Patent Application
20160298094
Method for Identifying a Cell having an Intracellular Concentration of a Particular Metabolite that is Increased Compared to the Wild Type of the Cell, wherein the Modification of the Cell is Achieved by Recombineering, to a Method for Producing a Production Cell that is Genetically Modified Compared to the Wild Type of the Cell and has Optimized Production of a Particular Metabolite, to a Method for Producing this Metabolite, and to Nucleic Acids Suited Therefor.
The invention relates to a method for identifying a cell having an intracellular concentration of a particular metabolite that is increased compared to the wild type of the cell, wherein the modification of the cell is achieved by recombineering, to a method for producing a production cell that is genetically modified compared to the wild type of the cell and has optimized production of a particular metabolite, to a method for producing this metabolite, and to nucleic acids suited therefor.
Microorganisms have been used on a large scale for decades to produce low molecular weight molecules. For example, low molecular weight molecules are natural bacterial metabolites such as amino acids (EP 1070132 B1, WO 2008/006680 A8), nucleosides and nucleotides (EP 2097512 C1, CA 2297613 C1), fatty acids (WO 2009/071878 C1, WO 2011/064393 C1), vitamins (EP 0668359 C1), organic acids (EP 0450491 B1, EP 0366922 B1) or sugars (EP 0861902 C1, U.S. Pat. No. 3,642,575 A). Low molecular weight molecules produced by bacteria are also molecules that are formed by the expression of heterologous genes stemming from plants, for example. These are plant active agents. These include, for example, taxol (WO 1996/032490 C1, WO 1993/021338 C1), artemisinin (WO 2009/088404 C1), and further molecules belonging to the classes of isoprenoids, phenylpropanoids or alkaloids (Marienhagen J, Bott M, 2012, J Biotechnol., doi.org/10.1016/j.jbiotec.2012.06.001). In addition to molecules, or precursors of molecules of plant origin, it is generally also possible to obtain such molecules by using microorganisms that are of commercial interest. These include, for example, hydroxyisobutyric acid to produce methacrylates (PCTIEP2007/055394), diamines to produce plastics (JP 2009-284905 A), or alcohols for use as fuel (WO 2011/069105 C2, WO 2008/137406 C1).
Gram-negative bacteria, gram-positive bacteria and yeasts are suitable microorganisms for producing low molecular weight molecules. Suitable bacteria are, for example, Escherichia species belonging to the genus Enterobacter, such as Escherichia coli, or Bacillus species belonging to the genus Firmicutes, such as Bacillus subtilis, or Lactococcus species belonging to the genus Firmicutes, such as Lactococcus lactis, or Lactobacillus species such as Lactobacillus casei, Saccharomyces species belonging to the genus Ascomycetes such as Saccharomyces cerevisiae, or Yarrowia species such as Yarrowia lipolytica, or Corynebacterium species belonging to the genus Corynebacterium.
Corynebacterium efficiens (DSM44549), Corynebacterium thermoaminogenes (FERM BP-1539) and Corynebacterium ammoniagenes (ATCC6871) are preferred among the corynebacteria, in particular Corynebacterium glutamicum (ATCC13032). Several species of Corynebacterium glutamicum are also known by different names in the related art. These include, for example, Corynebacterium acetoacidophilum ATCC13870, Corynebacterium lilium DSM20137, Corynebacterium melassecola ATCC 17965, Brevibacterium flavum ATCC14067, Brevibacterium lactofermentum ATCC13869, Brevibacterium divaricatum ATCC14020, and Microbacterium ammoniaphilum ATCC15354.
To achieve the formation and production of low molecular weight molecules, separate genes of the microorganism, or homologous genes or heterologous genes of the synthesis pathways of the low molecular weight molecules are expressed, or the expression thereof is intensified, or the mRNA stability thereof is increased. For this purpose, the genes can be introduced into the cell on plasmids or vectors, or they can be present on episomes or be integrated into the chromosome. It is also possible to increase the expression of the intracellular chromosomally encoded genes. This is achieved by appropriate mutations in the chromosome in the region of the promoter, for example. It is also possible to introduce other mutations resulting in product increases into the chromosome, which influence mRNA stability, for example, or which influence the osmotic stability or the resistance to pH fluctuations, or genes whose function is not known, but which favorably affect product formation. In addition, homologous genes or heterologous genes are inserted into the chromosome, or they are inserted so that they are present in the chromosome in multiple copies.
The deliberate insertion of mutations or genes into the genome necessitates the construction of a plasmid, which is produced by in vitro recombination of DNA sequences using restriction endonucleases and DNA ligases. The entire procedure for deliberately introducing chromosomal mutations further comprises the following steps to achieve the in vivo exchange, the test for successful exchange, and finally the test for increased product formation. This requires a plurality of steps, A1 to A8, which are schematically listed in
The deliberate insertion of mutations or genes into the chromosome necessitates the in vitro recombination of DNA sequences using restriction endonucleases and DNA ligases to produce a plasmid (
In this way a single clone is constructed, which thereafter is cultivated (
Of late, what is known as “recombineering” has been introduced as another method of deliberate genome mutation. Introducing mutations requires far fewer steps than the insertion of mutations by way of plasmids (
A further problem is that, so far, no general system exists to identify product-forming microorganisms in large cell populations directly after recombineering and to isolate the same from such cell populations. The method previously employed in recombineering involving the selection on petri dishes is, as mentioned above, limited to very special applications and additionally limited in terms of the number of recombinants that are obtained on petri dishes, which makes the method unsuitable for screening large recombinant libraries.
Recombineering is based on homologous recombination, which is mediated by proteins originating from phages or prophages. Two homologous systems are known for Escherichia coli. The RecE/RecT from the Rac prophage, and the Red operon, consisting of red gamma, red beta and red alpha from the bacteriophage lambda. Both systems allow the exchange of freely selectable DNA segments between two different DNA molecules. The exchange of DNA takes place via two homologous (similar or identical) regions that flank the target fragment and have lengths of 30 to 100 base pairs. So as to introduce chromosomal mutations, the DNA molecule carrying the mutation is commercially synthesized as a single strand (
The prior art with respect to product detection also includes metabolite sensors—also known as nanosensors—which can be used to detect increased product formation in individual bacteria. Such metabolite sensors use transcription factors or RNA aptamers to detect low molecular weight metabolites in bacteria and yeasts. Known transcription factor-based metabolite sensors are pSenLys, pSenArg, pSenSer, pSenOAS and pJC1-lrp-bmF-eyfp (WO2011138006; DPA 102012 016 716.4), for example. 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 containing the metabolite sensor can be any arbitrary microorganism. Bacteria, yeasts or enterobacteria, such as Escherichia coli, Corynebacterium glutamicum or Saccharomyces cerevisiae, can be mentioned by way of example.
