WO2017036903A1 - Improved vitamin production - Google Patents

Abstract

The present invention provides an improved biotechnological production of riboflavin (also referred herein as vitamin B2) using genetically engineered microorganisms, in particular microorganism selected from Bacillus, such as e.g. Bacillus subtilis. Using said modified microorganisms, the yield of riboflavin 5 production could be increased by at least 5%. The present invention relates to modified microorganisms, processes to generate said modified microorganisms and the use thereof for production of riboflavin.

Description

Improved vitamin production

The present invention provides an improved biotechnological production of riboflavin (also referred herein as vitamin B2) using genetically engineered microorganisms, in particular microorganism selected from Bacillus, such as e.g. Bacillus subtilis. Using said modified microorganisms, the yield of riboflavin production could be increased by at least 5%. The present invention relates to modified microorganisms, processes to generate said modified microorganisms and the use thereof for production of riboflavin.

Riboflavin is synthesized by all plants and many microorganisms but is not produced by higher animals. Riboflavin is essential for basic metabolism, because it is a precursor of coenzymes such as flavin adenine dinucleotide and flavin mononucleotide that are required in the enzymatic oxidation of carbohydrates. In higher animals, insufficient riboflavin supply can cause loss of hair, inflammation of the skin, vision deterioration, and growth failure. Biosynthesis of riboflavin starts from guanosine triphosphate (GTP) and ribulose- 5-phosphate. The genes involved in biosynthesis of riboflavin are known from various sources, such as e.g. , Bacillus subtilis, Ereothecium ashbyii, Ashbya gossypii, Candida flareri, Saccharomyces cerevisiae, E. coli (see e.g. EP 405370, Figure 2 in EP 1 186664 or Ullman^'s Encyclopedia of Industrial Chemistry, 7^th Edition, 2007, Chapter Vitamins).

With regards to the situation in Bacillus subtilis as one example of a riboflavin producing (micro)organism, the genes involved in riboflavin biosynthesis include ribG (ribD), ribB (ribE), ribA, and ribH. The ribA gene encodes two enzymatic activities, i.e. GTP cyclohydrolase II catalyzing the first step in riboflavin biosynthesis and 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS), which catalyzes the conversion of ribulose-5-phosphate to 3,4-dihydroxy-2-butanone 4- phosphate (DHBP). Deaminase and reductase are encoded by the first gene of the operon, ribG (ribD). The penultimate step in riboflavin biosynthesis is catalyzed by lumazine synthase, the gene product of ribH. Riboflavin synthase, which catalyzes the last step of the pathway, is encoded by the second gene of the operon, ribB (ribD). The function of ribT located at the 3^' end of the rib operon is, at present, unclear; however, its gene product is not required for riboflavin synthesis. Transcription of the riboflavin operon from the rib promoter (P_rib) is controlled by a riboswitch involving an untranslated regulatory leader region (hereinafter referred to as rib leader) of almost 300 nucleotides located in the 5^' region of the rib operon between the transcription start and the translation start codon of the first gene in the operon, ribG. Elongation of the nascent riboflavin RNA is dependent on the presence or absence of FMN or FAD: in the presence of these effectors, a transcription termination hairpin is formed (so-called rib terminator) wherein in their absence, the formation of a so-called anti-terminator results in read-through transcription of the rib operon.

The establishment of an industrial production process using microorganisms, such as e.g. strains of Bacillus, requires some modification of either the host strain and/or the process conditions (see e.g. Kil et al. , Mol Gen Genet 233, 483-486, 1992; Mack et a/. , J. Bacteriol. , 180:950-955, 1998).

Recently, it could be shown that sequences at the 3 '-end of the rib leader play an important role in riboflavin biosynthesis. Interestingly, deletion of only the so- called terminator did not let to significant increase in riboflavin production (see WO 2010/052319).

However, there is still a need of further optimizing the industrial production of riboflavin obtained by fermentation.

Thus, it is an ongoing task to look for improvements in the production strains, such as e.g. Bacillus, preferably strains of B. subtilis.

Surprisingly, it has been found that the transcription termination factor Rho plays an important role in the fermentative production of riboflavin, in particular in a process using a strain of Bacillus, preferably B. subtilis.

The transcription terminator factor Rho (EC 3.6.4. -) is the product of the rho gene and functions as a hexamer of a single polypeptide chain organized in an open ring structure. It has been isolated from e.g. Bacillus subtilis 168 and is publicly available under e.g. UniProtKB - Q03222 or BSU37080. Rho acts as ATP-dependent helicase that is able to bind nascent RNA to interfere with the transcription elongation complex and promote termination. A Blast search running on other species of Bacillus revealed an identity in the range of 79% (B. clausii DSM-K16) to 97% (B. licheniformis ATCC 14580; B. amyloliquefaciens FZB42) on the DNA level (see also Table 4).

In particular, the present invention is directed to a genetically-manipulated riboflavin-producing host cell, such as e.g. a microorganism selected from Bacillus, preferably B. subtilis, wherein the activity of Rho has been reduced or abolished, such as e.g. through genetic modification or mutation of the rho gene, including a knock-out of said gene.

Furthermore, a new process has been developed, wherein said modified host cell is cultivated under such conditions that the yield of biotechnologically produced riboflavin is increased by at least 5% compared to a process wherein a non- modified host cell carrying a wild-type rho gene encoding a protein with a wild- type activity of Rho, i.e. a non-modified or non-mutated Rho is used for fermentative production of riboflavin.

In particular, the invention is directed to a riboflavin-producing host cell, preferably a microorganism selected from Bacillus or Corynebacterium, more particular from B. subtilis, wherein the activity of the endogenous transcription termination factor Rho is reduced or abolished.

A suitable host cell according to the present invention may be any known riboflavin-producing strain encoding an endogenous transcription termination factor Rho, said host cell being capable of converting a given carbon source, such as e.g. glucose, into riboflavin including any known precursors and/or derivatives thereof and wherein the activity of said Rho-type transcriptional regulator is reduced or abolished such that the production of riboflavin by said host is increased. Preferably, the host cell is selected from a riboflavin-producing microorganism, such as e.g. a strain of Bacillus or Corynebacterium, preferably selected from B. subtilis, B. atrophaeus, B. licheniformis, B. amyloliquefaciens, B. pumilus, B. infantis, B. coagulans, B. megaterium, B. thuringiensis, B. cereus, B. halodurans, or C. glutamicum. More preferably, the host cell is selected from B. subtiiis, B. licheniformis, B. amyloliquefaciens, or B. megaterium, most preferably from B. subtiiis, in particular B. subtiiis 1A747 or B. subtiiis 168. These microorganisms might be publicly available from different sources, such as culture collections (e.g. DSMZ, ATCC, NRRL, BGSC, etc. ).

In connection with the present invention it is understood that the above- mentioned microorganisms also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes. The nomenclature of the microorganisms as used herein is the one officially accepted (at the filing date of the priority application) by the International Committee on Systematics of Prokaryotes and the Bacteriology and Applied Microbiology Division of the International Union of Microbiological Societies, and published by its official publication vehicle International Journal of Systematic and Evolutionary Microbiology (IJSEM).

Thus, the present invention is preferably related to a host cell according to the description above, wherein the activity of the endogenous Rho is reduced or abolished, in particular reduced by at least 20%, more preferably by at least 50, 60, 70, 80, 90%, most preferably Rho activity is abolished, i.e. reduced to zero activity. This might be achieved by e.g. knocking out the rho gene or parts of the gene as described herein.

In one embodiment, the present invention relates to a riboflavin-producing host cell as described herein, such as e.g. Bacillus, in particular from B. subtiiis, wherein the activity of Rho (compared to the activity of a non-modified Rho) is reduced via mutation(s) introduced in the ribosome binding site. Preferably, the part(s) to be mutated correspond to the putative ribosome binding site, e.g. nucleotides corresponding to positions -17 to -6 of SEQ ID NO: 1 (ATG-17bp to ATG- 6bp), preferably via deletion of said nucleotides (Shaw et al. , Biochim Biophys Acta. 1729(1 ): 10-3, 2005).

In a further embodiment, the present invention relates to a riboflavin-producing host cell as described herein, such as e.g. Bacillus, in particular from B. subtiiis, wherein the activity of Rho (compared to the activity of a non-modified Rho) is reduced via mutation(s) introduced in the cold-shock domain, e.g. nucleotides corresponding to positions +160 to +360 of SEQ ID NO:1 (ATG +160bp to ATG +360bp), preferably via deletion of said nucleotides. In a further embodiment, the present invention relates to a riboflavin-producing host cell as described herein, such as e.g. Bacillus, in particular from B. subtilis, wherein the activity of Rho (compared to the activity of a non-modified Rho) is reduced via mutation(s) introduced in the RNA binding site, e.g. nucleotides corresponding to positions +174 to +336 of SEQ ID NO:1 (ATG +174bp to ATG +336bp), preferably via deletion of said nucleotides. In a particularly preferred embodiment, the modified Rho transcriptional regulator comprises an amino acid substitution, e.g. a substitution on an amino acid corresponding to position 56 of SEQ ID NO: 2, preferably a substitution of the wild-type amino acid into aspartic acid, more preferably a replacement of glycine on a position corresponding to position 56 of SEQ ID NO:2 to aspartic acid, i.e. G56D mutation, which results in a non-functional Rho protein, i.e. wherein the activity of Rho in the host cell as specified herein is abolished.

