DE19937548A1 - Single or multicellular organisms for the production of riboflavin - Google Patents

Abstract

The present invention relates to a unicellular or multicellular organism, in particular a microorganism, for the biotechnological production of riboflavin, the enzyme activity of which is higher with respect to NAD (P) H formation than that of a wild type of the species Ashbya gossypii ATCC10895.

Description

The present invention relates to a single or multicellular organism for Production of riboflavin.

Vitamin B ₂ , also called riboflavin, is essential for humans and animals. With vitamin B ₂ deficiency, 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, reduced 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. Both chemical manufacturing processes, the riboflavin is usually in several stages Processes obtained as a pure end product, although also relatively costly raw materials - such as D-Ribose - can be used have to.

The production offers an alternative to the chemical production of riboflavin of this substance by microorganisms. The microbial production of riboflavin is particularly suitable for cases in which this is very pure Substance is not required. This is the case, for example, if that Riboflavin is to be used as an additive to feed products. In such Cases, the microbial production of riboflavin has the advantage that it Substance can be obtained in a one-step process. Can also as Starting products for the microbial synthesis of renewable raw materials, like at for example vegetable oils.  

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

In addition, processes with the yeast Candida famata are, for example, in the US 05231007.

Riboflavin overproducing bacterial strains are for example in the EP 405370 described, the strains by transformation of the riboflavin Biosynthetic genes from Bacillus subtilis were obtained. These prokaryotic genes but were for a recombinant riboflavin production process Eukaryotes such as Saccharomyces cerevisiae or Ashbya gossypii are unsuitable. Therefore, according to WO 93/03183 specific for riboflavin biosynthesis Genes isolated from a eukaryote, namely from Saccharomyces cerevisiae a recombinant production process for riboflavin in one to provide eukaryotic production organism. Such recombinant However, manufacturing processes for riboflavin production do not have any or limited success when providing substrate for those at the Riboflavin biosynthesis specifically involved enzymes is insufficient.

In 1967, Hanson (Hanson AM, 1967, in Microbial Technology, Peppler, HJ, pp. 222-250 New York) that the addition of the amino acid glycine to riboflavin Formation of Ashbya gossypii increases. However, such a method is disadvantageous because glycine is a very expensive raw material. For this reason, one was strives to increase riboflavin production by producing mutants optimize.  

DE 195 45 468.5 A1 describes another microbial process Production of riboflavin known in which the isocitrate lyase activity or Isocitrate lyase gene expression of a riboflavin-producing microorganism is increased. In addition, DE 198 40 709 A1 is a single or multi-cell Organism in particular a microorganism for the biotechnical production of Riboflavin known. This is characterized by the fact that one is changed Glycine metabolism shows that its riboflavin synthesis performance without external Glycine intake at least equal to that of a wild-type Ashbya species gossypii ATCC10892.

But even in comparison to these methods, there is still a need for one further optimization of riboflavin production.

The object of the present invention is accordingly a single or multi-cell Organism, preferably a microorganism, for the biotechnical Production of riboflavin to provide further optimization of riboflavin formation. In particular, an organism should Be made available, which enables a production that is compared to the prior art is more economical. Above all, the organism an increased riboflavin formation compared to previous organisms allow.

This task is solved by a single or multicellular organism, the Enzyme activity that is higher than that of NAD (P) H formation Wild types of the species Ashbya gossypii ATCC10895.

The goal of accelerated intracellular NAD (P) H supply can be achieved by Increasing the activity of a NAD (P) H-forming or lowering the activity of an enzyme consuming NAD (P) H or a change in specificity can be achieved. This can be done with the known methods of Strain improvement of organisms can be achieved. That is, in the simplest case appropriate strains can be made according to the standard in microbiology Establish selection using screening. The mutation is also followed by Selection can be used. The mutation can be both chemical and  by means of physical mutagenesis. Another method is Selection and mutation with subsequent recombination. Finally, produce the organisms according to the invention by means of genetic engineering.

