The microbiological method of obtaining amino acids by increased activity of export-media

 

The invention relates to biotechnology. The microbiological method of obtaining amino acids includes the increase in the activity of the export carrier and/or the expression of the export-gene of the microorganism producing the desired amino acid. According to the invention, one specific gene is responsible for the export of this amino acid and basic microbiological method of obtaining amino acids, including controlled increased expression of the export-gene and/or activity of the export carrier of the microorganism producing the amino acid that appears in the first period of time. Increased expression or activity of the export carrier, resulting this way, increases the rate of excretion and accelerates therefore, the transport of the desired amino acids. 6 C. and 24 C.p. f-crystals, 4 Il., 3 table.

The invention relates to a microbiological method of obtaining amino acids according to PP.1-20 claims, export-genes on PP.21-26, regulatory genes on PP.27 and 28, the gene structures PP.29 and 30, vectors PP.31-33, transformed cells on PP.34-40; membrane proteins PP.41 and 42, and applications PP.43-48.

Amino acids are of great economic interest, Prout as a feed additive, L-glutamate as a spice, L-isoleucine and L-tyrosine in the pharmaceutical industry, L-arginine and L-isoleucine in medicine or L-glutamate and L-phenylalanine as a starting substance for the synthesis of pure chemicals.

The preferred method of obtaining these various amino acids is a biotechnology obtain with the help of microorganisms, as this method is directly obtained biologically effective and optically active form of the corresponding amino acids, and it is possible to apply a simple and inexpensive raw materials. As the microorganisms used, for example, Corynebacterium glutamicum and its relatives ssp. flavum and spp. lactofermentum (Liebl and others)., Int J System Bacteriol (1991) 41:255-260) as Escherichia coli and related bacteria.

These bacteria produce usual amino acids, but only necessary for the growth of the quantity, so that, therefore, no excess amino acids are not formed and is not highlighted. This is justified by the fact that in the cell biosynthesis of amino acids by multiple controlled way. Therefore, already the most famous different ways to improve the formation of the product off of the control mechanisms. When these processes are applied, for example, analogs of amino acids to cyclocreatine against analogs of L-tyrosine and L-phenylalanine (Japan patent 19037/1976 and 39517/1978). It also describes the ways in which the use of resistant against analogs of L-lysine or L-threonine bacteria to overcome control mechanisms (EP 0205849 B1, Ukrainian patent, application for patent in the UK 2152509 A).

Further, known also constructed by recombinant DNA methods microorganisms, which also increases the regulation of biosynthesis of the fact that genes that are no longer encode break destination enzymes with feedback, clone and Express. So, for example, is known for producing L-lysine, recombinant bacterium encoded plasmid resistant to feedback aspartoacylase (EP 0381527). Also described is a recombinant, producing L-phenylalanine by a bacterium resistant to feedback preventdisease (Japan patent 124375/1986, EP 0488424). In addition, excessive expression of genes that do not encode sensitive to feedback enzymes for the synthesis of amino acids, reach also increased outputs amino acids. For example, the formation of lysine improve by increased synthesis dihydropyrimidinase (EP 0197335). Increased synthesis trainingsgerate also achieve improved education threonine (EP 0436886 A1).

Further try antralnogo metabolism. So, it is known that achieved by recombinant methods overexpression transketolase makes possible an improved formation of the product of L-tryptophan, L-tyrosine or L-phenylalanine (EP 0600463 A2). Further, the decreased activity of fosfoenolpiruvatcarboksilaza in Corynebacterium leads to enhanced formation of aromatic amino acids (EP 0331145).

These numerous attempts to improve the performance, in General, aims to overcome the limitation citicolina synthesis of amino acids. But as another limitation, in principle, take into account the export educated in the inner part of the cells of amino acids in the nutrient medium. Therefore, there are separate initial mixture to improve exports and profitability of production of amino acids. Thus, the permeability of cells in Corynebacterium increased by Biotin deficiency, treatment with detergents or treatment with penicillin. However, these additional measures were successful only in the production of glutamate, while the synthesis of other amino acids to improve in this way failed. Were also identified strains of bacteria, in which the activity of the secretion system was improved through chemical or physical mutations. The result Etten, in particular, for the production of L-lysine (patent Germany 4203320).