In another embodiment, the present invention relates to a riboflavin-producing host cell as described herein, such as e.g. Bacillus, in particular from B. subtilis, wherein the activity of Rho (compared to the activity of a non-modified Rho) is reduced via mutation(s) introduced in the ATP binding site, e.g. nucleotides corresponding to positions +538 to +1062 of SEQ ID NO:1 (ATG +538bp to ATG +1062bp), preferably via deletion of said nucleotides.

In one embodiment, the present invention relates to a riboflavin-producing host cell as described herein, such as e.g. Bacillus, in particular from B. subtilis, wherein the activity of Rho (compared to the activity of a non-modified Rho) is reduced via mutation(s) in a DNA fragment corresponding to positions +155 to +165 of SEQ ID NO:1 (ATG +155bp to ATG +165bp), preferably via deletion of said nucleotides. Furthermore, the present invention relates to a riboflavin-producing host cell as described herein, such as e.g. Bacillus, in particular from B. subtilis, wherein the activity of Rho (compared to the activity of a non-modified Rho) is reduced via mutation(s) introduced in the promoter region, e.g. nucleotides corresponding to positions -200 to +1 of SEQ ID NO: 1 (ATG -200bp to ATG +1 ), preferably via deletion of said nucleotides.

The skilled person knows how to genetically manipulate such a host cell resulting in reduction or abolishment of Rho activity. These genetic manipulation include, but are not limited to, e.g. gene replacement, gene amplification, gene disruption, transfection, transformation using plasmids, viruses, or other vectors. A genetically modified host/organism, e.g. genetically modified microorganism, is also often referred to as a recombinant host/organism, e.g. recombinant microorganism. The terms ^"modified^", ^"mutated^" or ^"recombinant^" in this connection are used interchangeably herein. Furthermore, the terms ^"host cell^" and ^"host organisms^" are used interchangeably herein.

Modifications in order to have the host cell produce less or no copies of the Rho gene and/or protein may include the use of a weak promoter, or the mutation (e.g. insertion, deletion or point mutation) of (parts of) the Rho gene (as described herein), in particular its regulatory elements. An example of such a genetic manipulation may for instance affect the interaction with DNA that is mediated by the N-terminal region of Rho or interaction with other effector molecules. In particular, modifications leading to reduced or abolished Rho-specific activity may be carried out in the ATP-binding site, RNA-binding site, the region of helicase activity or the region of RNA-dependent ATPase activity within Rho and as described herein and known in the art. Furthermore, reduction or abolishment of Rho-specific activity might be achieved by contacting Rho with specific inhibitors or other substances that specifically interact with Rho. In order to identify such specific inhibitors, the Rho protein may be expressed and tested for activity in the presence of compounds suspected to inhibit the activity of Rho. Potential inhibiting compounds may for instance be monoclonal or polyclonal antibodies against the Rho protein. Such antibodies may be obtained by routine immunization protocols of suitable laboratory animals. Additionally, Rho activity might be tested in a reporter strain, wherein a gene which is regulated via action of Rho (through binding to the Rho-utilization site) is fused to green fluorescence protein (GFP). Transcription of said gene can be measured in a Western Blot using anti-GFP antibodies. In case of non-activity of Rho, GFP is not translated and thus no signal can be detected in the Western Blot. The skilled person knows how to perform these experiments and which genes to choose that are regulated by the Rho transcriptional regulator.

The term ^"genetically-manipulated^" host cell refers to genetic modification or mutation or genetic alteration of the host cell in such a way that the activity of the transcription termination factor Rho is reduced or abolished, compared to the endogenous regulatory activity without such manipulation in the respective host cell. The term ^"genetically modified^" or ^"genetically engineered^" or ^"genetically altered^" are used interchangeably herein. A host cell carrying an intact endogenous gene encoding transcription termination factor Rho which exhibits 100% Rho-specific activity is called a wild-type or non-modified/non-mutated host cell (in contrast to a modified host cell as described herein), i.e. carrying a non- modified Rho.

For the purpose of the present invention, the term ^"activity^" in connection with Rho means in particular the activity to bind to nascent RNA in its function as ATP- dependent helicase and/or to activate the RNA-dependent ATPase activity of Rho and/or to release the mRNA from the DNA template. Rho-specific activity can be determined by measuring the transcription, in particular transcriptional elongation of regulated genes, such as e.g. transcription of genes of the rib- operon. Measurement of reduction of Rho-specific biological activity may be done as follows: prior to the genetic manipulation of the host cell, Rho-specific activity is measured and set as 100%. The same measurement is performed after modification/mutation of the host cell resulting in reduction or abolishment of Rho-specific activity, i.e. an activity of less than 100%, which measurement is known in the art (see, e.g. , Yakhnin et al. , J Bacteriol. 183(20):5918-26, 2001 or Ingham et al. , Mol Microbiol. 31 (2):651 -63, 1999).

A riboflavin-producing host cell as described herein might carry additional modification(s) either on the DNA or protein level, such as e.g. replacing the natural promoter of the rib operon by a strong (constitutive or inducible) promoter such as e.g. P_spoi 5 or P_veg, as long as such modification(s) has/have direct impact on the yield, production and/or efficiency of the production of riboflavin by the respective host from substrates such as e.g. glucose. The introduction of such a strong promoter results in an increase in riboflavin production which is at least 50%, 75%, 100%, 200%, 250%, 300%, 350%, 500% or even more than 1000% compared to a microorganism carrying only a modified rho. This may be furthermore increased by overexpression of one or more rib gene(s), in particular rib A or by introduction of multiple copies of the rib operon in the host cell, such as implemented in B. subtiiis strain RB50 (see e.g. EP 405370). Compared to the riboflavin production in B. subtiiis RB50, the riboflavin production can be increased by at least 100%, 200%, 250%, 500%, or even more than 750% by genetically altering a microorganism in such a way, i.e. via fusion of the rib genes to a strong promoter. A microorganism carrying a modified rho as defined herein optionally combined with the introduction of a strong promoter and/or multiple copy/copies of the rib operon may be furthermore altered via a decoupling of growth from production of riboflavin, such as e.g. via introduction of an auxotrophy such as described in EP 1 186664 for e.g. biotin, and/or furthermore combined with introduction of modified transketolase gene as e.g. described in WO 2007/051552, and/or furthermore combined with the use of modified rib leader sequences as e.g. described in WO 2010/052319, and/or furthermore combined with mutations of the flavokinase encoding ribC gene as e.g. described in US 5837528, all incorporated herein by reference.

A particular preferred strain for the production of riboflavin is B. subtiiis. A more preferred strain is B. subtiiis RB50: : [pRF69]_n containing multiple (n) copies (for example about 5 to about 20 copies) of pRF69 encoding a rib operon modified with the strong promoter P_spoi 5 to enhance transcription of the rib genes (see e.g. EP 405370 and Perkins et al. , J. Ind. Microbiol. Biotechnol. , 22:8-18, 1999 for construction of the strain and culture conditions to result in riboflavin production). B. subtiiis RB50 and plasmid pRF69 may be available from NRRL (accession number B 18502) and ATCC (accession number ATCC 68338), respectively. This strain might be further manipulated leading to reduction or abolishment of Rho activity as described above.

Thus, the present invention is related to a riboflavin-producing host cell as well as to the use of such host cell for production of riboflavin as described herein, such as e.g. Bacillus, in particular from B. subtiiis, wherein the activity of Rho (compared to the activity of a non-modified Rho) is reduced or abolished compared to the activity of a non-modified Rho, said endogenous rho gene comprising a polynucleotide selected from the group consisting of:

(a) polynucleotides comprising the nucleotide sequence according to SEQ ID NO: 1 ,

(b) polynucleotides encoding a polypeptide according to SEQ ID NO:2, (c) polynucleotides encoding a fragment or derivative of a polypeptide encoded by a polynucleotide according to (a) or (b), wherein in said fragment or derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said fragment or derivative has Rho-specific activity,

(d) polynucleotides the complementary strand of which hybridizes under stringent conditions to a polynucleotide as defined in (a) or (b) and which encode a polypeptide having Rho-specific activity,

(e) polynucleotides which are at least 75%, such as 77, 78, 79, 80, 81 , 82, 84, 85, 90, 95, 97 or 98% identical to a polynucleotide as defined in (a) or (b) and which encode a polypeptide having Rho-specific activity; or the complementary strand of such a polynucleotide.

The nucleic acids of the present invention are preferably provided in an isolated form, and preferably purified to homogeneity.

The term ^"isolated^" means that the material is removed from its original environment (e.g. , the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living microorganism is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides could be part of a composition and still be isolated in that such vector or composition is not part of its natural environment.

As used herein, the terms ^"polynucleotide^" or ^"nucleic acid molecule^" are intended to include DNA molecules (e.g. , cDNA or genomic DNA) and RNA molecules (e.g. , mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g. , inosine or phosphorothioate nucleotides). Such oligonucleotides may be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence may be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors.

The terms ^"homology^" or ^"percent identity^" are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. , gaps may be introduced in the sequence of a first nucleic acid sequence for optimal alignment with a second nucleic acid sequence). The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences [i.e. , % identity = number of identical positions/total number of positions (i. e. , overlapping positions) x 100] . Preferably, the two sequences are the same length.

The skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. 48, 444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / /www.accelrys.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1 , 2, 3, 4, 5 or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.accelrys.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1 , 2, 3, 4, 5 or 6. In another embodiment, the percent identity between two nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11 -17, 1989) which has been incorporated into the ALIGN program (version 2.0) (available at http://vega.igh.cnrs.fr/bin/align-guess.cgi) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

"Stringent conditions" for hybridization mean in the context of the present invention might be for instance overnight incubation (e.g., 15 hours) at 42°C in a solution comprising: 50% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 Mg/ml of denatured, sheared salmon sperm DNA, followed by washing in 0.1 x SSC at about 65 °C. Further specific conditions are known to the skilled person and described in e.g. by Sambrook et al., "Molecular Cloning", second edition, Cold Spring Harbor Laboratory Press 1989, New York.

