# Patent

## Title
Method for producing vectors containing a gene coding for an enzyme having reduced or deactivated feedback inhibition and the use thereof for producing amino acids and nucleotides

## Abstract

A method for producing vectors containing a gene coding for an enzyme having reduced or deactivated feedback inhibition and to the use thereof for producing amino acids and nucleotide. Genes coding for feedback-inhibited enzymes are mutagenized in vitro, —ligated into vectors in a further step—the vectors are each transformed in a microorganism containing a metabolite sensor in a further step, which brings about the synthesis of a fluorescent protein at an increased metabolite concentration, —whereupon microorganisms exhibiting increased or maximum fluorescence are selected; and —the vectors that contain a gene coding for an enzyme having reduced or deactivated feedback inhibition are isolated from microorganisms having increased or maximum fluorescence.

## Background
BACKGROUND OF THE INVENTION
The invention relates to a method for producing vectors containing a gene coding for an enzyme having reduced or deactivated feedback inhibition and to the use thereof for producing amino acids and nucleotides
Amino acids are used in human medicine, in the pharmaceutical industry, the food industry, and particularly in animal nutrition. Amino acids are known to be produced by the fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum, and of enterobacteria, in particular Escherichia coli. Due to their great significance, efforts are continually underway to improve production methods. Improvements to the process can relate to measures concerning fermentation techniques, such as stirring and the supply of oxygen, or to the composition of the nutrient media, such as the sugar concentration during fermentation or processing into product form by ion exchange chromatography, for example, or to the intrinsic performance characteristics of the microorganism itself.
Mutagenesis, selection, and mutant selection methods are employed to improve the performance characteristics of these microorganisms. In this way, strains are obtained that are resistant to antimetabolites or are auxotrophic for regulatorily significant metabolites and produce amino acids. Likewise, recombinant DNA methods are used to improve strains of L-amino acid producing strains by amplifying individual amino acid biosynthesis genes and analyzing the effect on amino acid production.
The synthesis of amino acids in microorganism takes place by way of synthesis pathways, which are referred to as amino acid biosynthesis pathways, amino acid synthesis pathways, or synthesis pathways. These synthesis pathways are composed of individual steps, in which the amino acid is synthesized from precursors. These precursors are provided in the central metabolism. They include, for example, pyruvate, alpha-ketoglutarate, oxaloacetate, pentose phosphate, acetyl-CoA, erythrose phosphate, phosphoenolpyruvate or phosphoglycerate. Moreover NADPH2, NH4+ and reduced tetrahydrofolate are required to synthesize amino acids from precursors using the synthesis pathways. The synthesis of amino acids from precursors using synthesis pathways in coryneform bacteria and enterobacteria is known to those skilled in the art. This relates to the amino acids L-alanine, L-valine, L-leucine, L-asparagine, L-aspartate, L-lysine, L-methionine, L-threonine, L-isoleucine, L-histidine, L-glutamate, L-glutamine, proline, glycine, L-arginine, L-tryptophan, L-tyrosine, L-phenylalanine, L-serine and L-cysteine. The synthesis of amino acids has been studied particularly well in Escherichia coli and Corynebacterium glutamicum. 
The synthesis pathways consist of a series of reactions catalyzed by enzymes and are subject to strict regulations. Particularly strict regulation is achieved by feedback-resistant enzymes. In wild type strains, strict regulating mechanisms prevent the production of metabolic products, such as amino acids, beyond the subsistence level needed and the delivery thereof to the medium. From a manufacturer's view, the construction of strains overproducing organic-chemical compounds therefore entails the need to overcome these metabolic regulations. These feedback-resistant enzymes preferably catalyze input reactions of the synthesis pathways or branch points on the synthesis pathways. The regulation often takes place via the end product of the synthesis pathway, which is to say the amino acids L-alanine, L-valine, L-leucine, L-asparagine, L-aspartate, L-lysine, L-methionine, L-threonine, L-isoleucine, L-histidine, L-glutamate, L-glutamine, proline, glycine, L-arginine, L-tryptophan, L-tyrosine, L-phenylalanine, L-serine and L-cysteine.
Intermediates can also intervene in a regulating manner, such as O-acetylserine, O-acetylhomoserine, O-succinylserine, O-succinylhomoserine or adenosylmethionine. Regulation is such that, at an increased concentration of the listed amino acids or intermediates, the respective enzyme of the synthesis pathway, or the feedback-resistant enzymes of the synthesis pathway, are inhibited.
Examples of enzymes of synthesis pathways according to the prior art, formed in the cell by feedback-controlled formation, and the genes coding for the same, are shown in Table 1. In addition, the EC numbers are indicated, which characterize the respective reaction with respect to the substrates and products of the mechanism of the key reaction. In addition, the accession numbers for sequences of the regulated feedback-resistant enzymes of the wild type of E. coli and C. glutamicum are indicated. The nucleotide sequences of these genes and the coded polypeptide sequences are stored in public databases. For C. glutamicum, for example, in the National Center for Biotechnology Information (NCBI) database of the National Library of Medicine (Bethesda, Md., USA) under accession numbers NC_003450.2 and BX927148.1 to BX927157.1. The nucleotide sequences of these genes and the coded polypeptide sequences of E. coli have been described by Blattner et al. (Science 277: 1453-1462 (1997)) and stored in the National Center for Biotechnology Information (NCBI) database of the National Library of Medicine (Bethesda, Md., USA) under accession number NC_000913.2. Access is also possible via the database UniProtKB/Swiss-Prot European Molecular Biology Laboratory, Heidelberg.







