Patent Application
20050176033
1. Technical Field
The present invention relates to a method for producing L-amino acid, such as L-histidine. More specifically, the present invention relates to a novel feedback-resistant enzyme involved in the biosynthesis of purines and L-histidine. More specifically, the present invention concerns a new feedback-resistant mutant phosphoribosylpyrophosphate synthetase (PRPP synthetase) from E. coli. The invention also relates to a method for producing L-histidine by fermentation using bacterial strains containing the novel feedback-resistant enzyme.
2. Background Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources or mutants thereof, which are modified to enhance production yields of L-amino acids.
Many techniques to enhance production yields of L-amino acids have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). Other techniques include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, Japanese Laid-open application No. 56-18596 (1981), WO 95/16042 or U.S. Pat. Nos. 5,661,012 and 6,040,160).
5-Phosphoribosyl-α-1-pyrophosphate (hereinafter, “PRPP”) and adenosine-5′-triphosphate (hereinafter, “ATP”) are the initial substrates in histidine biosynthesis. PRPP can sometimes induce the histidine biosynthesis to follow divergent pathways, resulting in the biosynthesis of pyrimidine nucleotides, purine nucleotides, pyridine nucleotides, and tryptophan (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C., 1996).
Many nucleotides competitively inhibit the activity of PRPP synthetase with ATP. However, the only potent nucleotide inhibitor is adenosine-5′-diphosphate (ADP); it competes with ATP and is an allosteric inhibitor that binds to a site other than the active site (Hove-Jensen, B. et al, J. Biol. Chem. 261:6765-6771 (1986)).
Mutants with altered PRPP synthetase have been obtained in both E. coli and S. typhimurium. One of the E. coli mutants produces a PRPP synthetase with a 27-fold increase in the Km value for ATP, and the enzyme is no longer inhibited by AMP. This mutation results from substitution of aspartic acid 128 by alanine (prsDA mutation). One S. typhimurium prs mutant is temperature-sensitive and has only 20% of the wild-type PRPP synthetase activity. This mutant enzyme had elevated Km values for ATP and ribose 5-phosphate and reduced sensitivity to inhibition by ADP. The mutation is the result of the replacement of arginine 78 by cysteine (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C., 1996).
It is well known that superactivity of human PRPP synthetase and resistance to purine nucleotide are associated with neurodevelopmental abnormalities in addition to hyperuricemia and gout (Becker M. A. et al, Arthritis Rheum, 18:6 Suppl: 687-94 (1975); Zoref E. et al, J. Clin. Invest., 56(5): 1093-9 (1975)). Uric acid overproduction in individuals with superactivity of PRPP synthetase results from increased production of PRPP and consequent acceleration of purine nucleotide synthesis de novo. It was shown that superactivity of PRPP synthetase is a result of an A to G mutation at nucleotide 341, which results in an asparagine to serine substitution at amino acid residue 113 of the mature enzyme. This mutant PRPP synthetase is resistant to purine nucleotides that inhibit the normal enzyme by a mechanism that is noncompetitive with respect to ATP (Roessler, B. J. et al. J. Biol. Chem., v. 268, No 35, 26476-26481 (1993); Becker, M. A. et al, J. Clin. Invest., 96(5): 2133-41 (1995)).
A process for producing purine nucleosides via fermentation of a microorganism belonging to the genus Escherichia and having purine nucleoside-producing ability, and containing a prsDA mutation is disclosed (European patent application EP1004663A1). However, there are no reports describing mutant bacterial PRPP synthetase which is feedback-resistant to purine nucleotides, or the use of such a mutant PRPP synthetase for improving L-histidine production using L-histidine-producing strains.
An object of the present invention is to provide a new mutant bacterial PRPP synthetase. Furthermore, it is an object of the present invention to provide an L-histidine-producing strain containing the mutant PRPP synthetase, which has enhanced production yields of L-histidine. Also, it is an object of the present invention to provide a method for producing L-histidine using the above-described strain.
It is an object of the present invention to provide a mutant bacterial phosphoribosylpyrophosphate synthetase (PRPP synthetase), wherein the aspartic acid at position 115 in a wild-type phosphoribosylpyrophosphate synthetase from Escherichia coli is substituted with another L-amino acid residue, and feedback inhibition by purine nucleotides is desensitized.