The invention also relates to processes for reduction or abolishment of endogenous Rho specific activity, to processes for the production of (modified) polynucleotides as defined above in a suitable host cell as described herein, such as a riboflavin- producing microorganism, and to processes for the production of such modified microorganisms capable of producing riboflavin.

Thus, in accordance with an object of the present invention, the above-described riboflavin-producing host cells are used for fermentative production of riboflavin, wherein through genetically manipulations within the rho gene as described above the yield of riboflavin is improved compared to the use of a host cell carrying a non-modified Rho (exhibiting 100% Rho-specific activity). As used herein, ^"improved yield of riboflavin^" means an increase of at least 5%, such as preferably at least 25%, 30%, 40%, 50%, 75%, 100%, 200% or even more than 500%, compared to a wild-type host (such as e.g. a microorganism), i. e. a host encoding a non-modified Rho. The terms ^"riboflavin^" and ^"vitamin B2^" are used interchangeably herein. The genes involved in biosynthesis of riboflavin as well as methods for fermentative production of riboflavin, in particular fermentative production using Bacillus strains, are known (see e.g. EP 405370 or Ullman^'s Encyclopedia of Industrial Chemistry, 7^th Edition, 2007, Chapter Vitamins). These methods may be also applied for production of riboflavin using mutant strains comprising modified rib leader sequences as described herein.

As used herein, the term ^"riboflavin^" also includes riboflavin precursors, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and derivatives thereof. Riboflavin precursors and derivatives of riboflavin, FMN or FAD include but are not limited to: DRAPP; 5-amino-6-ribosylamino-2,4 (1 H,3H)- pyrimidinedione-5^'-phosphate; 2,5-diamino-6-ribitylamino-4 (3H)-pyrimidinone-5^'- phosphate; 5-amino-6-ribitylamino-2,4 (1 H, 3H)-pyrimidinedione-5^'-phosphate; 5- amino-6-ribitylamino-2,4 (1 H,3H)-pyrimidinedione; 6,7-dimethyl-8- ribityllumazine (DMRL); and flavoproteins. Derivatives of riboflavin include, but are not limited to: riboflavin-5-phosphate and salts thereof, such as e.g. sodium riboflavin-5-phosphate.

Several substrates may be used as a carbon source in a process of the present invention, i. e. a process for production of riboflavin as mentioned above. Particularly suited carbon sources may be selected from compounds consisting of 3, 5 or 6 carbon atoms, such as e.g. D-glucose, fructose, lactose, cellulose, glycerol, thick juice, dextrose, starch, sucrose, ribose or unpurified mixtures from renewable feedstock. Preferably, the carbon source is D-glucose. The term ^"carbon source^", ^"substrate^" and ^"production substrate^" in connection with the above process is used interchangeably herein. A medium as used herein for the above process using a modified microorganism may be any suitable medium for the production of riboflavin. Typically, the medium is an aqueous medium comprising for instance salts, substrate(s), and a certain pH. The medium in which the substrate is converted into riboflavin is also referred to as the production medium. An example of a suitable medium for production of riboflavin is described in WO 04/ 1 13510 (VF-medium), which is particularly useful with regards to Bacillus. ^"Fermentation^" or ^"production^" or ^"fermentation process^" as used herein may be the use of growing cells using media, conditions and procedures known to the skilled person, or the use of non-growing so-called resting cells, after they have been cultivated by using media, conditions and procedures known to the skilled person, under appropriate conditions for the conversion of suitable substrates into riboflavin. Fermentation as used herein is not limited to whole-cellular fermentation processes as described above, but may also use, e.g. , permeabilized host cells, crude cell extracts, cell extracts clarified from cell remnants by, e.g. , centrifugation or filtration, or even reconstituted reaction pathways with isolated enzymes. Also combinations of such processes are in the scope of the present invention. In the case of cell-free biosynthesis (such as with reconstituted reaction pathways), it is irrelevant whether the isolated enzymes involved in the riboflavin production process have been prepared by and isolated from a host cell, by in vitro transcription /translation, or by still other means. An example of a suitable medium for production of riboflavin is described in WO 04/ 1 1 3510 (VF-medium), which is particularly useful with regards to Bacillus.

The produced riboflavin may be recovered from the cells by any suitable means. Recovering means for instance that the produced riboflavin may be separated from the production medium. Optionally, the thus produced fermentation product may be further processed, e.g. purified. In connection with the above process using a microorganism, in one aspect, the growing step can be performed in an aqueous medium, i. e. the growth medium, supplemented with appropriate nutrients for growth normally under aerobic conditions. The cultivation may be conducted, for instance, in batch, fed-batch, semi-continuous or continuous mode, wherein fed-batch or semi-continuous mode is preferred. Detailed fermentation methods are known to the skilled person or otherwise described in e.g. EP 405370. The cultivation period may vary depending on for instance the host, pH, temperature and nutrient medium to be used, and may be for instance about 10 h to about 10 days, preferably about 4 to about 7 days, more preferably about 2 to about 6 days, depending on the microorganism. The skilled person will know the optimal culture conditions of suitable microorganisms.

The cultivation may be conducted for instance at a pH of about 7.0, preferably in the range of about 6 to about 8, more preferably about 6.5 to 7.5. A suitable temperature range for carrying out the cultivation may be for instance from about 13 °C to about 70° C, preferably from about 35 °C to about 39 °C, more preferably from about 30°C to about 39°C, and most preferably from about 36°C to about 39 °C. The culture medium for growth usually may contain such nutrients as assimilable carbon sources, e.g. , D-glucose, glycerol, thick juice, dextrose, starch, sucrose or ribose; and digestible nitrogen sources such as organic substances, e.g. , peptone, yeast extract and amino acids. The media may be with or without urea and/or corn steep liquor and/or baker's yeast. Various inorganic substances may also be used as nitrogen sources, e.g. , nitrates and ammonium salts. Furthermore, the growth medium usually may contain inorganic salts, e.g. , magnesium sulfate, manganese sulfate, potassium phosphate, and calcium carbonate. Cells obtained using the procedures described above can then be further incubated at essentially the same modes, temperature and pH conditions as described above, in the presence of substrates such as described above in such a way that they convert these substrates into the desired target fermentation product. Incubation can be conducted in a nitrogen-rich medium, containing, for example, organic nitrogen sources, e.g. , peptone, yeast extract, baker's yeast, urea, amino acids, and corn steep liquor, or inorganic nitrogen sources, e.g. , nitrates and ammonium salts, in which case cells will be able to further grow while producing the desired fermentation product. Alternatively, incubation can be conducted in a nitrogen-poor medium, in which case cells will not grow substantially, and will be in a resting cell mode, or biotransformation mode. In all cases, the incubation medium may also contain inorganic salts, e.g. , magnesium sulfate, manganese sulfate, potassium phosphate, and calcium chloride.

The terms "production" or "productivity" are art-recognized and include the concentration of riboflavin formed within a given time and a given fermentation volume (e.g. , kg product per hour per liter). The term ^"efficiency of production^" includes the time required for a particular level of production to be achieved (for example, how long it takes for the cell to attain a particular rate of output of a fermentation product). The term ^"yield^" is art- recognized and includes the efficiency of the conversion of the carbon source into the product (i.e. , riboflavin). This is generally written as, for example, kg product per kg carbon source. By ^"increasing the yield and/or production /productivity^" of the compound it is meant that the quantity of recovered molecules, or of useful recovered molecules of that compound in a given amount of culture over a given amount of time is increased.

Analytical methods for determining the yield /productivity of riboflavin are known in the art. Such methods may include, but are not limited to HPLC or use of indicator strains (see e.g. Bretzel et al. , J. Ind. Microbiol. Biotechnol. 22, 19-26, 1999). Thus, the present invention includes, but is not limited to, a riboflavin-producing host cell selected from a strain of Bacillus or Corynebacterium, preferably selected from B. subtilis, B. atrophaeus, B. licheniformis, B. amyloliquefaciens, B. pumilus, B. infantis, B. coagulans, B. megaterium, B. thuringiensis, B. cereus, B. halodurans, or C. glutamicum, more preferably selected from B. subtilis, B. licheniformis, B. amyloliquefaciens, or B. megaterium, most preferably from B. subtilis, in particular B. subtilis 1A747 or B. subtilis 168, wherein the activity of Rho is reduced or abolished compared to the activity of a non-modified Rho, preferably reduced by at least 20%, more preferably by at least 50, 60, 70, 80, 90%, most preferably reduced by 100% (i.e. Rho activity is abolished); wherein in particular

(1 ) the activity of Rho is reduced via mutation(s) introduced in the ribosome binding site, preferably, one or more mutation(s) introduced in nucleotides corresponding to positions -17 to -6 of SEQ ID NO: 1 (ATG-17bp to ATG-6bp), more preferably deletion of said domain; and/or (2) the activity of Rho is reduced via mutation(s) introduced in the cold-shock domain, preferably, one or more mutation(s) in nucleotides corresponding to positions +160 to +360 of SEQ ID NO: 1 (ATG +160bp to ATG +360bp), more preferably deletion of said domain; and/or (3) the activity of Rho is reduced via mutation(s) introduced in the RNA-binding site, preferably, one or more mutation(s) in nucleotides corresponding to positions +174 to +336 of SEQ ID NO: 1 (ATG +174bp to ATG +336bp), more preferably deletion of said domain; and/or (4) the activity of Rho is reduced via mutation(s) introduced in the ATP-binding site, preferably, one or more mutation(s) in nucleotides corresponding to positions +538 to +1062 of SEQ ID NO: 1 (ATG +538bp to ATG +1062bp), more preferably deletion of said domain; and/or

(5) the activity of Rho is reduced via mutation(s) introduced in the promoter region, preferably, one or more mutation(s) in nucleotides corresponding to positions -200 to +1 of SEQ ID NO: 1 (ATG -200bp to ATG +1 ), more preferably deletion of said region; and/or

(6) the activity of Rho is reduced via introduction of one or more mutation(s) in nucleotides corresponding to positions +1 55 to +165 of SEQ ID NO: 1 (ATG +1 55bp to ATG +165bp), more preferably deletion of said nucleotides; and/or

(7) the activity of Rho is reduced via amino acid substitution in a residue corresponding to position 56 of SEQ ID NO:2, preferably, a substitution of the wild- type amino acid into aspartic acid, more preferably a replacement of glycine on a position corresponding to position 56 of SEQ ID NO:2 to aspartic acid, i.e. G56D mutation; and/or

(8) the activity of Rho is reduced via deletion of nucleotides corresponding to the rho gene according to SEQ ID NO: 1 .