TABLE 1







Examples of feedback-inhibited enzymes, the enzyme classification numbers


characterizing the same, the genes coding for the same, and the exemplary


accession numbers of the genes for E. coli and C. glutamicum














Accession number
Accession number


Enzyme
EC Number
Gene
E. coli
C. glutamicum





2-isopropylmalate synthase
EC 2.3.3.13
leuA
EG11226
YP_224548


Acetohydoxy acid synthase
EC 2.2.1.6
ilvN
EG10502
YP_225560.1


Acetohydoxy acid synthase
EC 2.2.1.6
ilvB
EG10494
YP_225561.1


Acetohydoxy acid synthase
EC 2.2.1.6
ilvI
EG10500
YP_225560.1


Acetohydoxy acid synthase
EC 2.2.1.6
ilvH
EG10499
YP_225561.1


Acetylglutamate synthase
EC 2.3.1.1
argA
EG10063
YP_225682.1


Anthranilate synthase
EC 4.1.3.27
trpD
EG11027
YP_227281.1


Anthranilate synthase
EC 4.1.3.27
trpE
EG11028
YP_227280.1


Asparagine synthetase B
EC 6.3.5.4
asnB
NP_415200.1
NC_006958.1


Aspartate transcarbamylase
EC 2.1.3.2
pyrB
NP_418666
Q8NQ38


Aspartate transcarbamylase
EC 2.1.3.2
pyrI
NP_418665.1
Q8NQ38


Aspartate kinase
EC 2.7.2.4
lysC
EG10550
P26512.2


Aspartate kinase
EC 2.7.2.4
metL
EG10590
P26512.2


Aspartate kinase
EC 2.7.2.4
thrA
EG10998
P26512.2


ATP-phosphoribosyl
EC 2.4.2.17
hisG
EG10449
Q9Z472.2


transferase


Carbamoyl phosphate
EC 6.3.5.5
carA
EG10134
Q8NSR1.1


synthetase


Carbamoyl phosphate
EC 6.3.5.5
carB
NP_414574.1
P58939.1


synthetase


Chorismate mutase I
EC 5.4.99.5
tyrA
EG11039
BAB98246.1


Chorismate mutase 11
EC 5.4.99.5
pheA
EG10707
YP_227138.1


Cysteine synthase
EC 2.5.1.47
cysK
NP_416909.1
CBV01938.1


Cysteine synthase
EC 2.5.1.47
cysM
NP_416916.1
CBF99279.1


D-3-phosphoglycerate
EC 1.1.1.95
serA
NP_417388.1
AEA48241.1


dehydrogenase


3-deoxy-D-arabino-
EC 4.1.2.15
aroG
NP_415275.1
CAC25920.1


heptulosonate-7-phosphate


synthase


Dihydrodipicolinate synthase
EC 4.2.1.52
dapA
NP_416973.1
CAA37940.1


Glutamate dehydrogenase
EC 1.4.1.4
gdh
NP_416275.1
NP_601279.1


Glutamate synthase
EC 1.4.1.13
gltB
NP_417679.2
YP_224481.1


Glutamate synthase
EC 1.4.1.13
gltD
NP_417680.1
YP_226992.1


Glutamine synthetase
EC 6.3.1.2
glnA
NP_418306.1
YP_226471.1


Glutamyl kinase
EC 2.7.2.11
proB
NP_414777.1
YP_226601.1


Homocysteine transmethylase
EC 2.1.1.14
metE
NP_418273.1
CAF19845.1


Homoserine O-
EC 2.3.1.46
metX
NP_417417.1
NP_600817.1


aceyltransferase


Homoserine O-
EC 2.3.1.46
metA
NP_418437.1
YP_225473.1


succinyltransferase


Homoserine dehydrogenase
EC 1.1.1.3
metL
EG10590
CAF19887.1


Homoserine dehydrogenase
EC 1.1.1.3
thrA
EG10998
NP_414543.1


Methionine synthase
EC 2.1.1.13
metH
NP_418443.1
NP_600723.1


Phosphoribosylpyrophosphate
EC 2.7.6.1
prs
NP_415725.1
YP_225235.1


synthetase


Prephenate dehydrogenase I
EC 1.3.1.12
pheA
NP_417090.1
CAF20922.1


Prephenate dehydrogenase II
EC 1.3.1.12
tyrA
EG11039
YP_227138.1


Pyrroline-5-carboxylate
EC 1.5.1.2
proC
NP_414920.1
NP_599858.1


reductase


Ribose 1,5-bisphosphokinase
EC 2.7.4.23
phnN
NP_418518.1
NP_600170


Serine acetyl(succinyl)
EC 2.3.1.30
cysE
NP_418064.1
NP_601761.1


transferase


Threonine ammonia-lyase
EC 4.3.1.19
ilvA
NP_418220.1
NP_601328.2


Tyrosine aminotransferase
EC 2.6.1.57
tyrB
NP_418478.1
NP_599471.2









Proceeding from the wild form of the feedback-regulated enzymes mentioned in Table 1 by way of example, there is a need to achieve deregulation of the enzyme activity so as to improve the production of target compounds, such as amino acids, nucleotides, amino acid derivatives or intermediates of the synthesis pathways.
It is known that such feedback-resistant enzymes, the regulation of which has been eliminated so as to contribute to improving the performance capability of microorganisms, is important, and feedback-resistant alleles carrying mutations as compared to the wild type have been described. For example, it is described in DE102008040352A1 and EP000002147972A1 that alleles of aroG coding for 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase led to improved formation of tryptophan. Moreover, a recombinant, L-lysine-producing bacterium is known, for example, containing an lysC allele with feedback-resistant aspartate kinase (EP0381527A1). In addition, a recombinant, L-phenylalanine-producing bacterium with feedback-resistant prephenate dehydrogenase is described (EP088424A2). Moreover, a leuA allele is described, which codes for a mutated isopropylmalate synthase and a microorganism producing L-leucine (EP000001568776B1). A mutant acetolactate synthase having a mutation in the IlvN subunit and a microorganism overproducing L-valine are described in EP07017918.9. An allele is also described for the enzyme acetylglutamate synthase, which is feedback-resistant to arginine as compared to the wild type enzyme, and strains that produce more L-arginine with the allele are described (U.S. Pat. No. 7,169,586 B2). Mutant alleles of phosphoribosyl pyrophosphate synthetase and methods for producing L-histidine are described (EP 1529839 A1). It is also known that L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine, and therefore the ability to produce L-histidine can also be effectively enhanced by introducing a mutation conferring resistance to feedback inhibition into ATP phosphoribosyl transferase (Russian patents nos. 2003677 and 2119536). Improved arginine production was achieved when the enzyme used was carbamoyl phosphate synthetase (EP000001026247A). The introduction of a cysE allele coding for a serine O-acetyltransferase having reduced feedback inhibition through L-cysteine also increased the production of cysteine (US6218168B1; Nakamori et al., 1998, Appl. Env. Microbiol. 64: 1607-1611; Takagi et al., 1999, FEBS Lett. 452: 323-327). A feedback-resistant CysE enzyme substantially decouples the production of O-acetyl-L-serine, the immediate precursor of L-cysteine, from the L-cysteine level of the cell.
Mutagenesis, selection, and mutant selection methods are employed to eliminate the control mechanisms and improve the performance characteristics of these microorganisms. In this way strains are obtained, which are resistant to antimetabolites, such as alpha-amino-beta-hydroxyvaleric acid (AHV), an analog of threonine, or are auxotrophic for regulatorily significant metabolites, and produce amino acids, such as L-threonine. Resistance to 5-methyl-DL-tryptophan (5-MT), a tryptophan analog, is characteristic of a strain producing L-tryptophan, for example (DE 102008040352 A1). In addition, strains that were resistant to 4-aza-D,L-leucine or 3-hydroxy-D,L-leucine, β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine and 5,5,5-trifluoroleucine included a feedback-resistant isopropylmalate dehydrogenase (EP 1568776A2, EP 1067191 A2). Strains are described having acetolactate synthases which exhibit reduced feedback inhibition through the use of the inhibitors sulfonylurea and imidazolinone (CA 2663711 A1). It has also been possible to obtain strains with argA alleles that are suitable for producing L-arginine by selecting slowly growing mutants (U.S. Pat. No. 7,169,586 B2). Likewise, arginine producers were obtained via the resistance-conferring substances 5-azauracil, 6-azauracil, 2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azacytosine, 6-azacytosine, arginine hydroxamate, 2-thiouracil and 6-azauracil (Japanese patent 49-126819). By utilizing analogs of pyrimidine, such as azidothymidine or azidodeoxyurldine, it is possible to obtain strains in which feedback-resistant enzymes, such as the aspartate transcarbamoylase of the shared synthesis pathway of L-histidine, purine and pyrimidine are mutated (U.S. Pat. No. 5,213,972). By utilizing analogs of lysine, such as S-(2-aminoethyl)-cysteine, it has been possible to obtain lysC alleles, or by utilizing analogs of methionine, such as alpha-methylmethionine, ethionine, norleucine, N-acetylnorleucine, S-trifluoromethylhomocysteine, 2-amino-5-heprenoit acid, selenomethionine, methionine sulfoximine, methoxine, 1-aminocyclopentane carboxylic acid, it has been possible to obtain strains with alleles that accumulate L-methionine at an increased level (EP 1745138 B1). Through the use of 1,2,4-triazole-D-alanine, it has been possible to eliminate the feedback inhibition of the HisG allele hisG13 and thereby obtain strains having enhanced performance characteristics (Mizukami T et al., Biosci Biotechnol Biochem. 1994 April, 58(4):635-8).
A major drawback in the existing production of enzymes having reduced feedback resistance is the use of analogs, such as alpha-amino-beta-hydroxyvaleric acid, 5-methyl-DL-tryptophan, 4-aza-D,L-leucine or 3-hydroxy-D,L-leucine, β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine, sulfonylurea, imidazolinone, 5-azauracil, 6-azauracil, 2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azacytosine, 6-azacytosine, arginine hydroxamate, 2-thiouracil, 6-azauracil, azidothymidine, azidodeoxyuridine, S-(2-aminoethyl)-cysteine, alpha-methyl-methionine, ethionine, norleucine, N-acetylnorleucine, S-trifluoromethylhomocysteine, 2-amino-5-heprenoit acid, selenomethionine, methionine sulfoximine, methoxine, 1-aminocyclopentane carboxylic acid, 1,2,4,-triazole-D-alanine and the isolation of mutants resistant to such analogs. Another major drawback of existing methods for producing feedback-resistant enzymes, and of the use thereof to enhance the performance capability of the microorganisms, is that many strains must be tested for enhanced performance capability following undirected mutagenesis and the use of analogs, since resistance to analogs can have a wide variety of causes, such as improved decomposition of the analog, or improved export of the analog, which can feign a feedback resistance and results in no new feedback-resistant enzymes being produced, and no strain having enhanced performance capability being present. Existing techniques for isolating the targeted production of feedback-resistant enzymes, which are directed to enhancing the performance capability of microorganisms for the production of amino acids, can therefore achieve the object only partially, incompletely or not at all.
SUMMARY OF THE INVENTION
It is therefore the object of the invention to create a method for producing vectors containing a gene coding for an enzyme having reduced or deactivated feedback inhibition, which allows feedback-resistant enzymes, or enzymes having reduced inhibition as compared to the wild type, and the deoxyribonucleic acid coding therefor, to be provided more reliably, more quickly, and better. Moreover, feedback-resistant enzymes, or enzymes having reduced inhibition, and the deoxyribonucleic acids coding for them, are to be provided.
The method according to the invention now makes it possible to provide or obtain feedback-resistant enzymes, or enzymes having reduced inhibition as compared to the wild type, and the deoxyribonucleic acid coding therefor and vectors containing this deoxyribonucleic acid. The method is more accurate, more reliable and faster than the methods according to the prior art, since the procedure does not feign feedback resistance, or a reduction in feedback resistance, but the same is established in direct causal relation with the methods that are carried out.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention will be explained hereinafter in the general form thereof.