In General, all studies so far experiments increase the secretion formed in the inner part of the cells of amino acids are distinguished by the fact that the increased outflow of amino acids on the basis of incorrect or non-specific methods could be achieved only by accident. Only in the German patent application No. 19523279.8-41 described method, which allows a completely purposefully to increase the secretion formed in the inner part of the cells of amino acids, and expression of coding the import of amino acids genes was increased. Underlying this principle is the conclusion that cells use imported proteins for export of amino acids, and the fact that microorganisms by nature do not form and do not emit any excess amino acids, suggests that specific transfer of amino acid export-genes or export-proteins do not exist, and what cells amino acids excretiruyutza through other systems export.

Known for still system of export export of toxic metal ions, toxic antibiotics and more high molecular weight toxins. These exports have a relatively complex structure: as p is th reaction exports, so presumably for transfer requires additional, extracytoplasmic auxiliary proteins (Dinh, T. and others, A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria. J. Bacteriol. 1994, 176:3825-3831). Then it is known that dependent secretion system to export to the extracellular proteins are essential at least 6 different components of proteins for export. This prior art suggests that relevant for the export of amino acids, but still unknown systems consist of many components of proteins or genes provide the export of amino acids. Reference to this could be described Vrljic, etc. (J Bacteriol (1995) 177:4021-4027) are different, defective in the export of lysine mutants.

Now unexpectedly been found that the export of amino acids are responsible, respectively, only one specific gene, so that according to the invention for the first time seems to be at the disposal of the microbiological method of obtaining amino acids, which purposefully increase the expression of the export-gene and/or activity of the export carrier, producing the corresponding amino acid of the microorganism. The resulting action methods increased the ith amino acids increased. Such modified microorganisms accumulate also an increased amount of the appropriate acid in the culture medium.

To increase the activity of export-media increase, in particular, the endogenous activity of producing amino acid of the microorganism.

Increasing the activity of the enzyme can, for example, be achieved by the fact that changes in the catalytic center is an increased conversion of the substrate or stops the action of enzyme inhibitors. Increased activity of the enzyme can cause increased synthesis of the enzyme, for example, gene amplifications or deletions of factors that inhibit the biosynthesis of the enzyme. Endogenous activity of export-media increase mainly by mutation of the endogenous export-gene. Such mutations can be made or classical methods decelerate, as, for example, by irradiation with ultraviolet rays or solvent mutations by chemicals, or purposefully using genetic engineering methods, as deletion (deletion), insertion(s) and/or exchange of nucleotides (nucleotide exchanges).

The expression of the export of the gene is increased by increasing the number of copies of the gene and/or strengthening of regulatory factors that polietileno at the level of transcription, however, what, in particular, increased transcriptional signals. This can be achieved, for example, due to the fact that by changing upstream of the structural gene sequence of the promoter the promoter improves its effectiveness or to the fact that the promoter is completely changed to a more efficient promoters. You can also gain transcription by the impact of slave exports gene regulatory gene, as explained further. But along with this it is also possible strengthening of translation, and improves the stability of the m-RNA.

To increase the number of copies of the gene export-gene built into the design of the gene or in a vector, preferably a vector with a lower number of copies. The design of the gene contains, in particular, the subordinate export-gene regulatory sequences of genes, preferably such that increase gene expression. Regulatory sequences of genes are especially given in table.1 the sequence of amino acids or their encoding variation in the alleles of a nucleotide sequence or a nucleotide sequence from nucleotide 954 82 table.2 or equally valid, in General, the DNA sequence. Variations of alleles or equally deistvuya), the insertion (insertion) and/or substitution (substitution) nucleotides of the corresponding sequences, with, however, the activity of regulatory proteins or regulatory function of the protein is retained or even improved. Thus, mutation of the regulatory sequence of a gene can have such an influence on the binding of the regulatory protein to DNA regulatory export-gene, resulting in increased transcription and, consequently, increases the expression of a gene. You can then give the export-gene as regulatory sequences are also so-called "enhancers" that through improved interaction between RNA polymerase and DNA also cause increased expression of the export-gene.

