DE4420785A1 - Riboflavin biosynthesis in fungi - Google Patents

Abstract

The invention concerns the riboflavin-biosynthesis genes in the fungus Ashbya gossypii as well as a method of producing riboflavin using these genes and gene products.

Description

The present invention relates to the genes for riboflavin bio synthesis in fungi, the proteins encoded with them and gen technical processes for the production of riboflavin under Ver use of these genes and gene products.

The production of riboflavin by fermentation of mushrooms such as Eremothecium ashbyii or Ashbya gossypii is known (The Merck Index, Windholz et al., Eds. Merck & Co., page 1183, 1983).

In EP 405370 are riboflavin overproducing bacteria strains described by transformation of the riboflavin bio Synthesis genes from Bacillus subtilis were obtained.

Because the genetics of riboflavin biosynthesis in bacteria and Eukaryotes is different, the genes mentioned above are made up of Bacillus subtilis not for a recombinant manufacturing process for riboflavin with eukaryotic production organisms such as Ashbya gossypii suitable.

In a filed on November 19, 1992 at the German Patent Office Patent application was the cloning of the riboflavin biosynthesis Genes of the yeast Saccharomyces cerevisiae are described.

A cloning of the Ashbya gossypii riboflavin biosynthesis genes using the S. cerevisiae RIB genes with common hybridi However, methods of settlement did not work; homology was evident the RIB genes from S. cerevisiae and A. gossypii for a hybridi not big enough.

It was therefore the task of the riboflavin biosynthesis genes a eukaryote to isolate a recombinant her Process for setting riboflavin in a eukaryotic product to provide onsorganism.

Accordingly, six genes were found in the Ascomycete Ashbya gossypii (rib genes), which are based on enzymes of riboflavin biosynthesis code from GTP, found and isolated.

The invention relates to the following DNA sequences:  

DNA sequences for a polypeptide with the in SEQ ID NO: 2 encode amino acid sequence shown or for an analog or derivative of the polypeptide according to SEQ ID NO: 2, wherein one or multiple amino acids deleted, added or by other ami have been substituted with no acidic effect significantly reduce the polypeptide.

DNA sequences for a polypeptide with the in SEQ ID NO: 4 encode amino acid sequence shown or for an analog or derivative of the polypeptide according to SEQ ID NO: 4, wherein one or multiple amino acids deleted, added or by other ami No acids have been substituted without the enzymatic effect significantly reduce the polypeptide.

DNA sequences for a polypeptide with that in SEQ ID NO: 6 encode amino acid sequence shown or for an analog or derivative of the polypeptide according to SEQ ID NO: 6, wherein one or multiple amino acids deleted, added or by other ami No acids have been substituted without the enzymatic effect significantly reduce the polypeptide.

DNA sequences for a polypeptide with the in SEQ ID NO: 8 encode amino acid sequence shown or for an analog or derivative of the polypeptide according to SEQ ID NO: 8, wherein one or multiple amino acids deleted, added or by other ami No acids have been substituted without the enzymatic effect significantly reduce the polypeptide.

DNA sequences which are for a polypeptide with the in SEQ ID NO: 10 encode amino acid sequence shown or for an analog or derivative of the polypeptide according to SEQ ID NO: 10, wherein one or multiple amino acids deleted, added or by other ami No acids have been substituted without the enzymatic effect significantly reduce the polypeptide.

DNA sequences which are for a polypeptide with the in SEQ ID NO: 12 encode amino acid sequence shown or for an analog or derivative of the polypeptide according to SEQ ID NO: 12, wherein one or multiple amino acids deleted, added or by other ami No acids have been substituted without the enzymatic effect significantly reduce the polypeptide.