Furthermore, the invention relates to a process, wherein a riboflavin-producing host cell selected from a strain of Bacillus or Corynebacterium, preferably selected from B. subtiiis, B. atrophaeus, B. licheniformis, B. amyloliquefaciens, B. pumilus, B. infantis, B. coagulans, B. megaterium, B. thuringiensis, B. cereus, B. halodurans, or C. glutamicum, more preferably selected from B. subtiiis, B. licheniformis, B. amyloliquefaciens, or B. megaterium, most preferably from B. subtiiis, in particular B. subtiiis 1A747 or B. subtiiis 168, wherein the activity of Rho is reduced or abolished compared to the activity of a non-modified Rho, preferably reduced by at least 20%, more preferably by at least 50, 60, 70, 80, 90%, most preferably reduced by 100% (i.e. Rho activity is abolished); wherein in particular the activity of Rho is reduced by one of the embodiments (1 ) to (8) described above. Figures

Figure 1 : Scheme of B. subtilis strain constructions.

Figure 2: Riboflavin production yields in % (y-axis) in the presence (i.e. BS4912 or BS7307) or absence (BS7301 or BS7309) of rho gene. For more details, see in particular Example 10 (Table 3).

Figure 3: Alignment generated with ClustalW2 with default settings. ECOLI : E. coli K12 (P0AG30), PSEPU: Pseudomonas putida S16 (F8FZD7), BACSU: B. subtilis 168 (Q03222), STAAE: Staphylococcus aureus Newman (A60JW5), DEIRA: Deinococcus radiodurans (P52153). The amino acid at position 56 of B. subtilis Rho (glycine) is underlined.

The following examples are illustrative only and are not intended to limit the scope of the invention in any way. The contents of all references, patent applications, patents and published patent applications, cited throughout this application are hereby incorporated by reference, in particular EP 405370, WO 04/ 106557, WO 07/051552 and EP1 186664.

Examples

The following media and general methods as referred to in the examples have been used:

VY medium: Yeast extract (Becton-Dickinson) (5 g/l), Veal infusion broth

(Sigma) (25 g/l).

TBAB medium: 33 g/l Tryptose blood agar base (Becton Dickinson).

10X Spizizen Salts (SS): 20 g/l (NH₄)₂S0₄; 140 g/l K₂HP0₄; 60 g/l KH₂P0₄; 10 g/l tri-sodium citrate; 2 g/l MgS0₄-7 H₂0.

10X BSS: 20 g/l (NH₄)₂S0₄; 140 g/l K₂HP0₄; 60 g/l KH₂P0₄; 2 g/l MgS0₄-2H₂0; pH 6.8.

100X Trace elements solution: 12.5 g/l MgCl₂-6 H₂0; 0.55 g/l CaCl₂; 1 .35 g/l Fe(lll )Cl₃-6 H₂0; 0.1 g/l MnCl₂-4 H₂0; 0.17 g/l ZnCl₂; 0.043 g/l CuCl₂-2 H₂0; 0.06 g/l CoCl₂-6 H₂0; 0.06 g/l Na₂Mo0₄-2 H₂0.

Spizizen Minimal Medium (SMM): 100 ml 10X SS, 10 ml 100X Trace elements solution, 10 ml 50% glucose solution. H₂0 added up to 1000 ml. For solid medium, a final concentration of 1 .5% agar (Becton Dickinson) was added.

Riboflavin screening medium (RSM): 100 ml 10X BSS; 10 ml 100X Trace elements solution; 50 mg/l Yeast Extract (Merck), supplemented with one slow release glucose FeedBeads 6 mm (Kiihner AG, Birsfelden, Switzerland).

10X MN medium: 136 g/l K₂HP0₄; 60 g/l KH₂P0₄; 8.8 g/l Sodium citrate-2 H₂0. MNGE medium: 0.9 ml 10X MN medium, 400 ml 50% glucose solution, 50 μΐ 40% sodium glutamate solution, 50 μΐ, ammonium iron(lll) citrate solution (2.2 mg/l), 30 μΐ 1M MgS0₄ solution. This medium is supplemented with 100 μΐ 10% Casamino acids (CAA) solution (Becton Dickinson) in the first stage of the preparation of the B. subtilis competent cells.

Expression Mix (EM): 7.5 ml 10% yeast extract; 3.75 ml 10% CAA; 18.75 ml H₂0.

Isolation of genomic DNA from B. subtilis: Genomic DNA was prepared using the High Pure PCR Template Preparation Kit (Roche, Switzerland) according to the description of the manufacturer. 1 ml of a 3 ml-overnight culture of B.

subtilis in VY liquid medium incubated at 37°C (250 rpm) was used as source for the bacterial cells. At the end, the genomic DNA was eluted in 200 μΐ of Tris-HCl buffer (supplied with the kit).

Transformation in B. subtilis: 2 ml of MNGE medium supplemented with CAA were inoculated with a single colony and incubated overnight at 37° C at 180 rpm. The next day, the culture was used to inoculate 10 ml MNGE supplemented with CAA to a starting OD₆oonm -0.1 . The diluted culture was incubated at 37° C (180 rpm) until it reached OD₆oonm -1 .2. The culture was diluted with the same volume of MNGE without CAA and incubated for an additional hour. After a centrifugation step (5 min, 4000 rpm, RT), the supernatant was decanted into a sterile tube. The pellet was suspended in a 1 19 of the saved supernatant.

Transformation was done using either freshly prepared or frozen competent cells. When frozen, glycerol was added to cells to a concentration of 13%. 400 μΐ aliquots were frozen down at -80° C. The aliquot of competent cells were thawed at 37° C and suspended in 1 .7 ml 1X MN, 100 μΐ 50% glucose solution, 34 μΐ 1M MgS0₄. DNA to be transformed was added to the competent cells, and then incubated at 180 rpm for 30 min at 37 °C. 100 μΐ of EM were added, and the cells were incubated at 180 rpm for 1 h at 37° C. The cells were spread onto the - In

appropriate selective agar plates (preceded of two washing steps with 1 ml 1 X SS, when selecting for recovery of prototrophy).

Transduction of B. subtilis with PBS1 bacteriophage lysate: the donor lysate was prepared by inoculating one single colony of the donor strain in 10 ml VY medium. The culture was incubated overnight at 30° C, with slow agitation. The next day, 10 ml of VY medium were inoculated with this 100 μΐ of the

preculture, and incubated at 37° C, 120 rpm up to OD₆oonm -0.5. This culture was then infected with 750 μΐ of PBS1 bacteriophage lysate. The infected culture was incubated at 37° C, 120 rpm for 6 h, and lysed at room temperature without agitation overnight. The next day, the lysed culture was centrifuged and the supernatant was filter-sterilized. The resulting donor lysate was stored at +4° C for further use. The recipient strain was prepared by inoculating one single colony of the donor strain in 10 ml VY medium. The culture was incubated overnight at 30° C, with slow agitation. The next day, 10 ml of VY medium were inoculated with 100 μΐ of the preculture, and incubated at 37° C, 120 rpm up to OD₆oonm ~1 .2. 2 ml of the culture were then infected with 150 μΐ of the donor lysate, and shaked at 37 ° C, 100 rpm for 30 min. Cells were then harvested by centrifugation (1 5 min, 3500 rpm), and washed twice with 10 ml of 1 X SS. The pellet was suspended in 150 μΐ of 1 X SS and spread on a selective agar medium, followed by incubation at 37° C for 1 -2 days.

Assay of riboflavin production in deep-well microtiter plates (MTP): an overnight culture was made from a single colony in 3 ml of VY containing selective antibiotics where appropriate. The preculture was incubated at 39 ° C, 550 rpm, 80% humidity. The next day, 3 ml of RSM were inoculated with the preculture with a starting OD₆oonm -0.05. Cultures in MTP were made in triplicate, covering the wells with a breath seal. MTP were incubated at 39 ° C, 550 rpm, 80% humidity for 48 hours. 250 μΐ of the 48 hour-culture were treated with 20 μΐ of 4M NaOH solution in order to solubilize the riboflavin crystals (shaked for 1 min at 300 rpm). 230 μΐ of a 1 M potassium phosphate buffer, pH 6.8 were added (shaked for 1 min at 300 rpm). Riboflavin was assayed by measuring OD_444nm of an appropriate dilution. One unit of OD_444nm corresponds to 33.05 mg/l of riboflavin. Additionally, the potential accumulation of glucose in the culture broth was analyzed by an Agilent 1 100 series HPLC system using a quaternary pump, an autosampler, a UV- and a refractive index detector. The separation was achieved on a CAPCELL PAK NH2 UG80 column (4.6 mm x 250 mm, 5μ; Shiseido). The optimal column temperature was 35 ° C. The mobile phase was a mixture of acetonitrile and deionized water at a ratio of 65: 35. The flow rate was 1 .0 ml/min and the injection volume set to 5 μΐ. The refractive index signal was monitored and used for detection. The calibration range for each compound was from 0.5 mg/ml to 30 mg/ml.