Plaster export-gene in gene structure export-gene, isolated mainly from a strain of microorganism of the genus Corynebacterium and the export-gene gene construct, transform to produce the corresponding amino acid strains of microorganisms, especially Corynebacterium. The selection and transformation of the corresponding transport gene carry out the conventional methods. In the case of selection and cloning of transport gene from Corynebacterium use, for example, the method homola strukturnoi gene, you may first conduct the insertion sequences of the vector in the transport gene, then select it via the "rescue plasmids in the form of inactive fragments. For the method according to the invention are particularly suitable genes from C. glutamicum ATCC 13032 or C. glutamicum ssp. flavum ATCC 14067 or C. glutamicum ssp. lactofermentum ATCC 13869. After isolation of genes and recombination in vitro with known vectors (Appl Environ Env (1989) 55:684-688; 102 Genes (1991) 93-98), in producing the amino acid strains carry out the transformation by electroporation (Lieb and others (1989) FEMS Environ Lett 65:299-304) or by conjugation (Schaefer and others (1990) J Bacteriol 172:1663-1666). To migrate preferably used carriers with a low number of copies. As cells of the hosts used are preferably such producers of amino acids in the synthesis of the corresponding amino acids deregulated and/or contain a high amount of metabolites of the Central metabolism.

After selecting the export-genes get with nucleotide sequences that encode the specified table.3 amino acid sequence, or variations of their alleles, respectively, which have a nucleotide sequence from nucleotide 1016 to 1725 table.2 or, basically acting the same after which lastnosti, functional derivatives of the above for the regulatory sequences of sense. These export-genes are used preferably in the method according to the invention.

To export gene with upstream promoter or without it, or with the corresponding regulatory gene or without it can be turned on and/or additionally include one or more DNA sequences, so that the gene is contained in the structural gene.

Cloning export genes receive the plasmid vector respectively, which contain export-gene and, as already mentioned above, were used for transformation of a producer of amino acids. Obtained by transformation of a cell in which it is preferably of transformed cells of Corynebacterium contain a gene in a replicated form, i.e., additional copies on the chromosome, and a copy of the gene is integrated by homologous recombination at any places on the genome, and/or plasmids, respectively - vector.

There are many sequences that encode membrane proteins of unknown function. The receipt of the export-genes according to the invention, as, for example, export-gene with the nucleotide sequence from nucleotide 1016 to 1725 you can now to identify membrane proteins, the function is the transport of amino acids by comparing the sequences. Identified in this way, the export-gene can be used then to improve the production of amino acids by the method according to the invention.

Known from the prior art membrane proteins typically have 12, partially 4 transmembrane helix. Now unexpectedly been found, the corresponding export amino acids, respectively suitable membrane proteins have 6 transmembrane helices (see, for example, are given in table.3 the amino acid sequence of the export protein, in which 6 transmembrane regions are indicated by underlining). Thereby presented here is not yet described so far and, therefore, a new class of membrane proteins.

Examples:

a) Cloning the export-gene and cloning of the regulator from Corynebacterium glutamicum.