The genes and their gene products (polypeptides) are in sequence protocol with their primary structure and have the following Assignment:

SEQ ID NO: 1: rib 1 gene
SEQ ID NO: 2: rib 1 gene product (GTP cyclohydrolase II)
SEQ ID NO: 3: rib 2 gene
SEQ ID NO: 4: rib 2 gene product (DRAP deaminase)
SEQ ID NO: 5: rib 3 gene
SEQ ID NO: 6: rib 3 gene product (DBP synthase)
SEQ ID NO: 7: rib 4 gene
SEQ ID NO: 8: rib 4 gene product (DMRL synthase)
SEQ ID NO: 9: rib 5 gene
SEQ ID NO: 10: rib 5 gene product (riboflavin synthase)
SEQ ID NO: 11: rib 7 gene
SEQ ID NO: 12: rib 7 gene product (HTP reductase)

Guanosine triphosphate (GTP) is by GTP-Cyclohydrolase II (rib 1 gene product) to 2,5-diamino-6-ribosylamino-4- (3H) -pyrimidine- 5-phosphate converted. This connection is then through rib 7 gene product to 2,5-diamino-ribitylamino-2,4 (1H, 3H) - pyrimidine-5-phosphate reduced and then by rib 2 gene product deaminated to 5-amino-6-ribitylamino-2,4 (1H, 3H) pyrimidinedione. It is then catalyzed in a rib 4 gene product tion added the C4 connection DBP and it is created 6,7-dimethyl-8-ribityllumazin (DMRL), from which in the rib 5 gene product catalyzed reaction riboflavin is formed. The C4 Ver Binding DBP (L-3,4-dihydroxy-2-butanone-4-phosphate) is made from D-Ribulose-5-phosphate catalyzed in a rib 3 gene product Reaction formed.

The DNA sequences described in SEQ ID NO: 1, 3, 5, 7, 9, 11 code ren for the polypeptides described in SEQ ID NO: 2, 4, 6, 8, 10, 12 are written.

In addition to the DNA sequences mentioned in the sequence listing are also those suitable due to the degeneration of the genetic Codes have a different DNA sequence, but for the same one Encode polypeptide.

Such DNA sequences are also the subject of the invention that for a gene product (polypeptide) with other than that in Code the primary structure listed as long as the gene product still essentially the same biological egg properties such as the gene product mentioned in the sequence listing sits. Among the biological properties are above all those Biosynthesis of riboflavin-causing enzymatic activities to understand.

Such modified gene products with essentially the same bio logical properties are by deletion or addition of one or more amino acids or peptides or by Aus  exchange of amino acids for other amino acids available or can be isolated from organisms other than Ashbya gossypii the.

The DNA sequences that code for the modified gene products are usually added to the DNA sequences according to the sequence listing 80 or more percent homologous. Such DNA sequences can be starting from those described in SEQ ID NO: 1, 3, 5, 7, 9, 11 DNA sequences, for example using conventional hybridization methods or the PCR technique from eukaryotes other than Ashbya gossypii isolate. These DNA sequences hybridize under standard conditions with those described in SEQ ID NO: 1, 3, 5, 7, 9, 11 DNA sequences.

For example, temperatures are under standard conditions between 42 and 58 ° C in an aqueous buffer solution with a Concentration between 0.1 and 1 × SSC (1 × SSC: 0.15 M NaCl, 15 mM Sodium citrate pH 7.2). The experimental conditions genes for DNA hybridizations are in textbooks on genetic engineering, for example in Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989.

The invention further relates to regulatory sequences, especially promoter sequences that are in the 5 direction before that for the corresponding DNA sequences encoding polypeptide. The Regulatory sequences are listed in the sequence listing and in following explained in more detail.

Regulatory sequence for rib 1 gene:
SEQ ID NO: 1 nucleotide 1-242
Regulatory sequence for rib 2 gene:
SEQ ID NO: 3 nucleotides 1-450
Regulatory sequence for rib 3 gene:
SEQ ID NO: 5 nucleotides 1-314
Regulatory sequence for rib 4 gene:
SEQ ID NO: 7 nucleotide 1-270
Regulatory sequence for rib 5 gene:
SEQ ID NO: 9 nucleotide 1-524
Regulatory sequence for rib 7 gene:
SEQ ID NO: 11 nucleotide 1-352

The regulatory sequences can also in 5'- and / or 3'-direction can be shortened without their function significantly subsides.

Fragments are generally essential for the regulatory effect from 30 to 100, preferably 40 to 70 nucleotides from the above given sequence areas.