It is a further object of the present invention to provide the mutant PRPP synthetase described above, wherein the aspartic acid residue at position 115 in a wild-type PRPP synthetase is substituted with an serine residue.
It is a further object of the present invention to provide the mutant PRPP synthetase as described above, wherein the wild-type PRPP synthetase is derived from Escherichia coli.
It is a further object of the present invention to provide the mutant PRPP synthetase as described above, which includes deletion, substitution, insertion, or addition of one or several amino acids at one or a plurality of positions other than position 115, wherein feedback inhibition by purine nucleotides is desensitized.
It is a further object of the present invention to provide a DNA encoding a mutant PRPP synthetase as described above.
It is a further object of the present invention to provide a bacterium of the Enterobacteriaceae family, which contains the DNA described above, and has an ability to produce L-histidine.
It is a further object of the present invention to provide the bacterium as described above, wherein the activity of the mutant PRPP synthetase is enhanced.
It is a further object of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Escherichia.
It is a further object of the present invention to provide the bacterium as described above, wherein the activity of the mutant PRPP synthetase is enhanced by increasing the expression of the mutant PRPP synthetase gene.
It is a further object of the present invention to provide the bacterium as described above, wherein the activity of the mutant PRPP synthetase is enhanced by increasing the copy number of the mutant PRPP synthetase gene, or modifying an expression control sequence of the gene so that the expression of the gene is enhanced.
It is a further object of the present invention to provide the bacterium as described above, wherein the copy number is increased by integration of additional copies of the mutant PRPP synthetase gene into the chromosome of the bacterium.
It is a further object of the present invention to provide a method for producing L-histidine comprising cultivating the bacterium as described above in a culture medium, allowing the L-histidine to accumulate in the culture medium, and collecting the L-histidine from the culture medium.
It is a further object of the present invention to provide the method as described above, wherein the bacterium has enhanced expression of the genes for histidine biosynthesis.
The above-described objects were achieved by constructing a new mutant PRPP synthetase from E. coli. Based on the high conservatism of the prsA gene (Taira M. et al., J. Biol. Chem., v. 262, No 31, pp.14867-14870 (1987)), the mutant E. coli PRPP synthetase having a mutation corresponding to the human mutation Asn-113 was constructed. It was shown that the use of such a mutant PRPP synthetase enhances L-histidine production when additional copies of the gene encoding the mutant PRPP synthetase are introduced into a L-histidine-producing strain. Thus, the present invention has been completed.
(1) Mutant PRPP Synthetase and Mutant prsA Gene.
The mutant PRPP synthetase of the present invention is referred to as “the mutant PRPP synthetase” hereinafter and is defined as having a substitution at the aspartic acid residue at position 115 of wild-type PRPP synthetase. A DNA coding for the mutant PRPP synthetase is referred to as “the mutant prsA gene” or “mutant PRPP synthetase gene,” and a PRPP synthetase without the above position 115 substitution is referred to as “wild-type PRPP synthetase.”
It is known that the genetic and functional basis of superactivity of human PRPP synthetase associated with resistance to purine nucleotide is caused by single base substitution in prsA gene (Roessler, B. J. et al. J. Biol. Chem., v. 268, No 35, 26476-26481 (1993)). Based on the high conservatism of prsA gene (Taira M. et al., J. Biol. Chem., v. 262, No 31, pp.14867-14870 (1987)), the mutant PRPP synthetase from E. coli having a mutation corresponding to the human Asn-113 mutation was constructed. This mutation has never been described for all bacterial PRPP synthetases. The phrase “bacterial PRPP synthetase” means the PRPP synthetase existing in the bacteria of Enterobacteriaceae family, corynebacteria, bacteria belonging to the genus Bacillus etc. Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Erwinia, Providencia and Serratia. The genus Escherichia is preferred.