The use of metabolite sensors for inserting cells having increased product formation is based on the increased production and extracellular accumulation of metabolite with increased formation of a metabolite, and the presence of an increased intracellular concentration of the metabolite compared to the wild type (A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level. Binder S, Schendzielorz G, Stabler N, Krumbach K, Hoffmann K, Bott M, Eggeling L. Genome Biol. 2012 May 28; 13(5):R40; Engineering microbial biofuel tolerance and export using efflux pumps. Dunlop M J, Dossani Z Y, Szmidt H L, Chu H C, Lee T S, Keasling J D, Hadi M Z, Mukhopadhyay A. Mol Syst Biol. 2011 May 10; 7:487).
Metabolite sensors are described for the detection of mutant libraries of microorganism mutants with increased product formation and for sorting these mutants by way of flow cytometry and automatic sorting devices (WO02011138006; DPA 102012 016 716.4). The mutant library in this case had been produced using chemical undirected mutagenesis of the chromosome or by inserting mutations into a plasmid-encoded gene using a faulty polymerase chain reaction. The present invention does not relate to chemical undirected mutagenesis or mutagenesis by way of a faulty polymerase chain reaction.
The drawback of existing techniques for strain development is that so far no technique is available for deliberately introducing mutations into a cellular gene or the chromosome, while also directly identifying an improved metabolite producer as a single cell in cell suspensions, and isolating it from the cell suspensions, without clonal cultivation (petri dishes) after introduction of the mutation.
It is thus the object of the invention to provide such a method for the accelerated development of microbial producers of smaller molecules and overcome the disadvantages of the state of the art.
This object is achieved according to the invention by a cell according to claim 1, by a method according to the other independent claims 6, 7 and 15, by a recombinase gene according to claim 18, by a recombinase according to claim 19, and by nucleic acids according to claim 20.
Advantageous refinements of the invention will be apparent from the dependent claims.
With the cell, the methods, the recombinase genes, the recombinase, the identified genes G1 to Gn and mutations M1 to Mm it is now possible, in a particularly fast way, to create cells for the increased production of metabolites that allow metabolites to be produced at an increased rate compared to the original cell.
The invention will be described hereafter in its general form.
According to the invention, a cell is provided that is genetically modified compared to the wild type thereof and that contains a gene sequence coding for a recombinase and additionally a gene sequence coding for a metabolite sensor.
The cell is preferably a microorganism, especially a bacterium, in particular of the genus Corynebacterium, Enterobacterium or the genus Escherichia, and particularly preferably Corynebacterium glutamicum or Escherichia coli.
The gene sequence coding for a recombinase can be a sequence that has improved functionality compared to a known recombinase in a desired microorganism. This is a gene sequence coding for a recombinase which codes for a protein that recombines extracellularly added DNA with intracellular DNA. The test for functionality can be carried out as shown schematically in
The gene sequence coding for the recombinase can be transformed in the cell and expressed by way of a vector, for example a plasmid, whereby the recombinase is formed.
The recombinase used in the method is characterized by recombining extracellularly added DNA with the intracellular DNA. The recombinase can originate from a larger gene pool, such as metagenome, for example, where possible recombinases are identified by way of sequence comparisons to known recombinases. Such sequence comparisons can also be used to identify possible recombinases in existing databases. Moreover, it is possible to detect proteins that reportedly have recombinase activity, or those suspected to have such activity, as recombinase by way of functional characterization. Recombinases can preferably be isolated from phages or prophages. For example, recombinases can be isolated from prophages of the biotechnologically relevant bacteria Leuconostoc, Clostridia, Thiobacillus, Alcanivorax, Azoarcus, Bacillus, Pseudomonas, Pantoea, Acinetobacter, Shewanella, or Corynebacterium, and the respective related species, and used. Preferred are recombinases homologous to the recombinase RecT of the Rac prophage, or to the recombinase Bet of the Lambda page. The recombinase RecT from the E. coli prophage Rac, the combinase Bet from the E. coli phage Lambda, and the recombinase rCau (Cauri_1962) from Corynebacterium aurimucosum are particularly preferred.
The used gene sequence coding for the metabolite sensor is the sequence of vectors, for example plasmids, coding for proteins that detect metabolites, such as amino acids, organic acids, fatty acids, vitamins or plant active agents and render these visible through fluorescence. The stronger the fluorescence, the higher is the intracellular metabolite concentration. In this way, it is possible to identify a cell having increased fluorescence compared to the genetically unmodified form, and thus increased product formation.
The cell thus modified is suitable for inserting externally supplied DNA molecules that carry the mutations M1 to Mm, or the mutated genes G1 to Gn, into the intracellular DNA, and for indicating increased production of a particular metabolite mediated by the insertion of the DNA by way of fluorescence. The metabolite sensor is selected so as to respond to the detection of the metabolite that is to be formed at an increased rate.
The invention further includes a method for identifying a cell having an intracellular concentration of a particular metabolite that is increased compared to the wild type of the cell, in a cell suspension, comprising the following method steps:
i) providing a cell suspension containing cells of the above-described type;
ii) genetically modifying the cells by recombineering while adding DNA that contains at least one modified gene G1 to Gn, or at least one mutation M1 to Mm, obtaining a cell suspension in which the cells differ in terms of the intracellular concentration of a particular metabolite; and
iii) identifying individual cells in the cell suspension having an increased intracellular concentration of a particular metabolite by fluorescence detection using a metabolite sensor.
The cells used are preferably microorganisms, especially bacteria, in particular of the genus Corynebacterium, Enterobacterium or the genus Escherichia, and particularly preferably Corynebacterium glutamicum, or Escherichia coli.
Recombineering involves methods that are known from the prior art and the methods disclosed in the specific description section, by way of example and without limitation. The recombinase gene is preferably inserted into the cell in a plasmid. It is particularly preferred when a gene according to SEQ ID No. 1 is inserted into the cell for a recombinase.
