Patent Grant
9045762
The present invention relates to a process for preparing fine chemicals with the use of genetically modified microorganisms in which variants of the promoter of the gap gene coding for glyceraldehyde-3-phosphate dehydrogenase enable various genes to be overexpressed.
Fine chemicals, meaning in particular amino acids, organic acids, vitamins, nucleosides and nucleotides, are employed in human medicine, in the pharmaceutical, cosmetic and food industries and in animal nutrition.
Many of these compounds are prepared by fermenting strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to the great importance, continuous efforts are made to improve the production processes. Said processes may be improved with respect to fermentation-related measures such as, for example, stirring and oxygen supply, or the composition of nutrient media, such as, for example, sugar concentration during fermentation, or the work-up into the product form, for example by means of ion exchange chromatography, or the intrinsic performance characteristics of the microorganism itself.
The performance characteristics of these microorganisms are improved by employing methods of mutagenesis, selection and choice of mutants. In this way strains are obtained which are resistant to anti-metabolites such as, for example, the lysine analogue S-(2-aminoethyl)-cysteine or the valine analogue 2-thiazolalanine, and produce chemical compounds, for example L-amino acids such as L-lysine or L-valine.
For some years now, methods of recombinant DNA technology have likewise been used in order to improve L-amino acid-producing Corynebacterium glutamicum strains by enhancing or attenuating individual amino acid biosynthesis genes, for example, and studying the effect on production of said chemical compound.
Summaries regarding the biology, genetics and biotechnology of Corynebacterium glutamicum can be found in “Handbook of Corynebacterium glutamicum” (Eds.: L. Eggeling and M. Bott, CRC Press, Taylor & Francis, 2005), in the Journal of Biotechnology special issue (Chief Editor: A. Pühler) entitled “A New Era in Corynebacterium glutamicum Biotechnology” (Journal of Biotechnology 104/1-3, (2003)), and in the book by T. Scheper (Managing Editor) “Microbial Production of L-Amino Acids” (Advances in Biochemical Engineering/Biotechnology 79, Springer Verlag, Berlin, Germany, 2003).
The nucleotide sequence of the Corynebacterium glutamicum genome is described in Ikeda and Nakagawa (Applied Microbiology and Biotechnology 62, 99-109 (2003)), in EP 1 108 790 and in Kalinowski et al. (Journal of Biotechnology 104/1-3, (2003)).
The nucleotide sequences of the Corynebacterium glutamicum genome are also available in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA), in the DNA Data Bank of Japan (DDBJ, Mishima, Japan), or in the nucleotide sequence database of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany, and Cambridge, UK).
The natural promoter of the Corynebacterium glutamicum gap gene coding for glyceraldehyde-3-phosphate dehydrogenase has been described and analysed by Patek et al (Journal of Biotechnology 104, 311-323 (2003)).
In US 2008/0050786, SEQ ID NO:20 depicts a 484 bp DNA molecule referred to as seqP-RBS—20 which comprises the promoter of the gap gene including the ribosome binding site. This promoter may be utilized advantageously for expressing various genes such as the lysC gene coding for aspartate kinase, for example. For better clarity, the sequence is listed under SEQ ID NO:1.
It was an object of the invention to provide variants of the promoter and/or the expression unit of the gap gene coding for glyceraldehyde-3-phosphate dehydrogenase, it being possible for various genes such as, for example, the lysC gene coding for aspartate kinase to be expressed advantageously under the control of said variants.
During the work on the present invention, the activity of the promoter of the Corynebacterium glutamicum ATCC13032 gap gene, depicted in SEQ ID NO:2, was found to have increased after replacement of the nucleobase guanine in position 362 of said promoter with the nucleobase adenine.
A further increase in promoter activity was found after additional replacement of the nucleobases:
Cytosine in position 265 with thymine,
Guanin in position 269 with cytosine,
Adenine in position 290 with thymine, and
Guanine in position 292 with adenine,
in the promoter of the Corynebacterium glutamicum ATCC13032 gap gene, depicted in SEQ ID NO:2.