    
    
        In a first step, the feedback-sensitive gene of the wild type of a microorganism, for example of a bacterium or a yeast, is mutagenized in vitro as the starting organism and ligated to a vector.
        As a result of the ligation approach, a microorganism is transformed, and a vector gene bank is obtained. The microorganism used for transformation is one that contains a metabolite sensor, which results in synthesis of a fluorescent protein at an increased metabolite concentration.
        In a subsequent step, microorganisms having the highest fluorescence are selected, and the vectors thereof are isolated. It is also possible to select microorganisms having fluorescence induced by the metabolite sensor, which is increased as compared to the starting organism containing the wild-type gene.
    
    


These method steps can be used to ascertain which mutant produces the highest, or increased, production of the desired product.
The vectors that are isolated according to this method contain genes coding for enzymes having reduced or deactivated feedback inhibition.
These genes contained in the vectors, which code for enzymes having reduced or deactivated feedback inhibition, can be used in methods known to a person skilled in the art for producing amino acids, nucleotides or intermediates. By way of example, the following method steps, which are known to a person skilled in the art, can be carried out for this purpose:

    
    
        The genes coding for enzymes having reduced or deactivated feedback inhibition can be introduced on the vector into the production strain, giving the same the ability to better produce the desired product.
        As an alternative, the genes coding for enzymes having reduced or deactivated feedback inhibition can be inserted in vectors that are already used to produce the desired product and result in an additional increase in the product formation.
        In a further embodiment, the genes coding for enzymes having reduced or deactivated feedback inhibition can be introduced into the chromosome of the production strain, giving this the ability to better produce the desired product.
    
    