For this purpose, the cells are preferably transformed using vectors, particularly preferably plasmids, according to the sequences with SEQ ID No. 3 to No. 9.
The metabolites occurring in increased intracellular concentration compared to the wild type can be amino acids, organic acids, fatty acids, vitamins or plant active agents, for example. These are desired products, the production of which is to be improved.
The cell suspension can be cells that are present in a saline aqueous solution, for example, and can optionally contain nutrients.
The DNA used for genetically modifying the cell by recombineering can be single-stranded or double-stranded DNA, or synthetic DNA, or DNA isolated from cells. The DNA can comprise 50 bp to 3 Mb, and DNA having a length of 50 to 150 bp is preferred. The DNA can code for proteins, or parts of proteins, of the producer that is to be genetically modified. It is also possible to use DNA that is homologous to promoter regions, or regions having unknown functions, of the producer that is to be genetically modified. Moreover, the DNA can code for genes or regulatory elements from other organisms than those of the producer to be genetically modified.
In addition to defined DNA molecules, it is also possible to use mixtures of DNA molecules, which is advantageous for creating large genetic diversity, for example.
The insertion may be made into the chromosome or into a plasmid.
Fluorescence detection methods by way of a metabolite sensor are known to a person skilled in the art.
In one embodiment, the invention also relates to a method for producing a production cell that is genetically modified compared to the wild type thereof and has optimized production of a particular metabolite, comprising the following steps:
i) providing a cell suspension containing cells of the above-described type;
ii) genetically modifying the cells by recombineering while adding DNA that contains at least one modified gene G1 to Gn, or at least one mutation M1 to Mm. Obtaining a cell suspension in which the cells differ in terms of the intracellular concentration of a particular metabolite;
iii) identifying individual cells in the cell suspension having an increased intracellular concentration of a particular metabolite by fluorescence detection using a metabolite sensor;
iv) separating the identified cells from the cell suspension;
v) identifying those genetically modified genes G1 to Gn, or those mutations M1 to Mm, in the identified and separated cells that are responsible for the increased intracellular concentration of the particular metabolite; and
vi) produing a production cell that is genetically modified compared to the wild type thereof and has optimized production of the particular metabolite, the genome of the cell comprising at least one of the genes G1 to Gn and/or at least one mutation M1 to Mm.
The same interrelationships that apply to the method for identifying a cell having an increased intracellular concentration of a particular metabolite compared to the wild type of the cell in a cell suspension also apply to the cells, the recombineering, the metabolites, the cell suspension, the methods of fluorescence detection, the vectors, and the DNA inserted into the cells from steps i), ii) and iii).
The separation of the identified cell can be carried out using known methods.
To produce a production cell that is modified compared to the wild type, the cells that were used to identify the increased production and indicate an increased production of metabolites by way of increased fluorescence are isolated.
In these cells, the mutation M1 to Mm and/or in the gene G1 to Gn, or the mutations M1 to Mm are identified in the genes G1 to Gn. This may be done by way of PCR amplification of the target genes in the genes G1 to Gn and/or the mutation types M1 to Mm, with subsequent sequencing. Likewise, sequencing of the genome can be carried out.
The identified product-increasing mutations M1 to Mm and/or genes G1 to Gn are subsequently transferred into the production cell. This may be done by methods that are known to the person skilled in the art from the prior art.
The designation-G1 to Gn is directed to at least one of the genes G1, G2, G3 to Gn that was added to the cell as part of the recombineering and is now considered to be the cause for a particularly good increase in the production of the metabolite.
The designation M1 to Mm-is directed to mutations M1, M2, M3 to Mm that are contained in the genes G1 to Gn and added to the cell in method step ii) and that is now considered to be the cause of a particularly good increase in the production of the metabolite.
These genes or these mutations are isolated from the cell and inserted into the genome of the production cell using known methods. The gene or the mutation, or the genes or the mutations, can be inserted into the chromosomal DNA or a plasmid of the production cell.
These genes or mutations are DNA segments that preferably code for proteins of the steps of a biosynthesis pathway of the desired metabolite, or optionally a metabolic process related thereto. This can also be DNA that is used to favorably influence the promoter activity of genes or the stability of mRNA of genes for product formation.
In particular, the genes according to sequences SEQ ID No. 33 to SEQ ID No. 44 were found, which are suitable for increasing the production of L-lysine.
The invention further relates to a method for producing metabolites, comprising the following method steps:
a) producing a production cell that is genetically modified compared to the wild type thereof and has optimized production of a particular metabolite using a method of the type described above; and
b) cultivating the cell in a culture medium containing nutrients under conditions in which the production cell produces the particular metabolite from the nutrients.
The metabolite thus produced is secreted into the culture medium and can be isolated from the culture medium.
The culture medium or fermentation medium to be used must satisfy the needs of the respective strains in a suitable manner. Suitable culture media are known to the person skilled in the art. Descriptions of culture media for different microorganisms can be found in the handbook „ Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981). The terms culture medium and fermentation medium, or medium, are mutually interchangeable.
The carbon source used can be sugar and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, sucrose-containing solutions from sugar beet or sugar cane processing, starch, starch hydrolysate and cellulose, oils and fats such as soy bean oil, sunflower oil, peanut oil and coconut fat, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol, methanol and ethanol, and organic acids such as acetic acid or lactic acid.
The nitrogen source used can be organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea, or organic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used individually or as mixtures.
The phosphorus source used can be phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium-containing salts.
The culture medium must additionally include salts, for example in the form of chlorides or sulfates or metals, such as sodium, potassium, magnesium, calcium and iron, for example magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth-promoting substances such as amino acids, for example homoserine, and vitamins, for example thiamine, biotin or pantothenic acid, can be used in addition to the above-mentioned substances.
The described charged substances can be added to the culture in the form of a single batch, or fed in an appropriate manner during cultivation.