Chromosomal DNA from C. glutamicum R127 (FEMS Environ Lett (1989) 65:229-304) have been described as the Shartsey and other (Bio/Technology (1990) 9:84-87) and highlighted. The latter was digested with restrictive enzyme Sau3 and shared gradient centrifugation sucrose, as described by Sambrook and others (Molecular Cloning, A Laboratory manual (1989) Cold Spring Harbour Laboratory Press). Individual fractions were analyzed for linkage with the vector pJCI. For this vector pJCI with BamHI was linearizable and dephosphorylated. 5 ng of this ligated with 20 ng of chromosomal fragment sizes 6-10 square With a total slivochnoi insert transformed the export-defective mutant NA8 (J Bacteriol (1995) 177:4021-4027) by electroporation (FEMS Environ Lett (1989) 65:299-304). Transformants selected on LBHIS (FEMS Environ Lett (1989) 65:299-304) with 15 μg of kanamycin per 1 ml of These transformants were subjected to extensive tests on plasmids, and chose 200 separately from all 4500 of the obtained clones, and determined the amount of plasmid and size. On average, approximately half of the investigated resistant to kanamycin clones were carrying recombinant plasmids with insert medium size 8 square thus obtained probability of 0.96 for the presence of each x-any gene from C. glutamicum formed in the gene Bank. All 4500 of the obtained transformants was tested separately on the return receipt secretion of lysine. Was used for this are described Vrljic system for the induction of excretion of L-lysine in Corynebacterium glutamicum (J Bacteriol (1995) 177:4021-4027). This produced the so-called indicator plates with minimal medium, 1 l contained 20 g (NH4)2SO4, 5 g urea, 1 g KN2RHO41 g2NRA4, 0.25 g gSO447H2Oh, 1 mg MnSO4H2Oh, 0.1 mg ZnSO47H2Oh, 0.02 mg CuSO4, 0.002 mg NiCl26N2Oh, 20 g agar-agar, and 107cells/ml lysine-auxotroph C. glutamicum mutant 49/3. All original 4500 transformed separately using a toothpick pinned on indicator plates, respectively, with the control of the original nevadalas NA8 (J Bacteriol (1995) 177:4021-4027) and the original strain R127. Simultaneously imparted, respectively, 2 plates, of which only one contained an additional 5 mmol L-methionine, in order thus to induce the secretion of lysine. Indicator plates were incubated at 30C and examined after 15, 24 and 48 hours Just received thus 29 clones that were shown at left with methionine indicator plate bezel growth of the indicator strain 49/3. Clones were disconnected and then again, as described above, was tested for reverse acquisition of rim growth. This method has received two clone NA8 pMV8-5-24 and NA8 pMV6-3, which again received the ability (1990) 9:84-87).

Retransformation in NA8 was confirmed associated with plasmids effect excretion of L-lysine. Both plasmids were subjected to restriction analysis. Plasmids pMV8-5-24 carries a box of 8.3 kV and pMV6-3 carries an insert of 9.5 square Physical map insert shows Fig.1.

b) Subclavian DNA fragment, which reconstituirea export lysine.

From the insert of plasmid pMV6-3, using specific locations of restriction segments, produced individual subclones. So, XhoI-SaII fragment of 3.7 kV, BamHI-fragment of 2.3 kV and BamHI-fragment 7.2 kV respectively ligated with the cut and processed by the vector pJCI (Mol Gen Genet (1990) 220:478-480). Products stitching transformed directly C. glutamicum NA8, transformants as described above, was tested for reverse acquisition excretion of lysine, and the presence of subclones were confirmed by obtaining plasmid and restriction fragments length polymorphism analysis. In this way received the strain with plasmid pMV2-3 as the smallest subclone (Fig.1). Last, encourage exports of lysine fragment contains as inserting BamHI-fragment of 2.3 kV from pMV6-3.

(C) Sequence export-gene lysine lys E and its regulator lys G.

The nucleotide sequence BamHI-fragments of 2.3 kV was carried out according to the method of dideoxy-breaking the chain of Sanger and foresry analysis was carried out using the automated laser fluorescence device for determining the DNA sequence (A. L. F) Pharmacia-LKB (Piscataway, NJ, USA). The obtained nucleotide sequence was analyzed using the software package HUSAR (document 3.0) German cancer research center (Heidelberg). The nucleotide sequence and the result of the analysis are given in table.2. The analysis provides two full open raster for reading (ORF) on the interval with which the sequence of DNA. ORFI encodes a protein with a length of 236 amino acids, ORF2 encodes a protein with a length of 290 amino acids. Made from ORFI protein shows a cluster of hydrophobic amino acids, which is typical for membrantechnik proteins. Detailed analysis of the distribution of hydrophobic and hydrophilic amino acids with the program PHD.HTM (Protein Science (1995) 4:521-533) shown in the table.3. This implies that the protein contains six hydrophobic regions of helices that cross the membrane. This protein we are talking about the sought exporter of the amino acid L-lysine. Therefore, the corresponding gene indicate below how lysE. He indicated respectively in table.2. ORF2 transcribers in the opposite direction to ORFI. The sequence analysis shows that ORF2 has high identity with regulatory genes, which unite as one family (Ann. Rev Environ (1993) 597-626). Genes of this family positive is the fact below ORF2 is designated as lysG (agreement alone govern=control). Because of this subordination and as lysE you can clone (see a)) and subclinical (see b)) only in conjunction with lysG, lysG controller lysE involved, therefore, also in the export of lysine. Gene lysG and his educated amino acid sequence is also shown in the table.2 or in table.1.

