EP1563057A1

EP1563057A1 - Method for producing riboflavin

Info

Publication number: EP1563057A1
Application number: EP03781159A
Authority: EP
Inventors: Aleksandr Sergeevich Mironov; Natalya Valentinovna Korolkova; Lopes Lyubov Errais; Lilia Emmanuilovna Semenova; Daniil Aleksandrovich Perumov; Rimma Aleksandrovna Kreneva; Aleksandr Viktorovich Glazunov; Raisa Illarionovna Akishina; Yurgis Antanas Vladovich Iomantas; Elena Georgievna Abalakina; Natalia Viktorovna Stoynova; Yuri Ivanovich Kozlov; Vladimir Georgievich Debabov
Original assignee: State Research Institute of Genetics and Selection of Industrial Microorganisms
Current assignee: State Research Institute of Genetics and Selection of Industrial Microorganisms
Priority date: 2002-11-15
Filing date: 2003-11-14
Publication date: 2005-08-17
Also published as: RU2261273C2; EP1563057A4; WO2004046347A1

Abstract

Riboflavin is produced by cultivating in a culture medium the recombinant bacterium belonging to the genus Bacillus which comprises depressed Bacillus amyloliquefaciens rib operon, is capable to use glycerophosphate is a sole carbon source, has a resistance to growth inhibition by glyoxylate and has ability to produce and a accumulate riboflavin in the culture, and recovering riboflavin therefrom.

Description

METHOD FOR PRODUCING RIBOFLAVIN

Technical field

The present invention relates to microbiological industry, specifically to a method for producing riboflavin. More specifically, the present invention concerns a method for producing riboflavin using bacterium belonging to the genus Bacillus.

Background Art Riboflavin (vitamin B₂) is an essential compound for higher animals including humans. Deficiency in riboflavin results in several types of diseases, such as loss of hair, inflammation of the skin and similar skin damage, conjunctivitis, vision deterioration, growth failure etc.

Therefore riboflavin is commercially used as a vitamin preparation for use in vitamin deficiency and as a feed additive. In addition, it is also employed as a food dye, for example in mayonnaise, ice cream etc.

Riboflavin is prepared either chemically or microbiologically. In the chemical methods of preparation, the riboflavin is usually obtained as a pure end product in complicated multistep processes using relatively expensive starting compounds such as D- ribose.

The preparation of riboflavin by means of fermenting fungi such as Ashhya gossypii or Eremothecium ashbyii has been disclosed (The Merck Index, Windholz et al., eds. Merck & Co., page 1183, 1983). Yeasts, such as Candida or Saccharomyces are also suitable for producing riboflavin (WO 93/03183, JP63098399A2, US Pat. No. 4794081). Fungus from the Ashhya genus with increased isocitrate lyase (ICL) activity having ability to produce and accumulate riboflavin are also disclosed (US Pat. No. 5,976,844)

Mutants of Bacillus subtilis, selected by exposure to the purine analogs azaguanine and azaxanthine, have been reported to produce riboflavin in recoverable amounts (U.S. Pat. No. 3,900,368, Enei et al, 1975). In general, exposure to purine or riboflavin analogs selects for deregulated mutants that exhibit increased riboflavin biosynthesis, because the mutations allow the microorganism to "compete out" the analog by increased production (Matsui et al., Agric. Biol. Chem. 46:2003, 1982). Mutant strains of Bacillus subtilis having an activity of liberating phosphoric acid from 5 ' -guanylic acid of not higher than 0.081 μmol/min/mg -protein have been reported to produce riboflavin (EP0531708B1).

Mutants of Bacillus subtilis, selected by resistance roseoflavin and 8-azaguanine, and having mutation in ribC gene, have been described as a riboflavin producing strains (USSR patent 908092). There are Bacillus subtilis strains VNIIgenetika-304 and 304a. Further the Bacillus subtilis strain 304/pMX45, comprising plasmid with Bacillus subtilis rib operon, having increased riboflavin productivity (up to 3.5 g/1) has been obtained (FR Pat. 2546907).