These regulatory sequences can also be directed Mutagenesis compared to the natural sequences in your Function can be optimized.

The regulatory sequences according to the invention are suitable for the Overexpression of genes in Ashbya, especially genes that are responsible for the riboflavin biosynthesis.

The invention furthermore relates to expression vectors which contain one or more of the DNA sequences according to the invention. Such expression vectors are obtained by using the invention modern DNA sequences with suitable functional regulations signals. Such regulatory signals are DNA sequences responsible for expression, for example Promoters, operators, enhancers, ribosomal binding sites, and that are recognized and operated by the host organism.

If necessary, other regulatory signals, which at for example, replication or recombination of the recombinant DNA control in the host organism, part of the expression vector his.

Likewise belong those with the DNA sequences according to the invention or Expression vectors transformed host organisms to the opposite state of the invention. Are preferred as host organisms eukaryotic organisms, particularly preferably those of the genus Saccharomyces, Candida, Pichia, Eremothecium or Ashbya used. Saccharomyces are particularly preferred species cerevisiae, Candida flaveri, Candida famata, Eremothecium ashbyii and Ashbya gossypii.

A recombinant preparation also belongs to the invention Procedure for riboflavin in which the trans formed host organisms in a manner known per se Fermentation are grown and that during the fermentation Riboflavin formed is isolated from the fermentation medium and is cleaned if necessary.  

The rib genes and gene products can be as in the example and in Isolate and characterize the sequence listing.

example 1 Isolation of the Ashbya gossypii riboflavin biosynthesis genes (rib- Genes) a. Construction of an Ashbya gossypii cDNA library

Total RNA was overproducing from the mycelium of riboflavin Strain Ashbya gossypii ATCC 10195 after cultivation on YEPD medium (Sherman et al., "Methods in yeast genetics," Cold Spring Harbor, New York, 1989) in the late logarithmic growth phase ex trahed.

Poly (A) ⁺ RNA was purified by double adsorption and elution on oligo (dT) cellulose (Aviv and Leder, Proc. Natl. Acad. Sci. USA 69, 1972, 1408-1412). The cDNA was isolated according to the general procedure of Gubler and Hoffmann (Gene 25, 1983, 263) and synthetic EcoRI adapters were added to the ends of the blunt end cDNA molecules. The EcoRI-cut cDNA fragments were then phosphorylated using T4 polynucleotide kinase and cloned into the dephosphorylated EcoRI-cut vector pYEura3 ( FIG. 1). pYEura3 (Clonetech Laboratories, Inc., California) is a yeast expression vector that contains the galactose-inducible GAL1 and GAL10 promoters and URA, CEN4 and ARS1. These yeast elements allow the transformation and expression of cloned DNA fragments in yeast cells.

Aliquots of the ligation reaction were used to make highly competent (Hanahan, DNA Cloning, ed. D.M. Glover; IRL Press, Oxford 1985, 109) E. coli XL1-Blue (Bullock et al., Biotechniques 5 (1987) 376-378) and transformants were based selected their ampicillin resistance.

Approximately 3 × 10⁵ ampicillin resistant cells were pooled amplified and plasmid DNA isolated therefrom (Birnboim and Doly, Nucleic Acids Res. 7, 1979, 1513).

b. Isolation of Ashbya gossypii cDNA clones required for ribofla encode vin-forming enzymes

Ashbya gossypii cDNA clones for riboflavin-forming enzymes were encoded by functional complementation of Saccharo myces cerevisiae mutants that are involved in riboflavin biosynthesis are isolated.

The strains AJ88 (Mata leu2 his3 rib1 :: URA3 ura3-52), AJ115 (Ma talpha leu2 inos1 rib2 :: URA3 ura3-52), AJ71 (Matalpha leu2 inos1 rib3 :: URA3 ura3-52), AJ106 (Matalpha leu2 inos1 rib4 :: URA3 ura3-52), AJ66 (Mata canR inos1 rib5 :: URA3 ura3-52) and AJ121 (Matalpha leu2 inos1 rib7 :: URA3 ura3-52) are mutated strains, by destroying one of the six genes (RIB1 to RIB5 and RIB7) involved in riboflavin biosynthesis at Saccharomyces cerevisiae are involved.