d) Identification of the unknown membrane protein from Escherichia coli by comparing the sequences.

Sustainable sequences according to table.3 you can search for existing banks sequences to identify the action formed from sequentronic regions of proteins. Accordingly compared the amino acid sequence of the lysine exporter of C. glutamicum using the HUSAR program package (document 3.0) German cancer research center (Heidelberg) formed by sequences of all proteins deposited there DNA sequences. For a single sequence of previously unknown function of E. coli was high homology to 39.3% identical amino acids and 64,9% similar amino acids. The comparison shown in Fig.2. Not sure still open raster reading of E. coli identified through this method as the export-gene amino acids.

e) Increases the ovale with plasmids pMV2-3 and compared the secretion of L-lysine strains. For this purpose, respectively, were separately inoculated NA8 and NA8pMV2-3 in a complex environment described Vrljic, etc. (J Bacteriol (1995) 177:4021-4027), and fermentation medium CGXII (J Bacteriol (1993) 175:5595-5603). Medium containing 5 mmol L-methionine to induce the biosynthesis of intracellular L-lysine. After culturing for 24 h at 30With on a rotation apparatus for shaking at 140 rpm was performed for the determination of intracellular and external L-lysine. For intracellular determination was carried out by centrifugation with silicone oil (Methods Enzymology LV (1979) 547-567); determination of amino acids was performed using liquid chromatography high pressure (J Chromat (1983) 266:471-482). These definitions were carried out in different periods of time, as shown in Fig.3. In accordance with the used method accumulated intracellular lysine through pMV2-3 is secreted in higher amounts and accumulated. As was to be expected, consequently greatly reduces the number of available inside cells L-lysine. Therefore, the use of open and described exporter is a way to significantly improve the formation of L-lysine.

f) Increased accumulation of L-lysine by lysE or lysEG.

The sub is the sequence ligated bearing lysE 1173 BP PvuII-HindII fragment in pZ1 (Appl Environ Env (1989) 55:684-688), and received in this way, the plasmid plysE. This plasmid and bearing lysElysG plasmids pMV2-3 was introduced by electroporation into C. glutamicum strain d, and the chromosomal region subjected to the division. The obtained strains C. glutamicum d pMV2-3, C. glutamicum d, plysE, C. glutamicum pJCI was chosen, as described in paragraph (e), first on a complex medium, then cultured in production minimal medium CGXII with 4% glucose and 5 mmol L-methionine, and samples were taken to determine the accumulated L-lysine. As can be seen from Fig.4, due lysElysG achieve greater accumulation of lysine compared to the control definition. This way plysE reaches extremely high accumulation of L-lysine from 4.8 to 13.2 mmol.

Notes to tables and drawings.

Table 1: amino acid Sequence controller lysine exporter of Corynebacterium glutamicum, with a typical DNA-binding protein Helix-Turn-Helix Motif.

Table 2 (three pages): a nucleotide Sequence encoding the lysine exporter and control export lysine region of C. glutamicum.

Table 3: amino acid Sequence of the lysine exporter of Corynebacterium glutamicum, identified transmembrane helices TMHI-TMH6.

Fig.2: Comparison of the formed amino acid sequence LysE of C. glutamicum (above), with gene-product of previously unknown function of Escherichia coli (below), which is identified as export-media.

Fig.3: Increased export of lysine by pMV2-3 from C. glutamicum NA8. Above the control with a small selection and accumulation of intracellular lysine approximately 150 mmol. Below caused due pMV2-3 high allocation of only a small accumulation of intracellular lysine about 30 mmol.