Bacillus subtilis strain 62/pMX30ribO186 producing up to 12.5 g/1 of riboflavin in 42 hours of fermentation has been disclosed (Russian patent 2081906). The strain

62/pMX30ribO186 has been obtained from Bacillus subtilis strain RK6121 as a mutant resistant to 8-azaguanine, methionine sulphoxide, diacetyl, and psikofuranine and contained, in addition, plasmid with mutant (ribO mutation) Bacillus subtilis rib operon.

Bacterial strains obtained by introducing the riboflavin biosynthesis genes from Bacillus subtilis into bacterial chromosome also can produce riboflavin (US Pat. Nos. 5,837,528, 5,925,538 and 6,322,995). The best known strain, RB50::[pRF69]₆o(Ade⁺) containing a transcriptionally-modified riboflavin operon containing two SPO1-15 promoters, produced 13.0-14.0 g/1 riboflavin in 48 hours and 15 g/1 in 56 hours (US Pat. No. 5,925,538) during cultivation in standard commercial batch and feed conditions. Though the productivity of riboflavin has considerably been improved by breeding of such microorganisms as mentioned above or production processes have been improved, it is still desired to develop more efficient processes for producing riboflavin in order to meet the expected markedly increased future demand of the vitamin.

Disclosure of the invention

An object of present invention is to enhance the productivity of riboflavin by riboflavin producing strains and to provide a method for producing riboflavin using the strains.

To enhance the riboflavin productivity the present inventors constructed the strain comprising derepressed Bacillus amyloliquefaciens rib operon. Further the mutant strain has ability to utilize glycerophosphate and resistant to growth inhibition by glyoxylate has been bred.

Thus the present invention has been completed. The present invention provides a bacterium belonging to the genus Bacillus and having riboflavin producing ability. Specifically, the present invention provides the bacterium with improved riboflavin producing ability conditioned by presence of derepressed Bacillus amyloliquefaciens rib operon, ability to use glycerophosphate and/or a mutation, which confers resistance to glyoxylate.

The present invention further provides a method for producing riboflavin by fermentation comprising the steps of cultivating the aforementioned bacterium in the culture medium, and collecting riboflavin from the culture medium. The present inventions are as follows: 1. A bacterium Bacillus subtilis which has ability to produce riboflavin comprising a heterologous rib operon in the chromosome of the bacterium, wherein the rib operon is Bacillus amyloliquefaciens rib operon. 2. The bacterium Bacillus subtilis according to 1 , wherein said Bacillus amyloliquefaciens rib operon is deregulated. 3. A bacterium Bacillus subtilis which has ability to produce riboflavin and has ability to utilize glycerophosphate.

4. A bacterium Bacillus subtilis which has ability to produce riboflavin and has a resistance to growth inhibition by glyoxylate.

5. A bacterium Bacillus subtilis which has ability to produce riboflavin and has one or more of following characteristics:

- comprises Bacillus amyloliquefaciens rib operon in the chromosome of the bacterium;

- has ability to utilize glycerophosphate; and

- has a resistance to growth inhibition by glyoxylate. 6. A method for producing riboflavin comprising the steps of cultivating the bacterium according to any of 1 to 5 in the culture medium, and collecting riboflavin from the culture medium.

The present invention described in details below.

(1) Bacterium of the present invention

The bacterium of the present invention is a riboflavin producing bacterium Bacillus subtilis, wherein riboflavin production by the bacterium is enhanced by introducing into chromosome of the bacterium heterologous rib operon from Bacillus amyloliquefaciens. The term "bacterium which has ability to produce riboflavin" used herein means a bacterium, which is able to produce and accumulate riboflavin in a culture medium in an amount of larger than a wild type or parental strain of B. subtilis, such as B. subtilis strain 168, and preferably means that the microorganism is able to produce and accumulate in a medium an amount of not less than 0.5 g/L, more preferably not less than 1.0 g/L of riboflavin.

