# Patent

## Title
Modified transketolase and use thereof

## Abstract

The present invention relates to a improved process for the biotechnological production of compounds for which ribose-5-phosphate, ribulose-5-phosphate or xylulose-5-phosphate is biosynthetic precursor like riboflavin (vitamin B2), FAD, FMN, pyridoxal phosphate (vitamin B6), guanosine, GMP, adenosine, AMP. The invention further pertains to the generation of the organism producing those compounds. It furthermore relates to the generation of mutated transketolases that allow normal growth on glucose but reduced growth on gluconate when introduced into the production strains and to polynucleotides encoding them.

## Background
This application is the U.S. national phase under 35 USC 371 of Int'l Application No. PCT/EP2006/010270, filed 25 Oct. 2006, which designated the U.S. and claims priority to European Patent Application No. 05023813.8, filed 2 Nov. 2005; the entire contents of each of which are hereby incorporated by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 2, 2011, is named 4662-788.txt and is 67.628 bytes in size.
The present invention provides modified transketolase enzymes. Microorganisms synthesizing one of the modified transketolases instead of the wild type transketolase are prototroph for aromatic amino acids and impaired in using carbon sources that are assimilated via the pentose phosphate pathway. The modified enzymes and polynucleotides encoding the same can be used in the fermentation process for substances that use ribose-5-phosphate, ribulose-5-phosphate, or xylulose-5-phosphate as substrate for the biosynthesis such as e.g. riboflavin, riboflavin precursors, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and derivatives thereof. They also can be used for the production of pyridoxal phosphate (vitamin B6), guanosine and adenosine and derivatives of these nucleotides.
BACKGROUND OF THE INVENTION
Riboflavin (vitamin B2) is synthesized by all plants and many microorganisms but is not produced by higher animals. Because it is a precursor to coenzymes such as flavin adenine dinucleotide and flavin mononucleotide that are required in the enzymatic oxidation of carbohydrates, riboflavin is essential to basic metabolism. In higher animals, insufficient riboflavin can cause loss of hair, inflammation of the skin, vision deterioration, and growth failure.
The enzymes required catalyzing the biosynthesis of riboflavin from guanosine triphosphate (GTP) and ribulose-5-phosphate are encoded by four genes (ribG, ribB, ribA, and ribH) in B. subtilis. These genes are located in an operon, the gene order of which differs from the order of the enzymatic reactions catalyzed by the enzymes. For example, GTP cyclohydrolase II, which catalyzes the first step in riboflavin biosynthesis, is encoded by the third gene in the operon, ribA. The ribA gene also encodes a second enzymatic activity, i.e., 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS), which catalyzes the conversion of ribulose-5-phosphate to the four-carbon unit 3,4-dihydroxy-2-butanone 4-phosphate (DHBP). Deaminase and reductase are encoded by the first gene of the operon, ribG. The penultimate step in riboflavin biosynthesis is catalyzed by lumazine synthase, the product of the last rib gene, ribH. Riboflavin synthase, which controls the last step of the pathway, is encoded by the second gene of the operon, ribB. The function of the gene 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 ribP1 promoter is controlled by an attenuation mechanism involving a regulatory leader region located between ribP1 and ribG. ribO mutations within this leader region result in deregulated expression of the riboflavin operon. Deregulated expression is also observed in strains containing missense mutations in the ribC gene. The ribC gene has been shown to encode the flavin kinase/FAD synthase of B. subtilis (Mack, M., et al., J. Bacteriol., 180:950-955, 1998). Deregulating mutations reduce the flavokinase activity of the ribC gene product resulting in reduced intracellular concentrations of flavin mononucleotide (FMN), the effector molecule of the riboflavin regulatory system.
Engineering of riboflavin production strains with increased production rates and yields of riboflavin has been achieved in the past in a number of different ways. For instance, (1) classical mutagenesis was used to generate variants with random mutations in the genome of the organism of choice, followed by selection for higher resistance to purine analogs and/or by screening for increased production of riboflavin. (2) Alternatively, the terminal enzymes of riboflavin biosynthesis, i.e., the enzymes catalyzing the conversion of guanosine triphosphate (GTP) and ribulose-5-phosphate to riboflavin, were over-expressed, resulting also in a higher flux towards the target product. The metabolic flux into and through a biosynthetic pathway, e.g. the riboflavin biosynthetic pathway, is determined by the specific activities of the rate-limiting enzymes of this particular pathway and by the intracellular concentrations of the substrates for these enzymes. Only at or above saturating substrate concentrations an enzyme can operate at its maximal activity. The saturating substrate concentration is a characteristic feature for each enzyme. For example, the metabolic flux into the riboflavin pathway may be increased or kept at a high level by keeping the intracellular concentrations of ribulose-5-phosphate above or as close as possible to the saturating substrate concentration of the 3,4-dihydroxy-2-butanone 4-phosphate synthase, a presumed rate limiting enzyme for the riboflavin biosynthetic pathway. High intracellular concentrations of ribulose-5-phosphate may, for example, be reached by preventing or interfering with drainage of ribulose-5-phosphate into the central metabolism via the non-oxidative part of the pentose phosphate pathway.
A key enzyme in the non-oxidative part of the pentose phosphate pathway is the transketolase enzyme, which catalyzes the reversible conversion of ribose-5-phosphate and xylulose-5-phosphate to seduheptulose-7-phosphate and glyceraldehyde-3-phosphate. In addition, transketolase catalyzes also the conversion of fructose-6-phosphate and glyceraldehyde-3-phosphate to xylulose-5-phosphate and erythrose-4-phosphate (Kochetov, G. A. 1982, Transketolase from yeast, rat liver, and pig liver, Methods Enzymol., 90:209-23).
It has previously been reported that transketolase deficient Bacillus subtilis strains carrying knock-out mutations in the transketolase encoding gene produces ribose, which accumulates in the fermentation broth (De Wulf, P., and E. J. Vandamme. 1997. Production of D-ribose by fermentation, Appl. Microbiol. Biotechnol. 48:141-148; Sasajima, K., and Yoneda, M. 1984, Production of pentoses by microorganisms. Biotechnol. and Genet. Eng. Rev. 2: 175-213). Obviously, increased intracellular C5 carbon sugar pools can be reached in transketolase knock-out mutants up to a level that exceeds the physiological requirements of the bacteria and leads to secretion of excess ribose.
As mentioned above, transketolase catalysed reactions are also required to produce erythrose-4-phosphate, from which the three proteinogenic aromatic amino acids are derived. Therefore, transketolase deficient microorganisms are auxotroph for these amino acids. They can only grow if these amino acids or their biosynthetic precursors, for instance shikimic acid, can be supplied via the cultivation medium.
In addition to the unfavorable auxotrophy for aromatic amino acids or shikimic acid, transketolase-deficient B. subtilis mutants show a number of severe pleiotropic effects like very slow growth on glucose, a defective phosphoenolpyruvate-dependent phosphotransferase system, deregulated carbon catabolite repression, and altered cell membrane and cell wall composition (De Wulf, P., and E. J. Vandamme. 1997).
An other transketolase-deficient riboflavin secreting B. subtilis strain was described by Gershanovich et al. (Gershanovich V N, Kukanova A I a, Galushkina Z M, Stepanov A I (2000) Mol. Gen. Mikrobiol. Virusol. 3:3-7).
Furthermore, U.S. Pat. No. 6,258,554 B1 discloses a riboflavin overproducing Corynebacterium glutamicum strain in which transketolase activity is deficient. It can be noted form the disclosure of the U.S. Pat. No. 6,258,554 B1 that the deficiency in transketolase activity and the resulting amino acid auxotrophy was essential for the improved riboflavin productivity, since a prototrophic revertant produced riboflavin in amounts similar to a C. glutamicum strain with a wild-type transketolase background.
These disadvantages, i.e. auxotrophy for aromatic amino acids and further pleiotropic effects discussed above, make a transketolase deficient mutant a less preferable production strain for stable industrial processes, such as, e.g. the industrial production of riboflavin within such strain.
SUMMARY OF THE INVENTION
It is in general an object of the present invention to provide a transketolase mutant strain which is modified in such a way that the catalytic properties of the modified transketolase allowing higher intracellular ribulose-5-phosphate and ribose-5-phosphate concentrations than those of the non-modified transketolase, but which does not have the disadvantages of the transketolase-deficient strains mentioned above.
Surprisingly, it has now been found that by genetically altering a microorganism such as for instance B. subtilis, by replacing the wild-type gene by a mutated gene encoding a modified transketolase that allows some residual flux through the pentose phosphate pathway by having modulated specific activities, the production of a fermentation product such as e.g. riboflavin can be significantly improved without loosing the prototrophic properties.
The present invention relates to modified transketolases, polynucleotide sequences comprising a gene that encodes a modified transketolase with properties described above, a host cell which has been transformed by such a polynucleotide sequence, and a process for the biotechnological production of a fermentation product such as for instance riboflavin, a riboflavin precursor, FMN, FAD, pyridoxal phosphate or one or more derivatives thereof based on a host cell in which the wild-type transketolase gene has been stably replaced by a polynucleotide coding for the mutated transketolase.
As a first step to isolate mutants, in which the wild-type transketolase is replaced by one of such modified transketolases, a deletion mutant may be generated that is auxotroph for the proteinogenic aromatic amino acids and cannot grow with carbon sources assimilated via the pentose phosphate pathway, e.g. gluconate. The transketolase deletion mutant may then be transformed with a mixture of DNA fragments encoding various transketolase mutants. Prototrophic transformants may be isolated, from which those are selected which show a reduced growth rate on gluconate. Mutants isolated according to this method may synthesize modified transketolase enzymes that allow sufficient erythrose-4-phosphate biosynthesis to prevent auxotrophic growth, but act as a bottle neck for assimilation of gluconate. In addition, the undesired pleiotropic effects typically observed with B. subtilis transketolase deletion mutants may be prevented. U.S. Pat. No. 6,258,554 B1 indicates that together with the reversion of the auxotrophic to the prototrophic growth riboflavin secreting C. glutamicum transketolase mutants lost their ability to produce more riboflavin than a similar strain containing a wild-type transketolase gene. As shown in the examples of the present invention, prototrophic B. subtilis transketolase mutants isolated as outlined above unexpectedly produced more riboflavin than the transketolase wild-type parent strain, whereas a transketolase deletion mutant had partly lost their riboflavin production capabilities.
Methods for the introduction of mutations into DNA fragments are well known in the art. Transketolase mutants can be generated for instance by protein engineering using one of the available 3D structures of e.g. the yeast transketolase (Lindqvist, Y., G. Schneider, U. Ermler, and M. Sundstrom. 1992. Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution. Embo. J. 11:2373-9) for selecting suitable positions of the amino acid sequence or by random mutagenesis. The selection process in both cases may be done as described above. The modified transketolase—when it is used as substitution for the wild-type transketolase—exhibits catalytic properties, i.e. modulated specific activities, which allow the growth of a host cell on a carbon source that is metabolized exclusively by the pentose phosphate pathway (for example gluconate) with a reduced growth rate in comparison to a host cell containing the wild-type transketolase. These properties result in higher intracellular ribulose-5-phosphate and ribose-5-phosphate concentrations and a residual flux through the pentose phosphate pathway, so that sufficient erythrose-4-phosphate can be produced to prevent auxotrophic growth.