Alkaline compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to control the pH value of the culture. The pH value is generally set to a value of 6.0 to 8.5, and preferably 6.5 to 8. To control foam development, it is possible to use anti-foaming agents, such as fatty acid polyglycol ester. So as to maintain the stability of plasmids, it is possible to add appropriate, selective acting substances, such as antibiotics, to the medium. The fermentation is preferably carried out under aerobic conditions. Oxygen or oxygen-containing gas mixtures, such as air, are added to the culture to maintain these conditions. It is likewise possible to use liquids that are enriched with hydrogen peroxide. The fermentation is optionally carried out at positive pressure, for example at a positive pressure of 0.03 to 0.2 MPa. The temperature of the culture is normally 20° C. to 45° C., preferably 25° C. to 40° C., and particularly preferably 30° C. to 37° C. In batch processes, cultivation preferably continues until a sufficient amount for the measure of obtaining the desired metabolite, such as an amino acid, organic acid, a vitamin or a plant active agent, has formed. This goal is normally reached within 10 to 160 hours. Longer cultivation times are possible with continuous processes. The activity of the microorganisms results in an enrichment (accumulation) of the metabolite in the fermentation medium and/or in the cells of the microorganisms.
Examples of suitable fermentation media can be found in the patent specifications U.S. Pat. No. 5,770,409, U.S. Pat. No. 5,990,350, U.S. Pat. No. 5,275,940, WO 2007/012078, U.S. Pat. No. 5,827,698, WO 2009/043803, U.S. Pat. No. 5,756,345 or U.S. Pat. No. 7,138,266, among others.
The method according to the invention for producing metabolites can be used to particularly effectively produce amino acids, organic acids, vitamins, carbohydrates or plant active agents, for example.
This method is preferably used to produce L-amino acids, nucleotides and plant active agents, and particularly preferably L-lysine.
The invention also relates to a recombinase gene according to SEQ ID no. 1 and the alleles thereof, displaying homology of at least 70%, preferably 80%, particularly preferably 85% and/or 90%, and most preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
The invention also relates to a recombinase according to SEQ ID no. 2 and the homologous proteins thereof, displaying homology of 95%, 96%, 97%, and preferably of 98% or 99%.
Moreover, nucleic acids according to the sequences of SEQ ID no. 33 to SEQ ID no. 44 form part of the invention, which code for genes that allow particularly productive production strains to be obtained and originate from the cell for the identification of mutations.
The invention will be described hereafter in the specific description section, in more detail but without limitation.
A method, in which a recombinase is identified and used, helps to achieve the object. So as to identify recombinases for biotechnologically relevant bacteria such as Leuconostoc, Clostridia, Thiobacillus, Alcanivorax, Azoarcus, Bacillus, Pseudomonas, Pantoea. Acinetobacter, Shewaniella, and Corynebacterium species, and more particularly Corynebacterium glutamicum, genome databases are analyzed, according to known methods, for proteins which are homologous to known recombinases and which are expected to provide, or which are hoped will provide, an improved function in the desired organism, over that of the known recombinases. Genome databases are readily accessible, for example the database of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK), the database of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA), the database of the Swiss Institute of Bioinformatics (Swissprot, Geneva, Switzerland), or the Protein Information Resource Database (PIR, Washington, D.C., USA), and the DNA Data Bank of Japan (DDBJ, 111 1 Yata, Mishima, 411-8540, Japan).
The aforementioned databases are used to search for proteins that are homologous to known recombinases (
According to the invention, the sequences according to the invention also comprise those sequences that display 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) to 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 identity, in percent, to the amino acid sequences indicated in the sequence listing 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 sequences indicated in the sequence listing 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 probes, 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, U K, 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 amino acid sequences, 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).
The new DNA sequence from Corynebacterium aurimucosum thus determined, which includes the recombinase gene recT (SEQ ID No. 1) and codes for the functional recombinase rCau (SEQ ID No. 2), forms part of the present invention.
Identified recombinases are cloned in a vector, for example a plasmid, that allows the inducible expression of the recombinase gene in the host in which the recombination is carried out (
According to a preferred embodiment of the vectors according to the invention, the vectors are pCLTON2-bet (SEQ ID No. 3), pCLTON2-recT (SEQ ID No. 4), pCL-TON2-gp43 (SEQ ID No. 5), pCLTON2-gp61 (SEQ ID No. 6), pCLTON2-rCau (SEQ ID No. 7), pEKEx3-recT (SEQ ID No. 8), and pEKEx3-bet (SEQ ID No. 9).
The vectors thus produced are tested for activity of the recombinase in the respective host (
If the desired phenotype is produced according to the described method, thereafter the recombineering process is preferably optimized (
The DNA used for recombineering is single-stranded DANN, which is synthesized by commercial providers and can be up to 300 base pairs long. The desired mutation to be introduced into the chromosome is present at the center of the DNA and, flanking the same, the DNA includes sequences that are homologous to the chromosomal sequence of the host (U.S. Pat. No. 7,144,734). The optimization includes the test of DNA of varying lengths. The DNA used is DNA having a length of 20 to 300 base pairs, and preferably of 100 base pairs. The optimization includes the test of DNA of varying quantities, wherein 0.2 to 30 micrograms is used for transformation, and preferably 10 micrograms. The optimization further includes the test of DANN that is either homologous to the sense strand or to the antisense strand, wherein preferably the DNA that is homologous to the complementary strand is used (U.S. Pat. No. 7,674,621). The individual optimization steps are known to the person skilled in the art and, for example, are described for E. coli (Rekombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Sawitzke J A, Thomason L C, Costantino N, Bubunenko M, Datta S, Court D L. Methods Enzymol. 2007; 421:171-99), Bacillus subtilis (Bacillus subtilis genome editing using ssDNA with short homology regions. Wang Y, Weng J, Waseem R, Yin X, Zhang R, Shen Q. Nucleic Acids Res. 2012 July; 40(12):e91), or Lactococcus (High efficiency Rekombineering in lactic acid bacteria. van Pijkeren J P, Britton R A. Nucleic Acids Res. 2012, 40(10):e76).
To carry out the recombineering so as to obtain a microbial producer (
The DNA used for recombination is synthesized or produced by way of PCR amplification. It is 30 to 3000 base pairs long and has the organizational structure A-B-C. B is the desired mutation located at the center. In the case of an insertion, this may be a sequence of 1 to 3000 base pairs, preferably one of 1 to 1000, more preferably one of 1 to 100, and particularly preferably one of 1 base pair. The sequences A and C are homologous to chromosomal sequences. In the synthesized DNA, they are in each case 20 to 100 base pairs long. In the case of a deletion desired in the chromosome, B is zero base pairs long, and A and C are homologous to sequences in the chromosome that directly adjoin the region to be deleted. In the synthesized DNA, the sequences A and C are 20 to 100 base pairs long. The deletion in the chromosome can be 1 base pair or up to 10 kb. For exchange of bases in the chromosome, B represents the region to be exchanged, which can comprise 1 to 50 base pairs. The sequences A and C are homologous to chromosomal sequences adjoining the region to be exchanged. In the synthesized DNA, they are 20 to 100 base pairs long. DNA syntheses are carried out, for example, by Genescript (GenScript USA Inc., 860 Centennial Ave., Piscataway, N.J. 08854, USA), or Eurofins (Eurofins MWG Operon, Anzingerstr. 7a, 85560 Ebersberg, Germany), or DNA 2.0 (DNC2.0; DNA 2.0] Headquarters, 1140 O'Brien Drive, Suite A, Menlo Park, Calif. 94025, USA).