According to the invention, the organism is changed such that it is intracellular NAD (P) H produces in an amount greater than its need for the Maintaining its metabolism. This increase in intracellular According to the invention, NAD (P) H generation can preferably be achieved by that an organism is produced in which the enzyme activity of the isocitrate Dehydrogenase is increased. This can be achieved, for example, in that by changing the catalytic center there is an increased substrate turnover or by canceling the action of enzyme inhibitors. Also one can increased enzyme activity of isocitrate dehydrogenase by increasing the Enzyme synthesis, for example by gene amplification or by switching off are caused by factors that repress enzyme biosynthesis.

According to the invention, the isocitrate dehydrogenase activity can preferably be achieved by Mutation of the isocitrate dehydrogenase gene can be increased. Such mutations can either be generated undirected using classic methods, such as for example by UV radiation or mutation-triggering chemicals, or targeted using genetic engineering methods such as deletion, insertion and / or Nucleotide exchange.

Isocitrate dehydrogenase gene expression can be achieved by incorporating isocitrate Dehydrogenase gene copies and / or by enhancing regulatory factors, that positively affect isocitrate dehydrogenase gene expression become. For example, regulatory elements can preferably be reinforced Transcription level take place, in particular by increasing the transcription signals become. In addition, an increase in translation is also possible by for example, the stability of the m-RNA is improved.

To increase the number of gene copies, the isocitrate Dehydrogenase gene is inserted into a gene construct or into a vector, the regulatory gene preferably assigned to the isocitrate dehydrogenase gene  Contains gene sequences, especially those that increase gene expression. Then a riboflavin-producing microorganism with which the Isocitrate dehydrogenase gene-containing gene construct transformed.

According to the invention, the overexpression of isocitrate dehydrogenase can also can be achieved by exchanging the promoter. It is possible to choose the higher one alternatively by incorporating gene copies or by enzymatic activity Exchange of the promoter. Equally, however, it is also possible by simultaneously exchanging the promoter and installing gene copies to achieve the desired change in enzyme activity.

The change in the isocitrate dehydrogenase gene leads to an accelerated NAD (P) H formation and at the same time to a surprisingly high increase in Riboflavin formation that was previously unattainable.

The isocitrate dehydrogenase gene is preferably made up of microorganisms, particularly preferably isolated from mushrooms. Here are mushrooms of the genus Ashbya again preferred. The species Ashbya gossypii is most preferred.

For the isolation of the gene there are also all other organisms, their Cells that contain the sequence for the formation of isocitrate dehydrogenase, also plant and animal cells. The isolation of the gene can be done by homologous or heterologous complementation of an isocitrate dehydrogenase Gen defective mutant or by heterologous probing or PCR heterologous primers. The insert of the complementing plasmids with appropriate steps Restriction enzymes can be minimized in size. After sequencing and The putative gene is identified by a precise subcloning PCR. Plasmids which carry the fragments thus obtained as an insert are inserted into the Isocitrate dehydrogenase gene defects introduced in mutant that affect functionality of the isocitrate dehydrogenase gene is tested. Become functional constructs finally used to transform a riboflavin producer.  

After isolation and sequencing, the isocitrate dehydrogenase genes are available with nucleotide sequences which code for the specified amino acid sequence or its allele variation. Allelic variations include, in particular, derivatives which can be obtained from corresponding sequences by deletion, insertion and substitution of nucleotides, the isocitrate dehydrogenase activity being retained. A corresponding sequence is shown in Fig. 2b from nucleotide 1 to 1262.

The isocitrate dehydrogenase genes are in particular a promoter of the nucleotide sequence from nucleotide -661 to -1 in accordance with. Fig. 11 or an essentially identical DNA sequence upstream. 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, but without the functionality or the effectiveness of the promoter being impaired. Furthermore, the effectiveness of the promoter can be increased by changing its sequence or can be completely replaced by effective promoters.

The isocitrate dehydrogenase gene can furthermore contain regulatory gene Sequences or regulator genes can be assigned, which in particular the isocitrate Increase dehydrogenase gene activity. So the isocitrate dehydrogenase Gen, for example, so-called "enhancers" can be assigned, which have an improved Interaction between RNA polymerase and DNA an increased isocitrate Effect dehydrogenase expression.