“Wild-type enzyme” or “wild-type transketolase” which can be used for the present invention may include any transketolase as defined above that is used as starting point for designing mutants according to the present invention. The wild-type transketolase may be of eukaryotic or prokaryotic, preferably fungal or bacterial origin, in particular selected from Escherichia, Bacillus, Corynebacterium, Saccharomyces, Eremothecium, Candida or Ashbya, preferably from E. coli, B. subtilis, B. licheniformis, B. halodurans, S. cerevisiae, E. gossypii, C. flareri or A. gossypii or any transketolase having an amino acid sequence which is homologous to an amino acid sequence as shown in FIG. 1. Most preferably the transketolase is from B. subtilis. “Homologous” refers to a transketolase that is at least about 50% identical, preferably at least about 60% identical, more preferably at least about 70%, 80%, 85%, 90%, 95% identical, and most preferably at least about 98% identical to one or more of the amino acid sequences as shown in FIG. 1. “Wild-type” in the context of the present invention may include both transketolase sequences derivable from nature as well as variants of synthetic transketolase enzymes (as long as they are homologous to any one of the sequences shown in FIG. 1), The terms “wild-type transketolase” and “non-modified transketolase” are used interchangeably herein.
The term “% identity”, as known in the art, means the degree of relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” can be readily determined by known methods, e.g., with the program BESTFIT (GCG Wisconsin Package, version 10.2, Accelrys Inc., 9685 Scranton Road, San Diego, Calif. 92121-3752, USA) using the following parameters: gap creation penalty 8, gap extension penalty 2 (default parameters).
A “mutant”, “mutant enzyme”, “mutated enzyme” or “mutant transketolase” or a “modified transketolase” as used herein means any variant derivable from a given wild-type enzyme/transketolase (according to the above definition) according to the teachings of the present invention and, when used for replacing the wild-type gene of a host organism/cell should have an effect on the growth on e.g. gluconate and/or ribose. For the scope of the present invention, it is not relevant how the mutant(s) are obtained; such mutants may be obtained, e.g., by site-directed mutagenesis, saturation mutagenesis, random mutagenesis/directed evolution, chemical or UV mutagenesis of entire cells/organisms, etc. These mutants may also be generated, e.g., by designing synthetic genes, and/or produced by in vitro (cell-free) translation. For testing of specific activity, mutants can may be (over-) expressed by methods known to those skilled in the art. The terms “mutant transketolase”, “modified transketolase” or “mutated transketolase” are used interchangeably herein.
“Host cell” is a cell capable of producing a given fermentation product and containing the wild-type transketolase, or a nucleic acid encoding the modified transketolase according to the invention. Suitable host cells include cells of microorganisms.
As used herein, the term “specific activity” denotes the reaction rate of the wild-type and mutant transketolase enzymes under properly defined reaction conditions as described in Kochetov (Kochetov, G. A. 1982. Transketolase from yeast, rat liver, and pig liver. Methods Enzymol 90:209-23). The “specific activity” defines the amount of substrate consumed and/or product produced in a given time period and per defined amount of protein at a defined temperature. Typically, “specific activity” is expressed in μmol substrate consumed or product formed per min per mg of protein. Typically, μmol/min is abbreviated by U (=unit). Therefore, the unit definitions for specific activity of μmol/min/(mg of protein) or U/(mg of protein) are used interchangeably throughout this document. It is understood that in the context of the present invention, specific activity must be compared on the basis of a similar, or preferably identical, length of the polypeptide chain.
Many mutations may change a wild-type transketolase in such a way that growth on gluconate is affected as described above.
It is an object of the present invention to provide a modified transketolase having the properties defined above, wherein the amino acid sequence of the modified transketolase contains at least one mutation when compared with the amino acid sequence of the corresponding non-modified transketolase.
The at least one mutation may be an addition, deletion and/or substitution.
Preferably, the at least one mutation is at least one amino acid substitution wherein a given amino acid present in the amino acid sequence of the non-modified transketolase is replaced with a different amino acid in the amino acid sequence of the modified transketolase of the invention. The amino acid sequence of the modified transketolase may contain at least one amino acid substitution when compared with the amino acid sequence of the corresponding non-modified transketolase. Particularly, a modified transketolase as of the present invention contains at least one mutation on an amino acid position which corresponds to amino acid position 357 of the B. subtilis transketolase amino acid sequence as depicted in SEQ ID NO:2.
In further embodiments, the modified transketolase contains at least two, at least three, at least four or at least five substitutions when compared with the amino acid sequence of the corresponding transketolase. For example, the modified transketolase contains one to ten, one to seven, one to five, one to four, two to ten, two to seven, two to five, two to four, three to ten, three to seven, three to five or three to four amino acid substitutions when compared with the amino acid sequence of the corresponding non-modified transketolase.
In a preferred embodiment of the invention the non-modified transketolase is obtainable from Bacillus, preferably B. subtilis, as depicted in SEQ ID NO:2. The corresponding DNA sequence is shown in SEQ ID NO:1. The modified transketolase contains at least one mutation on position 357 of SEQ ID NO:2, leading to a modified transketolase having the above described properties.
The at least one amino acid substitution in the non-modified transketolase located on a position corresponding to amino acid 357 as shown in SEQ ID NO:2 may be selected from substitution R357H, R357A, R357S, R357N, R357T, R357K, R3571, R357V, R357G, and R357L.
In a particularly preferred embodiment, the mutated transketolase consists of one substitution which affects the amino acid position corresponding to amino acid position 357 of the amino acid sequence as shown in SEQ ID NO:2 and which may be selected from substitution R357H, R357A, R357S, R357N, R357T, R357K, R3571, R357V, R357G, and R357L.
In an other preferred embodiment, the modified transketolase contains at least two amino acid substitutions when compared with the amino acid sequence of the corresponding non-modified transketolase, wherein at least one mutation corresponding to amino acid position 357 of the amino acid sequence as shown in SEQ ID NO:2 and which may be selected from substitution R357H, R357A, R357S, R357N, R357T, R357K, R3571, R357V, R357G, and R357L.
The amino acid present in the non-modified transketolase is preferably arginine at position 357. The amino acid in the sequence of the non-modified transketolase may be changed to histidine, alanine, serine, asparagine, lysine, threonine, leucine, glycine, isoleucine or valine at position 357. Preferably, the substitution at the amino acid position corresponding to position 357 of the sequence as shown in SEQ ID NO: 2 consists of the replacement of arginine with histidine, arginine with alanine, arginine with serine, arginine with leucine, arginine with lysine, arginine with asparagine, arginine with threonine, arginine with glycine, arginine with isoleucine, arginine with valine.
The modified transketolase of the invention may comprise foreign amino acids, preferably at its N- or C-terminus. “Foreign amino acids” mean amino acids which are not present in a native (occurring in nature) transketolase, preferably a stretch of at least about 3, at least about 5 or at least about 7 contiguous amino acids which are not present in a native transketolase. Preferred stretches of foreign amino acids include but are not limited to “tags” that facilitate purification of the recombinantly produced modified transketolase. Examples of such tags include but are not limited to a “His6” tag (SEQ ID NO: 31), a FLAG tag, a myc tag, and the like. For calculation of specific activity, the values need to be corrected for these additional amino acids (see also above).
In another embodiment the modified transketolase may contain one or more, e.g. two, deletions when compared with the amino acid sequence of the corresponding non-modified transketolase. Preferably, the deletions affect N- or C-terminal amino acids of the corresponding non-modified transketolase and do not significantly reduce the functional properties, e.g., the specific activity, of the enzyme.
The invention further relates to a polynucleotide comprising a nucleotide sequence which codes for a modified transketolase according to the invention. “Polynucleotide” as used herein refers to a polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA. Polynucleotides include but are not limited to single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. The term “polynucleotide” includes DNA or RNA that comprises one or more unusual bases, e.g., inosine, or one or more modified bases, e.g., tritylated bases.
The polynucleotide of the invention can easily be obtained by modifying a polynucleotide sequence which codes for a non-modified transketolase. Examples of such polynucleotide sequences encoding non-modified transketolase enzymes include but are not limited to the amino acid sequences of FIG. 1. Preferably, the non-modified transketolase is originated from Bacillus, in particular B. subtilis, more preferred is a polynucleotide encoding a non-modified transketolase as depicted in SEQ ID NO:2.
Methods for introducing mutations, e.g., additions, deletions and/or substitutions into the nucleotide sequence coding for the non-modified transketolase include but are not limited to site-directed mutagenesis and PCR-based methods.
DNA sequences of the present invention may be constructed starting from genomic or cDNA sequences coding for transketolase enzymes known in the state of the art, as are available from, e.g., Genbank (Intelligenetics, California, USA), European Bioinformatics Institute (Hinston Hall, Cambridge, GB), NBRF (Georgetown University, Medical Centre, Washington D.C., USA) and Vecbase (University of Wisconsin, Biotechnology Centre, Madison, Wis., USA) or from the sequence information disclosed in FIG. 1 by methods of in vitro mutagenesis [see e.g. Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York]. Another possibility of mutating a given DNA sequence which may also be suitable for the practice of the present invention is mutagenesis by using the polymerase chain reaction (PCR). DNA as starting material may be isolated by methods known in the art and described, e.g., in Sambrook et al. (Molecular Cloning) from the respective strains/organisms. It is, however, understood that DNA encoding a transketolase to be constructed/mutated in accordance with the present invention can also be prepared on the basis of a known DNA sequence, e.g. by construction of a synthetic gene by methods known in the art (as described, e.g., in EP 747483).
Once complete DNA sequences of the present invention have been obtained, they can be integrated into vectors or directly introduced into the genome of a host organism by methods known in the art and described in, e.g., Sambrook et al. (s.a.) to (over-) express the encoded polypeptide in appropriate host systems. However, a man skilled in the art knows that also the DNA sequences themselves can be used to transform the suitable host systems of the invention to get (over-) expression of the encoded polypeptide.
In a preferred embodiment the present invention provides