The substitution of the aspartic acid at position 115 of wild-type PRPP synthetase [EC 2.7.6.1] from E. coli with any amino acid, preferably with serine, leads to formation of a mutant protein which is feedback-resistant to purine nucleotides, such as purine di- and mononucleotides, mainly guanosine-5′-diphosphate (GDP), adenosine-5′-diphosphate (ADP) and adenosine-5′-monophosphate (AMP).
The mutant PRPP synthetase can be obtained by introducing mutations into a wild-type prsA gene using known methods. The prsA gene of E. coli (nucleotide numbers 1260151 to 1261098 in the sequence of GenBank Accession NC—000913, gi: 16129170, SEQ ID NO: 1) is one example of a wild-type prsA gene. The prsA gene is located between the ychM and ychB ORFs on the chromosome of E. coli strain K-12. Therefore, the prsA gene can be obtained by PCR (polymerase chain reaction; see White, T. J. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene. Genes coding for PRPP synthetase of other microorganisms can be obtained in a similar manner.
The mutant PRPP synthetase may include deletion, substitution, insertion, or addition of one or several amino acids at one or a plurality of positions other than 115, provided that the activity of PRPP synthetase is not lost. The phrase “activity of PRPP synthetase” means an activity catalyzing the reaction of ribose-5-phosphate and ATP with release of AMP to form 5-phosphoribosyl-α-1-pyrophosphate (PRPP). The PRPP synthetase activity of the extracts and degrees of inhibition by ADP can be measured using the partially modified method of K. F. Jensen et al. (Analytical Biochemistry, 98, 254-263 (1979)). Specifically, [α-32P]ATP can be used as the substrate and [32P]AMP produced by the reaction should be measured.
The number of “several” amino acids differs depending on the position or type of amino acid in the three dimensional structure of the protein. This is because some amino acids have high homology to one another and therefore do not greatly affect the three dimensional structure of the protein. Therefore, the mutant PRPP synthetase of the present invention may be one which has homology of not less than 30 to 50%, preferably 50 to 70%, more preferably 70% to 90%, and most preferably 95% or more, with respect to the entire PRPP synthetase amino acid sequence, and which retains the PRPP synthetase activity.
In the present invention, “position 115” means position 115 in the amino acid sequence of SEQ ID NO: 2. In the PRPP synthetase from E. coli, the amino acid residue in position 115 is aspartic acid. A position of an amino acid residue may change, for example, if an amino acid residue is inserted at the N-terminus portion, the amino acid residue inherently located at position 115 becomes position 116. In this situation, the amino acid residue corresponding to the original position 115 is to mean the amino acid residue at position 115 in the present invention.
To determine the L-amino acid residue corresponding to position 115 of PRPP synthetase from E. coli, it is necessary to align the amino acid sequence of PRPP synthetase from E. coli (SEQ ID NO: 2) and an amino acid sequence of PRPP synthetase from the bacterium of interest.
The DNA of the present invention, which codes for the substantially the same protein as the mutant PRPP synthetase described above, may be obtained, for example, by modifying the nucleotide sequence, for example, by means of site-directed mutagenesis so that one or more amino acid residues at a specified site are deleted, substituted, inserted, or added. The DNA modified as described above may be obtained by conventionally known mutation treatments. Mutation treatments include a method for treating in vitro a DNA containing the mutant prsA gene, for example, with hydroxylamine, and a method for treating a microorganism, for example, a bacterium, belonging to the genus Escherichia containing the mutant prsA gene with ultraviolet irradiation or a mutating agent usually used for the such treatment, such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid.
The substitution, deletion, insertion, or addition of a nucleotide as described above also includes mutation, which naturally occurs (mutant or variant), for example, on the basis of the individual difference or the difference in species or genus of bacterium, which contains PRPP synthetase.
The DNA, which codes for substantially the same protein as the mutant PRPP synthetase, can be obtained by isolating a DNA which hybridizes as a probe to DNA having a known prsA gene sequence or part of it, under stringent conditions, and which codes for a protein having the PRPP synthetase activity. The DNA may be isolated from a cell containing the mutant PRPP synthetase which is subjected to mutation treatment.