Primers used for the B. subtilis strain construction are described in the examples and listed in Table 1 . For more information check the corresponding text in the examples. Table 1 : sequences as used in the present invention, including the SEQ ID NOs and names ("P" means primer; "rho" means the polynucleotide sequence of rho; "Rho" means the amino acids sequence of the Rho protein).

Name Sequence (5" - 3") NO:

ATGAAAGACGTATCTATTTCCTCTTTGGAAAATATGAAATTGAAAGA

1 GCTTTATGAACTTGCAAGACATTATAAAATCTCCTATTACAGCAAAC T GAC AAAAAAAGAAC T C AT T T T C GC CAT T CT GAAAGC GAAT GC AGAA CAGGAAGATCTGCTGTTTATGGAAGGCGTTCTCGAGATCATCCAGTC TGAAGGTTTCGGATTCCTGAGACCGATCAACTACTCTCCAAGCTCAG AAGACATTTACATCTCAGCTTCACAAATCCGCCGTTTCGATTTGCGG AACGGAGACAAAGTATCTGGCAAGGTTCGCCCGCCAAAAGAAAATGA GCGTTACTATGGACTTTTGCACGTTGAAGCAGTAAATGGGGATGATC CCGAATCTGCAAAAGAGCGTGTGCATTTCCCGGCTCTTACGCCACTT TATCCGGATCGTCAAATGGTGCTTGAAACAAAGCCGAACTTCTTGTC TACAAGAATTATGGACATGATGGCGCCGGTTGGATTTGGGCAGCGCG GATTGATTGTTGCGCCGCCGAAAGCCGGAAAAACGATGTTGCTGAAG

rho GAAATTGCCAACAGCATTACAGCGAACCAGCCTGAAGCAGAGCTGAT

CGTGCTTTTAATTGACGAAAGACCTGAGGAAGTAACCGATATCGAGC GCTCTGTAGCTGGGGATGTCGTCAGCTCAACGTTTGATGAAGTGCCG GAAAACCATATCAAAGTGGCCGAGCTTGTGCTTGAACGTGCGATGCG TCTCGTGGAACACAAAAAAGACGTCATTATCCTGATGGACAGCATCA CACGTCTTGCCCGCGCCTACAACTTAGTGATTCCGCCAAGTGGAAGA ACGCTTTCCGGGGGGATTGACCCAGCGGCGTTCCACCGTCCGAAACG CTTCTTTGGGGCTGCGAGAAATATCGAAGAGGGCGGCAGCTTAACCA TCCTTGCTACGGCTCTGGTCGATACAGGTTCACGTATGGATGATGTC ATTTATGAAGAATTCAAGGGAACAGGCAACATGGAGCTCCATCTTGA CCGCTCTCTTGCCGAGCGCCGCATCTTCCCTGCCATCGATATCCGCC GTTCAGGAACGCGCAAAGAAGAGCTGCTTGTGCCTAAAGAGCATCTT GATCGTTTATGGTCTATCCGCAAAACGATGTCTGATTCACCTGATTT CGCAGAAAAGTTCATGAGAAAAATGAAAAAAACCAAAACAAACCAGG AATTTTTCGATATTCTCAATCAAGAATGGAAACAGGCAAATCTATCA TCTGCAAGAAGGTAA

MKDVS I S SLENMKLKELYELARHYKI S YYSKLTKKEL I FAI LKANAE QEDLLFMEGVLE I IQSEGFGFLRPINYSPSSEDIY I SASQIRRFDLR NGDKVSGKVRPPKENERYYGLLHVEAVNGDDPE SAKERVHFPALTPL YPDRQMVLETKPNFLSTRIMDMMAPVGFGQRGL IVAPPKAGKTMLLK

Rho E IANS I TANQPEAEL IVLL I DERPEEVTDIERSVAGDWSST FDEVP 2

ENH IKVAELVLERAMRLVEHKKDVI I LMDS I TRLARAYNLVI PPSGR TLSGGI DPAAFHRPKRFFGAARNIEEGGSLT I LATALVDTGSRMDDV I YEEFKGTGNMELHLDRSLAERRI FPAI DIRRSGTRKEELLVPKEHL DRLWS IRKTMS DS PDFAEKFMRKMKKTKTNQEFFDI LNQEWKQANLS SARR

P1 CGGATATGGATACATATCGGTTC 3

P2 CGACTAAAGCAGAAAGAGGAAGAG 4

P3 TTCAACTAACGGGGCAGGTTA 5

P4 GGTTATCACCTGTGAAATAGG 6

P5 CAGAGGATCAAACCGGAGAAACGG 7

P6 GCTCTTGGACCCGGGATCCTTATTTCCGCAAATTGCTG 8

P7 CAGCAATTTGCGGAAATAAGGATCCCGGGTCCAAGAGC 9

TAGAATAAAATTTGCGTGCGTTGCAAGCCTTGGAAGCTGTCAGTAGT

P8 10

ATACC

GGTATACTACTGACAGCTTCCAAGGCTTGCAACGCACGCAAATTTTA

P9 1 1

TTCTA

P10 GTTTCAACGGTAAGCGTTCTTCCG 12

P1 1 GCCCACAGGTGTATATG 1 3

P12 GCTCAGTTAATTCTTTGATGCC 14

P1 3 AGAGAAT GAAG AG AC TGCAGAGTG 1 5

P14 TTCTGCCTCGTAATCTCCCGAAG 16

P1 5 CTGAAAGCTTAGTTATCCGTGC 17

P16 GATCTCGACCTGCAGCCCAAGCCACCACGCTTTTCATAGTCAATATC 18

P17 CTTGGGGCTGCAGGTCGAGATC 19

P18 GTTCAAAATGGTATGCGTTTTGACAC 20

GTGTCAAAACGCATACCATTTTGAACGGCAAATCTATCATCTGCAAG

P19 21

AAGG

P20 CTGATCAGCTCTTCAGATTTCC 22

P21 CTCAGGTGGAATCAGATTGGC 23

P22 GATTCGGTCTGTCCTTCG 24

P23 ACATATTCCCGTTATGCATCG 25

P24 ACTGGCACGGTTGTTGCGTCC 26

P25 GATAATAAGGGTAACTATTGCCGAGTCGCTCCAGTTGCAAACG 27

P26 CGGCAATAGTTACCCTTATTATC 28

P27 TTATAAAAGCCAGTCATTAGGCC 29

P28 GTTTTTATATTACAGCTCCAGATCGCTGGACGGACGAAGAAATTG 30

P29 CATCCTCTACAACATAAACGG 31

P30 GATCTGGAGCTGTAATATAAAAAC 32 The following microorganisms have been generated throughout the invention (see Table 2).

Table 2: construction of microorganisms used for performing the present invention.

Example 1 : Generation of riboflavin phototrophic strains

For constructions of phototrophic strains, Bacillus subtilis BS168-SP1 , a tryptophan-prototroph derivative of the Marburg strain 168, was first generated by replacing trpC2 mutation by a non-mutated trpC gene amplified by PCR on DNA isolated from strain B. subtilis ATCC 6051 obtained from The American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108 USA. DNA fragments were generated as follows: 1 μΐ of a 100 μΜ solution of primers P1 together with P2 were added to 0.1 μg B. subtilis ATCC 6051 chromosomal DNA in a 50 μΐ reaction volume containing 1 μΐ of 10 mM dNTPs, 5 μΐ of 10X buffer and 0.5 μΐ Pfu polymerase (Stratagene). The PCR reaction was performed in 35 cycles of three sequential steps: (i) denaturing step at 94°C for 30 sec; (ii) annealing step at 53°C for 30 sec; (iii) elongation step at 72°C for 2 min. The PCR cycles were preceded by a denaturation step at 95 °C for 2 min. The 1425 bp-long trpC PCR products was purified by agarose gel electrophoresis and extracted from the gel using the MinElute Gel Extraction Kit (Ojagen). Strain B. subtilis 168 (CIP106309), originated from the collection of strains of the Pasteur Institute (Paris), was transformed with 1 μg of trpC PCR products of ATCC6051 , according to the method described above. Tryptophan prototroph (Trp+) transformants were selected on SMM agar plates. One of the transformants, designated BS168-SP1 , was confirmed for tryptophan prototrophy, since it was able to grow on SMM agar plates even if not supplemented with tryptophan 20 μg/ml. In addition, the genotype was confirmed by sequencing of BS168-SP1 . Example 2: Construction of a riboflavin-auxotroph B. subtilis BS1 68-SP1