    (i) a DNA sequence which codes for a modified transketolase carrying at least one mutation as defined above and which hybridizes under standard conditions with any of the DNA sequences of the specific modified transketolase enzymes, for example which hybridizes with the DNA sequences according to SEQ ID NO:1, or
    (ii) a DNA sequence which codes for a modified transketolase carrying at least one mutation as defined above but, because of the degeneracy of the genetic code, does not hybridize but which codes for a polypeptide with exactly the same amino acid sequence as a DNA sequence which hybridizes under standard conditions with any of the DNA sequences of the specific modified transketolase enzymes of the present invention, or
    (iii) a DNA sequence which is a fragment of such modified DNA sequences which maintains the activity properties of the polypeptide of which it is a fragment.


“Standard conditions” for hybridization mean in the context of the present invention the conditions which are generally used by a man skilled in the art to detect specific hybridization signals and which are described, e.g. by Sambrook et al., “Molecular Cloning”, second edition, Cold Spring Harbor Laboratory Press 1989, New York, or preferably so-called stringent hybridization and non-stringent washing conditions or more preferably so-called stringent hybridization and stringent washing conditions a man skilled in the art is familiar with and which are described, e.g., in Sambrook et al. (s.a.). A specific example of stringent hybridization conditions is overnight incubation (e.g., 15 hours) at 42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at about 65° C.
In another preferred embodiment the invention further provides a DNA sequence which can be obtained by the so-called polymerase chain reaction method (“PCR”) by PCR primers as shown in FIG. 2, designed on the basis of the specifically described DNA sequences.
The polypeptides and polynucleotides 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 or polypeptide present in a living microorganism is not isolated, but the same polynucleotide or polypeptide, 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 or polypeptides could be part of a composition and still be isolated in that such vector or composition is not part of its natural environment.
An isolated polynucleotide or nucleic acid as used herein may be a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′-end and one on the 3′-end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, a nucleic acid includes some or all of the 5′-non-coding (e.g., promoter) sequences that are immediately contiguous to the coding sequence. The term “isolated polynucleotide” therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an “isolated nucleic acid fragment” is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.
As used herein, the term isolated polypeptide refers to a polypeptide that is substantially free of other polypeptides. An isolated polypeptide is preferably greater than 80% pure, more preferably greater than 90% pure, even more preferably greater than 95% pure, most preferably greater than 99% pure. Purity may be determined according to methods known in the art, e.g., by SDS-PAGE and subsequent protein staining. Protein bands can then be quantified by densitometry. Further methods for determining the purity are within the level of ordinary skill.
As mentioned above, the modified transketolases and the corresponding polynucleotides of the invention may be utilized in the genetic engineering of a suitable host cell to make it better and more efficient in the fermentation process for substances that use ribose-5-phosphate, ribulose-5-phosphate, or xylulose-5-phosphate as substrate for the biosynthesis. The presence of said modified transketolase within a suitable host cell may result in higher intracellular ribulose-5-phosphate and ribose-5-phosphate concentrations and a residual flux through the pentose phosphate pathway within said recombinant host, so that sufficient erythrose-4-phosphate can be produced to prevent auxotrophic growth.
Appropriate host cells are for example fungi, like Aspergilli, e.g. Aspergillus niger or Aspergillus oryzae, or like Trichoderma, e.g. Trichoderma reesei, or Ashbya, e.g. Ashbya gossypii, or Eremothecium, e.g. Eremothecium ashbyii, or yeasts like Saccharomyces, e.g. Saccharomyces cerevisiae, or Candida, like Candida flareri, or Pichia, like Pichia pastoris, or Hansenula polymorpha, e.g. H. polymorpha (DSM 5215). Bacteria which can be used are, e.g., Bacilli as, e.g., Bacillus subtilis or Streptomyces, e.g. Streptomyces lividans. E. coli which could be used are, e.g., E. coli K12 strains, e.g. M15 or HB 101.
Thus, the present invention relates to a microorganism wherein the activity of a transketolase is modified in such a way that the microorganism is capable of growing on a carbon source that is metabolized exclusively by the pentose phosphate pathway (for example gluconate) with a reduced growth rate in comparison to a host cell containing the wild-type transketolase. It is in general possible to introduce an obtained transketolase mutant originating from a certain organism e.g. B. subtilis in the same organism again and now used as a host cell or to introduce any obtained mutant into any other relevant host cells.
As used herein, the term “growth rate” denotes to the following: Bacterial cells reproduce by dividing in two. If growth is not limited, doubling continues at a constant rate so both the number of cells and the rate of population increase doubles with each consecutive time period. For this type of exponential growth, plotting the natural logarithm of cell number against time (preferably in hours) produces a straight line. The slope of this line is the specific growth rate of the organism, which is a measure of the number of divisions per cell per unit time. In food, bacteria cannot grow continuously as the amount of nutrient available will be finite and waste products will accumulate. In these conditions growth curves tend to be sigmoid.
It is an object of the present invention to provide a recombinant host cell wherein the growth rate of said recombinant host cell (e.g. microorganism) according to the present invention carrying a modified transketolase on a carbon source that is metabolized exclusively by the pentose phosphate pathway, in particular gluconate, is less than 100% when compared to the wild-type organism. In particular, the growth rate may be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or even 90% and more compared to the growth rate of a wild-type organism. Preferably, the growth rate reduction is between 10% and 90%, more preferably between 20% and 80%, still more preferably between 25% and 75% compared to a cell containing a wild-type transketolase gene.
Especially, the invention relates to a genetically engineered/recombinantly produced host cell (also referred to as recombinant cell or transformed cell) in which the wild-type transketolase gene has been replaced by a modified transketolase gene encoding an enzyme that allows a slightly or non-reduced growth on a carbon source that is not exclusively metabolized by the pentose phosphate pathway, but shows a clearly reduced growth rate when the organism grows on a carbon source that is metabolized exclusively by the pentose phosphate pathway. Such genetically engineered host cells show an improvement of the yield of the fermentation product and of the efficiency of the production process with the advantages that undesired auxotrophic growth and pleiotropic effects can be prevented.
The invention further relates to a process for producing a host cells capable of expressing a transketolase according to the invention, comprising the steps of