The phrase “stringent conditions” in the present invention means conditions under which so-called specific hybrids are formed, and non-specific hybrids are not formed. It is difficult to express this condition precisely by a numerical value. However, for example, stringent conditions include conditions under which DNAs having high homology, for example, DNAs having homology of not less than 50% with each other hybridize to each other, and DNAs having homology lower than the above will not hybridize with each other.
To evaluate the degree of protein or DNA homology, several calculation methods, such as BLAST search, FASTA search and CrustalW, can be used.
BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin, Samuel and Stephen F. Altschul (“Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes”. Proc. Natl. Acad. Sci. USA, 1990, 87:2264-68; “Applications and statistics for multiple high-scoring segments in molecular sequences”. Proc. Natl. Acad. Sci. USA, 1993, 90:5873-7). FASTA search method described by W. R. Pearson (“Rapid and Sensitive Sequence Comparison with FASTP and FASTA”, Methods in Enzymology, 1990 183:63-98). ClustalW method described by Thompson J. D., Higgins D. G. and Gibson T. J. (“CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice”, Nucleic Acids Res. 1994, 22:4673-4680).
Alternatively, stringent conditions are exemplified by conditions under which DNAs hybridize with each other at a salt concentration corresponding to ordinary conditions of washing in, Southern hybridization, i.e., 60° C., 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS. As a probe for the DNA that codes for variants and hybridizes with prsA gene, a partial sequence of the nucleotide sequence of SEQ ID NO: 1 can also be used. Such a probe may be prepared by PCR using oligonucleotides based on the nucleotide sequence of SEQ ID NO: 1 as primers, and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1 as a template. When a DNA fragment of about 300 bp is used as the probe, the washing conditions for the hybridization consist of, for example, 50° C., 2×SSC, and 0.1% SDS. Duration of the washing procedure depends on the type of membrane used for blotting and, as a rule, is recommended by manufacturer. For example, recommended duration of washing the Hybond™ N+ nylon membrane (Amersham) under stringent conditions is 15 minutes.
The gene, which is hybridizable under conditions as described above, includes those having a stop codon generated within a coding region of the gene, and those having no activity due to mutation of the active center. However, such inconveniences can be easily removed by ligating the gene with a commercially available expression vector, and investigating the PRPP synthetase activity of the expressed protein.
(2) Bacterium of the Present Invention.
The bacterium of the present invention is an L-histidine-producing bacterium of the Enterobacteriaceae family containing DNA encoding the mutant PRPP synthetase of the present invention. Furthermore, the bacterium of the present invention is an L-histidine-producing bacterium of the Enterobacteriaceae family having increased activity of mutant PRPP synthetase of the present invention. More specifically, the bacterium of the present invention is an L-histidine-producing bacterium of Enterobacteriaceae family, wherein L-histidine production by the bacterium is enhanced by enhancing an activity of the protein of the present invention in the bacterium. More preferably, the bacterium of the present invention is an L-histidine-producing bacterium belonging to the genus Escherichia, wherein L-histidine production by the bacterium is enhanced by enhancing an activity of the protein of the present invention, namely mutant PRPP synthetase, in the bacterium. More preferably, the bacterium of present invention contains the DNA encoding the mutant prsA gene, which is overexpressed by the chromosome or by a plasmid in the bacterium. As a result, the bacterium of the present invention has enhanced ability to produce L-histidine.
“Bacterium, which has an ability to produce L-histidine” means a bacterium which has an ability to cause accumulation of L-histidine in a medium, when the bacterium of the present invention is cultured in the medium. The L-histidine-producing ability may be imparted or enhanced by breeding. The term “bacterium, which has an ability to produce L-histidine” used herein also means a bacterium, which is able to produce and cause accumulation of L-histidine in a culture medium in an amount larger than a wild-type or parental strain, and preferably means that the bacterium is able to produce and cause accumulation of L-histidine in a medium in an amount of not less than 0.5 g/L, more preferably not less than 1.0 g/L.
Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Erwinia, Providencia and Serratia. The genus Escherichia is preferred.
The term “a bacterium belonging to the genus Escherichia” means the bacterium which is classified as the genus Escherichia according to the classification known to a person skilled in the art of microbiology. Examples of microorganisms belonging to the genus Escherichia used in the present invention include, but are not limited to Escherichia coli (E. coli).