A feedback-sensitive gene within the meaning of the invention shall be understood to mean a deoxyribonucleic acid coding for an enzyme in which feedback inhibition is caused. It does not matter whether the feedback inhibition is the result of the product that is formed or of an intermediate created during production.
The term “wild type” within the meaning of the invention shall be understood to mean a gene, or a gene from a strain, which has not yet been modified by way of the method according to the invention, or which already underwent a modification according to the invention, and the coding enzyme activity of which is to be further increased, which is to say which is to be used as the starting gene for the method according to the invention. This can be a naturally occurring gene, or a gene from a strain that was genetically modified, which is used as the starting material for the mutagenesis that is carried out according to the invention.
The mutagenization can preferably be carried out by way of undirected methods which are known from the prior art, such as by way of a faulty polymerase chain reaction, for example.
The starting organism from which the gene to be modified is procured can be any arbitrary microorganism containing a gene for a protein subject to feedback regulation or feedback inhibition, for example a bacterium, such as a Corynebacterium or an Enterobacterium, or a yeast, such as Saccharomyces cerevisiae. In particular Corynebacterium glutamicum or E. coli can be used as the starting organism.
By way of example, and without limitation, the organisms mentioned in Table 1, and the genes and enzymes thereof, can be used for modification according to the invention.
According to the invention, the amino acid sequences of feedback-controlled enzymes disclosed in Table 1, and the nucleic acid sequences of the genes coding for the same, serving as starting materials for the method according to the invention, also comprise such sequences that exhibit homology (at the amino acid level) or identity (at the nucleic acid level, exclusive of the natural degeneration) of greater than 70%, preferably 80%, more preferably 85% (based on the nucleic acid sequence) or 90% (also based on the polypeptides), preferably greater than 91%, 92%, 93% or 94%, more preferably greater than 95% or 96%, and particularly preferably greater than 97%, 98% or 99% (based on both types of sequences) with one of these sequences, as long as the mode of action or function and purpose of such a sequence are preserved. The term “homology” (or identity) as used herein can be defined by the equation H (%)=[1-V/X]×100, where H denotes homology, X is the total number of nucleobases/amino acids of the comparison sequence, and V is the number of different nucleobases/amino acids of the sequence to be examined based on the comparison sequence. In any case, the term ‘nucleic acid sequences’ coding for polypeptides encompasses all sequences that appear possible according to the proviso of degeneration of the genetic code. The same also applies to all other possible genes or enzymes that are to be modified according to the invention and not listed in Table 1.
The identity, in percent, with the amino acid sequences indicated in this description by the accession number (Accession No.) in Table 1 can be readily ascertained by a person skilled in the art using methods known in the prior art. A suitable program that can be used according to the invention is BLASTP (Altschul et al. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25(17): 3389-3402).
According to the invention, the nucleic acid sequences of the gene coding for feedback-sensitive enzymes disclosed in Table 1 also comprise nucleic acid sequences hybridized with those listed. A person skilled in the art can find instructions on hybridization, among other things, in “The DIG System Users Guide for Filter Hybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology 41: 255-260 (1991)). The hybridization takes place under stringent conditions, which is to say only hybrids are formed, in which the probe, for example the nucleotide sequence complementary to the gene, and the target sequence, which is to say the polynucleotides treated with the probe, are at least 70% identical. It is known that the stringency of the hybridization process, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is generally carried out at relatively low stringency in comparison with the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996). For example, a buffer corresponding to 5×SCC buffer at a temperature of approximately 50° C. to 68° C. can be used for the hybridization reaction. Probes can also hybridize with polynucleotides having an identity lower than 70% with the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by lowering the salt concentration to 2×SCC, and optionally subsequently 0.5×SCC (The DIG System User's Guide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany, 1995), wherein the temperature is set to approximately 50° C. to 68° C., approximately 52° C. to 68° C., approximately 54° C. to 68° C., approximately 56° C. to 68° C., approximately 58° C. to 68° C., approximately 60° C. to 68° C., approximately 62° C. to 68° C., approximately 64° C. to 68° C., or approximately 66° C. to 68° C. The washing steps are preferably carried out at temperatures of approximately 62° C. to 68° C., preferably 64° C. to 68° C., or approximately 66° C. to 68° C., and particularly preferably 66′C to 68° C. Optionally, it is possible to lower the salt concentration to a concentration corresponding to 0.2×SCC or 0.1×SSC. By incrementally increasing the hybridization temperature in steps of approximately 1 to 2° C. from 50° C. to 68° C., it is possible to isolate polynucleotide fragments coding for feedback-sensitive enzymes, which have, for example, at least 70%, or at least 80%, or at least 90% to 95%, or at least 96% to 98%, or at least 99% identity with the sequence of the probe that is used. Further hybridization instructions are available on the market in the form of so-called kits (such as DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558). Analogously, it is also possible to use other amino acid sequences not mentioned in Table 1, and genes coding for the same, in hybridized form as starting materials for the feedback-resistant genes modified according to the invention.
All known plasmid bodies, transposons, insertion elements or phages can be used as raw materials for the vectors, in which the mutagenized genes were inserted. By way of example, and without limitation, vectors can be used that are derived from pACYC184 (Bartolome et al.; Gene 102, 75-78 (1991)), or pTrc99A (Amann at al.: Gene 69: 301-315 (1988)) or pSC101 (Vocke and Bastia, Proceedings of the National Academy of Sciences USA 80 (21): 6557-6561 (1983)). Such genetic systems are described in the patent specifications of U.S. Pat. No. 4,822,738, U.S. Pat. No. 5,804,414 and U.S. Pat. No. 5,804,414, for example. Similarly, it is also possible to use vectors such as pZ1, pXZ10142, pEKEx2, pEKEx3, or pEC-t19mob2 (cited in “Handbook of Corynebacterium glutamicum” (publisher: L. Eggeling and M. Bott)), which are used for the method according to the invention.
Known metabolite sensors, such as pSenLys, pSenArg, pSenSer, pSenOAS or pJC1-lrp-bmF-eyfp, can be used as metabolite sensors. The metabolite sensor includes a gene sequence coding for an autofluorescent protein, wherein the expression of the autofluorescent protein is dependent on the intracellular concentration of a particular metabolite. The expression of the gene sequence coding for the autofluorescent protein is controlled as a function of the intracellular concentration of the particular metabolite at the transcription level. Depending on the intracellular concentration of the respective metabolite, more or less mRNA is therefore produced, which can be translated by the ribosomes, forming the autofluorescent protein.
The microorganism used for transformation can be any arbitrary microorganism. Bacteria, yeasts or enterobacteria, such as E. coli, Corynebacterium glutamicum or Saccharomyces cerevisiae, can be mentioned by way of example. The microorganism used for transformation is a microorganism that contains a metabolite sensor, which results in synthesis of a fluorescent protein at a metabolite concentration that is higher than the metabolite concentration present in the starting organism.
Deposit of the following biological material (i.e., plasmids containing named metabolite sensor(s)) has been made at Leibuiz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraβe 7B, 38124 Braunschweig, GERMANY, a depository accredited according to the Budapest Treaty, in a manner by which such biological material is readily and irrevocably available to the public: pSenArg (Accession Number DSM 32346) deposited on Jul. 22, 2016; pSenLys (Accession number 32347) deposited on Jul. 22, 2016; pSenSer (Accession number 32351) deposited on Jul. 22, 2016; and pSenOAS (Accession number 32350) deposited on Jul. 22, 2016.
For transformation, the vectors containing genes that code for enzymes having reduced or deactivated feedback inhibition can be introduced. These can be obtained directly from selection by way of metabolite sensors.
The embodiment, in which the genes coding for enzymes having reduced or deactivated feedback inhibition can be introduced in other vectors that are already used to produce the desired product, can involve vectors that are already used to produce the desired product and that already contain genes that enhance the production characteristic of the microorganism used for production.
In the embodiment, in which the genes coding for enzymes having reduced or deactivated feedback inhibition can be introduced into the chromosome of the production strain, which is then given the ability to better produce the desired product, the insertion can take place at any arbitrary position on the chromosome according to methods known to a person skilled in the art.