    (i) generating mutated transketolases displaying modulated activities in comparison to the respective wild-type enzyme, i.e.
    a) providing a polynucleotide encoding a first or non-modified transketolase with catalytic properties that should be adapted;
    b) introducing one or more mutations into the polynucleotide sequence such that the mutated polynucleotide sequence encodes a new or modified transketolase which contains at least one amino acid mutation when compared to the first transketolase wherein the at least one amino acid mutation may be at amino acid corresponding to position 357 of the amino acid sequence as shown in SEQ ID NO: 2;
    c) optionally inserting the mutated polynucleotide in a vector or plasmid;
    (ii) replacing the wild-type transketolase(s) of the host cell by a transketolase variant from the same organisms or another organism that allows normal or slightly reduced growth on a carbon source that is not exclusively metabolized by the pentose phosphate pathway, but shows an effect on the growth rate when the organism grows on a carbon source that is metabolized exclusively by the pentose phosphate pathway, i.e.
    a) replacing the wild-type transketolase of a suitable wild-type host cell without altering the regulatory sequences of the gene;
    b) determining the growth rate on gluconate in minimal medium and comparing it to the wild-type host strain; and
    c) selecting transketolase mutants that allow a growth rate on gluconate which is less than 100% of the wild-type strain.


The invention further relates to a method for the production of substances that are secondary products of ribose-5-phosphate, ribulose-5-phosphate, or xylulose-5-phosphate comprising:

    a) culturing a genetically engineered/recombinantly produced host cell in which the wild-type transketolase gene has been replaced by a modified transketolase gene encoding an enzyme that allows a slightly or non-reduced growth on a carbon source that is not exclusively metabolized by the pentose phosphate pathway, but which shows a reduced growth rate when the organism grows on a carbon source that is metabolized exclusively by the pentose phosphate pathway, in a suitable medium under conditions that allow expression of the modified transketolase; and
    b) separating the fermentation product from the medium.


The “fermentation product” as used herein may be any product produced by a suitable host cell as defined above the biosynthesis of which uses ribose-5-phosphate, ribulose-5-phosphate, or xylulose-5-phosphate as substrate. Examples of such fermentation products include but are not limited to riboflavin, riboflavin precursors, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and derivatives thereof, pyridoxal phosphate (vitamin B6), guanosine, adenosine and derivatives of these nucleotides.
“Riboflavin precursor” and “derivatives of riboflavin, FMN or FAD” in the context of this invention shall include any and all metabolite(s) requiring ribulose-5-phosphate or ribulose-5-phosphate as an intermediate or substrate in their (bio-) synthesis. In the context of this patent application, it is irrelevant whether such (bio-) synthesis pathways are natural or non-natural (i.e., pathways not occurring in nature, but engineered biotechnologically). Preferably, the synthesis pathways are biochemical in nature. Riboflavin precursors and derivatives of riboflavin, FMN or FAD include but are not limited to: DRAPP; 5-amino-6-ribosylamino-2,4 (1H,3H)-pyrimidinedione-5′-phosphate; 2,5-diamino-6-ribitylamino-4 (3H)-pyrimidinone-5′-phosphate; 5-amino-6-ribitylamino-2,4 (1H,3H)-pyrimidinedione-5′-phosphate; 5-amino-6-ribitylamino-2,4 (1H,3H)-pyrimidinedione; 6,7-dimethyl-8-ribityllumazine (DMRL); and flavoproteins. The term “riboflavin” also includes derivatives thereof, such as e.g. riboflavin-5-phosphate and salts thereof, such as e.g. sodium riboflavin-5-phospate.
The polynucleotides, polypeptides, recombinant host cells and methods described herein may be used for the biotechnological production of either one or more of the fermentation products as defined above.
Methods of genetic and metabolic engineering of suitable host cells according to the present invention are known to the man skilled in the art. Similarly, (potentially) suitable purification methods for e.g. riboflavin, a riboflavin precursor, FMN, FAD, pyridoxal phosphate or one or more derivatives thereof are well known in the area of fine chemical biosynthesis and production.
It is understood that a method for biotechnological production of a fermentation product such as for instance riboflavin, a riboflavin precursor, FMN, FAD, pyridoxal phosphate or one or more derivatives thereof according to the present invention 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 have been prepared by and isolated from a host cell, by in vitro transcription/translation, or by still other means.
Fermentation media must contain suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose or fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks. It is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
The various embodiments of the invention described herein may be cross-combined.



BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will now be illustrated in more detail by the following non-limiting examples. These examples are described with reference to the Figures.
FIG. 1-1, FIGS. 1-2, and FIGS. 1-3 shows—as already mentioned above—examples of polynucleotide sequences which code for a non-modified transketolase, and
FIG. 2 shows a set of primers, in which tkt 1S is SEQ ID NO:3, tkt 1AS is SEQ ID NO:13, tkt 1ASohne is SEQ ID NO:6, tkt 2S is SEQ ID NO:4, tkt 2AS is SEQ ID NO:4, tkt 3S is SEQ ID NO:9, tkt 4S is SEQ ID NO:10, tkt 5S is SEQ ID NO:11, tkt 6S is SEQ ID NO:12, tkt 357AS is SEQ ID NO:14, tkt Rec 1S is SEQ ID NO:27, tkt Rec 1AS is SEQ ID NO:28, tkt Rec 2S is SEQ ID NO:29, tkt Rec 2AS is SEQ ID NO:30, tkt 357A-S is SEQ ID NO:17, tkt 357N-S is SEQ ID NO:15, tkt 357K-S is SEQ ID NO:18, tkt 357Q-S is SEQ ID NO:16, tkt 357S-S is SEQ ID NO:19, tkt 357T-S is SEQ ID NO:20, tkt 357H-S is SEQ ID NO:21, tkt 357V-S is SEQ ID NO:22, tkt 3571-S is SEQ ID NO:23, tkt 357L-S is SEQ ID NO:24, tkt 357M-S is SEQ ID NO:25, tkt 357G-S is SEQ ID NO:26, rpi MutS is SEQ ID NO:5.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
In particular, FIG. 1 shows multiple sequence alignment calculated by the program clustalW (1.83) of the transketolase amino acid sequences from Escherichia coli (TKT_ECOLI), Bacillus subtilis (TKT_BACSU), Bacillus licheniformis (TKT_BACLD), Bacillus halodurans (TKT_BACHD), Corynebacterium glutamicum (TKT_CORGL), Saccharomyces cerevisiae (TKT_YEAST), and Ashbya gossypii (TKT_ASHGO). Positions that are homologous/equivalent to the amino acid residue 357 of the B. subtilis transketolase that are discussed in one of the following examples are in bold letters. The numbering used for those positions is done according to the B. subtilis wild-type amino acid sequence. This type of alignment can be done with CLUSTAL or PILEUP using standard parameters. As shown, the amino acid sequence of transketolases is highly conserved. In particular, in all transketolases shown and in much more transketolases not shown, arginine 357 (numbering according to the B. subtilis transketolase) is conserved. Therefore, the type of experiment using the concepts and mutations reported here can also be done with other transketolases having an arginine at a position homologous to position 357 of the amino acid sequence of B. subtilis transketolase like Ashbya gossypii for improving the production of riboflavin, riboflavin derivatives or compounds having ribose-5-phosphate, xylulose-5-phosphate, or ribulose-5-phosphate as a precursor. It is also possible to replace an original transketolase gene of an organism by a B. subtilis transketolase mutant gene mutated at position 357 with or without adaptation of the DNA sequence to the new organism. It is not essential that the transketolase mutant genes originate from an organism in which it is going to be introduced. The practical steps required for another host organism are published and known to an expert in the field and outlined somewhere else.
EXAMPLE 1
Isolation of Genomic DNA from Bacillus subtilis 
gDNA was prepared using the DNeasy Tissue Kit from Qiagen (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany) according to the description of the supplier. 1 ml of a 3 ml overnight culture of B. subtilis in VY liquid medium (Becton Dickinson, Sparks, Md. 21152, USA) incubated at 37° C. (250 rpm) was used as source for the bacteria cells. At the end, the gDNA was eluted in 200 μl of AE buffer (supplied with the Kit).
EXAMPLE 2
Amplification of the Transketolase Gene from Bacillus subtilis 
gDNA from B. subtilis PY79 (P. Youngman, J. Perkins, and R. Losick (1984), Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression on the transposon-borne erm gene. Plasmid 12:1-9; see Example 1) was used for amplification of the tkt gene. According to the genomic DNA sequence, the tkt gene contains one Eco RI site inside of its coding sequence (SEQ ID NO: 1). Since the Eco RI restriction site is generally used for cloning into E. coli expression vectors such as pQE80 (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany), the site was deleted by replacing C315 by a T, which is a silent mutation changing the phenylalanine codon from TTC to TTT. For this, two separate PCRs A and B were performed. The following PCR conditions were used for PCR A: 2 μM of primer tkt 1S (according to SEQ ID No: 3, see also FIG. 2) and tkt 2AS (according to SEQ ID No: 4, FIG. 2), 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5 U of a proof-reading DNA polymerase (Stratagene, Gebouw California, 1101 CB Amsterdam Zuidoost, The Netherlands), 100 ng genomic DNA (Example 1) in the appropriate buffer as supplied together with the DNA polymerase.
Temperature regulation was as follows:

    Step 1: 3 min at 95° C.
    Step 2: 30 sec at 95° C.
    Step 3: 30 sec at 52° C.
    Step 4: 30 sec at 72° C.
    Step 5: 5 min at 72° C.


Steps 2 to 4 were repeated 35-times.
PCR B was done under the following conditions: 2 μM of primer tkt 2S (according to SEQ ID No: 8, FIG. 2) and tkt 1AS (according to SEQ ID No: 13, FIG. 2), 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5 U of a proof-reading DNA polymerase (Stratagene, Gebouw California, 1101 CB Amsterdam Zuidoost, The Netherlands), 100 ng genomic DNA (Example 1) in the appropriate buffer as supplied together with the DNA polymerase.
Temperature regulation was as follows:

    Step 1: 3 min at 95° C.
    Step 2: 30 sec at 95° C.
    Step 3: 30 sec at 52° C.
    Step 4: 2 min at 72° C.
    Step 5: 5 min at 72° C.


Steps 2 to 4 were repeated 35-times.
The two PCR products A and B were purified by Agarose gel electrophoresis and a following extraction out of the gel using the MinElute Gel Extraction Kit from Qiagen (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany). Using the overlapping region of PCR products A and B, it was possible to assemble them by a third PCR: 2 μM of primer Rpi MutS (according to SEQ ID No: 5, FIG. 2) and tkt 1ASohne (according to SEQ ID No: 6, FIG. 2), 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5 U of a proof-reading DNA polymerase (Stratagene, Gebouw California, 1101 CB Amsterdam Zuidoost, The Netherlands), 100 ng of PCR product A and PCR product B in the appropriate buffer as supplied together with the DNA polymerase.

    Step 1: 3 min at 95° C.
    Step 2: 30 sec at 95° C.
    Step 3: 30 sec at 53° C.
    Step 4: 2.5 min at 72° C.
    Step 5: 5 min at 72° C.


Steps 2 to 4 were repeated 35-times.
The PCR products were purified with the help of the Qiagen PCR purification Kit (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany) and eluted in 50 μl elution buffer. The PCR product was confirmed by an Eco RI digestion. For further confirmation, it was sequenced with the primers tkt IS, tkt 2S, tkt 2AS, tkt 3S (according to SEQ ID No: 9, FIG. 2), tkt 4S (according to SEQ ID No: 10, FIG. 2), tkt 5S (according to SEQ ID No: 11, FIG. 2), tkt 6S (according to SEQ ID No: 12, FIG. 2), tkt 1AS.
EXAMPLE 3
Construction of Tkt Mutants
The 3D structure of the yeast transketolase was available together with a selection of mutations that showed influence on substrate binding of the yeast transketolase (Nilsson, U., L. Meshalkina, Y. Lindqvist, and G. Schneider. 1997. At position R359 (number 357 in the B. subtilis transketolase), the original arginine was replaced by nearly all other amino acids. The construction of the mutants was basically done as described in example 1. An amino acid sequence alignment comprising the transketolases from yeast, B. subtilis and from other organisms is shown in FIG. 1.
Using the Eco RI-free tkt gene as template (Example 2), the mutations were introduced as already described for the deletion of the Eco RI site: The following PCR conditions were used for PCR A and B: 2 μM of primer Rpi MutS (A) or tkt 357nnn-S (B) and tkt 357AS (A) (according to SEQ ID No: 14, FIG. 2) or tkt 1ASohne (B), 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5 U of a proof-reading DNA polymerase (Stratagene, Gebouw California, 11011 CB Amsterdam Zuidoost, The Netherlands), 100 ng of the Eco RI-free tkt gene (Example 2) in the appropriate buffer as supplied together with the DNA polymerase. In the case of PCR B the sense primer was chosen according to the amino acid that was introduced: tkt 357N-S (according to SEQ ID No: 15, FIG. 2) for asparagine, tkt 357Q-S (according to SEQ ID No: 16, FIG. 2) for glutamine, tkt 357A-S (according to SEQ ID No: 17, FIG. 2) for alanine, tkt 357K-S (according to SEQ ID No: 18, FIG. 2) for lysine, tkt 357S-S (according to SEQ ID No: 19, FIG. 2) for serine, tkt 357T-S (according to SEQ ID No: 20, FIG. 2) for threonine, tkt 357H-S (according to SEQ ID No: 21, FIG. 2) for histidine, tkt 357V-S (according to SEQ ID No: 22, FIG. 2) for valine, tkt 3571-S (according to SEQ ID No: 23, FIG. 2) for Isoleucine, tkt 357L-S (according to SEQ ID No: 24, FIG. 2) for leucine, tkt 357M-S (according to SEQ ID No: 25, FIG. 2) for methionine, and tkt 357G-S (according to SEQ ID No: 26, FIG. 2) for the introduction of glycine at position 357 of the B. subtilis transketolase.
Temperature regulation was as follows:

    Step 1: 3 min at 95° C.
    Step 2: 30 sec at 95° C.
    Step 3: 30 sec at 52° C.
    Step 4: 60 sec at 72° C.
    Step 5: 5 min at 72° C.