The isocitrate dehydrogenase gene with or without an upstream promoter or with or without a regulatory gene, one or more DNA sequences can be and / or downstream, so that the gene is contained in a gene structure. By cloning the isocitrate dehydrogenase gene are plasmids or vectors available that contain the isocitrate dehydrogenase gene and for transformation of a riboflavin producer are suitable. The ones obtainable through transformation Cells contain the gene in replicable form, i.e. H. in additional copies the chromosome, where the gene copies by homologous recombination  arbitrary sites of the genome and / or on a plasmid or Vector.

In the single or multicellular organisms obtained according to the invention, it can are any cells that can be used for biotechnical processes. For this count for example mushrooms, yeasts, bacteria as well as vegetable and animal Cells. According to the invention, they are preferably transformed cells of mushrooms, particularly preferably of mushrooms of the genus Ashbya. Here is the Ashbya gossypii species are particularly preferred.

In the following the invention is explained in more detail by means of examples without so that a limitation to the subject of the examples should be connected:

The isocitrate dehydrogenase (IDP3) gene was cloned by PCR and then sequenced (see Figure 11 for the sequence). The partial deletion of the gene by genetic engineering by exchange mutagenesis with a geneticin resistance gene ( FIG. 1) was confirmed by Southern blot ( FIG. 2). This disruption, ie destruction of the gene in the genome of the fungus, means that the fungus can no longer form the isocitrate dehydrogenase encoded by it. Fig. 3 shows the decrease in enzyme activity in the disruption strain AgΔDP3b compared to the wild type ATCC 10895. It was shown in preparations of the peroxisomes that this enzyme is localized in these organelles ( Fig. 10). While the enzyme activity is clearly measurable in wild-type peroxisomes, there is no more activity in the peroxisomes of the disruption strain.

The disruption of the gene leads to a significant reduction in vitamin formation compared to the parent strain ( Fig. 4). If, on the other hand, the gene is brought into Ashbya cells under the control of the strong TEF promoter on a plasmid ( Fig. 6), a significant increase in the enzyme activity and the riboflavin formation can be measured ( Fig. 5).

Fig. 7 shows that when unsaturated fatty acids are metabolized, NADPH is required as a reducing agent in two of three alternative reaction pathways. The 2,4-dienoyl-CoA reductase involved was also localized in peroxisomes in Ashbya cells ( Fig. 8). The disruption of the IDP3 gene should now lead to a reduced growth of the cells on linoleic acid or linolenic acid. This could also be measured ( Fig. 9). This shows that the importance of IDP3 for cell metabolism lies in the formation of NADPH.

Fig. 1

Scheme of the construction of the vector pIDPkan for the gene exchange of the chromosomal AgIDP3 gene against a gene copy inactive by deletion and insertion of the G418 ^R gene

Fig. 2

Verification of the partial deletion and simultaneous insertion of the geneticin resistance cassette at the AgIDP locus by means of Southern blot analysis. Genomic, SphI-digested DNA was hybridized with a digoxygenin-labeled probe

Fig. 3

Comparison of the enzyme activities of the NADP-specific ICDH from the Ashbya wild type, the mutant A. g. ΔIDP3b and the AgIDP overexpressors A. g. pAGIDP3a and A. g. pAGIDP3b when growing on complete glucose medium

Fig. 4

Comparison of Ashbya wild-type growth and riboflavin formation and the mutant A. g. ΔIDP3b when growing on complete soybean oil medium

Fig. 5

Comparison of Ashbya wild-type growth, riboflavin formation and NADP-specific ICDH and AgIDP3 overexpressors A. g. pAGIDP3a and A. g. pAGIDP3b when cultivated on soybean oil full medium

Fig. 6 Plasmid for overexpression of the AgIDP3 gene under the control of TEF- Promoter and terminator

For the introduction of the SphI interface there was a change for the second nucleic acid coding sequence necessary. It was a conservative replacement of the amino acid glycine in leucine performed.