Genomic DNA isolated from strain RB55 (described in US20030232403) was used to construct a riboflavin-auxotroph BS168-SP1 strain (see Example 1 ). In RB55, the 7.2kb-section, containing the rib operon (ribG, ribB, ribA, ribH, and ribT) and its leader region, is removed and essentially replaced with a chloramphenicol resistance (cat) cassette. 1 μg of RB55 genomic DNA was used for transformation of competent B. subtilis BS168-SP1 (see Example 1 ). Chloramphenicol-resistant (Cmr) transformants were selected on TBAB plates containing 5 μg/ml chloramphenicol. One of the transformants, designated BS4842, was confirmed for riboflavin auxotrophy, since it was able to grow on SMM agar plates only if supplemented with riboflavin 500 μΜ. The correct genotype of the riboflavin- auxotrophic and Cmr BS4842 strain was confirmed by PCR using primers P3 together with P4, and chromosomal DNA of BS4842 as template DNA. The PCR reaction was performed using standard reaction conditions as described above. Example 3: Replacing the native rib promoter by a strong constitutive promoter in B. subtilis BS4842

In order to replace the cat gene in the strain B. subtilis BS4842 (see Example 2) by a modified version of the rib leader region (Pspo15_triple ribO_del mro175) and the ribGBAHT genes, resulting in the constitutive expression of the rib operon, 1 μg of genomic DNA isolated from strain BS3922 (described in W010052319) was used for transformation of competent B. subtilis BS4842 cells. The cells were plated onto SMM plates supplemented with roseoflavin 100 Mg/ml. One of the transformants, designated BS4903, was confirmed for riboflavin prototrophy, since it was able to grow on SMM agar plates even if not supplemented with riboflavin 500 μΜ. BS4903 grew only on TBAB agar plates that did not contain 5 Mg/ml chloramphenicol. In addition, the genotype was confirmed by sequencing of BS4912 (see Example 6), a derivative strain of BS4903.

Example 4: Introduction of the ribC820 lesion in the ribC gene of B. subtilis

BS4903

The ribC820 lesion originally identified in a riboflavin-overproducing mutant (Mack et al., J Bacteriol. 180(4):950-5, 1998) was inserted in the chromosome of strain B. subtilis BS4903 (see Example 3). First, an erythromycin resistance marker (erm) from plasmid pMUTIN4, obtained from The Bacillus Genetic Stock Center, The Ohio State University, USA (Vagner et al., J Bacteriol. 180(4):950-5, 1998), was inserted in the intergenic region between ribC and rpsO genes, immediately downstream ribC (Sequence 2). Long Flanking Homology Polymerase Chain Reaction (LFH-PCR) was used to generate a DNA fragment containing the 1163 bp erm resistance cassette flanked with the 1183 bp upstream region and the coding sequence of the ribC gene (flank 5') and the 660 bp downstream region of the ribC gene (flank 3'). Therefore, 3 DNA fragments flank 5', the erm resistance cassette and flank 3', were first amplified by PCR: for the flank 5' (bearing ribC820) 1 μΐ of a 100 μΜ solution of respectively primers P5 and P6 were added to 0.1 μg B. subtilis BS3922 (described in W010052319 A1 ) chromosomal DNA in a 50 μΐ reaction volume containing 1 μΐ of 10 mM dNTPs, 5 μΐ of 10X buffer and 0.5 μΐ Pfu polymerase (Stratagene); for the erm resistance cassette, primers P7 and P8 were added to 0.1 μg pMUTIN2 plasmid DNA in a 50 μΐ reaction volume as described earlier; for the flank 3', 1 μΐ of a 100 μΜ solution of respectively primers P9 and P10 were added to 0.1 μg B. subtilis BS168-SP1 (see Example 1 ) chromosomal DNA in a 50 μΐ reaction volume as described earlier. The PCR reactions were performed in 35 cycles of three sequential steps: (i) denaturing step at 94 ° C for 30 sec; (ii) annealing step at 53 ° C for 30 sec; (iii) elongation step at 72° for 2 min. The PCR cycles were preceded by a denaturation step at 95 ° C for 2 min. The three PCR products were separated by agarose gel electrophoresis and extracted from the gel using the QIAquick Gel Extraction Kit (Qiagen). Due to the overlapping regions of the flank 5' and flank 3' with the erythromycin cassette, it is possible to assemble them by a final LFH-PCR reaction: 1 μΐ of a 100 μΜ solution of primers P5 together with P10, 2 μΐ flank 5^' PCR product, 2 μΐ flank 3^' PCR product and 2 μΐ erm resistance cassette were added to give a final reaction volume of 50 μΐ containing 1 μΐ of 10 mM dNTPs, 5 μΐ of 10X buffer and 0.5 μΐ Pfu polymerase (Roche). The LFH-PCR reaction was performed in 10 cycles of three sequential steps: (i) denaturing step at 9 ° C for 30 sec; (ii) annealing step at 63 ° C for 30 sec; (iii) elongation step at 68 ° C for 4 min, followed by 20 cycles of three sequential steps: (i) denaturing step at 94 ° C for 30 sec; (ii) annealing step at 63 ° C for 30 sec; (iii) elongation step at 68° C for 4 min, incrementing 20 sec/cycle. The PCR cycles were preceded by a denaturation step at 95 ° C for 2 min. The assembled LFH-PCR product was purified by agarose gel electrophoresis and extracted from the gel using the QIAquick Gel Extraction Kit (Qiagen). The purified LFH-PCR product (1 μg) was used for transformation of competent B. subtilis BS4903 (see Example 3). Erythromycin-resistant (Erm) transformants were selected on TBAB plates containing 1 μg/ml erythromycin and 25 μg/ml lincomycin. The correct genotype of some Erm transformants was confirmed by a PCR reaction using primers P5 together with P10 (by comparison with the same amplicon made on BS168-SP1 genomic DNA, being 1 .1 kb shorter than on the Erm transformants). The PCR reaction was performed using standard reaction conditions as described above for the LFH-PCR. For one of the transformants, designated BS4905, the presence of the ribC820 lesion was controlled thanks to the associated creation of an Alul restriction site in a PCR fragment generated using primers P1 1 together with P12 (by comparison with the same amplicon made on BS168-SP1 genomic DNA, which does not harbor the Alul restriction site). The 1 5 μΐ-restriction mixture contained 10 μΐ of the PCR fragment, 1 .5 μΐ 10X buffer, and 1 .5 μΐ of Alul restriction enzyme (New England Biolabs, USA), and was incubated for 1 hour at 37° C. In addition, the genotype was confirmed by sequencing of BS4912 (see Example 6), a derivative strain of BS4905.

Example 5: Construction of a transketolase-deficient B. subtilis BS4905 strain

Preliminarily to the marker-free introduction of a mutated transketolase gene into the original tkt locus of the B. subtilis genome, a transketolase-deficient strain was constructed. A PCR fragment, containing a neomycin resistance cassette from pUB110 (Itaya et al., Nucleic Acids Res. 17(11 ):4410, 1989), between the base pairs 1043 and 1561 of the B. subtilis transketolase gene, was generated as follows: 1 μΐ of a 100 μΜ solution of primers P13 together with P14 were added to 0.1 μg B. subtilis BS3402 (described in WO2007051552) chromosomal DNA in a 50 μΐ reaction volume containing 1 μΐ of 10 mM dNTPs, 5 μΐ of 10X buffer and 0.5 μΐ Pfu polymerase (Roche). The PCR reaction was performed in 10 cycles of three sequential steps: (i) denaturing step at 94°C for 30 sec; (ii) annealing step at 63°C for 30 sec; (iii) elongation step at 68 °C for 4 min, followed by 20 cycles of three sequential steps: (i) denaturing step at 94° C for 30 sec; (ii) annealing step at 63 °C for 30 sec; (iii) elongation step at 68°C for 4 min, incrementing 20 sec/cycle. The PCR cycles were preceded by a denaturation step at 95 °C for 2 min. The 5kb-long tkt: :neo PCR products was purified by agarose gel electrophoresis and extracted from the gel using the MinElute Gel Extraction Kit (Qiagen). Strain B. subtilis BS4905 (see Example 4) was transformed with 1 μg of the tkt: :neo PCR product of BS3402, according to the method described above. Neomycin-resistant (Nmr) transformants were selected on TBAB plates containing 5 μg/ml neomycin supplemented with 500 μg/ml shikimic acid (Sigma). For one of the Nmr transformants, designated BS4909, the genomic DNA was isolated as previously described and the correct replacement of the transketolase DNA fragment from base pairs 1043 to 1561 by the neomycin resistance cassette was confirmed by a standard PCR using primers P13 together with primer P14. As expected for a transketolase deletion mutant, the BS4909 strain could grow on SMM agar plates only if supplemented with shikimic acid 500 μg/ml. Example 6: Introduction of the tktR357A lesion in the transketolase gene in strain B. subtilis BS4909 A PCR fragment, containing a modified transketolase gene (resulting in the mutation R357A), was generated as follows: 1 μΐ of a 100 μΜ solution of primers P13 together with P1 were added to 0.1 μg B. subtilis BS3922 (described in WO2010052319) chromosomal DNA in a 50 μΐ reaction volume containing 1 μΐ of 10 mM dNTPs, 5 μΐ of 10X buffer and 0.5 μΐ Pfu polymerase (Stratagene) in a 50 μΐ reaction volume as described earlier. The PCR reaction was performed in 35 cycles of three sequential steps: (i) denaturing step at 9 °C for 30 sec; (ii) annealing step at 53 °C for 30 sec; (iii) elongation step at 72 °C for 3 min. The PCR cycles were preceded by a denaturation step at 95 °C for 2 min. The 3kb-long tktR357A PCR products was purified by agarose gel electrophoresis and extracted from the gel using the MinElute Gel Extraction Kit (Qiagen). Strain B. subtilis BS4909 (see Example 5) was transformed with 1 μg of the tktR357A PCR product of BS3922, according to the method described above. The cells were plated onto SMM plates. One of the transformants, designated BS4912, was confirmed for prototrophy, since it was able to grow on SMM agar plates even if not supplemented with shikimic acid 500 μg/ml. BS4912 grew only on TBAB agar plates that did not contain 5 μg/ml neomycin. In addition, the genotype was confirmed by sequencing of BS4912.