Fig.4: Increased accumulation of lysine in C. glutamicum thanks lysElysG (pMV2-3) (middle curve), and due to lysE (plysE) accumulation (upper curve).

Claims

1. The microbiological method for L-lysine and/or L-arginine, involving the cultivation of Corynebacterium glutamicum and secretion of L-lysine and/or L-arginine, wherein the Corynebacterium glutamicum modify this obrotu nucleotides from 1016 until 1725 according to table 2, and/or increase the expression of the specified single export-gene.

2. The method according to p. 1, characterized in that increase the endogenous activity of the export carrier of the microorganism.

3. The method according to p. 2, characterized in that the mutation of endogenous export-gene create media with increased export activity.

4. The method according to one of paragraphs.1-3, characterized in that the expression of a gene export-media increase by increasing the number of copies of a gene.

5. The method according to p. 4, characterized in that to increase the number of copies of the gene export-gene is inserted into the gene construct.

6. The method according to p. 5, characterized in that the export-gene inserted into the vector with a low number of copies.

7. The method according to p. 5 or 6, characterized in that the export-gene is inserted into a gene construct, which contains subordinate export-gene regulatory genes.

8. The method according to p. 7, characterized in that slave exports gene in the composition of the gene construct regulatory gene has a nucleotide sequence encoding specified in table 1 amino acid sequence, taking into account the degeneracy of the genetic code.

9. The method according to p. 8, characterized in that slave exports gene in the composition of the gene construct regulatory gene has the sequence u is the resultant of PP.5-9, wherein the producing the corresponding amino acid of the microorganism transform contains export-gene gene construct.

11. The method according to p. 10, characterized in that coryneform bacteria transform contains export-gene gene construct.

12. The method according to one of the p. 10 or 11, characterized in that the transformation using a microorganism in which the enzymes involved in the synthesis of the corresponding amino acids, deregulated.

13. The method according to one of paragraphs.10-12, characterized in that the transformation using a microorganism, which has a high content of metabolites of the Central metabolism.

14. The method according to one of paragraphs.4-13, characterized in that the export-gene isolated from coryneform bacteria.

15. The method according to one of paragraphs.1-14, characterized in that the sequence of the export of the gene identified by comparison with the sequence already known export-gene.

16. The method according to p. 15, characterized in that derived on the basis of identifiable sequence export-gene sequence of amino acids compared to the amino acid sequence shown in table 3.

17. The method according to one of paragraphs.1-16, characterized in that the expression of export as the export-gene using the gene with the nucleotide sequence, the coding specified in table 3 amino acid sequence.

19. Export the gene encoding the exporting carrier with the nucleotide sequence from nucleotide 1016 to 1725 according to table 2, taking into account the degeneracy of the code.

20. Export-gene p. 19, characterized in that it encodes the exporting carrier with the amino acid sequence shown in table 3.

21. Export-gene p. 19, characterized in that it is governed by a regulatory gene, encoding the amino acid sequence listed in table 1.

22. Export-gene p. 19 or 21, characterized in that it is regulated by the regulatory gene having the nucleotide sequence from nucleotide 954 to 82 according to table 2, taking into account the degeneracy of the genetic code.

23. Regulatory gene for the regulation of the encoder export-media-export of the gene with the nucleotide sequence from nucleotide 954 to 82 according to table 2, taking into account the degeneracy of the genetic code.

24. Regulatory gene in p. 23 with a nucleotide sequence that encodes the amino acid sequence shown in table 1.

25. Gene structure containing at least one export-gene on one of the PP.19-22, and also operatively associated with a regulatory

27. The plasmid vector containing at least one export-gene on one of the PP.19-22 or genetic structure in p. 25, and an additional nucleotide sequence for breeding and/or replication in a cell-host or for integration into the genome of the host cell.

28. Plasmid vector for p. 27 with a low number of copies.

29. Plasmid vector for p. 27 containing additional regulatory gene in p. 23 or 24 or genetic structure in p. 26.

30. Export-media transport of lysine and/or arginine, representing a membrane protein with 6 transmembrane helices and the amino acid sequence shown in table 3.

 

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