The phrase “activity of the mutant PRPP synthetase is enhanced” means that the activity per cell is higher as compared to a non-modified strain, for example, a wild-type strain. For example, this meaning includes increasing the number of mutant PRPP synthetase molecules per cell, increasing the specific activity per mutant PRPP synthetase molecule, and so forth. Furthermore, Escherichia coli K-12 is an example of a wild-type strain that may serve as control. As a result of enhancement of intracellular activity of the mutant PRPP synthetase, an increase in the amount of L-histidine accumulation in a medium is observed.
Enhancement of the mutant PRPP synthetase activity in a bacterial cell can be attained by enhancement of expression of a gene coding for the mutant PRPP synthetase. The mutant PRPP synthetase gene of the present invention may encompass any of the genes encoding mutant PRPP synthetase derived from bacteria of Enterobacteriaceae family as well as the genes derived from other bacteria such as coryneform bacteria. Among these, genes derived from bacteria belonging to the genus Escherichia are preferred.
Transformation of a bacterium with a DNA encoding a protein means introduction of the DNA into a bacterium cell using conventional methods. As a result, expression of the gene encoding the protein of present invention is increased and the activity of the protein is enhanced in the bacterial cell.
Methods of enhancement of gene expression include increasing the gene copy number. Introduction of a gene into a vector that is able to function in a bacterium belonging to the genus Escherichia will increase the copy number of the gene. For such purposes, multi-copy vectors are preferably used. The multi-copy vector is exemplified by pBR322, pUC19, pBluescript KS+, pACYC177, pACYC184, pAYC32, pMW119, pET22b or the like. Other methods of gene expression enhancement can be achieved by introduction of multiple copies of the gene into the bacterial chromosome by, for example, methods of homologous recombination, or the like.
Other methods of gene expression enhancement can be achieved by placing the DNA of the present invention under the control of a more potent promoter instead of the native promoter. The strength of a promoter is defined by the frequency of RNA synthesis initiation. Methods for evaluating the strength of a promoter and examples of potent promoters are described by Deuschle, U., Kammerer, W., Gentz, R., Bujard, H. (Promoters in Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO J. 1986, 5, 2987-2994). For example, the PR promoter is known as a potent constitutive promoter. Other known potent promoters are PL promoter, lac promoter, trp promoter, trc promoter, of lambda phage and the like.
The enhancement of translation can be achieved by introducing a more efficient Shine-Dalgarno sequence (SD sequence) into the DNA of the present invention in place of the native SD sequence. The SD sequence is typically a region upstream of the start codon of the mRNA interacting with the 16S RNA of ribosome (Shine J. and Dalgarno L., Proc. Natl. Acad. Sci. U S A, 1974, 71, 4, 1342-6).
Use of a more potent promoter can be combined with multiplication of gene copies.
Preparation of chromosomal DNA, hybridization, PCR, plasmid DNA preparation, DNA digestion and ligation, transformation, selection of an oligonucleotide as a primer, and the like, are all methods well known to one skilled in the art. These methods are described in Sambrook, J., and Russell D., “Molecular Cloning A Laboratory Manual, Third Edition”, Cold Spring Harbor Laboratory Press (2001), and the like.
The bacterium of the present invention can be obtained by introduction of the aforementioned DNAs into a bacterium inherently having the ability to produce L-histidine. Alternatively, the bacterium of present invention can be obtained by imparting an ability to produce L-histidine to a bacterium already containing the DNAs.
The parent strain to be enhanced in activity of the protein of the present invention includes but is not limited to a bacteria belonging to the genus Escherichia having L-histidine producing ability, the L-histidine producing bacterium strains belonging to the genus Escherichia, such as E. coli strain 24 (VKPM B-5945, Russian patent 2003677); E. coli strain 80 (VKPM B-7270, Russian patent 2119536); E. coli strains NRRL B-12116-B12121 (U.S. Pat. No. 4,388,405); E. coli strains H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Pat. No. 6,344,347); E. coli strain H-9341 (FERM BP-6674) (European patent application 1085087A2); E. coli strain A180/pFM201 (U.S. Pat. No. 6,258,554), and the like.