The term "bacterium Bacillus subtilis" means that the bacterium is classified as the genus Bacillus subtilis according to the classification known to a person skilled in the microbiology. And the term "Bacillus amyloliquefaciens" means that the bacterium is classified as the genus Bacillus amyloliquefaciens according to the classification known to a person skilled in the microbiology.

The term "rib operon" means chromosomal fragment containing genes coding for proteins essential to production of riboflavin. Rib operon in the bacteria belonging to the genus Bacillus includes following genes: ribO gene coding for control element, ribG gene coding for deaminase / reductase, ribB gene coding for riboflavin synthase (α-subunit), ribA gene coding for GTP-cyclohydrolase / 3,4-dihydroxy-2-butanon-4-phosphate synthase, ribH gene coding for lumasine synthetase, and ribT gene coding for a protein with unknown function (Morozov et al. Mol. Genet. Mik. Nirusol. no. 7:42 (1984)). Νucleotide sequences of ribG, ribB, ribA, ribH and ribH genes of Bacillus subtilis are presented in the GenBank under accession numbers gi:16079385, gi:16079384 gi:16079383 gi:16079382 and gi:16079381, respectively (nucleotides 2429492..2430577, 2428834..2429481, 2427623..2428819, 2427126..2427590 and 2426639..2427013 in the accession ΝC_000964.1).

The term "heterologous operon", used herein, means that the operon has been isolated from the chromosome of organism other then Bacillus subtilis. Bacterium comprising a heterologous operon in the chromosome could be constructed by standard recombinant DNA technology, transformation, and transfection.

Term "deregulated" means that the level of riboflavin production is greater than that observed in a bacterium with natural riboflavin regulatory systems (i.e., a wild type bacterium). Examples of such bacteria having deregulated rib operon include those, which are resistant to various purine analogs or antagonists, or riboflavin analogs.

Deregulation of riboflavin regulatory system could be performed by altering the regulatory region of operon, substitution the regulatory region with other constitutive strong region, inactivation of gene coding for repressor, producing mutations in the repressor protein etc. The said deregulation can be perrormeα by conventional methods, such as mutagenesis treatment using UV irradiation or nitrosoguanidine (N-methyl-N'-nitro-N- nitrosoguanidine) treatment, site-directed mutagenesis, gene disruption using homologous recombination or/and insertion-deletion mutagenesis. The term "has ability to utilize glycerophosphate" means an ability to metabolize glycerophosphate more efficiently than the parental strain, for example, an ability of 5. subtilis strain used for the present invention to grow faster than the parental strain when the strain is cultivated in a medium containing glycerophosphate as a sole carbon source. More concretely, it can be said that B. subtilis strain has ability to utilize glycerophosphate if the strain grows faster than the parental strain when the strains are cultivated in a medium containing glycerophosphate as a sole carbon source, for example, the liquid medium containing 0.1% of glycerophosphate under an appropriate condition. Most concretely, it can be said that B. subtilis strain has ability to utilize glycerophosphate if the strain forms a colony within 2 days at 37°C when the strain is cultivated on an agar medium containing glycerophosphate as a sole carbon source, for example, the medium containing 0.1% of glycerophosphate and agar under an appropriate condition. The term "an appropriate condition" refers to temperature, pH, air supply or optional presence of essential nutrients or the like for the B. subtilis strain which is to be cultivated.

It is known that gluconeogenesis enzymes have level of activity, which is not sufficient to reutilize products of glycolysis, such as glycerophosphates. It is also Icnown that B. subtilis strains grow very poorly on glycerophosphate as soul carbon source. So it was suggested the isolation of mutants capable to grow on glycerophosphate as a sole carbon source allows to select mutants with increased activities of enzymes involved in gluconeogenesis and thereby to increase the riboflavin production during fermentation process on sucrose.

