V-ATPase gene according to any one of items 1 to 12. 14. A gene for producing a riboflavin-producing unicellular or multicellular organism, which does not have a V-ATPase gene. 15. A vector containing the gene according to any one of claims 10 to 14. 16. A transformed organism for producing riboflavin containing the gene according to any one of claims 10 to 14 in a replicable form. 17. A transformed organism containing the vector according to claim 15. 18. A method for producing riboflavin, comprising using the organism according to any one of claims 1 to 9. 19. The method according to claim 18, wherein a chemical inhibitor is added to the fermentation medium. 20. The method according to claim 19, wherein conkanamycin, bafilomycin, N-ethylmaleimide, and nitrate are added to the fermentation medium as inhibitors. 21. A method for producing a riboflavin-producing unicellular or multicellular organism, which comprises altering a unicellular or multicellular organism so that a major amount of the produced riboflavin accumulates extracellularly. 22. The method according to claim 21, wherein the change of the organism is carried out by a genetic engineering method. 23. A modification or deletion of the V-ATPase gene to cause ATP
23. Blocking at least partially or completely the activity of ase.
The method according to any one of the preceding claims. 24. Use of an organism according to any one of claims 1 to 9 and 16 and 17 for producing riboflavin. 25. Use of the ATPase gene according to any one of claims 10 to 13 for producing the organism according to any one of claims 1 to 9 and 16 and 17. 26. Use of the vector according to claim 14 for producing the organism according to any one of claims 1 to 9 and 15 and 16.