These strains were each with 25 µg cDNA from Ashbya gossypii cDNA library transformed and on solid galactose-containing medium plated without riboflavin. After about a week of growth Rib + transformants were isolated from the culture dishes.

One transform from each transformed mutant (Rib1 +, Rib2 +, Rib3 +, Rib4 +, Rib5 + and Rib7 +) was analyzed and in all cases the Rib + phenotype was found only in Galactose medium, but was not expressed in glucose medium.

These results show that the Rib + phenotype under the Kon trolls of the plasmid-bound galactose-inducible GAL10 promo tors was expressed.

Plasmid DNA was generated from the Rib1 +, Rib2 +, Rib3 +, Rib4 +, Rib5 + and Rib7 + transformants isolated by transforming E. coli and called pJR715, pJR669, pJR788, pJR733, pJR681 and pJR827.

Partial sequencing of the cDNA-In contained in these plasmids Sertions confirmed that they code for proteins that are analog to proteins of the Rib gene products from Saccharomyces.

c. Isolation of Ashbya gossypii genomic clones used for ri encode boflavin-forming enzymes

The genomic copies of Ashbya's riboflavin-forming genes Isolating gossypii became an Ashbya gos genomic library sypii ATCC 10195 in the cosmid superCos1 (Stratagene Cloning Sy stems, California) and with 32 P-labeled samples from the cDNA copies of RIB1, RIB2, RIB3, RIB4, RIB5 and RIB7 Genes derived from Ashbya gossypii were screened.  

Cosmid clones with RIB1, RIB2, RIB3, RIB4, RIB5 and RIB7 DNA were isolated by colony hybridization (Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 72, 1975, 3961-3965). More Southern Analyzes of enzymatically cleaved cosmid DNA using the same RIB-specific cDNA samples allowed identification ornamentation of defined restriction fragments that the RIB1, RIB2, RIB3, RIB4, RIB5 and RIB7 genes from Ashbya gossypii contained.

A 3.1 kb BamHI-ClaI DNA fragment was found which contained the entire RIB1 gene from Ashbya gossypii, coding for GTP-Cyclohydrolase II. This fragment was isolated from an agarose gel and cloned into the BamHI and ClaI cut pBluescript KS (+) phagemid (Stratagene Cloning Systems) and thus gave the plasmid pJR765 ( FIG. 2).

A 1329 bp DNA sequence was obtained (SEQ ID NO: 1), which the RIB1 open reading frame of 906 bp, 242 bp from the 5′-not coding region and 181 bp from the 3′-non-coding region contained.

The entire Ashbya gossypii RIB2 gene coding for the DRAP deaminase was found on a 3.0 kb EcoRI-PstI fragment which, when cloned into pBluescript KS (+), gave the plasmid PJR758 ( Fig. 3).

A 2627 bp region of the EcoRI-PstI insertion with the open NEN reading frame from RIB2 of 1830 bp, 450 bp the 5'-untranslated region and 347 bp of the 3′-untranslated region was se quoted (SEQ ID NO: 3).

The entire Ashbya gossypii RIB3 gene, which codes for the DBP synthase, was found on a 1.5 kb long PstI-HindIII fragment which, when cloned into pBluescript KS (+), gave the plasmid PJR790 ( FIG. 4).

A 1082 bp region of the PstI-HindIII insertion with the open reading frame of RIB3 of 639 bp, 314 bp of 5′-untransla region and 129 bp of the 3′-untranslated region sequenced (SEQ ID NO: 5).

The Ashbya gossypii RIB4 gene, which codes for the DMRL synthase, was found on a 3.2 kb PstI-PstI fragment which, when cloned in pBluescript KS (+), gave the plasmid PJR762 ( FIG. 5).

A 996 bp region of the PstI-PstI insert with the open RIB4 reading frame of 519 bp, 270 bp of the 5′-untranslated Region and 207 bp of the 3'-untranslated region was sequenced adorned (SEQ ID NO: 7).