Steps 2 to 4 were repeated 35-times.
The two PCR products A and B were purified by Agarose gel electrophoresis and a following extraction out of the gel using the MinElute Gel Extraction Kit from Qiagen (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany). Assembling of PCR product
A and B was done in a third PCR: 2 μM of primer Rpi MutS and tkt 1ASohne, 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5 U of a proof-reading DNA polymerase (Stratagene, Gebouw California, 1101 CB Amsterdam Zuidoost, The Netherlands), 100 ng of PCR product A and PCR product B in the appropriate buffer as supplied together with the DNA polymerase.

    Step 1: 3 min at 95° C.
    Step 2: 30 sec at 95° C.
    Step 3: 30 sec at 53° C.
    Step 4: 2.5 min at 72° C.
    Step 5: 5 min at 72° C.


Steps 2 to 4 were repeated 35-times.
The PCR products of the transketolase were purified with the Qiagen PCR purification Kit (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany) and eluted in 50 μl elution buffer. The PCR products were used for the transformation of B. subtilis. 
EXAMPLE 4
Construction of a Transketolase-Deficient B. subtilis Strain
For 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. Two DNA fragments obtained by PCR comprising base pair 452 to 1042 and base pair 1562 to 2001 of the B. subtilis transketolase gene (SEQ ID NO: 2) were combined with the neomycin resistance gene cassette (M. Itaya, K. Kondo, and T. Tanaka. 1989. A neomycin resistance gene cassette selectable in a single copy state in the Bacillus subtilis chromosome. Nucleic Acids Res 17:4410). The following PCR conditions were used for PCR A: 2 μM of primer tkt Rec1S (according to SEQ ID No: 27, FIG. 2) and tkt Rec1AS (according to SEQ ID No: 28, FIG. 2), 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5 U of a proof-reading DNA polymerase (Stratagene, Gebouw California, 1101 CB Amsterdam Zuidoost, The Netherlands), 100 ng of the amplified tkt gene of Example 2 in the appropriate buffer as supplied together with the DNA polymerase.
Temperature regulation was as follows:

    Step 1: 3 min at 95° C.
    Step 2: 30 sec at 95° C.
    Step 3: 30 sec at 52° C.
    Step 4: 30 sec at 72° C.
    Step 5: 5 min at 72° C.


Steps 2 to 4 were repeated 30-times.
PCR B was done under the following conditions: 2 μM of primer tkt Rec 2S (according to SEQ ID No: 29, FIG. 2) and tkt Rec 2AS (according to SEQ ID No: 30, FIG. 2), 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5 U of a proof-reading DNA polymerase (Stratagene, Gebouw California, 1101 CB Amsterdam Zuidoost, The Netherlands), 100 ng of the amplified tkt gene of Example 2 in the appropriate buffer as supplied together with the DNA polymerase.
Temperature regulation was as follows:

    Step 1: 3 min at 95° C.
    Step 2: 30 sec at 95° C.
    Step 3: 30 sec at 52° C.
    Step 4: 2 min at 72° C.
    Step 5: 5 min at 72° C.


Steps 2 to 4 were repeated 30-times.
The two PCR products A and B were purified by Agarose gel electrophoresis and a following extraction out of the gel using the MinElute Gel Extraction Kit from Qiagen (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany). Due to the overlapping regions of the two PCR products A and B with the sequence of the neomycin resistance cassette, it is possible to assemble them by a third PCR: 2 μM of primer tkt Rec 1S and tkt Rec 2AS, 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5 U of a proof-reading DNA polymerase (Stratagene, Gebouw California, 1101 CB Amsterdam Zuidoost, The Netherlands), 100 ng of PCR product A, 100 ng of PCR product B, and 100 ng of neomycin resistance cassette in the appropriate buffer as supplied together with the DNA polymerase.

    Step 1: 3 min at 95° C.
    Step 2: 30 sec at 95° C.
    Step 3: 30 sec at 55° C.
    Step 4: 2.5 min at 72° C.
    Step 5: 5 min at 72° C.