The production strain used can be any one of the strains used in biotechnology, in which plasmids are introduced in the mutagenized genes coding for feedback-resistant enzymes, or enzymes having reduced feedback sensitivity. Coryneform bacteria, enterobacteria or yeasts, such as E. coli, Corynebacterium glutamicum or Saccharomyces cerevisiae, can be mentioned by way of example.
The method according to the invention is generally used to provide any enzyme that is of interest and the deoxyribonucleic acid coding therefor, the inhibition of which is either completely eliminated or, at a minimum, reduced.
By way of example, and without limitation, the enzymes mentioned in Table 1 and every deoxyribonucleic acid coding for them can be mentioned as materials to be modified according to the invention.
The method according to the invention can be used to enhance the production of amino acids and the derivatives thereof, nucleotides or intermediates of synthesis pathways as products compared to the prior art. The method according to the invention allows the production of amino acids and the derivatives thereof, nucleotides or Intermediates of synthesis pathways, which were not produced, or produced only to a low degree, by the corresponding microorganisms prior to the modification according to the invention.
The nucleic acids and enzymes obtained by the method according to the invention can be used for producing these desired products and are more active with respect to the function thereof. The invention will be described in more detail hereafter by way of example, and without limitation.
The invention relates to alleles of genes coding for enzymes having reduced or deactivated feedback inhibition over the wild form, which are obtained by way of metabolite sensors, wherein the wild type alleles are present in plasmid-encoded manner, which after in vitro mutagenesis are introduced in microorganisms, preferably coryneform bacteria and enterobacteria, containing the metabolite sensor, and individual cells are generated or isolated, which are recognized as cells containing an enzyme that is no longer regulated.
Metabolite sensors allow the direct detection of increased intracellular amino acid concentrations in individual cells of coryneform bacteria, such as Coryneform bacteria, and in enterobacteria, such as Escherichia coli (WO2011138006). Metabolite sensors moreover allow the detection of increased intracellular amino acid concentrations of L-valine, L-leucine, L-asparagine, L-lysine, L-methionine, L-threonine, L-isoleucine, L-histidine, L-glutamate, L-glutamine, proline, glycine, L-arginine, L-tryptophan, L-tyrosine, L-phenylalanine, L-serine and L-cysteine, and particularly preferably those of L-histidine, L-arginine, L-lysine and L-leucine.
Metabolic sensors also allow the detection of increased concentrations of intermediates, such as intermediates of the amino acid or nucleotide synthesis, such as O-acetylserine, O-acetylhomoserine, cystathionine, orotidine-5′-phosphate, 5-phosphoribosyl diphosphate, or inosine-5′-phosphate.
The method for producing the enzymes modified according to the invention is characterized in that the genes or alleles in which the feedback inhibition is to be eliminated or reduced are present in a vector-encoded, and preferably plasmid-encoded, manner and are not chromosomally encoded. The genes or alleles of the enzymes that are to be modified according to the invention are in particular those of the amino acid synthesis pathways leuA, ilvN, ilvB, ilvI, ilvH, argA, trpD, trpE, asnA, asnB, pyrB, pyrI, lysC, metL, thrA, hisG, carA, carB, tyrA, pheA, cysK, cysM, serA, aroG, dapA, gdh, gltB, gltD, gInA, proB, metE, metX, metA, metL, gnd, zwf, thrA, metH, prs, pheA, tyrA, proC, prs, cysE, ilvA, tyrB, preferably lysC, hisG, argB, cysE and leuA. These genes are mutagenized in vitro according to known methods. The nucleotide sequences of these genes and the coded polypeptide sequences of C. glutamicum are described in EP-A-1108790 and are also stored in the National Center for Biotechnology Information (prs) database of the National Library of Medicine (Bethesda, Md., USA) under accession numbers NC_003450.2 and BX927148.1 to BX927157.1. The nucleotide sequences of these genes and the coded polypeptide sequences of E. coli have been described by Blattner et al. (Science 277: 1453-1462 (1997)) and stored in the National Center for Biotechnology Information (NCBI) database of the National Library of Medicine (Bethesda, Md., USA) under accession number NC_000913.2. The peptides in which, according to the invention, feedback inhibition is to be reduced or deactivated also include those that are at least 90 to 95%, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the polypeptides of the enzymes LeuA, IlvN, IlvB, Ilvl, IlvH, ArgA, TrpD, TrpE, AsnA, AsnB, PyrB, Pyrl, LysC, MetL, ThrA, HisG, CarA, CarB, TyrA, PheA, CysK, CysM, SerA, AroG, DapA, Gdh, GltB, Gnd, Zwf, OpcA, GltD, GlnA, ProB, MetE, MetX, MetA, MetL, Gnd, Zwf, ThrA, MetH, Prs, PheA, TyrA, ProC, Prs, CysE, IlvA, TyrB, preferably LysC, HisG, ArgB, CysE, and LeuA. The present invention also relates to the nucleotide sequences ilvN, ilvB, ilvI, ilvH, argA, trpD, trpE, asnA, asnB, pyrB, pyrI, lysC, metL, thrA, hisG, carA, carB, tyrA, pheA, cysK, cysM, serA, aroG, dapA, gdh, gltB, gftD, gInA, proB, metE, metX, metA, metL, gnd, zwf, thrA, metH, prs, pheA, tyrA, proC, prs, cysE, ilvA, tyrB, preferably lysC, hisG, argB, cysE and leuA, and such sequences exhibiting homology of at least 70%, preferably 80%, more preferably 85% (based on the nucleic acid sequence) or 90%, preferably greater than 91%, 92%, 93% or 94%, more preferably greater than 95% or 96%, and particularly preferably greater than 97%, 98% or 99% with one of these sequences, as long as the mode of action or function and purpose of such a sequence are preserved or are identical thereto.
To introduce the site-undirected mutations in the plasmid-encoded genes of the enzymes, mutagenesis, which is preferably in vitro, is carried with the aid of a faulty polymerase chain reaction (PCR) and an amplification technique. To this end, the gene to be mutated is first subjected to PCR using a polymerase, which incorrectly inserts individual nucleotides in the synthesized genes compared to the wild form as a function of the conditions of the reaction (Tindall, K. R. and T. A. Kunkel, Fidelity of DNA synthesis by the Thermus aquaticus DNA Polymerase. Biochemistry, 1988. 27(16): p. 6008-13). A common variant of this method includes the use of manganese(II) ions or of nucleotide analogs in the PCR batch (Cadwell R. C et al. (1992) PCR Methods Appl. 2:28-33./Leung D. W. et al. (1989) Techniques 1:11-15). These techniques for introducing mutations are referred to as “Error-prone PCR (epPCR)” (Labrou N E. Random mutagenesis methods for in vitro directed enzyme evolution. Curr Protein Pept Sci. 2010; 11:91-100). The mutations can be point mutations, for example; and it is possible to generate substitutions, deletions or insertions, for example, by way of polymerase. The mutation rate ranges between 1 and 40 mutations per 1 kb, and preferably between 1 and 5 mutations per 1 kb.
The vectors, preferably plasmids containing the mutations obtained according to the invention in genes of the enzymes, are subsequently introduced in a microorganism, such as E. coli, C. glutamicum or Saccharomyces cerevisiae, by way of transformation. The term transformation encompasses all methods for transferring polynucleotides, in particular DNA, in a desired microorganism. This includes, among other things, the use of isolated DNA during the transformation, electrotransformation or electroporation, transfer by cell contact, as in the case of conjugation, or transfer of DNA by way of particle bombardment.
During the subsequent method step of the method according to the invention, individual transformed cells in the cell suspension having an increased intracellular concentration of metabolites, as compared to the wild form of the metabolite detectable by the metabolite sensor, are identified by detection of the intracellular fluorescence. For this purpose, the cell suspension is exposed to electromagnetic radiation at that frequency which excites the autofluorescent protein of the metabolite sensor to emit light. Autofluorescent proteins are preferably proteins that are read from gene sequences which code for fluorescent proteins, for example of the Aequorea species, such as the green fluorescent protein (GFP), and variants thereof fluorescing in a different wavelength range (such as yellow fluorescent protein, YFP: blue fluorescent protein, BFP; cyan fluorescent protein, CFP) or the fluorescence of which is enhanced (enhanced GFP or EGFP, or EYFP, EBFP or ECFP). According to the invention, moreover it is also possible to use gene sequences that code for other autofluorescent proteins, such as DsRed, HcRed, AsRed, AmCyan, ZsGreen, AcGFP, ZsYellow, as they are known from BD Biosciences, Franclin Lakes, USA. The particularly preferred autofluorescent protein is EYFP or the gene coding therefor.
According to one particular embodiment of the method according to the invention, thereafter, and preferably immediately after identification of the cells, a further method step takes place, in which the identified cells are separated from the cell suspension, wherein this separation is preferably carried out by way of flow cytometry (FACS=fluorescence-activated cell sorting), most particularly preferably by way of high-throughput flow cytometry (HT-FACS=high throughput fluorescence-activated cell sorting). Details regarding the analysis of cell suspensions by way of flow cytometry can be found in Sack U, Tamok A, Rothe G (publisher): Zelluläre Diagnostik. Grundlagen, Methoden und klinische Anwendungen der Durchflusszytometrie (Cellular Diagnostics. Fundamentals, Methods and Clinical Applications of Flow Cytometry), Basel, Karger, 2007, pages 27-70, for example.
By way of the method according to the invention, it is thus possible to deliberately isolate those cells, in which feedback-resistant enzymes result in an increased intracellular concentration of a particular metabolite, in a cell suspension in which transformants are present, which contains a gene bank of plasmid-encoded enzymes.