The entire Ashbya gossypii RIB5 gene, which codes for the riboflavin syn thase, was found on a 2.5 kb long PstI-PstI fragment which, when cloned in pBluescript KS (+), gave the plasmid PJR739 ( FIG. 6).

A 1511 bp region of the PstI-PstI insertion with the open RIB5 reading frame of 708 bp, 524 bp of the 5'-untranslated Region and 279 bp of the 3'-untranslated region was sequenced adorned (SEQ ID NO: 9).

Finally, the Ashbya gossypii RIB7 gene, which codes for the HTP reductase, was found on a 4.1 kb long EcoRI-EcoRI fragment which, cloned in pBluescript KS (+), gave the plasmid PJR845 ( FIG. 7).

A 1596 bp region of the EcoRI-EcoRI insertion with the open NEN reading frame of RIB7 of 741 bp, 352 bp of 5′-untranslated region and 503 bp of the 3′-untranslated region was se quoted (SEQ ID NO: 11).

Example 2 mRNA analysis of the Ashbya gossypii RIB genes

In order to identify the RIB specific transcripts Northern analyzes performed. Total RNA was from Ashbya gossypii strain ATCC 10195 as described in Example 1, iso liert. The RNA samples of the strain (5 µg) were electrophoresed separated on 0.8% agarose formaldehyde gels together with RNA Size markers and blotted under vacuum on nylon membrane (Tho mas, proc. Natl. Acad. Sci. USA, 77, 1980, 5201-5205).

The nylon membranes were independently hybridized with 32 P-labeled RIB-specific DNA samples at 42 ° C in 5 x SSC and in the presence of 50% formamide. The Ashbya gossypii RIB1 gene is expressed as a unique message of approximately 1150 nucleotides, which was detected in both strains by a 0.7 kbp SmaI-SacI sample from the plasmid pJR765 ( FIG. 8).

Analogously, unique 1900 nucleotides were RIB2, 900 nucleotides long RIB3, 800 nucleotides long RIB4, 1050 nucleotides long RIB5 and 1000 nucleotide long RIB7 transcripts in the blots  Using a 0.5 kbp SmaI-SmaI fragment from pJR758, one 0.6 kbp HindIII-KpnI fragment from pJR790, a 0.5 kbp long ScaI-HindIII fragment from pJR739 and a 0.3 kbp long PstI-PstI fragments from pJR845 as a specific sample grasslands.

Example 3 Expression of the Ashbya gossypii RIB genes in Saccharomyces cerevisiae

As described in Example 1, mutants can be well studied of Saccharomyces cerevisiae, which in one stage of riboflavin biosynthesis are defective, awake on culture media without riboflavin if they carry a plasmid that is complementary Enzymes encoded by Ashbya. To the function of Ashbya gossypii RIB gene products were used to test flavin-forming enzyme activities measured in cell-free extracts of S. cerevisiae mutants which one of the expression plasmids pJR715, pJR669, pJR788, pJR733, pJR681 and pJR827 wore.

These pYEura3-derived Plas described in Example 1 Ashidea gossypii contain RIB-specific cDNA fragments under the control of the galactose-inducible GAL10 promoter.

Cell-free protein extracts from S. cerevisiae were obtained from cultures obtained in liquid medium up to an optical density of about 2 OD had grown.

The cells were harvested with cold 20 mM Tris HCl, pH 7.5 washed and in the same buffer containing 1 mM phenylethyl sulfo nyl fluoride was supplemented, resuspended.

Cell lysates were vortexed in the presence of glass balls and Centrifugation at 3000 g for 20 min. made at 4 ° C.

GTP cyclohydrolase II, DRAP deaminase, DBP synthase, DMRL syn thase, riboflavin synthase and HTP reductase enzyme activities were determined as described in the literature (Shavlovsky et al, Arch. Microbiol. 124 1980, 255-259; Richter et al., J. Bace riol. 175, 1993, 4045-4051; Klein and Bacher, Z. Naturforsch. 35b, 1980, 482-484; Richter et al. J. Bacteriol. 174, 1992, 4050-4056; Nielsen et al. J. Biol. Chem. 261, 1986, 3661; Plaut and Harvey, Methods Enzymol. 18B, 1971, 515-538; Hollander and Brown, Biochem. Biophys. Res. Commun. 89, 1979, 759-763; Shavlov ski et al., Biochim. Biophys. Acta, 428, 1976, 611-618).  