Example 7: Deletion of the rho gene in strain B. subtilis BS4912 The production of Rho protein in BS4912 was prevented in a rho-null mutant. The strain was constructed by replacing the rho gene in B. subtilis BS4912 strain by a neomycin resistance cassette from pUB110 (Itaya et al., see above). The chromosomal integration of the neomycin resistance cassette results in a complete deletion of the rho gene, from 5 base pairs upstream the start codon, to 11 base pairs downstream the stop codon (Sequence 3). Long Flanking Homology Polymerase Chain Reaction (LFH-PCR) was used to generate a DNA fragment containing the 1234 bp- neomycin resistance cassette flanked with the 499 bp upstream region (flank 5') and the 515 bp downstream region (flank 3') of the rho gene. Therefore, 3 DNA fragments; flank 5', the neo resistance cassette and flank 3', were first amplified by PCR: for the flank 5', 1 μΐ of a 100 μΜ solution of respectively primers P15 and P16 were added to 0.1 μg B. subtilis BS168-SP1 (see Example 1 ) chromosomal DNA in a 50 μΐ reaction volume containing 1 μΐ of 10 mM dNTPs, 5 μΐ of 10X buffer and 0.5 μΐ Pfu polymerase (Stratagene); for the neo resistance cassette, primers P17 and P18 were added to 0.1 μg B. subtilis BS3402 (described in WO2007051552/A1 ) chromosomal DNA in a 50 μΐ reaction volume as described earlier; for the flank 3', 1 μΐ of a 100 μΜ solution of respectively primers P19 and P20 were added to 0.1 μg B. subtilis BS168-SP1 (see Example 1 ) chromosomal DNA in a 50 μΐ reaction volume as described earlier. The PCR reactions were performed in 35 cycles of three sequential steps: (i) denaturing step at 9 °C for 30 sec; (ii) annealing step at 53 °C for 30 sec; (iii) elongation step at 72°C for 2 min. The PCR cycles were preceded by a denaturation step at 95°C for 2 min. The three PCR products were separated by agarose gel electrophoresis and extracted from the gel using the QIAquick Gel Extraction Kit (Qiagen). Due to the overlapping regions of the flank 5' and flank 3' with the neomycin resistance cassette, it is possible to assemble them by a final LFH-PCR reaction: 1 μΐ of a 100 μΜ solution of primers P15 together with P20, 2 μΐ flank 5' PCR product, 2 μΐ flank 3' PCR product and 2 μΐ neo resistance cassette were added to give a final reaction volume of 50 μΐ containing 1 μΐ of 10 mM dNTPs, 5 μΐ of 10X buffer and 0.5 μΐ Pfu polymerase (Roche). The LFH-PCR reaction was performed in 10 cycles of three sequential steps: (i) denaturing step at 94°C for 30 sec; (ii) annealing step at 63°C for 30 sec; (iii) elongation step at 68 °C for 3 min, followed by 20 cycles of three sequential steps: (i) denaturing step at 94°C for 30 sec; (ii) annealing step at 63°C for 30 sec; (iii) elongation step at 68°C for 3 min, incrementing 20 sec/cycle. The PCR cycles were preceded by a denaturation step at 95 °C for 2 min. The assembled LFH-PCR product was purified by agarose gel electrophoresis and extracted from the gel using the QIAquick Gel Extraction Kit (Qiagen). The purified LFH-PCR product (1 μg) was used for transformation of competent B. subtilis BS168-SP1 (see Example 1 ). Neomycin-resistant (Nmr) transformants were selected on TBAB plates containing 5 μg/ml neomycin. The genomic DNA of one of the Nmr transformants, designated BS7180, was isolated as previously described and the correct deletion of the complete rho coding sequence by the neomycin resistance cassette was confirmed by a standard PCR using primers P20 together with primer P21. Transduction of the rho deletion construct was performed with PBS1 phage according to the method described above, wherein a lysate of BS7180 was used to transduce the strain B. subtilis BS4912 (see Example 6). Nmr transductants were selected on TBAB plates containing 5 μg/ml neomycin. The genomic DNA of one of the Nmr transductants, designated BS7301 , was isolated as previously described and the correct deletion of the complete rho coding sequence by the neomycin resistance cassette was confirmed by a standard PCR using primers P20 together with primer P21.

Example 8: Replacement of pRF69 and pRF93 in strain B. subtilis BS5596

Examples of the strains used in the present invention derive from the adenine prototroph B. subtilis strain BS5596, also named RB50::[pRF69]60:: [pRF93]120 (construction described in EP821063 and US6190888), known to be capable of producing more than 14.0 g/l of riboflavin under optimized jar fermentation conditions. In addition to a constitutively expressed copy of the rib operon in each plasmid, pRF69 harbors a chloramphenicol resistance (cat) cassette and pRF93 harbors a tetracycline (tet) resistance cassette. In this riboflavin overproducing strain, the pRF69 plasmids integrated at the rib locus (207.6° in the chromosome) and the pRF93 plasmids integrated at the bpr locus (136.5° in the chromosome) were respectively substituted by the rib operon of BS3922 (described in WO2010052319), which expression is driven by the Pspol 5_triple ribO_del mro175 leader region, and a chloramphenicol (cat) resistance cassette. For the strain construction, BS5596 was infected as described above with a PBS1 lysate of BS4664, a derivative of B. subtilis strain BS3922 (described in WO10052319 A1 ) harboring the spoOA12 non-sense mutation (Hoch JA, 1971 ) in the spoOA gene. For the selection, a spectinomycin (spec) resistance cassette from plasmid pDG1728, obtained from The Bacillus Genetic Stock Center, The Ohio State University, USA (Guerout-Fleury et al., see above) was inserted in the intergenic region between spoOA and yqiG genes, 221 base pairs downstream spoOA (Sequence 4). Spectinomycin resistant (Specr) clones resulting from the transduction of RB50: : [pRF69]60: : [pRF93] 120 Ade+ with BS4664 lysate were selected on TBAB agar plates containing 100 Mg/ml spectinomycin. As a consequence of the substitution of the pRF69 plasmid by Pspol 5_triple ribO_del mro175 rib, chloramphenicol- sensitive (CmS) transductants were screened on TBAB agar plates containing 5 Mg/ml chloramphenicol. The resulting strain was designated BS7331. The genomic DNA of BS7331 was isolated as previously described and the correct replacement of the pRF69 by the Pspol 5_triple ribO_del mro175 rib was confirmed by a standard PCR using primers P22 together with primer P23 (by comparison with the same amplicon made on BS168-SP1 genomic DNA, being 103 bp shorter). In order to have only one copy of the rib operon in BS7331 , pRF93 plasmid at bpr locus of the BS7331 chromosome was replaced by a chloramphenicol resistance (cat) cassette from plasmid pSac-Cm obtained from The Bacillus Genetic Stock Center, The Ohio State University, USA (Middleton and Hofmeister, Plasmid. 51 (3):238-45, 2004). For the strain construction, Long Flanking Homology Polymerase Chain Reaction (LFH-PCR) was used to generate a DNA fragment containing the 1035 bp chloramphenicol resistance cassette flanked with the 581 bp upstream region (flank 5') and the 564 bp downstream region (flank 3') of the bpr gene. Therefore, 3 DNA fragments flank 5' , the cat resistance cassette and flank 3' were first amplified by PCR: for the flank 5' , 1 μΐ of a 100 μΜ solution of respectively primers P24 and P25 were added to 0.1 Mg B. subtilis BS168-SP1 (see Example 1 ) chromosomal DNA in a 50 μΐ reaction volume containing 1 μΐ of 10 mM dNTPs, 5 μΐ of 10X buffer and 0.5 μΐ Pfu polymerase (Stratagene); for the cat resistance cassette, primers P26 and P27 were added to 0.1 μg of pSac-Cm plasmid DNA in a 50 μΐ reaction volume as described earlier; for the flank 3' , 1 μΐ of a 100 μΜ solution of respectively primers P28 and P29 were added to 0.1 μg B. subtilis BS168-SP1 (see Example 1 ) chromosomal DNA in a 50 μΐ reaction volume as described earlier. The PCR reactions were performed in 35 cycles of three sequential steps: (i) denaturing step at 94 ° C for 30 sec; (ii) annealing step at 53 ° C for 30 sec; (iii) elongation step at 72° C for 2 min. The PCR cycles were preceded by a denaturation step at 95 ° C for 2 min. The three PCR products were separated by agarose gel electrophoresis and extracted from the gel using the OJAquick Gel Extraction Kit (Qiagen). Due to the overlapping regions of the flank 5' and flank 3' with the chloramphenicol resistance cassette, it is possible to assemble them by a final LFH-PCR reaction: 1 μΐ of a 100 μΜ solution of primers P24 together with P29, 2 μΐ flank 5' PCR product, 2 μΐ flank 3' PCR product and 2 μΐ cat resistance cassette were added to give a final reaction volume of 50 μΐ containing 1 μΐ of 10 mM dNTPs, 5 μΐ of 10X buffer and 0.5 μΐ Pfu polymerase (Roche). The LFH-PCR reaction was performed in 10 cycles of three sequential steps: (i) denaturing step at 94° C for 30 sec; (ii) annealing step at 63 ° C for 30 sec; (iii) elongation step at 68 ° C for 3 min, followed by 20 cycles of three sequential steps: (i) denaturing step at 94° C for 30 sec; (ii) annealing step at 63 ° C for 30 sec; (iii) elongation step at 68 ° C for 3 min, incrementing 20 sec/cycle. The PCR cycles were preceded by a denaturation step at 95° C for 2 min. The assembled LFH-PCR product was purified by agarose gel electrophoresis and extracted from the gel using the OJAquick Gel Extraction Kit (Qiagen). The purified LFH-PCR product (1 μg) was used for transformation of competent B. subtilis BS168-SP1 (see Example 1 ). Chloramphenicol-resistant (Cmr) transformants were selected on TBAB plates containing 5 °g/ml chloramphenicol. The genomic DNA of one of the Cmr transformants, designated BS4566, was isolated as previously described and the correct deletion of the bpr coding sequence by the chloramphenicol resistance cassette was confirmed by standard PCR using primers P24 together with primer P30. Transduction of the bpr deletion construct was performed with PBS1 phage according to the method described above, wherein a lysate of BS4566 was used to transduce the strain B. subtilis BS7331. Cmr transductants were selected on TBAB plates containing 5 Mg/ml chloramphenicol. The genomic DNA of one of the Cmr transductants, designated BS7307, was isolated as previously described and the correct deletion of the bpr coding sequence by the chloramphenicol resistance cassette was confirmed by standard PCR using primers P24 together with primer P30. Example 9: Deletion of the rho gene in strain B. subtilis BS7307