The term "bacterium has a resistance to growth inhibition by glyoxylate" means a bacterium derived from the parent strain by modification in genetic properties so that it can grow in a medium containing glyoxylate. Concretely, resistance to glyoxylate means ability for bacterium to grow on a minimal medium containing glyoxylate in concentration under which the wild type or the parental strain of the bacterium cannot grow, or ability for bacterium to grow faster on a medium containing glyoxylate than the wild type or the parental strain of the bacterium. For example, a bacterium which can form colonies within 3- 5 days of cultivation at 34oC on an agar plate containing 0.5 mg/ml or more, preferably 1.0 mg/ml or more of glyoxylate is resistant to glyoxylate. Glyoxylate is very important intermediate of glyoxylate bypass, which is essential for growth on carbon sources such as acetate or fatty acids because this pathway allows the net conversion of acetyl-CoA to metabolic intermediates. The pathway involves the synthesis of three enzymes. Two of them, isocitrate lyase and malate synthase A, convert some intermediates of TCA cycle from isocitrate to malate. It was shown that, in contrast to E. coli, activities of enzymes from glyoxylate shunt are very low in B. subtilis cells. Furthermore, glyoxylate inhibits growth of wild type B. subtilis cells at concentration 1 mg/ml. In order to activate glyoxylate bypass enzyme activities it was decided to isolate mutants resistant to glyoxylate. The bacterium of the present invention may be obtained from bacterium inherently having an ability to produce riboflavin by imparting thereto the specified resistance and/or ability to utilize glycerophosphate. Alternatively, the bacterium of present invention may be also obtained by imparting an ability to produce riboflavin to bacterium having the specified resistance and/or ability to utilize glycerophosphate. Thus, it is possible to subject any Icnown strain belonging to genus Bacillus inherently having an ability to produce riboflavin to one of the above mutation procedure for obtaining a mutant strain, and then to test the mutant strain to determine whether it satisfies the above-requirement of the present invention concerning utilization of glycerophosphate and resistance to glyoxylate, and it is therefore suitable for use in the present invention. Strains obtained are screened by culturing in a nutrient medium, and a strain having the ability to produce riboflavin in greater yields than its parent strain is selected and used in this invention.

As a parent strain which is to be improved to obtain the bacterium of the present invention, the riboflavin producing bacterium strain belonging to the genus Bacillus, such as Bacillus subtilis strains VNIIgenetika-304 and 304a (USSR patent 908092), Bacillus subtilis strain 304/pMX45 (TsMPM B-2694) (FR Pat. 2546907), Bacillus subtilis strain 62/pMX30ribO186 (VKPM B-6797) (Russian patent 2081906), Bacillus subtilis strain RB50::[pRF69]₆₀(Ade⁺) (US Pat. No. 5,925,538), Bacillus subtilis strains FΕRM BP-3855 and FΕRM BP-3855 (ΕP 0531708B1), and the like may be used.

(2) The method for producing riboflavin.

The method of present invention includes method for producing riboflavin comprising the steps of cultivating the bacterium of present invention in the culture medium, and collecting riboflavin from the culture medium. In the method of present invention, the cultivation of the bacterium belonging to the genus Bacillus, the collection and purification of riboflavin from the liquid medium may be performed in a manner similar to those of the conventional method for producing riboflavin by fermentation using a bacterium. Culture medium for riboflavin production may be either a synthetic medium or a natural medium provided that it contains a carbon source, a nitrogen source, inorganic ions and other organic components as required. As the carbon source, saccharides such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose and hydrolysates of starches; alcohols such as glycerol, mannitol and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid and succinic acid and the like can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as of soybean hydrolysates; ammonia gas; aqueous ammonia and the like can be used. It is desirable that vitamins such as vitamin Bi, required substances, for example, nucleic acids such as ade ine and RNA, or yeast extract and the like are contained in appropriate amounts as trace amount organic nutrients. Other than these, small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese ions and the like may be added, if necessary.