Protein was quantified using the Peterson method (Anal. Biochem. 83, 1977, 346-356). As can be seen from Table 1, causes the plasmid pJR715 the expression of GTP cyclohydrolase II Activity in the S. cerevisiae mutant AJ88. Furthermore, this is Activity only present in cells that are on galactose medium are growing, indicating that the RIB1 cDNA expression by Ashbya gossypii under the control of galactoseinduzierba ren GAL10 promoter takes place.

Therefore, these results demonstrate that RIB1 for the GTP cyclohydro lase II encoded in Ashbya gossypii. It was shown in an analogous way that RIB2 for DRAP deaminase, RIB3 for DBP synthase, RIB4 for DMRL synthase, RIB5 for riboflavin synthase and RIB7 for HTP- Reductase encoded in this fungus.

Table 1

GTP-Cyclohydrolase II activity of the S. cerevisiae RIB1 mutant AJ88 and its transformants

Table 2

DRAP deaminase activity of the S. cerevisiae RIB2 mutant AJ115 and its transformants

Table 3

DBP synthase activity of the S. cervisiae RIB3 mutant AJ71 and its transformants

Table 4

GTP-Cyclohydrolase II activity of the S. cervisiae RIB4 mutant A106 and its transformants

Table 5

Riboflavin synthase activity of the S. cerevisiae RIB5 mutant AJ66 and its transformants

Table 6

HTP reductase activity of the S. cerevisiae RIB7 mutant AJ121 and its transformants

SEQUENCE LOG

Claims (9)

1. DNA sequences for a polypeptide with the in SEQ ID NO: 2 encode amino acid sequence shown or for an analog ges or derivative of the polypeptide according to SEQ ID NO: 2, wherein deleted or added one or more amino acids have been substituted by other amino acids without the To substantially reduce the enzymatic effect of the polypeptide ren. 2. DNA sequences which are for a polypeptide with the in SEQ ID NO: 4 encode amino acid sequence shown or for an analog ges or derivative of the polypeptide according to SEQ ID NO: 4, wherein deleted or added one or more amino acids have been substituted by other amino acids without the To substantially reduce the enzymatic effect of the polypeptide ren. 3. DNA sequences that are for a polypeptide with that in SEQ ID NO: 6 encode amino acid sequence shown or for an analog ges or derivative of the polypeptide according to SEQ ID NO: 6, wherein deleted or added one or more amino acids have been substituted by other amino acids without the To substantially reduce the enzymatic effect of the polypeptide ren. 4. DNA sequences for a polypeptide with the in SEQ ID NO: 8 encode amino acid sequence shown or for an analog ges or derivative of the polypeptide according to SEQ ID NO: 8, wherein deleted or added one or more amino acids have been substituted by other amino acids without the To substantially reduce the enzymatic effect of the polypeptide ren. 5. DNA sequences necessary for a polypeptide with that in SEQ ID NO: 10 encode amino acid sequence shown or for an Ana loges or derivative of the polypeptide according to SEQ ID NO: 10, wherein deleted or added one or more amino acids have been substituted by other amino acids without the To substantially reduce the enzymatic effect of the polypeptide ren.   6. DNA sequences necessary for a polypeptide with that in SEQ ID NO: 12 encode amino acid sequence shown or for an Ana loges or derivative of the polypeptide according to SEQ ID NO: 12, wherein deleted or added one or more amino acids have been substituted by other amino acids without the To substantially reduce the enzymatic effect of the polypeptide ren. 7. Expression vector containing one or more DNA sequences according to claims 1 to 6. 8. Host organism with an expression system according to An saying 7 has been transformed. 9. Recombinant manufacturing process for riboflavin, thereby ge indicates that a host organism according to claim 8 is used.