The invention relates to use of feedback-resistant enzymes or of enzymes having reduced feedback sensitivity thus obtained, and the use of the genes coding for the same, to enhance the performance characteristics of microorganism. This is achieved in that vectors obtained by the method, for which hereinafter plasmids are listed here by way of example, and without limitation, and that code for feedback-resistant enzymes or for enzymes having reduced feedback sensitivity, are introduced directly into the microorganism. For this purpose, the plasmids are isolated according to known methods, and the microorganism to be enhanced is transformed with these plasmids. The term transformation encompasses all methods for transferring polynucleotides, in particular DNA, into a desired bacterium. This includes, among other things, the use of isolated DNA during the transformation, electrotransformation or electroporation, transfer by cell contact, as in the case of conjugation, or transfer of DNA by way of particle bombardment.
In a further embodiment, the genes that are obtained by the method and code for feedback-resistant enzymes or for enzymes having reduced feedback sensitivity are initially introduced into other plasmids, which already contain additional genes, for example, that already enhance the performance characteristic of the microorganism, or which also contain a replication origin that allows the plasmid to be replicated in the microorganism to be enhanced. Such plasmids are known to those skilled in the art, such as plasmid vectors that can be replicated in Enterobacteriaceae, such as cloning vectors derived from pA-CYC184 (Bartolomé et al.; Gene 102: 75-78 (1991)), pTrc99A (Amann et al.; Gene 69: 301-315 (1988)) or pSC101 derivatives (Vocke und Bastia; Proceedings of the National Academy of Sciences USA 80(21): 6557-6561 (1983)) or plasmids that replicate in Corynebacteria and related organisms and are listed in the Handbook of Corynebacterium glutamicum (eds. Eggeling and Bott), in Section 23 on pages 535-66, or in EP 1097998 B1, or the commercially available plasmids for Bacillus strains available from MoBiTech GmbH, Lotzestrasse 22a, 37083 Goettingen or those described for Lactobacillus (Wang T T, Lee B H, 1997, Crit Rev Biotechnol. Plasmids in Lactobacillus. 17(3):227-72) or commercially available plasmids (MoBiTech GmbH, Lotzestrasse 22a, 37083 Goettingen).
In a further embodiment, the genes that are obtained by the method and code for feedback-resistant enzymes or for enzymes having reduced feedback sensitivity are introduced into the genome of the microorganism that is to be enhanced. This is described by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) or in WO03/040373. In this method, the gene is cloned into a plasmid vector, which is not able to replicate in the host, the performance characteristics of which are to be enhanced. Possible vectors include, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schafer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega Corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; U.S. Pat. No. 5,487,993), pCR®Blunt (Firma Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Spratt at al., 1986, Gene 41: 337-342). The plasmid vector coding for the feedback-resistant enzyme, or for the enzyme having reduced feedback sensitivity, and optionally including the expression and/or regulation signals, and the boundary regions of the genes, are subsequently transferred in the desired strain, the performance characteristic of which is to be enhanced by way of conjugation or transformation. The conjugation method is described in Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)), for example. Methods for transformation are described in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican und Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiology Letters 123, 343-347 (1994)), for example. Following homologous recombination by way of a “crossover” event, the resulting strain contains a copy of the heterologous gene, including the plasmid vector, at the desired gene chromosomal locus of Corynebacterium glutamicum or E. coli, for example, which was predetermined via the homologous nucleotide sequences on the plasmid. By way of a second suitable “crossover” event in the target gene or in the target sequence bringing about excision, the insertion of only the gene of the feedback-resistant enzyme, or of the enzyme having reduced feedback sensitivity, is achieved.
The invention further relates to enzymes having reduced or deactivated feedback inhibition and the genes coding therefor, which result in an increased intracellular concentration of certain metabolites, in particular amino acids such as L-valine, L-leucine, L-asparagine, L-lysine, L-methionine, L-threonine, L-isoleucine, L-histidine, L-glutamate, L-glutamine, proline, L-arginine, L-tryptophan, L-tyrosine, L-phenylalanine, L-serine and L-cysteine. The invention relates in particular to enzymes of L-lysine synthesis, and most particularly also feedback-resistant aspartate kinases resulting in L-lysine. The invention further relates to enzymes having reduced or deactivated feedback inhibition and genes coding therefor, which result in an increased concentration of intermediates of the amino acid or nucleotide synthesis, such as O-acetylhomoserine, cystathionine, orotidine-5′-phosphate, 5-phosphoribosyl diphosphate or inosine-5′-phosphate.
In this way, by way of example, it is possible to obtain mutations in the aspartate kinase gene lysC at nucleotide positions 60, 61, 233, 556 and 982 as indicated in SEQ. ID. NO. 9, with the amino acid exchanges 20, 21, 78, 186, 328 as indicated in SEQ. ID. NO. 10, or at nucleotide positions 60, 61, 932, 1196 as indicated in SEQ. ID. NO. 11, with the amino acid exchanges at positions 20, 21, 311, 399 as indicated in SEQ. ID. NO. 12, or at nucleotide 61 as indicated SEQ. ID. NO. 13, with the amino acid exchanges at positions 21 as indicated in SEQ. ID. NO. 14, or at nucleotide positions 61, 833, 839 as indicated in SEQ. ID. NO. 15, with the amino acid exchanges at positions 21, 278, 280 as indicated in SEQ. ID. NO. 16, or at nucleotide positions 61, 833, 839, 1172 as indicated in SEQ. ID. NO. 33, with the amino acid exchanges at positions 21, 278, 280, 391 as indicated in SEQ. ID. NO. 34, or at nucleotide positions 61 and 932 as indicated in SEQ. ID. NO. 17, with the amino acid exchanges at positions 21 and 311 as indicated in SEQ. ID. NO. 18, or at nucleotide positions 61, 932, 1196 as indicated in SEQ. ID. NO. 19, with the amino acid exchanges at positions 21, 311, 399 as indicated in SEQ. ID. NO. 20, or at nucleotide positions 61 and 1094 as indicated in SEQ. ID. NO. 21, with the amino acid exchanges at positions 21 and 365 as indicated in SEQ. ID. NO. 22, or at nucleotide positions 61, 908 as indicated in SEQ. ID. NO. 29, with the amino acid exchanges at positions 21, 303 as indicated in SEQ. ID. NO. 30, or at nucleotide positions 715 as indicated in SEQ. ID. NO. 31, with the amino acid exchanges at positions 239 as indicated in SEQ. ID. NO. 32, or at nucleotide positions 71, 932, as indicated in SEQ. ID. NO. 23, with the amino acid exchanges at positions 24, 311 as indicated in SEQ. ID. NO. 24, or at nucleotide positions 205, 370, 631, 680, 703, 1049, 1120, as indicated in SEQ. ID. NO. 25, with the amino acid exchanges at positions 69, 124, 211, 227, 235, 350, 374 as indicated in SEQ. ID. NO. 26, as nucleotide positions 476, 1010, 1021 as indicated in SEQ. ID. NO. 27, and with the amino acid exchanges at positions 159, 337, 341 as indicated in SEQ. ID. NO. 28.
It is also possible in each case for only one of the nucleotide and amino acid exchanges per allele and enzyme to be present, or for a plurality to be present, which is to say up to seven, as indicated in SEQ. ID. NO. 25 and SEQ. ID. NO. 26.
Disclaimer:
The invention does not relate to mutations that are known from the prior art listed below, such as mutation lysC A279T described in the application JP 1994062866-A (sequence 1), or lysC A279V from JP 1994261766-A (sequence 3), lysC S301 F from JP 1994261766-A (sequence 4), lysC T3081 from JP 1994261766-A (sequence 5), lysC G345D (Follettie and Sinskey, database entry L16848), lysC R320G, lysC G345D (Jetten et al., database entry L27125), lysC S301 F (U.S. Pat. No. 3,732,144), lysC S381F (EP0435132), lysC S317A (U.S. Pat. No. 5,688,671 (sequence 1)) and also lysC T3801 (WO 01/49854).
The invention also relates to other amino acid exchanges at the positions identified by metabolite sensors and at comparable positions of the enzymes. In the aspartate kinase enzyme of SEQ. ID. NO. 28, for example, where Val can be exchanged with Gly at position 159, preferably any other proteinogenic amino acid is also present, such as Lys, Asn, Arg, Ser, Thr, Ile, Met, Glu, Ala, Val, Gln, His, Pro, Leu, Tyr. Trp, Cys or Phe. In the same enzyme, Asn is exchanged with Ser at position 337, and any other proteinogenic amino acid may also be present at this position, such as Lys, Asn, Arg, Thr, Ile, Met, Glu, Asp, Ala, Val, Gin, His, Pro, Leu, Tyr, Trp, Cys or Phe, or any other proteinogenic amino acid such as Lys, Arg, Ser, Thr, Ile, Met, Glu, Asp, Ala, Val, Gln, His, Pro, Leu, Tyr, Trp, Cys or Phe may be present at position 341 instead of the exchange of Asp with Asn.
According to the invention, the expression “comparable position” shall be understood to mean a position that, by way of comparison of the starting sequence with the comparison sequence using a sequence comparison program (BLAST, Altschul et al. J. Mol. Biol. 1990, 215, 403-410) at the considered position of the starting sequence, supplies an amino acid position in the comparison sequence that differs from the position to be compared by more than ±5, preferably ±4, further preferably ±3, still further preferably ±2, extremely preferably ±1, and most extremely preferably by no position.
The invention will be described in greater detail hereafter based on non-limiting examples.