It is preferable that the L-histidine-producing bacterium be further modified to have enhanced expression of L-histidine biosynthesis. Genes effective for L-histidine biosynthesis include hisG gene and genes of hisBHAFI operon, preferably hisG gene encoding ATP phosphoribosyl transferase wherein feedback inhibition by L-histidine is desensitized (Russian patents 2003677 and 2119536).
(3) Method of the Present Invention.
The method of present invention includes a method for producing L-histidine, including the steps of cultivating the bacterium of the present invention in a culture medium, allowing the L-histidine to accumulate in the culture medium, and collecting the L-histidine from the culture medium.
In the present invention, the cultivation, collection, and purification of L-histidine from the medium and the like may be performed by conventional fermentation methods for producing amino acids using a microorganism. The medium used for culture may be either synthetic or natural, so long as the medium includes a carbon source, a nitrogen source, minerals and, if necessary, appropriate amounts of nutrients which the microorganism requires for growth. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation used by the chosen microorganism, alcohol including ethanol and glycerol may be used. The nitrogen source may include various ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, a natural nitrogen source, such as peptone, soybean-hydrolysate, and digested fermentative microorganisms. Minerals may include potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like. Some additional nutrients may be added to the medium, if necessary. For instance, if the microorganism requires proline for growth (proline auxotrophy), a sufficient amount of proline may be added to the cultivation medium.
The cultivation is performed preferably under aerobic conditions such as a shaking culture, and stirring culture with aeration, at a temperature of 20 to 42° C., preferably 37 to 40° C. The pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1- to 5-day cultivation leads to accumulation of the target L-amino acid in the liquid medium.
After cultivation, solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the target L-amino acid can be collected and purified by ion-exchange, concentration and crystallization methods.
The present invention will be more concretely explained below with reference to the following non-limiting examples. In the examples, an amino acid is of L-configuration unless otherwise noted.
The entire nucleotide sequence of E. coli strain K-12 has been reported (Science, 277, 1453-1474, 1997). Based on the reported nucleotide sequence, the primers depicted in SEQ ID No. 3 (primer 1) and SEQ ID No.4 (primer 2) were synthesized and used for amplification of prsA gene. The primer 1 contains a BglII recognition site introduced at the 5′ thereof. The primer 2 contains a XbaI recognition site introduced at the 5′-end thereof.
Chromosomal DNA of E. coli K12 was used as template for PCR, and was prepared by an ordinary method. PCR was carried out on the Applied Biosystems GeneAmp PCR System 2400 under the following conditions: initial DNA denaturation at 95° C. for 3 min; followed by 30 cycles of denaturation at 95° C. for 30 sec, annealing at 60° C. for 60 sec and elongation at 72° C. for 120 sec; and the final polymerization for 7 min at 72° C. using Taq polymerase (Fermentas, Lithuania). The resulting PCR fragment containing prsA gene without a promoter was treated with BglII and XbaI and inserted under PR promoter into the integrative vector pMW 119-PR previously treated with the same enzymes. Vector pMW119-PR was constructed from a commercially available vector pMW119 by insertion of PR promoter from phageλ and attR and attL sites necessary for further Mu-integration. Thus, plasmid pMW-PR-prsA was obtained.
Mutant prsDA gene (substitution of aspartic acid 128 with alanine in the PRPP synthetase coded by mutant prsDA gene) was obtained by PCR as described above using primers 1 (SEQ ID No. 3) and 2 (SEQ ID No. 4), and using plasmid pUCprsDA as a template. Plasmid pUCprsDA is described in detail in the European patent application EP1004663A1. The resulting PCR product was treated with BglII and XbaI and inserted under the control of the PR promoter into the integrative vector pMW119-PR previously treated with the same enzymes. Thus, plasmid pMW-PR-prsDA was obtained.