The synthesized DNA, or the DNA produced by way of PCR amplification, is inserted by transformation into the microorganism that expresses the recombinase and contains a metabolite sensor (
However, it is also possible to use defined mixtures of different DNA sequences for transformation and recombination. These mixtures are preferably used during the exchange of bases in the chromosome in the region “B,” where “B” represents the region to be exchanged in the organizational structure A-B-C of the DNA sequence. For example, it is possible to simultaneously exchange various amino acids in one position in the polypeptide in a gene in a population of microorganisms. It is also possible to simultaneously exchange various amino acids in different positions in the polypeptide. It is also possible to simultaneously exchange various nucleotides in a promoter region. The corresponding DNA mixtures can be directly produced by mixing individual defined DNA sequences, or they can already be synthesized by the manufacturer as mixtures, whereby up to several thousand different DNA molecules are present in a batch, which are then also used in a batch for transformation and recombination (
Moreover, it is also possible to used undefined DNA sequences for transformation and recombination. This is genomic DNA from existing producers, for example. In this way, it is possible to identify DNA segments and/or mutations and/or genes that favor product formation.
Subsequent to transforming the recombinase- and nanosensor-containing microorganisms, regeneration is carried out in a complex medium, as is known to the person skilled in the art and described, for example, for E. coli (Hanahan, D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983; 166(4): 557-80), or Corynebacterium (Tauch A, Kirchner O, Ldffler B, Götker S, Pühler A, Kalinowski J. Efficient electrotransformation of corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGC1. Curr Microbiol. 2002; 45(5):362-7). Following the regeneration, the cells are optionally transferred into a minimal medium for segregation whereupon, in accordance with the method according to the invention, the product analysis is carried out directly in individual cells by way of flow cytometry and selection of the producer (
Subsequent to the producer isolation (
The invention will now be described in more detail based on figures and non-limiting example.
Using the sequence of RecT from the Rac prophage of Escherichia coli stored under accession number CAD61789.1 in the database of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA), a homology search was carried out by way of the Blast program, BLAST 2.2.27+(Wheeler, David; Bhagwat, Medha (2007). “Chapter 9, BLAST QuickStart”. In Bergman, Nicholas H. Comparative Genomics Volumes 1 and 2. Methods in Molecular Biology. 395-396. Totowa, N.J.: Humana Press). The homology search was carried out with comparison to all proteins coded in the genomes of the following Corynebacteria species: C. accolens, C. ammoniagenes, C. amycolatum, C. aurimucosum, C. bovis, C. diphtheriae, C. efficiens, C. genitalium, C. glucuronolyticum, C. glutamicum, C. jeikeium, C. kroppenstedtii, C. lipophiloflavum, C. matruchotii, C. nuruki, C. pseudogenitalium, C. pseudotuberculosis, C. resistens, C. striatum, C. tuberculostearicum. C. ulcerans, C. urealyticum, and C. variabile.
The result obtained was the sequence cauri_1962, which codes for a protein having a length of 272 amino acids, of which 41% are identical to, and 61% similar to, the sequence of RecT. The DNA sequence from C. aurimucosum thus determined, which contains the recombinase gene recT, is indicated as SEQ ID No. 1 and the protein sequence is indicated as SEQ ID No. 2.
Recombinases were cloned in the expression vector pCLTON2 (A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression. Lausberg F, Chattopadhyay A R, Heyer A, Eggeling L, Freudl R. Plasmid. 2012 68(2):142-7), and in the vector pEKEx3 (The E2 domain of OdhA of Corynebacterium glutamicum has succinyltransferase activity dependent on lipoyl residues of the acetyltransferase AceF. Hoffelder M, Raasch K, van Ooyen J, Eggeling L. J Bacteriol. 2010; 192(19):5203-11).
To clone Bet, the vector pSIM8 (Rekombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Sawitzke J A, Thomason L C, Costantino N, Bubunenko M, Datta S, Court DL. Methods Enzymol. 2007; 421:171-99) was isolated from E. coli using the QIAGEN Plasmid Plus Maxi Kit (order no. 12963). This plasmid served as a template for PCR amplification using the primer pairs bet-F and bet-R.
The resulting fragment of 0.8 kb was isolated by way of gel isolation using the Minielute Extraction Kit (order no. 28704) from Quiagen, filled with the Klenow fragment, and subsequently phosphorylated with T4 polynucleotide kinase from Fermentas (order no. EK0031). The vector pCLTON2 (A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression. Lausberg F, Chattopadhyay A R, Heyer A, Eggeling L, Freudl R. Plasmid. 2012 68(2): 142-7) was cut S times and dephosphorylated using shrimp alkaline phosphatase from Fermentas (order no. EF0511). The fragment and the vector were ligated using the Rapid DNA Ligation Kit from Roche (order no. 11 635 379 001) and used to transform E. coli DH5. Transformed cells were plated out onto 100 ng/ml spectinomycin-containing complex medium.
To test for desired ligation products, a colony PCR was carried out using the primer pairs PcI_fw and Pcl_rv-pEKEx2_fw.
From a clone, which yielded a PCR product having the size 1.17 kb, a plasmid was prepared on a larger scale using the QIAGEN Plasmid Plus Maxi Kit (order no. 12963). The plasmid was labeled pCLTON2-bet, and the sequence thereof was labeled as SEQ ID No. 3.
To clone recT, the vector pRAC3 (Roles of RecJ, RecO, and RecR in RecET-mediated illegitimate recombination in Escherichia coli. Shiraishi K, Hanada K, Iwakura Y, Ikeda H, J Bacteriol. 2002 September; 184(17):4715-21) was isolated from E. coli using the QIAGEN Plasmid Plus Maxi Kit (order no. 12963). This plasmid served as a template for PCR amplification using the primer pairs recT-F and recT-R.