Cultivation is preferably performed under an aerobic condition for 16 to 72 hours, and culture temperature during the cultivation is controlled within 30 to 45 °C and pH within 5 to 8. The pH can be adjusted by using an inorganic or organic acidic or alkaline substance as well as ammonia gas.

Riboflavin can be recovered from the fermentation liquor by any or any combination of conventional methods such as techniques utilizing ion exchange resin and precipitation.

Best Mode for Carrying Out the Invention Example 1. Construction of Bacillus subtilis strain, comprising B. amyloliquefaciens riboflavin operon in chromosome.

It is known the direct homologous recombination of rib operon from B. amyloliquefaciens into chromosome of B. subtilis is not successful.

To clone genes of riboflavin biosynthesis, the chromosome of B. amyloliquefaciens strain A50 was treated with Hindlll restrictase. Obtained mixture of DNA fragments was ligated into vector pACYC184 (NBL Gene Sciences Ltd., UK, GenBank/EMBL accession number X06403) previously treated with the same restrictase. Resulted product was used for transformation of E. coli strain BSN821. Transformants having ability to grow on the medium containing chloramphenicol were selected. Plasmid from such transfomants was isolated and fragment containing rib operon was recloned to the vector pCB20. Resulted plasmid was named as pBX2. Plasmid pBX2 complemented mutations in all genes of rib operon except ribTD gene.

To breed the riboflavin constitutive mutants, plasmid pBX2 was treated with hydroxylamine under the standard procedure for mutagenesis. Laboratory auxotrophic strain B. subtilis ribG850 was transformed with the mutated DΝA plasmid and ability to produce riboflavin by the resulted transformants was evaluated. It was determined by PCR, that the riboflavin production was caused by mutations in the ribO gene. Mutation ribOl (T-»C substitution at position +87) was identical to the known mutation in B. subtilis operon. Mutation rib02 is new mutation C— T at position +149. Plasmid containing ribO2 mutation was named as pBX21. B. subtilis strain ribG850 transformed with plasmid pBX21 produced up to 65 mg/1 of riboflavin whereas the same strain transformed with plasmid pBX2 did not produce any amount of riboflavin.

Plasmidless Bacillus subtilis strain 304 (USSR patent 908092) has been used as a parental strain for construction of Bacillus subtilis strain GM49 comprising deregulated B. amyloliquefaciens riboflavin operon in the chromosome. For integration of deregulated B. amyloliquefaciens riboflavin operon into chromosome of Bacillus subtilis strain 304, the integrative vector pΕK14 (Km^R, Ap^R, Rib⁺) have been constructed. Nector pEK14 consists of the E. coli plasmid pUC19, a kanamycin nucleotidyl transferase (kan) gene from pUBl 10 plasmid, and desensitized (ribO2 mutation) riboflavin operon from B. amyloliquefaciens derived from plasmid pBX21. The plasmid pEK14 was unable to replicate in B. subtilis. To integrate pEK14 into chromosome, "random integration" method was used. At first, the RαmHI-fragment of pEK14 was ligated to BamHl fragments of chromosomal ON A from B. subtilis strain 168. Plasmid carrying a region homologous to the genome of B. subtilis was able to integrate into the chromosome by homologous recombination. Then, the ligation mixture was used for the transformation of laboratory auxotrophic strain B. subtilis ribG850. The Km^R Rib⁺ transformants were selected. Frequency of transformation was about 100 per 1 γ of DΝA. It was analyzed 60 Km^R Rib⁺ clones of integrants. The most of integrants were unstable or caused auxotrophy. They lost Km^R and Rib⁺ markers during growth in media without antibiotic. One stable integrant clone named as BK53 was found. The B. subtilis BK53 ribG850 tfβb: ;pEK14 (Rib_Bam+ Km^R) strain contained B. amyloliquefaciens riboflavin operon (Rib_Bam+) and Km^R resistance gene (kaή) integrated into B. subtilis chromosome in locus named as "aab".