Fig. 7

Degradation pathways of unsaturated fatty acids with double bonds on even (A) and odd (B, C) C atoms in peroxisomes according to Henke et al. (1998)

Fig. 8 Separation of organelles isolated from Ashbya wild-type in Percoll Density gradients

Activities [U / ml] of the marker enzymes catalase (peroxisomes) and fumarase (mitochondria), the NAD- and NADP-specific ICDH and the 2,4-dienoyl-CoA reductase required for the breakdown of unsaturated fatty acids and Δ ³ , Δ ² - Enoyl-CoA isomerase.

Fig. 9

Comparison of the radial growth of Ashbya wild type, the mutants A. g. ΔIDP3a and A. g. ΔIDP3b and the overexpressors A. g. pAGIDP3a and A. g. pAGIDP3b on various fatty acids (A: 18: 1 cis 9, B: 18: 2 cis 9, 12, C: 18: 3 cis 9, 12, 15)

Fig. 10

Distribution of the enzymes catalase and ICDH in the Percoll density gradient after centrifugation of organelles from wild type mycelium (A) and the mutant A. g. ΔIDP3b (B)

Fig. 11

Nucleotide sequence and amino acid sequence derived therefrom of the A. gossypii AgIDP3 gene coding for the peroxisomal NADP-specific isocitrate dehydrogenase

Claims (21)

1. Single or multi-cell organism, in particular microorganism, for the biotechnical production of riboflavin, characterized in that its enzyme activity with respect to NAD (P) H formation is higher than that of a wild type of the species Ashbya gossypii ATCC10895. 2. Single or multi-cell organism according to claim 1 characterized in that it has an increased isocitrate Has dehydrogenase activity. 3. Single or multi-cell organism according to one of claims 1 or 2, characterized in that it is a mushroom. 4. Single or multi-cell organism according to one of claims 1 to 3, characterized in that it is a mushroom of the genus Ashbya is. 5. Single or multi-cell organism according to one of claims 1 to 4 characterized in that it is a fungus of the species Ashbya gossypii is. 6. Isocitrate dehydrogenase gene with a nucleotide sequence coding for the amino acid sequence shown in FIG. 11 and its allele variation. 7. isocitrate dehydrogenase gene according to claim 6 with the nucleotide sequence of nucleotide 1 to 1262 acc. of Fig. 11 or a substantially equal-acting DNA sequence. 8. isocitrate dehydrogenase gene according to one of claims 6 or 7 with an upstream promoter of the nucleotide sequence with nucleotide -661 to -1 acc. of Fig. 11 or a substantially equal-acting DNA sequence. 9. isocitrate dehydrogenase gene according to any one of claims 6 to 8 with this assigned regulatory gene sequences. 10. Gene structure containing an isocitrate dehydrogenase gene according to one of the Claims 6 to 9. 11. Vector containing an isocitrate dehydrogenase gene according to one of the Claims 6 to 9 or a gene structure according to claim 10. 12. Transformed organism for the production of riboflavin containing in replicable form an isocitrate dehydrogenase gene according to one of the Claims 6 to 9 or a gene structure according to claim 10. 13. Transformed organism according to claim 12 containing a vector according to claim 11. 14. Process for the preparation of riboflavin, characterized in that an organism acc. one of claims 1 to 5 is used. 15. A process for producing a riboflavin-producing egg or multicellular organism, characterized in that it is changed so that whose enzyme activity with respect to NAD (P) H formation is higher than that of a wild type of Ashbya gossypii ATCC10895.   16. The method according to claim 15, characterized in that the change in Organism takes place using genetic engineering methods. 17. The method according to any one of claims 15 or 16, characterized in that the change in Organism by exchanging the promoter and / or increasing the Gene copy number is achieved. 18. The method according to any one of claims 15 to 17, characterized in that by changing the endogenous isocitrate dehydrogenase gene is an enzyme with increased activity is produced. 19. Use of the organism according to one of claims 1 to 5 and 12 and 13 for the production of riboflavin. 20. Use of the isocitrate dehydrogenase gene according to one of the claims 6 to 9 and the gene structure according to claim 10 for the production of a Organism according to one of claims 1 to 5 and 12 and 13. 21. Use of the vector according to claim 11 for the production of a Organism according to one of claims 1 to 5 and 12 and 13.