Mutant prsDS gene (substitution of aspartic acid 115 with serine in the PRPP synthetase coded by mutant prsDS gene) was constructed by two successive PCR runs. First, two fragments of the gene were synthesized using primers 1 (SEQ ID No. 3) and 3 (SEQ ID No. 5) for the first fragment, and primers 2 (SEQ ID No. 4) and 4 (SEQ ID No. 6) for the second one. Chromosomal DNA of E. coli K12 was used as a template. Then the resulting PCR products were separated by electrophoresis and eluted from gel. In the second PCR run, these two DNA fragments were annealed and the mutant prsDS gene was completed. The resulting PCR fragment containing prsDS gene without a promoter was treated with BglII and XbaI and inserted under the control of the PR promoter into the integrative vector pMW119-PR previously treated with the same enzymes. Thus, plasmid pMW-PR-prsDS was obtained.
Three L-histidine-producing plasmid-less strains containing additional copies of the prsA, prsDA or prsDS genes integrated into the bacterial chromosome were constructed. The L-histidine producing E. coli strain 80 was used as a parental strain for integration of the prsA, prsDA and prsDS genes into the bacterial chromosome. The strain 80 is described in Russian patent 2119536 and deposited in the Russian National Collection of Industrial Microorganisms (Russia, 113545 Moscow, 1st Dorozhny proezd, 1) under accession number VRPM B-7270.
Integration of the genes into the chromosome of strain 80 was performed in two steps. For the first step, the histidine-producing strain 80 was transformed with a helper plasmid containing replicon rep(p15A), transposase gene (genes cts62, ner, A, B from phage Mu-cts) and containing TetR marker. For the second step, the resulting strain was transformed with plasmid pMW-PR-prsA, pMW-PR-prsDA or pMW-PR-prsDS. For integration of the gene into the chromosome the heat-shocked cells were transferred to 1 ml of L-broth, incubated at 44° C. for 20 minutes, then at 37° C. for 40 minutes, and then were spread onto L-agar containing 10 μg/ml of tetracycline and 100 μg/ml of ampicillin. Colonies grown within 48 hours at 30° C. were inoculated in 1 ml of L broth and incubated for 72 hours at 42° C. in tubes. About 10 colonies from every tube were checked for ampicillin and tetracycline resistance. Colonies sensitive to both antibiotics were tested for presence of additional copies of the prs gene in the chromosome by PCR using primers 1 (SEQ ID No 3) and primer 5 (SEQ ID No 7). Primer 5 contains a sequence complementary to the attR site of phage Mu. For that purpose, a freshly isolated colony was suspended in 50 μl of water and then 1 μl was subject to PCR. PCR conditions were the following: initial DNA denaturation at 95° C. for 5 minutes; then 30 cycles of denaturation at 95° C. for 30 sec, annealing at 57° C. for 60 sec and elongation at 72° C. for 120 sec; the final polymerization at 72° C. for 7 min. A few of the antibiotic-sensitive colonies tested contained the necessary 1515 bp DNA fragment. Thus, strains 80::PR-prsA, 80::PR-prsDA, and 80::PR-prsDS were obtained.
For mini-jar batch-fermentation one loop of each strain grown on L-agar was transferred to L-broth and cultivated at 30° C. with rotation (140 rpm) to reach an optical density of culture OD540≈2.0. Then 25 ml of seed culture was added to 250 ml of medium for fermentation and cultivated at 29° C. for with rotation (1500 rpm). Duration of the batch-fermentation was approximately 35-40 hours. After the cultivation, the amount of histidine which accumulated in the medium was determined by paper chromatography. The paper was developed with a mobile phase: n-butanol : acetic acid: water=4:1:1 (v/v). A solution of ninhydrin (0.5%) in acetone was used as a visualizing reagent.
The composition of the fermentation medium (pH 6.0) (g/l):
Obtained data are presented in the Table 1:
As it seen from the Table 1, the use of mutant prsDS gene coding for PRPP synthetase feedback resistant to purine nucleotides improved histidine productivity of the E. coli strain 80.
While the invention has been described with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents, including the foreign priority documents RU2003132412 and RU2004120501, is incorporated by reference herein in its entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003132412 | Nov 2003 | RU | national |
| 2004120501 | Jul 2004 | RU | national |
This application claims the benefit of provisional application 60/587,492, filed Jul. 14, 2004.
| Number | Date | Country | |
|---|---|---|---|
| 60587492 | Jul 2004 | US |