The integrated B. amyloliquefaciens operon was transferred into chromosome of recipient B. subtilis plasmidless strain 304 producing riboflavin from the B. subtilis BK53 ribG850 σ δ::pEK14 (RibB_am+ Km^R) strain by transduction using phage AR9. The kanamycin resistance gene, integrated in the B. subtilis chromosome together with B. amyloliquefaciens riboflavin operon, was used as the selective marker. The constructed strain was named GM49. The strain GM49 (aab::pEK14 Rib _Bam₊ Km^R) contains two riboflavin operons in the chromosome of one bacterial cell: the first is its own and the second is from B. amyloliquefaciens.

The ability to produce riboflavin by B. subtilis strain GM49 was evaluated in flasks using cultural medium described below in the section "Fermentation conditions". Fermentation (batch process) was performed in the 850 ml flasks. Volume of culture was 150 ml. The GM49 strain produced 3 g/1 of riboflavin in 72 hours in flasks.

Example 2. Construction of Bacillus subtilis strain, comprising both B. amyloliquefaciens riboflavin operon in chromosome and Bacillus subtilis riboflavin operon on plasmid.

B. subtilis GM51/pMX45 strain was constructed from the plasmidless riboflavin producing B. subtilis strain GM49. At first, the recombination deficient B. subtilis strain was constructed.

To construct the GM51 strain with insertion mutation recEr. cat in the recE gene, plasmid pBT69 was used. The plasmid pBT69, which is derivative of plasmid pBT61 harboring recE gene from Bacillus subtilis (Gassel M. and Alonso J.C., "Expression of the recE gene during induction of the SOS response in Bacillus subtilis recombination-deficient strains", Mol. Biology, 1989, 3(9), 1269-1276), contains chloramphenicol resistance gene of chloramphenicol acethyl transferase (cat). Plasmid pBT69 was integrated into the recE gene in it's Clal site (recEr.cat). The mutated recEr.cat gene was integrated into the chromosome of B. subtilis GM49 strain by double crossing recombination event. Thus, the B. subtilis strain GM51 (aab::pEK14 recEr.cat (Rib_Bam+ Km^R Cm^R)) was constructed. Then, recombination deficient (recEr.cat) Cm^R B. subtilis strain GM51 was transformed with plasmid pMX45 containing the riboflavin operon from B. subtilis strain rib0335 The plasmid pMX45 carries the large (lOkb) DNA fragment of B. subtilis. The pMX45 plasmid is low copy plasmid (1-2 copies per cell). Plasmid pMX45 is described in detail in French Patent No 2546907. The GM51/pMX45 strain produced 5.8 g/1 riboflavin in 72 hours in flasks and 15 g/1 riboflavin in 70 hours fermentation in the 1 liter jar fermentors.

Example 3. Breeding the Bacillus subtilis strain capable to use glycerophosphate as sole carbon source.

B. subtilis strain GM41/pMX45 was obtained from the riboflavin producing B. subtilis strain GM51/ρMX45 as spontaneous mutant capable to use glycerophosphate (0.1%) as sole carbon source. B. subtilis strain GM41/pMX45 was selected on the Spizizen minimal medium containing glycerophosphate (0.1%) instead of glucose. The strain GM41/pMX45 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1^st Dorozhny proezd, 1) according to the Budapest treaty on September 17, 2002 under accession number VKPM B-8386.

The GM41/pMX45 strain produced 8.8 g/1 riboflavin after 72 hours of fermentation in flasks and 18 g/1 riboflavin after 70 hours of fermentation in the 1 liter jar fermentors.

Example 4. Breeding the Bacillus subtilis strain resistant to glyoxylate.