The resulting fragment of 0.8 kb was isolated as described in Example 2a, ligated to pCLTON2 and used to transform E. coli DH5. Transformed cells were plated out onto 100 ng/ml spectinomycin-containing complex medium.
To test for desired ligation products, a colony PCR was carried out as described in Example 2a. From a clone, which yielded a PCR product having the size 1.194 kb, a plasmid was prepared on a larger scale. The plasmid was labeled pCLTON2-recT, and the sequence thereof was labeled as SEQ ID No. 4.
To clone gp43, the gene was synthesized by Eurofins-MWG-Operon (Anzingerstr. 7a, 85560 Ebersberg, Germany). The sequence of the synthesized fragment is indicated as SEQ ID No. 10. The fragment was prepared as a 1407 bp fragment using the restriction enzymes Bglll and EcoRI, treated with the Klenow fragment, and subsequently phosphorylated with T4 polynucleotide kinase from Fermentas (order no. EK0031). The fragment was isolated as described in Example 2a, ligated to pCLTON2 and used to transform E. coli DH5. Transformed cells were plated out onto 100 ng/ml spectinomycin-containing complex medium.
To test for desired ligation products, a colony PCR was carried out as described in Example 2a. From a clone, which yielded a PCR product having the size 1.79 kb, a plasmid was prepared on a larger scale. The plasmid was labeled pCLTON2-gp43, and the sequence thereof was labeled as SEQ ID No. 5.
To clone gp61, the gene was synthesized by Eurofins-MWG-Operon (Anzingerstr. 7a, 85560 Ebersberg, Germany). The sequence of the synthesized fragment is indicated as SEQ ID No. 11. The fragment was prepared as 1082 bp using the restriction enzymes BglII and MunI, treated with the Klenow fragment, and subsequently phosphorylated with T4 polynucleotide kinase from Fermentas (order no. EK0031). It was isolated as described in Example 2a, ligated to pCLTON2 and used to transform E. coli DH5. Transformed cells were plated out onto 100 ng/ml spectinomycin-containing complex medium.
To test for desired ligation products, a colony PCR was carried out as described in Example 2a. From a clone, which yielded a PCR product having the size 1.45 kb, a plasmid was prepared on a larger scale. The plasmid was labeled pCLTON2-gp61, and the sequence thereof was labeled as SEQ ID No. 6.
To clone rCau (cauri_1962), the gene was synthesized by Eurofins-MWG-Operon (Anzingerstr. 7a, 85560 Ebersberg, Germany). The sequence of the synthesized fragment is indicated as SEQ ID No. 1. The fragment was prepared as 839 bp using the restriction enzymes Bglll and MunI, treated with the Klenow fragment, and subsequently phosphorylated with T4 polynucleotide kinase from Fermentas (order no. EK0031). It was isolated as described in Example 2a, ligated to pCLTON2 and used to transform E. coli DH5. Transformed cells were plated out onto 100 ng/ml spectinomycin-containing complex medium.
To test for desired ligation products, a colony PCR was carried out as described in Example 2a. From a clone, which yielded a PCR product having the size 1.22 kb, a plasmid was prepared on a larger scale. The plasmid was labeled pCLTON2-rCau, and the sequence thereof was labeled as SEQ ID No. 7.
To clone recT in pEKEx3, pCLTON2-recT from Example 2b was used as a template for PCR amplification. The gene was amplified using the primer pairs BglII-RBS-RecT-F and EcoRI-RecT-R.
The resulting fragment of 0.84 kb was isolated by way of gel isolation using the Minielute Extraction Kit (order no. 28704) from Quiagen), treated with the Klenow fragment, and subsequently phosphorylated with T4 polynucleotide kinase from Fermentas (order no. EK003). The vector pEKEx3 was cut with EcoRI and BamHI, and the resulting fragment of 8298 bp was dephosphorylated using shrimp alkaline phosphatase from Fermentas (order no. EF0511). The fragment and the vector were ligated using the Rapid DNA Ligation Kit from Roche (order no. 11 635 379 001) and used to transform E. coli DH5. Transformed cells were plated out onto 100 ng/ml spectinomycin-containing complex medium.
To test for desired ligation products, a colony PCR was carried out using the primer pairs col-pEKEx3-F and col-pEKEx3-R.
From a clone, which yielded a PCR product having the size 1.71 kb, a plasmid was prepared on a larger scale using the QIAGEN Plasmid Plus Maxi Kit (order no. 12963). The plasmid was labeled pEKEx3-recT, and the sequence thereof was labeled as SEQ ID No. 8.
To clone the recombinase Bet, pCLTON2-rCau from Example 2e was used as a template for PCR amplification. The gene was amplified using the primer pairs BglII-RBS-bet-F and EcoRI-bet-R.
The resulting fragment of 0.81 kb was isolated by way of gel isolation using the Minielute Extraction Kit (order no. 28704) from Quiagen), treated with the Klenow fragment, and subsequently phosphorylated with T4 polynucleotide kinase from Fermentas (order no. EK0031). The vector pEKEx3 was cut with EcoRI and BamHI, and the resulting fragment of 8298 bp was dephosphorylated using shrimp alkaline phosphatase from Fermentas (order no. EF0511). The fragment and the vector were ligated using the Rapid DNA Ligation Kit from Roche (order no. 11 635 379 001) and used to transform E. coli DH5. Transformed cells were plated out onto 100 ng/ml spectinomycin-containing complex medium.
To test for desired ligation products, a colony PCR was carded out as in Example 2e using the primer pairs col-pEKEx3-F and col-pEKEx3-r. From a clone, which yielded a PCR product having the size 1.08 kb, a plasmid was prepared on a larger scale using the QIAGEN Plasmid Plus Maxi Kit (order no. 12963). The plasmid was labeled pEKEx3-bet, and the sequence thereof was labeled as SEQ ID No. 9.
So as to insert a non-functional copy of a kanamycin resistance-imparting gene into the chromosome of C. glutamicum ATCC13032, initially the primer pairs ScaI-KanR-F/Kan(−)-L-R and MunI-R-R/Kan(−)-R-F were used to produce two PCR fragments to be fused as a template using the vector pJC1 (Cremer J, Treptow C, Eggeling L, Sahm H. Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum. J Gen Microbiol. 1988; 134(12):3221-9).