Steps 2 to 4 were repeated 35-times.
Five assembling PCRs were pooled and purified with the Qiagen PCR purification Kit (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany) and eluted in 50 μl elution buffer. The correct PCR product was confirmed by agarose gel electrophoresis and used for transformation of B. subtilis PY79. Preparation of competent B. subtilis cells was done according to Kunst et al., 1988 (F. Kunst, M. Debarbouille, T. Msadek, M. Young, C.
Mauel, D. Karamata, A. Klier, G. Rapoport, and R. Dedonder. 1988. Deduced polypeptides encoded by the Bacillus subtilis sacU locus share homology with two-component sensor-regulator systems. J Bacteriol 170: 5093-101). 2 ml MNGE+Bacto Casamino Acid (CAA) (9 ml MN-medium (13.6 g/l K2HPO4, 6.0 g/l KH2PO4, 0.88 g/l sodium citrate*2H2O), 1 ml glucose (20%), 40 μl potassium glutamate (40%), 50 μl, ammonium iron(III) citrate (2.2 mg/l, freshly prepared), 100 μl tryptophan (8 mg/l), 30 μl MgSO4 (1 M), +/−50 μl Bacto Casamino Acid (20%, Becton Dickinson AG, Postfach, CH-4002 Basel, Switzerland) were inoculated with a single colony and incubated overnight at 37° C. and 250 rpm. This culture was used to inoculate 10 ml MNGE+CAA (start OD500 nm of 0.1) and was incubated at 37° C. under shaking (250 rpm) until it reached an OD500 nm of 1.3. The culture was diluted with the same volume of MNGE w/o CAA and was incubated for another hour. After a centrifugation step (10 min, 4000 rpm, 20° C.), the supernatant was decanted into a sterile tube. The pellet was re-suspended in 1⁄8 of the kept supernatant. 300 μl of cells were diluted in 1.7 ml MN (1×), 43 μl glucose (20%) and 34 μl MgSO4 (1 M). 10 and 20 μl of the prepared PCR product was added to 400 μl of the diluted competent cells and shaked for 30 min at 37° C. 100 μl expression mix (500 μl 5% yeast extract (Becton Dickinson AG, Postfach, CH-4002 Basel, Switzerland), 125 μl CAA (20%), 1/100 of the final antibiotic concentration (2 μg/ml neomycin), if used for selection, and 750 μl sterile bidest. water) were added and the cells were shaked for 1 h at 37° C. At the end, the cells were spun down, suspended in 200 μl of the supernatant and plated onto TBAB plates (Becton Dickinson AG, Postfach, CH-4002 Basel, Switzerland) containing 2 μg/ml neomycin.
Two transformants were grown in VY medium (5 g/l yeast extract (Becton Dickinson AG, Postfach, CH-4002 Basel, Switzerland), 25 g/l veal infusion broth (Sigma)). From one of the transformants, designated BS3402, the genomic DNA was isolated as described in Example 1 and the correct replacement of the transketolase DNA fragment from base pair 1043 to 1561 by the neomycin gene cassette was confirmed by a standard PCR using tkt Rec 1 S and tkt Rec 2AS as primers. As expected for a transketolase deletion mutant, the strain could not grow on ribose or gluconate as sole carbon source and required all three aromatic amino acids or shikimic acid for growth.
EXAMPLE 5
Transformation of the Transketolase-Deficient B. subtilis Strain BS3402 with the Genes of the Transketolase Variants
0.5 and 1 μg DNA of the amplified transketolase gene and its variants (Example 2 and 3) were used to transform BS3402 as described in Example 4. Positive colonies were identified by growth on minimal medium (2 g/l glucose and sorbitol in SMS-medium (2 g/l (NH4)2SO4, 14 g/l K2HPO4, 6 g/l KH2PO4, 1 g/l tri-sodium citrate, 0.2 g/l MgSO4.7H2O; 1.5% agar (Becton Dickinson AG, Postfach, CH-4002 Basel, Switzerland) and trace elements (500-times concentrate: 5.0 g/l MnSO4×1 H2O, 2.0 g/l COCl2.6H2O, 0.75 g/l (NH4)6Mo7O24.4H2O, 0.5 g/l AlCl3.6H2O, 0.375 g/l CuCl.2H2O)). Colonies were visible after 24 to 48 h. All transformants were sensitive to neomycin indicating the replacement of the neomycin gene by the introduced wild-type and mutated tkt genes.
Genomic DNA was isolated from the transformants and the tkt gene was amplified by PCR as described in Example 1. The introduced mutations were confirmed by sequencing. No further nucleotide exchanges were observed. The generated B. subtilis strains were called:
R357A-BS3403, R357H-BS3482, R357K-BS3484, R357G-BS3512, R357V-BS3487, R357I-BS3509, R357L-BS3507, R357T-BS3492, R357S-BS3490, R357M-BS3505, R357N-BS3486, R357Q-BS3488.
EXAMPLE 6
Transduction of B. subtilis RB50::[pRF69] (EP 0405 370) with Bacteriophage PBS-1 Lysate of the Transketolase-Deficient Wild-Type Strain BS3402
Transduction work with phage PBS-1 was done as described in Henkin et al., 1984 (Henkin, T. M., and G. H. Chambliss. 1984. Genetic mapping of a mutation causing an alteration in Bacillus subtilis ribosomal protein S4. Mol Gen Genet 193:364-9). For preparation of the PBS-1 lysate, the strain BS3402 was grown on TBAB plates (5 μg/ml neomycin) at 37° C. overnight. The cells were used to inoculate 25 ml LB medium (Becton Dickinson AG, Postfach, CH-4002 Basel, Switzerland) to an OD of Klett 20-30 (using the green filter). When 50% of the cells were motile, 0.2 ml of the PBS-1 phage lysate (Henkin, T. M., and G. H. Chambliss. 1984. Genetic mapping of a mutation causing an alteration in Bacillus subtilis ribosomal protein S4. Mol Gen Genet 193:364-9) were added to 0.8 ml of the culture broth. After 30 min incubation at 37° C. under shaking, 9 ml LB-medium were added. This was followed by another 30 min incubation step at 37° C. Then 4 μg/ml chloramphenicol were added, and the incubation was continued for another 2 hours. Finally, the tubes were transferred into a 37° C. dry incubator where they were left overnight. On the next morning, the culture was filtered through a 0.45 μm filter and stored at 4° C. or directly used for transduction.
For the transduction of the riboflavin overproducing strain RB50::[pRF69], the strain was grown on a TBAB plate at 37° C. overnight. Cells of this plate were used to inoculate 25 ml LB-medium (Klett 20-30). When the culture reached Klett 175, 0.8 ml of the cells were mixed with 0.2 ml of a PBS-1 phage lysate from strain BS3402 prepared as described above. After 30 min incubation at 37° C. under shaking, cells were spun down and suspended in 1 ml VY medium. This was followed by 1 h incubation under the identical conditions. 200 to 1000 μl of the transduced cells were plated on a selection plate containing 2 μg/ml neomycin. Grown colonies were tested for neomycin resistance. After gDNA isolation (Example 1), a standard PCR using primer tkt 1S and Rec 2AS was done to confirm the replacement of the tkt wild-type gene by the construct of Example 4. A confirmed strain was called BS3523.
EXAMPLE 7
Introduction of the Modified Transketolase Genes into Strain BS3523
For preparation of PBS-1 lysates of strains BS3403, BS3482, BS3484, BS3486, BS3490, and BS3512, the respective strains were grown on TBAB plates (5 μg/ml neomycin) overnight at 37° C. Cells from those plates were used to inoculate 25 ml LB medium to an OD of Klett 20-30 (using the green filter). When 50% of the cells were motile (around Klett 150), 0.2 ml of the PBS-1 phage lysate (Henkin, T. M., and G. H. Chambliss. 1984.
Genetic mapping of a mutation causing an alteration in Bacillus subtilis ribosomal protein S4. Mol Gen Genet 193:364-9) were added to 0.8 ml of the culture broth. After 30 min incubation at 37° C. under slight shaking or turning (roller drum), 9 ml LB-medium were added to the cells. They were incubated for another 30 min under the same conditions. Chloramphenicol was added to a concentration of 4 μg/ml, and the incubation was continued for another 2 hours. The tubes were incubated overnight at 37° C. without shaking. On the next morning, the culture was filtered through a 0.45 μm filter and stored at 4° C. or directly used for subsequent transduction. For this, the transketolase-deficient strain BS3523 (see example 6) was grown on a TBAB plate overnight at 37° C. Cells from the plate were used to inoculate 25 ml LB-medium (Klett 20-30). The culture was incubated at 37° C. under shaking. When the culture reached Klett 175, 0.8 ml of the cells were mixed with 0.2 ml of PBS-1 phage lysate of each of the strains BS3403, BS3482, BS3484, BS3486, BS3490, and BS3512 as described above. After 30 min incubation at 37° C. under shaking, cells were spun down and suspended in 1 ml VY medium. After 1 h incubation under the identical conditions, the cells were spun down again, suspended in 0.2 ml 1×SMS medium and plated onto selection plates (1×SMS as described above with 1 g/l glucose, 1 g/l Sorbitol, and 15% agarose). Grown colonies were tested for loss of neomycin resistance. After gDNA isolation (Example 1), a standard PCR using primer tkt 1S and Rec 2AS was done to amplify the tkt gene from the genomic DNA. The tkt gene of colonies that showed a replacement of the inactivated tkt gene by an intact one, were sequenced to confirm the existence of the mutations. The generated strains were called BS3525 (BS3484 lysate), BS3528 (BS3482 lysate), BS3530 (BS3486), BS3534 (BS3403 lysate), BS3535 (BS3490 lysate), BS3541 (BS3512 lysate).
EXAMPLE 8
Growth of the Transketolase Mutant Strains on Glucose and Gluconate
To evaluate the effect of the transketolase mutations on viability and growth of B. subtilis, the maximal growth rate of the generated strains was determined on 2 g/l glucose or gluconate. The following medium was used: 1×SMS (2 g/l (NH4)2SO4, 14 g/l K2HPO4, 6 g/l KH2PO4, 1 g/l tri-sodium citrate, 0.2 g/l MgSO4.7H2O), 2 g/l glucose or gluconate, 500 μg/l yeast extract and trace elements solution as described in Example 5. 25 ml of the described medium in a 300 ml flask with baffles were inoculated from an overnight culture (5 ml VY, resuspended in 1 ml fresh VY) to an OD of Klett 20 to 30. They were incubated at 37° C. under shaking (220 rpm). The OD of the cultures were followed in one hour intervals during the lag phase. During the logarithmic phase the interval was reduced to 30 min. At least four data points during the logarithmic phase were used for the determination of the maximal growth rate.