Example 1
Production of a cell according to the invention based on the first embodiment using the example of a cell in which a gene sequence coding for an enzyme is mutated and selected, so that feedback-resistant enzymes and cells according to the invention containing feedback-resistant enzymes having enhanced performance characteristics are obtained, the enzyme being aspartate kinase.
a) Construction of the Vector pUC18-lysC and Error-Prone PCR of the Aspartate Kinase Gene lysC
Using the primer pairs rspl (SEQ. ID. NO. 1) and univ (SEQ. ID. NO. 2) as well as chromosomal DNA of the wild type of C. glutamicum ATCC 13032 as templates, the lysC gene coding for the enzyme aspartate kinase was amplified.









(SEQ. ID. NO. 1):



GATGGATCCGTGGCCCTGGTCGTACAGAAATATGG



 



(SEQ. ID. NO. 2):



GATGTCGACTTAGCGTCCGGTGCCTGCATAAACG






The chromosomal DNA of C. glutamicum was isolated as described in Tauch et al. (Tauch et al., 1995, Plasmid 33:168-179). The amplificate was treated with BamHI and SalI and ligated with likewise treated pUC18, and pUC18-lysC was obtained. 10 ng pUC18-lysC was used as the template in each reaction of the error-prone PCR for inserting the mutations, as was 0.1 to 0.8 mM Mn2+, wherein at the lower concentration of below <0.2 mM Mn2+ with Mg2+ an overall concentration of at least 0.2 mM was adjusted. In each reaction, x μl Taq polymerase from Fermentas (Catalog No.: EP0401) was added. The polynucleotides









(SEQ. ID. NO. 3): CACAGGAAACAGCTATGACCATG



 



(SEQ. ID. NO. 4): CGCCAGGGTTTTCCCAGTCACGAC






were used as primers. The reactions were incubated for 30 minutes. Thereafter, the reaction products were treated with BamHI and SalI and ligated to the likewise treated vector pSen-LysTK(b)-lysC. The ligation products were used to transform E. coli DH5amcr (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649). So as to construct the vector pSenLysTK(b)-lysC, the gene lysC was amplified with the preceding 520 bp using the primer pairs LysC-PSLTKC-fw (SEQ. ID. NO. 5) and LysC-PSLTKC-rv (SEQ. ID. NO.6) as well as chromosomal DNA of the wild type of C. glutamicum ATCC 13032 as templates. The amplificate was cleaved with XhoI and SalI and ligated to pSenLysTK (WO2011138006) cleaved in SalI.








(SEQ. ID. NO. 5):


GCCCTCGAGAAAACAAAAGGCTCAGTCGGAAGACTGGGCCTTTTGTTTTG


 


GTACCAGCGGCAGCGTGAACATC


 


(SEQ. ID. NO. 6):


GCCGTCGACACGGAATTCAATCTTACGGCCTGCGGAACG






The E. coli DH5amcr cells transformed with the ligation products were plated out on LB agar plates (Lennox, 1955, Virology, 1:190) containing 40 mg per liter kanamycin.
b) Preparation of the lysC Gene Bank and Transformation of Corynebacterium glutamicum 
Following overnight incubation of the E. coli DH5amcr cells transformed with the ligation products, LB agar plates were washed with 1 ml 0.9% NaCl and the plasmid DNA was prepared using ordinary methods (Sambrook et al., Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA). This plasmid DNA preparation was used to transform electrocompetent cells of C. glutamicum ATCC 13032, as is described in Kirchner, O. and Tauch, A. (J Biotechnol. 2003 Sep. 4; 104(1-3):287-99). The transformed cells were regenerated for 2 hours, then 25 mg per liter kanamycin was added, and 1:10 dilutions of this suspension were incubated for 15 hours in CGXII medium. The CGXII medium is described in Keilhauer et al. in J Bacteriol. 1993 September; 175(17):5595-603.
c) Selection of lysC Muteins
So as to identify and separate the cells with mutated aspartate kinases, the cell suspension was adjusted in CGXII medium to an optical density value of less than 0.1 and directly supplied to the FACS ARIA II high-speed cell sorter (Becton Dickinson GmbH, Tullastr. 8-12, 69126 Heidelberg). The analysis was carried out using excitation wavelengths of 488 and 633 nm, and the detection was carried out at emission wavelengths of 530±15 nm and 660±10 nm at a test pressure of 70 psi. The data was analyzed by way of the software Version BD DIVA 6.1.3 associated with the device. The sheath fluid used was BD FACSflow. Electronic gating was adjusted based on the forward and backward scatter so as to exclude non-bacterial particles. So as to sort EYFP-positive cells, the next stage of electronic gating was selected so as to exclude non-fluorescent cells. Fluorescent cells were sorted on Petri dishes containing Brain Heart Infusion Agar (Difco).
Example 2
Production of a cell according to the invention with feedback-resistant enzymes having enhanced performance characteristics, the enzyme being aspartate kinase and new aspartate kinase sequences.
a) Lysine Formation from Isolated Cells Using Mutated Aspartate Kinase as the Enzyme.
EYFP-positive individual cells sorted on Petri dishes were inoculated in 0.75 ml CGXII medium in microtiter plates of the Flowerplate type. The Flowerplates were procured from m2p-labs GmbH (Aachen) and incubated in a Microtron high-capacity microplate incubator (Infors AG) overnight using a shaking speed of 990 rpm and a shaking radius of 3 mm. Thereafter, a new CGXII medium was inoculated in microtiter plates of the Flowerplate type, and samples were removed after 48 for lysine determination.
The lysine determination was carried out as o-phthaldialdehyde derivative by way of high-pressure liquid chromatography using an UHPLC 1290 Infinity system (Agilent) on a Zorbax Eclipse AAA C18 3.5 micron 4.6×75 mm reversed-phase column and a fluorescence detector. The eluent used was a gradient of 0.01 M Na borate pH 8.2 with increasing methanol concentration, and the detection of the fluorescent isoindole derivatives was carried out at an excitation wavelength of 230 nm and an emission wavelength of 450 nm. The L-lysine values shown in Table 2 were determined, which show an improvement in the L-lysine production compared to the starting strain.







TABLE 2







Strains in which the gene sequence coding for aspartate kinase


were mutated with a plasmid, and which were selected for enhanced


performance characteristics by way of the metabolite sensor,


and excrete L-lysine at an increased rate.










Strain
L-Lysine (mM)














Starting strain ATCC13032
0.2



Strain lysC-ls42
21.1



Strain lysC-ss77
44.9



Strain lysC-ss12
20.0



Strain lysC-is90
39.0



Strain lysC-ls2
24.9



Strain lysC-ss92
44.3



Strain lysC-ls15
27.1



Strain lysC-ls57
29.5



Strain lysC-ss91
14.0



Strain lysC-ls1
19.7



Strain lysC-ls8
23.0



Strain lysC-ss63
32.5



Strain lysC-ls56
30.9










b) Determination of the Sequences of the Aspartate Kinases

A colony PCR was carried out on the strains shown in Table 2, as described in Lee and Cooper (Improved screen for bacterial colonies, Biotechniques 18(2):225-226). For this purpose, the following two primers were used:









(SEQ. ID. NO. 7): TGAGACGCATCCGCTAAAGCC



 



(SEQ. ID. NO. 8): ATCTTACGGCCTGCGGAACGTG






The amplificates were purified with the QIAquick PCR Purification Kit from Qiagen (Catalog No. 28104) and sequenced by the company GATC. The resulting DNA sequences were then analyzed using known algorithms or the GCG sequence analysis program from Butler (Methods of Biochemical Analysis 39, 74-97 (1998)), the FASTA algorithm from Pearson and Lipman (Proceedings of the National Academy of Sciences USA 85, 2444-2448 (1988)) or the BLAST algorithm from Altschul et al. (Nature Genetics 6, 119-129 (1994)) and compared to the sequence entries present in publicly accessible databases (EMBL, Heidelberg, Germany; NCBI, Bethesda, Md., USA).
The sequence identification numbers (SEQ. ID. NO.) of lysC alleles from the respective strains mentioned in Table 2 which contain the plasmids with the lysC alleles in pSenLysTK(b) are indicated in Table 3. In addition, the sequence identification numbers (SEQ. ID. NO.) of the aspartate kinase polypeptide sequences are also listed there. Furthermore the nucleotide exchanges of the lysC alleles with information as to the position in the polynucleotide sequence are listed, as are the amino acid exchanges of the lysC polypeptides with information as to the position in the polypeptide sequence.