The two resulting PCR fragments were purified using the Minielute Extraction Kit (order no. 28704) from Quiagen and fused in a fusion PCR with the primer pairs ScaI-KanR-F/MunI-R-R to yield the defective kanamycin resistance gene. This includes a cytosine as an additional nucleotide in position 234, resulting in a frame shift such that the gene is not read completely. The resulting product was restricted using ScaO and Muni and subsequently cloned in the pK18mobsacB-lysOP7 cut in EcoRI and Seal (Acetohydroxyacid synthase, a novel target for improvement of L-lysine production by Corynebacterium glutamicum. Blombach B, Hans S, Bathe B, Eikmanns B J. Appl Environ Microbiol. 2009 January; 75(2):419-427). In this vector, the defective kanamycin resistance gene is flanked by two non-coding regions of the C. glutamicum genome, by way of which the homologous integration into the genome takes place. Thereafter, the entire cassette was integrated into the C. glutamicum genome between positions 1.045.503 and 1.045.596 using known methods by way of double positive selection (Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Schafer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A Gene. 1994 Jul. 22; 145(1):69-73). The correct integration of the defective kanamycin resistance gene into the chromosome was checked using the primer pairs colNCR-L2 and colNCR-R2. The size of the PCR fragment was 3937 bp.
The transformation of the test strain was carried out as described by Tauch et al. for Corynebacterium diphtheriae and C. glutamicum (Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGC1. Tauch A, Kirchner O, LOffler B, Götker S, Pühler A, Kalinowski J. Curr Microbiol. 2002 November; 45(5):362-367). The strain was rendered competent, and in each case 0.5 micrograms of the vector coding for the recombinase was used for electroporation.
Spectinomycin-resistant clones were selected on the complex medium, brain heart infusion sorbitol, BHIS, (High efficiency electroporation of intact Corynebacterium glutamicum cells. Liebl W, Bayed A, Schein B, Stillner U, Schleifer K H. FEMS Microbiol Lett. 1989 December; 53(3):299-303), which contained 100 micrograms of spectinomycin (BHIS-Spec1OO). One clone each of the test strain containing the vector pCLTON2-bet, pCLTON2-recT, pCLTON2-gp43, pCLTON2-gp61, pCLTON2-rCau, pEKEx3-recT, or pEKEx3-bet was inoculated in 50 ml BHIS-Spec1OO and cultivated over night at 130 rpm and 30° C. in Erlenmeyer flasks. The next morning, 500 ml BHIS-Spec1OO+IPTG (0.5 mM when using pEKEx3-recT, pEKEx3-bet) or tetracycline (250 ng/ml when using pCLTON2-bet, pCLTON2-recT, pCLTON2-gp43, pCLTON2-gp61, pCLTON2-rCau) was inoculated with 10 ml of medium incubated overnight and cultivated for 4 to 6 hours until an OD of 1.5 to 2 was reached. Thereafter, the culture was cooled for 30 minutes on ice, washed twice with 50 ml 10% glycerol, 1 mM Tris pH 8, and subsequently twice with 10% glycerol. The cell pellet was 10% resuspended in the return flow and an additional 1 ml glycerol, aliquotted into 150 μl each, flash-frozen in liquid nitrogen, and stored at −75° C. until use. For use, the cells were gently thawed on ice within 20 minutes and mixed with 1 μg DNA.
The DNA used was the oligo Kan100*, having the sequence ATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAG CCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGC. This DNA was synthesized by Eurofins-MWG-Operon (Anzingerstr. 7a, 85560 Ebersberg, Germany).
The suspension of cells and 1 microgram DNA was transferred into electroporation cuvettes and carefully coated with 800 μl ice cold 10% glycerol and subsequently electroporated. For regeneration, the cells were immediately transferred into 4 ml of BHIS, which had been precontrolled to a temperature of 46° C., and incubated for 6 minutes at 46° C. Subsequently, a 1- to 6-hour cultivation was carried out at 30° C. and 170 rpm in a 15 ml flacon. Then the cells were transferred to BHIS, which contained 50 micrograms per milliliter of kanamycin. The result is shown in Table 1. It is apparent that, per batch, a maximum of 57 cells are spontaneously resistant to kanamycin; the maximum recombination frequency of 20054 clones was obtained with pEKEx3-recT; and decreasing recombinase activity occurs with pCLTON2-recT, pEKEx3-bet, pCLTON2-rCau, and pCLTON2-gp61. The recombinase activity of pCLTON2-gp43 is barely above background, and pCLTON2-bet is not active.
The test strain containing pEKEx3-recT was inoculated in 50 ml BHIS-Spec100 and cultivated overnight at 130 rpm and 30° C. in an Erlenmeyer flask. The next morning, 50 ml BHIS-Spec100+0.5 mM IPTG was inoculated with 10 ml of medium incubated overnight and cultivated for 0, 1, or 4 hours. The test strain with pCLTON2-recT was inoculated in 50 ml BHIS-Spec100 and cultivated overnight at 130 rpm and 30° C. in an Erlenmeyer flask. The next morning, 50 ml BHIS-Spec100+250 nanograms tetracycline was inoculated with 10 ml of medium incubated overnight and cultivated for 0, 1, or 4 hours. Thereafter, the culture was cooled for 30 minutes on ice, washed twice with 50 ml 10% glycerol, 1 mM Tris pH 8, and subsequently twice with 10% glycerol. The cell pellet was 10% resuspended in the return flow and an additional 1 ml glycerol, aliquotted into 150 μl each, flash-frozen in liquid nitrogen, and stored at −75° C. until use. For use, the cells were gently thawed on ice within 20 minutes and mixed with 1 microgram DNA.
The electroporation and regeneration were carried out as described in Example 4. Table 2 shows that the maximum recombination frequency is achieved in the vector pEKEx3-recT after 4 hours of induction when using the recombinase recT.
For further optimization, cells of the test strain containing pEKEx3-recT were used as previously, but increasing amounts of DNA were added. Table 3 shows that the maximum recombination frequency is achieved in the vector pEKE3-recT when using the recombinase recT at a concentration of 10 micrograms DNA.