TABLE 1





B. subtilis
Growth rate
% wild
Growth rate
% wild



mutant
on glucose
type
on gluconate
type
ratio




















Wild type
0.480
100% 
0.351
100% 
1.42/1  


PY79


R357G
0.384
80%
0.300
86%
1.28/0.93


R357S
0.372
78%
0.254
72%
1.46/1.08


R357T
0.342
71%
0.240
68%
1.43/1.04


R357N
0.366
76%
0.231
66%
1.58/1.15


R357A
0.381
79%
0.225
64%
1.69/1.23


R357L
0.324
68%
0.189
54%
1.71/1.25


R357H
0.324
68%
0.174
50%
1.86/1.36


R357K
0.348
73%
0.171
49%
2.04/1.48


R357I
0.243
51%
0.108
31%
2.25/1.65


R357Q
0.228
48%
0.09
26%
2.53/1.83


R357V
0.297
62%
0.101
24%
2.94/2.58


R357M
0.258
54%
0.066
19%
3.91/2.84


R357Y
0.222
46%
0.06
17%
3.70/2.71


R357F
0.174
36%
0.038
11%
4.58/3.27


R357D
0.156
33%
0
 0%
—









The wild-type strain PY79 showed as expected the highest growth rate on both substrates.
By introducing the different mutations at transketolase position 357, the growth on gluconate was, as expected, much more affected than the growth on glucose. Reduction of the maximal growth rate on gluconate was used as a measurement for the effect of the transketolase mutation on the flux through the non-oxidative pentose phosphate shunt and on the accumulation of the pentose phosphates. A wide range of growth rates were covered by the shown mutations.
EXAMPLE 9
Riboflavin Production in Shake Flasks
5 ml VY containing chloramphenicol (10 μg/ml) were inoculated with the riboflavin production strains RB50::[pRF69], BS32525, BS3528, BS34530, BS3434, BS34335, and BS3441 (see Example 7). After overnight incubation, the cells were spun down (15 min, 4000 rpm) and suspended in 1 ml screening medium (2×SMS, 10 g/l glucose, 1 g/l yeast extract, and trace elements as described in example 5). A 200 ml flask with baffles containing 25 ml screening medium was inoculated with 0.25 ml of the re-suspended cells. The cultures were incubated for 48 h at 37° C. in a water-saturated atmosphere. After 48 h incubation time, during which the supplied glucose was used up in all of the cultures, a sample of 0.5 ml was taken from the cultures, 35 μl 4 N NaOH was added and the mixture was vortexed for 1 min. 465 μl 1 M potassium phosphate buffer, pH 6.8, was added directly afterwards. The mixture was cleared by 5 min centrifugation at 14000 rpm (Eppendorf centrifuge 5415D). The supernatant was transferred into a new tube. Two different methods for riboflavin determination were used. For the calorimetric determination, 200 μl of the supernatant was diluted with 800 μl water. The absorption at 444 nm was multiplied with the factor of 0.03305 to obtain gram riboflavin per liter medium. For the final results, the obtained values were corrected for volume differences. The riboflavin concentration was also determined by HPLC according to Example 10. The results are shown in Table 2:











TABLE 2








HPLC




UV results
% of
results
% of



Riboflavin
RB50::
(riboflavin
RB50::


Strain
[mg/l]
[pRF69]
[mg/l]
[pRF69]



















BS3528(R357H)
179
171%
139
193%


BS3535(R357S)
173
166%
136
188%


BS3534(R357A)
144
138%
124
172%


BS3525(R357K)
148
142%
112
155%


BS3559(R357Q)
134
129%
103
143%


BS3530(R357N)
126
120%
93
129%


RB50::[pRF69]
104
100%
72
100%


BS3541(R357G)
92
 89%
66
 92%


BS3523(deletion)
90
 87%
58
 81%









Nearly all Bacillus strains containing a transketolase mutation showed a clearly increased riboflavin production, while the transketolase negative strain produced less riboflavin than the control strain. In the case of the R357H mutation, the riboflavin concentration was nearly doubled.
EXAMPLE 10
Riboflavin Fermentation
Fermentation runs were performed as described in EP 405370.
Fermentations were run with strains (1) RB50::[pRF69], (2) BS3534 (R357A), and (3) BS3528 (R357H). At 24 hours and 48 hours fermentation time, concentrations of riboflavin and biomass (cell dry weight) were measured in the culture broth. As shown in Table 3, parent strain RB50::[pRF69] produced 9.8 g/l riboflavin in 48 h with a yield on substrate of 3.59% (w/w). Biomass was produced with a yield on substrate of 20.3% (w/w). Derivatives of RB50::[pRF69] expressing a modified transketolase gene showed significant increases in riboflavin production. BS3528 and BS3534 produced 11.7 g/l and 14.6 g/l, respectively. This corresponds to a yield on glucose of 4.23% with BS3528 and 5.14% with BS3534, respectively (Table 3). These results demonstrate that the modification of transketolase activity leads to an increase in riboflavin productivity.







TABLE 3







Riboflavin and biomass yield on substrate


after 48 h fermentation time












B2 Yield

Biomass Yield




[%] (w/w)
Difference
[%] (w/w)
Difference















RB50::[pRF69]
3.59 ± 0.27

20.26 ± 0.80



BS3534
5.14 ± 0.09
+43%
18.92 ± 0.37
 −7%


BS3528
4.23 ± 0.19
+18%
17.78 ± 1.73
−12%









EXAMPLE 11
Analytical Methods for Determination of Riboflavin
For determination of riboflavin, the following analytical method can be used (Bretzel et al., J. Ind. Microbiol. Biotechnol. 22, 19-26, 1999).
The chromatographic system was a Hewlett-Packard 1100 System equipped with a binary pump, a column thermostat and a cooled auto sampler. Both a diode array detector and a fluorescence detector were used in line. Two signals were recorded, UV at 280 nm and fluorescence trace at excitation 446 nm, emission 520 nm.
A stainless-steel Supercosil LC-8-DB column (150×4.6 mm, 3 μm particle size) was used, together with a guard cartridge. The mobile phases were 100 mM acetic acid (A) and methanol (B). A gradient elution according to the following scheme was used:












Time [min]
% A
% B

















0
98
2


6
98
2


15
50
50


25
50
50









The column temperature was set to 20° C., and the flow rate was 1.0 ml/min. The run time was 25 min.
Fermentation samples were diluted, filtered and analyzed without further treatment.
Riboflavin was quantitated by comparison with an external standard. The calculations were based on the UV signal at 280 nm. Riboflavin purchased from Fluka (9471 Buchs, Switzerland) was used as standard material (purity≧99.0%).

## Claims

1. A microorganism selected from the group consisting of Bacillus sp. and Corynebacterium glutamicum genetically engineered with a polynucleotide encoding a modified transketolase having an amino acid sequence containing at least one mutation corresponding to a mutation at position 357 as shown in SEQ ID NO:2, wherein the polynucleotide encoding the transketolase is shown in SEQ ID NO:1, and wherein the microorganism's growth rate on a carbon source that is metabolized exclusively by the pentose phosphate pathway is reduced between 10 to 90% in comparison with a non-genetically engineered microorganism containing a non-modified transketolase and wherein the genetically-engineered microorganism is prototrophic for aromatic amino acids.

2. The microorganism according to claim 1 wherein the transketolase is a Bacillus transketolase.

3. A process for production of a fermentation product for which ribose-5-phosphate, ribulose-5-phosphate, or xylulose-5-phosphate is a biosynthetic precursor, the process comprising:
(a) culturing a microorganism selected from the group consisting of Bacillus sp. and Corynebacterium glutamicum genetically engineered with a polynucleotide encoding a modified transketolase having an amino acid sequence containing at least one mutation corresponding to a mutation at position 357 as shown in SEQ ID NO:2, wherein the polynucleotide encoding the transketolase is shown in SEQ ID NO:1, and wherein the genetically-engineered microorganism's growth rate on a carbon source that is metabolized exclusively by the pentose phosphate pathway is reduced between 10 to 90% in comparison with a non-genetically-engineered microorganism containing a non-modified transketolase and wherein the genetically-engineered microorganism is prototrophic for aromatic amino acids, in a suitable medium under conditions that allow expression of the modified transketolase and biosynthesis of the fermentation product; and
(b) separating the fermentation product from the medium.

4. The process according to claim 3 wherein the fermentation product is selected from the group consisting of riboflavin, a riboflavin precursor, FMN, FAD, pyridoxal phosphate, and one or more derivatives thereof.

5. The process according to claim 3 wherein the fermentation product is riboflavin.

6. A process for production of a fermentation product, wherein the fermentation product is selected from the group consisting of riboflavin, a riboflavin precursor, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), pyridoxal phosphate, and one or more derivatives of the aforementioned compounds, in a microorganism selected from the group consisting of Bacillus sp. and Corynebacterium glutamicum having a modified transketolase, wherein the amino acid sequence of the transketolase contains at least one mutation corresponding to a mutation at position 357 as shown in SEQ ID NO:2, wherein the polynucleotide encoding the transketolase is shown in SEQ ID NO:1, so that the specific activity of the modified transketolase is modulated in comparison to the corresponding non-modified wild-type enzyme, and wherein the microorganism is prototrophic for aromatic amino acids, the process comprising:
(a) culturing said microorganism in a medium containing glucose or gluconate as a carbon source under conditions that allow expression of the modified transketolase and biosynthesis of the fermentation product and
(b) isolating the fermentation product.

7. The process according to claim 6 wherein the fermentation product is riboflavin.

8. The process according to claim 3 wherein the transketolase is a Bacillus transketolase.

9. The process according to claim 6 wherein the microorganism's growth rate on glucose or gluconate is reduced between 10 to 90% in comparison with a non-genetically-engineered microorganism containing a non-modified transketolase.