TABLE 3







Nucleotide sequences and polypeptide sequences of the enzyme aspartate


kinase, which result in enhanced performance characteristics.














Nucleotide


Amino acid



Allele
exchange(s)

Polypeptide
exchange(s)
















SEQ. ID.
lysC-ls42
a60c, a61g, a233g,
SEQ. ID.
LysC-ls42
R20S, N21D, N78S,


NO. 9

a556g, a982g
NO. 10

I186V, K328E


SEQ. ID.
lysC-ss77
a60c, a61g, c932t,
SEQ. ID.
LysC-ss77
R20S, N21D, T311I,


NO. 11

q1196t
NO. 12

R399L


SEQ. ID.
lysC-ss12
a61g
SEQ. ID.
LysC-ss12
N21D


NO. 13


NO. 14


SEQ. ID.
lysC-ls90
a61g, a833g, c839t
SEQ. ID.
LysC-ls90
N21D, E278G,


NO. 15


NO. 16

A280V


SEQ. ID.
lysC-ss92
a61g, c932t
SEQ. ID.
LysC-ss92
N21D, T311I


NO. 17


NO. 18


SEQ. ID.
lysC-ls15
a61g, c932t, g1196t
SEQ. ID.
LysC-ls15
N21D, T311I,


NO. 19


NO. 20

R399L


SEQ. ID.
lysC-ls57
a61g, t1094a
SEQ. ID.
LysC-ls57
N21D, M365K


NO. 21


NO. 22


SEQ. ID.
lysC-ls8
a71g, c932t
SEQ. ID.
LysC-ls8
E24G, T311I


NO. 23


NO. 24


SEQ. ID.
lysC-ss63
c205t, g370c, a631g,
SEQ. ID.
LysC-ss63
L69F, 124P, 211V,


NO. 25

t680c, a703g, t1049c,
NO. 26

L227P, 235D,




a1120g


350A, 374D


SEQ. ID.
lysC-ls56
t476g, a1010g,
SEQ. ID.
LysC-ls56
V159E, N337S,


NO. 27

g1021a
NO. 28

D341N


SEQ. ID.
lysC-ss91
a61g, t908c
SEQ. ID
LysC-ss91
N21D, V303A


NO. 29


NO. 30


SEQ. ID.
lysC-ls1
a715g
SEQ. ID.
LysC-ls1
T239A


NO. 31


NO. 32


SEQ. ID.
lysC-1s2
a61g, a833g, c839t,
SEQ. ID.
LysC-ls2
N21D, E278G,


NO. 33

a1172g
NO. 34

A280V, E391G

## Claims

1. A method for producing vectors encoding a feedback inhibited enzyme, the enzyme comprising a mutation resulting in the enzyme having reduced or deactivated feedback inhibition by a metabolite, wherein the method comprises
mutating genes encoding the feedback-inhibited enzyme in vitro,
ligating the mutated genes into vectors wherein the ligated mutated genes are each operably linking to an expression control sequence,
transforming the ligated vectors into a population of microorganisms that each contain a fluorescent metabolite sensor which synthesizes a fluorescent protein in the presence of the metabolite wherein microorganisms comprising the ligated vectors with the mutations resulting in the enzyme having reduced or deactivated feedback inhibition synthesize the fluorescent protein at increased concentration compared to the concentration of fluorescent protein in a non-transformed cell presence of the vector comprising and isolating said plurality of microorganisms exhibiting the increased fluorescent protein concentration, and
wherein the vectors encoding the enzyme having reduced or deactivated feedback inhibition are isolated from the plurality of microorganisms having the increased fluorescence, and wherein the metabolite sensor comprises at least one metabolite sensor selected from the group of pSenLys, pSenArg, pSenSer, pSenOAS and pJC1-lrp-brnF-eyfp.

2. The method according to claim 1, wherein said mutating comprises random mutagenesis.

3. The method according to claim 1 wherein the vectors comprise at least one of a plasmid, an insertion element, a transposon and a phage.

4. The method according to claim 1, wherein the gene encoding the feedback-inhibited enzyme is from a yeast or a bacterium.

5. The method according to claim 1, wherein the feedback inhibited enzyme is 2-isopropylmalate synthase, acetohydroxy acid synthase, acetylglutamate synthase, anthranilate synthase, asparagine synthetase, aspartate transcarbamylase, aspartate kinase, ATP-phosphoribosyl transferase, carbamoyl phosphate synthetase, chorismatemutase I, chorismatemutase II, cysteine synthase, D-3-phosphoglycerate dehydrogenase, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, dihydrodipicolinate synthase, glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamyl kinase, homocysteinetransmethylase, homoserine O-acetyltransferase, homoserine O-succinyltransferase, homoserine dehydrogenase, methionine synthase, phosphoribosylphrophosphate synthase, prephenate dehydrogenase I, prephenate dehydrogenase II, pyrroline-5-carboxylate reductase, ribose 1,5-bisphosphokinase, serinace-tyl(succinyl) transferase, threonine ammoniumlyase or tyrosine aminotransferase.

6. The method according to claim 1, wherein isolating said plurality of microorganisms exhibiting the increase fluorescent protein concentration comprises selecting for isolation microorganisms from among said plurality of microorganisms that have relatively highest fluorescence.

7. The method according to claim 1, wherein production of the isolated vectors is achieved by transforming the isolated vectors into a population of second microorganisms.

8. The method according to claim 1, wherein the genes encoding the enzyme with reduced or deactivated feedback inhibition are isolated from the vectors and sub-cloned into a second set of vectors for improved expression of the enzymes wherein the second set of ligated vectors are transformed into a production strain.

9. The method according to claim 1, wherein the genes encoding the enzyme having reduced or deactivated feedback inhibition are introduced into the chromosome of a production strain.

10. The method according to claim 1, wherein the population of microorganism containing the gene encoding the enzyme having reduced or deactivated feedback inhibition is a bacterium or a yeast.

11. The method for producing vectors according to claim 1, wherein the isolated plurality of microorganisms further comprises a biosynthesis pathway for an amino acid or a nucleotide wherein the mutation results in enhanced production of the amino acid or the nucleotide.

12. The method according to claim 11, wherein the produced amino acid comprises at least one component from the group comprising L-valine, L-leucine, L-asparagine, L-lysine, L-methionine, L-threonine, L-isoleucine, L-histidine, L-glutamate, L-glutamine, proline, L-arginine, L-tryptophan, L-tyrosine, L-phenyalanine, L-serine and L-cysteine.

13. The method according to claim 11, wherein the produced nucleotide comprises at least one component from the group comprising orotidine-5′-phosphate, 5-phosphoribosyl diphosphate or inosine-5′-phosphate.