For the direct isolation of a strain producing increased amounts of lysine, starting from a starting strain, C. glutamicum ATCC13032 was transformed using the nanosensor pSenLys. The nanosensor pSenLys is described in WO2011138006. Cells of the resulting strain were transformed using pEKEx3-recT, and the recombinase was induced as described in Example 4. The DNA lysC-100* was synthesized by Eurofins-MWG-Operon (Anzingerstr. 7a, 85560 Ebersberg, Germany). It is stored as SEQ ID No. 32.
10 micrograms of the DNA lysC-100* were transferred into the strain by way of electroporation, as described in Example 4 (
For the flow cytometry analysis and sorting of the cells having high fluorescence, the cell suspension was adjusted in CGXII glucose medium to an optical density value of less than 0.1 and directly supplied to the 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. 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. In this way, 51 fluorescent cells were sorted out on petri dishes containing BHIS medium.
The petri dish was incubated for 30 hours at 30 degrees Celsius, and subsequently each of the 46 reaction vessels of the microtiter plate Flowerplate (48-well) of the BioLector cultivation system (m2plabs GmbH, Aachen, Germany) was inoculated with a respective clone. Each reaction vessel contained 0.7 microliters CGXII glucose. One of the reaction vessels was inoculated with a negative control, and one was inoculated with a positive control. Thereafter, the microtiter plate was incubated for 2 days at 30° C., 1200 rpm, and a shaking radius of 3 mm. In the BioLector cultivation system, the growth was recorded online as scattered light at 620 nm, and the fluorescence of the culture was recorded continuously at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.
After 2 days, the specific fluorescence of the cultures was determined based on the recorded data. It was elevated in 33 clonal cultures at least four-fold compared to the negative control. The lysC sequence in the genome was determined in 12 of these cultures. In all instances, the cytosine in position 932 of the gene had been exchanged with a thymidine. The sequence thus corresponded to the sequence part that was present on the synthesized oligo lysC-100* and results in the lysine formation with C. glutamicum (Binder et al. Genome Biology 2012, 13:R40).
For the direct isolation of a strain producing increased amounts of lysine, starting from a starting strain using murE mutations, the starting strain C. glutamicum ATCC13032 described in Example 6 was used with pSenLys and pEKEx3-recT. The individual murE DNA oligos were synthesized by Eurofins-MWG-Operon (Anzingerstr. 7a, 85560 Ebersberg, Germany). The following murE sequences were used: murEG81amb*, SEQ ID No. 12; murEG81A*, SEQ ID No. 13; murEG81C*, SEQ ID No. 14; murE G81D*, SEQ ID No. 15; murEG81E*, SEQ ID No. 16; murEG81F*, SEQ ID No. 17; murEG81H*, SEQ ID No. 18; murEG81I*, SEQ ID No. 19; murEG81K*, SEQ ID No. 20; murEG81L*, SEQ ID No. 21; murEG81M*, SEQ ID No. 22; murEG81N*, SEQ ID No. 23; murEG81P*, SEQ ID No. 24; murEG81Q*, SEQ ID No. 25; murEG81R*, SEQ ID No. 26; murEG81S*, SEQ ID No. 27; murEG81T*, SEQ ID No. 28; murEG81V*. SEQ ID No. 29; murEG81W*, SEQ ID No. 30; murEG81Y*, SEQ ID No. 31.
1 microgram of these DNA oligos was removed in each case, and the resulting 20 micrograms were mixed with an aliquot of cells and transferred in the strain by way of electroporation, as described in Example 4 (
In this way, 62 fluorescent cells were sorted out on petri dishes containing BHIS medium. The petri dish was incubated for 30 hours at 30 degrees Celsius, and subsequently each of the 46 reaction vessels of the microtiter plate Flowerplate (48-well) of the BioLector cultivation system (m2plabs GmbH, Aachen, Germany) was inoculated with a respective clone. Each reaction vessel contained 0.7 microliters CGXII glucose. One of the reaction vessels was inoculated with a negative control, and one was inoculated with a positive control. Thereafter, the microtiter plate was incubated for 2 days at 30° C., 1200 rpm, and a shaking radius of 3 mm. In the BioLector cultivation system, the growth was recorded online as scattered light at 620 nm, and the fluorescence of the culture was recorded continuously at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.
After 2 days, the specific fluorescence of the cultures was determined based on the recorded data. It was elevated in 33 clonal cultures at least twelve-fold compared to the negative control. An L-lysine determination in the medium was carried out for 21 cultures to verify the product formation (
The murE sequence in the genome was determined for these 21 clones. Sequencing was carried out after PCR amplification by the company Eurofins-MWG-Operon (Anzingerstr. 7a, 85560 Ebersberg, Germany). The resulting mutations are summarized in Table 4. It is apparent that in this way 10 different murE mutations were obtained starting from the starting strain, of which nine resulted in increased lysine formation compared to the starting strain. The sequences of the murE alleles obtained are SEQ ID No. 33, murEG81W; SEQ ID No. 34, murEG81Y; SEQ ID No. 35, murEG81N; SEQ ID No. 36, murEG81C; SEQ ID No. 37, murEG81S; SEQ ID No. 38, murEG81F; SEQ ID No. 39, murEG81V; SEQ ID No. 40, murEG81L; SEQ ID No. 41, murEG81H; SEQ ID No. 42, murEG81I; SEQ ID No. 43, murEG81T; and SEQ ID No. 44, murEG81R.
acfu (rec) indicates the number of kanamycin-resistant clones that resulted from recombineering with the Kan* oligo; cfu (spont) is the number of spontaneously kanamycin-resistant clones that resulted from a control batch, which contained water instead of the Kan* oligos. It is clearly apparent that high recombination efficiency is achieved with the recombinase recT in pEKEx3 recT or pCLTON2-recT. Very high recombination efficiency is also achieved with the recombinase rCau from Corynebacterium aurimucosum, which clearly exceeds that of the spontaneously resistant clones.
acfu (rec) and cfu (spont) are the same as in Table 1. The influence of the induction time for expression of the recombinase on the recombination efficiency is clearly apparent.
acfu (rec) and cfu (spont) are the same as in Table 1. It is clearly apparent how the DNA amount added to the recombineering batch increases the recombineering frequency. The maximum recombineering frequency is reached at approximately 10 micrograms DNA.
Sequences according to sequence listing:
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2012 024 435.5 | Dec 2012 | DE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/DE2013/000683 | 11/15/2013 | WO | 00 |