The production of Rho protein in BS7307 was prevented in a rho-null mutant. The strain was constructed by replacing the rho gene in B. subtilis BS7307 strain by a neomycin resistance cassette from pUB110 (Itaya et al., see above). The chromosomal integration of the neomycin resistance cassette results in a complete deletion of the rho gene, from 5 base pairs upstream the start codon, to 11 base pairs downstream the stop codon (Sequence 3). The construction of the rho deletion was detailed in Example 7. Transduction of the rho deletion construct was performed with PBS1 phage according to the method described above, wherein a lysate of BS7301 was used to transduce the strain B. subtilis BS7307 (see Example 8). Nmr transductants were selected on TBAB plates containing 5 Mg/ml neomycin. The genomic DNA of one of the Nmr transductants, designated BS7309, was isolated as previously described and the correct deletion of the complete rho coding sequence by the neomycin resistance cassette was confirmed by a standard PCR using primers P20 together with primer P21. Example 10: Riboflavin production assay in presence/absence of rho gene

The production of Rho protein in BS7307 was prevented in a rho-null mutant. The strain was constructed by replacing the rho gene in B. subtilis BS7307 strain by a neomycin resistance cassette from pUB1 10 (Itaya et al. , see above). The chromosomal integration of the neomycin resistance cassette results in a complete deletion of the rho gene, from 5 base pairs upstream the start codon, to 1 1 base pairs downstream the stop codon (Sequence 3). The construction of the rho deletion was detailed in Example 7. Transduction of the rho deletion construct was performed with PBS1 phage according to the method described above, wherein a lysate of BS7301 was used to transduce the strain B. subtilis BS7307 (see Example 8). Nmr transductants were selected on TBAB plates containing 5 Mg/ml neomycin. The genomic DNA of one of the Nmr transductants, designated BS7309, was isolated as previously described and the correct deletion of the complete rho coding sequence by the neomycin resistance cassette was confirmed by a standard PCR using primers P20 together with primer P21 .

Table 3: Riboflavin production with various B. subtilis strains having different genotypes as indicated. Strains B. subtilis BS4912 and BS7301 share the same genotype background (except for rho-deletion), strains B. subtilis BS7307 and BS7309 share the same genotype background (except for rho-deletion). For more explanation see text.

The riboflavin production yield of BS7301 was improved by ca. 33% compared to its direct parent BS4912 (see Figure 2). Consistently, the riboflavin production yield of BS7309 was improved by ca. 36% compared to its direct parent BS7307 (see Figure 2). These results showed a positive impact of the rho gene deletion (i.e. the Rho protein inactivation) on the riboflavin production in riboflavin overproducing strains of B. subtilis with different genetic backgrounds.

Example 1 1 : Mutation of the rho gene in strain B. subtilis

According to the rho deletions described in Example 7 and 9, partial deletions are introduced in the rho gene, i.e. deletions of the ribosome binding site (i.e. nucleotides corresponding to positions -17 to -6 of SEQ ID NO:1 (ATG-17bp to ATG- 6bp), the cold-shock domain (i.e. nucleotides corresponding to positions +160 to +360 of SEQ ID NO: 1 (ATG +160bp to ATG +360bp), the RNA-binding domain (i.e. nucleotides corresponding to positions +174 to +336 of SEQ ID NO: 1 (ATG +174bp to ATG +336bp), the ATP-binding site (i.e. nucleotides corresponding to positions +538 to +1062 of SEQ ID NO: 1 (ATG +538bp to ATG +1062bp), the promoter region (i.e. nucleotides corresponding to positions -200 to +1 of SEQ ID NO:1 (ATG -200bp to ATG +1 ), or deletions of a fragment corresponding to positions +155 to +165 of SEQ ID NO: 1 (ATG +155bp to ATG +165bp, or a deletion leading to a replacement of glycine on a position corresponding to position 56 of SEQ ID NO:2 to aspartic acid, i.e. G56D. When tested for riboflavin production, an increase in the range of 20 to 30% can be detected compared to the corresponding strains without modification in the endogenous rho gene.

Example 1 2: Generation of strains other than B. subtilis carrying Rho mutation for riboflavin production

The constructs as described in the Examples above can be used to identify/ generate corresponding modifications in host strains other than B. subtilis which are suitable for riboflavin production.

Generation of Rho mutations/deletions are performed as described in the above Examples. The identity of Rho proteins in similar Bacillus species is depicted below. The comparison of the B. subtilis rho coding sequence with public databases was made with the BLAST (Basic Local Alignment Search Tool) algorithm of the NCBI (National Center for Biotechnology Information, USA), and revealed that rho gene is highly conserved among Bacillus genus. Furthermore, non-Bacillus strains were tested by an alignment generated with ClustalW2 with default settings, showing the high conservation of G56 identified in the B. subtilis Rho. Exampl homology are listed in Table 4 and Figure 3.

Table 4: Homology for rho-coding sequence within various Bacillus species.

The strains according to Table 4 and Figure 3 can be used for manipulations as described herein, in particular Examples 7 to 1 1 . Thus a genetically-modified strain is constructed, wherein the activity of Rho is reduced or abolished as exemplified above. Measurement of riboflavin production is performed as shown, with an increase in riboflavin production according to the results depicted in Table 3.

Claims

Claims

1. A riboflavin-producing host cell selected from a strain of Bacillus or Corynebacterium, wherein the activity of endogenous Rho is reduced compared to the activity of a non-modified Rho by at least 20%, preferably reduced by at least 50, 60, 70, 80, 90%, most preferably reduced by 100%.

2. The host cell of claim 1 , wherein one or more mutation(s) have been introduced in the rho gene, said one or more mutation(s) being located in the ribosome binging site, the cold-shock domain, the RNA-binding site, the ATP- binding site or the promoter region, preferably located in a region selected from the group consisting of nucleotides corresponding to positions -17 to -6 of SEQ ID NO: 1 (ATG-17bp to ATG-6bp), positions +160 to +360 of SEQ ID NO:1 (ATG +160bp to ATG +360bp), +174 to +336 of SEQ ID NO:1 (ATG +174bp to ATG +336bp), positions +538 to +1062 of SEQ ID NO:1 (ATG +538bp to ATG +1062bp), and -200 to +1 of SEQ ID NO:1 (ATG -200bp to ATG +1 ).

3. The host cell of claim 2, wherein the one or more mutation(s) is a deletion of nucleotides corresponding to the ribosome-binding site, the cold-shock domain, the RNA-binding site, the ATP-binding site or the promoter region.

4. The host cell according to claim 1 , wherein one or more mutation(s) have been introduced in a fragment corresponding to positions +155 to +165 of SEQ ID NO:1 (ATG +155bp to ATG +165bp), preferably wherein said fragment is deleted.

5. The host cell according to any one of claims 1 , 2 or 4, comprising an amino acid substitution in a residue corresponding to position 56 of SEQ ID NO:2, preferably a substitution of the wild-type amino acid into aspartic acid, more preferably a replacement of glycine on a position corresponding to position 56 of SEQ ID NO:2 to aspartic acid.

6. The host cell of claim 1 , wherein the activity of Rho is reduced compared to the activity of a non-modified Rho via knocking out the rho gene.

7. The host cell according to any one of claims 1 to 6, wherein the strain is selected from the group consisting of B. subtilis, B. atrophaeus, B. licheniformis, B. amyloliquefaciens, B. pumilus, B. infantis, B. coagulans, B. megaterium, B. thuringiensis, B. cereus, B. halodurans, or C. glutamicum, more preferably selected from B. subtilis, B. licheniformis, B. amyloliquefaciens, and B. megaterium, most preferably from B. subtilis, in particular B. subtilis 1A747 or B. subtilis 168.

8. A process for production of riboflavin with a host cell according to any one of claims 1 to 7.

9. The process according to claim 8 comprising the steps of:

(a) culturing the host cell under fermentation conditions suitable for riboflavin production and optionally

(b) isolation of riboflavin from the culture medium.

10. Use of a host cell according to any one of claims 1 to 7 for production of riboflavin, wherein the yield of riboflavin is increased by at least 5% compared to the use of a host cell wherein the Rho specific activity is 100%.

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