B.subtilis strain GM44/pMX45 was obtained from the strain GM41/pMX45 as a spontaneous mutant resistant to 1 mg/ml glyoxylate. B. subtilis strain GM44/pMX45 was selected on the Spizizen minimal medium containing 1 mg/ml of glyoxylate and glycerophosphate (0.1 %) instead of glucose.

The strain GM44/pMX45 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1^st Dorozhny proezd, 1) according to the Budapest treaty on September 17, 2002 under accession number VKPM B- 8387. The GM44/pMX45 strain produced 9.6 g/1 riboflavin after 72 hours of fermentation in flasks and 21 g/1 riboflavin in 70 hours fermentation in the 1 liter jar fermentors.

Section: Fermentation conditions

The seed culture (50 ml) was prepared by incubated strain GM49 in flasks at the rotaty shaker (200-220 rpm) for 18 hours at 37 °C. Seeding medium:

Sucrose 20.0 g/1

Yeast autolysate 10.0 g/1 MgSO₄ x 6H₂O 1.0 g/i erytromycine lO γ/ml pH before sterilization 7.1-7.2

The process of riboflavin production was performed in laboratory fermentors

Marubishi with capacity 1 L equipped by bioprocessor. Cultivation conditions was following: agitation 1100 rpm, temperature 39 °C, aeration 1:1 (V:V), initial volume 300 ml, inoculum is 10% of initial fermentation medium volume, pH was controlled 7.0+0.2 by 6% ammonium water and 5% sulfuric acid. Mixture of sugars was added with feeding during the fermentation.

The rest concentrations of sugars (fructose, glucose, sucrose and maltose) were controlled during fermentation. Normally, the regime of feeding was selected to maintain the concentration of total sugars not more than 10 g/1.

Initial medium content (g/1):

Sucrose 30

Yeast Powder 32

Corn extract 10.8

KH₂PO₄ 3.0

K₂HPO₄ 9.0

MgSO₄ x 7H₂O 0.6

Urea 6.0

Feeding content (g/i)--

Molasse (type C) 227

NHM syrop 227

Sucrose 419

Feeding contains approximately 720 g/1 of total sugar (glucose, sucrose, maltose, etc).

Preparation of initial medium.

Yeast powder for medium was sterilized in boiling water bath for 40 min. 9.6 g of yeast powder was suspended in 100 ml sterile water at 45-55 °C. Procedure was performed in sterile flask. Suspension was kept in boiling water bath for 40 min. All the rest medium components were sterilized by the standard method in autoclave.

Volume at start (AS) 300 ml

Inoculum 30

Feeding (0-120h) about 170

Water ( 28-48h ) 90

Ammonia 1 : 1 about 20-30

Antifoam 20%> in water as necessary

Volume (final theoretical ) 620

( real ) 550-570

Feeding: 1.2 ml/h (7-24h)

1.5 ml/h (25-72h)

Duration of fermentation is up to 120 hr.

Amount of produced riboflavin was determined by spectrophotometric analysis at λ

464 nm and confirmed by reverse-phase HPLC (eluent - 34% methanol).

Claims

What is claimed is:

1. A bacterium Bacillus subtilis which has ability to produce riboflavin comprising a heterologous rib operon in the chromosome of the bacterium, wherein the rib operon is Bacillus amyloliquefaciens rib operon.

2. The bacterium Bacillus subtilis according to claim 1, wherein said Bacillus amyloliquefaciens rib operon is deregulated.

3. A bacterium Bacillus subtilis which has ability to produce riboflavin and has ability to utilize glycerophosphate.

5. A bacterium Bacillus subtilis which has ability to produce riboflavin and has two or more of following characteristics:

- has ability to utilize glycerophosphate; and

- has a resistance to growth inhibition by glyoxylate.

6. A method for producing riboflavin comprising the steps of cultivating the bacterium according to any of 1 to 5 in the culture medium, and collecting riboflavin from the culture medium.