The invention therefore relates to an isolated polynucleotide having promoter activity, which comprises a polynucleotide having the nucleotide sequence depicted in SEQ ID NO:3 or SEQ ID NO:34.
The invention also relates to an isolated polynucleotide having promoter activity, which essentially consists of a polynucleotide having the nucleotide sequence depicted in SEQ ID NO:3 or SEQ ID NO:34. The term “essentially” in this context means that a polynucleotide of no more than (≦) 1000, no more than 800, no more than (≦) 700, no more than (≦) 600, no more than (≦) 500 or no more than (≦) 400 nucleotides in length and a polynucleotide of no more than (≦) 15000, no more than (≦) 10000, no more than 7500, no more than (≦) 5000, no more than 2500, no more than (≦) 1000, no more than (≦) 800, no more than (≦) 700, no more than (≦) 600, no more than (≦) 500 or no more than (≦) 400 nucleotides in length have been added to the 5′ end and 3′ end, respectively, of the polynucleotides of SEQ ID NO:3 or SEQ ID NO:34.
Any useful combination of the features from the two lists is in accordance with the invention here.
“Useful combination” means, for example, a combination of features which results in an efficient recombination being carried out. The use of additions of the same length flanking a DNA region to be replaced facilitates the transfer of the region by homologous recombination in the experimental procedure. Relatively long flanking homologous regions are advantageous for efficient recombination between circular DNA molecules but cloning of the replacement vector is made more difficult with increasing length of the flanks (Wang et al., Molecular Biotechnology 32:43-53 (2006)). Therefore preference is given to the specific combination of additions of in each case 600 to no more than (≦) 1000 nucleotides, with additions of in each case 750 to 850 nucleotides being particularly preferred.
The invention furthermore relates to an isolated polynucleotide having promoter activity, which consists of the nucleotide sequence depicted in SEQ ID NO:3 or SEQ ID NO:34.
Details regarding the biochemistry and chemical structure of polynucleotides as present in living things such as microorganisms, for example, can be found inter alia in the text book “Biochemie” [Biochemistry] by Berg et al (Spektrum Akademischer Verlag Heidelberg Berlin, Germany, 2003; ISBN 3-8274-1303-6).
Polynucleotides consisting of deoxyribonucleotide monomers containing the nucleobases or bases adenine (A), guanine (G), cytosine (C) and thymine (T) are referred to as deoxyribo-polynucleotides or deoxyribonucleic acid (DNA). Polynucleotides consisting of ribonucleotide monomers containing the nucleobases or bases adenine (A), guanine (G), cytosine (C) and uracil (U) are referred to as ribo-polynucleotides or ribonucleic acid (RNA). The monomers in said polynucleotides are covalently linked to one another by a 3′→5′-phosphodiester bond.
A “polynucleotide having promoter activity” or a “promoter” means a polynucleotide, preferably deoxyribo-polynucleotide, or a nucleic acid, preferably deoxyribonucleic acid (DNA), which is functionally linked to a polynucleotide to be transcribed and determines the point and frequency of initiation of transcription of said polynucleotide, thereby enabling the strength of expression of the controlled polynucleotide to be influenced.
Owing to the double-stranded structure of DNA, the strand complementary to the strand in SEQ ID NO:3 or SEQ ID NO:34 of the sequence listing is likewise a subject of the invention.
“Transcription” means the process by which a complementary RNA molecule is produced starting from a DNA template. This process involves proteins such as RNA polymerase, “sigma factors” and transcriptional regulatory proteins. The synthesized RNA (messenger RNA, m-RNA) then serves as template in the process of translation which subsequently yields the polypeptide or protein.
From a chemical point of view, a gene is a polynucleotide. A polynucleotide encoding a protein/polypeptide is used herein synonymously with the term “gene”. Accordingly, the two terms “gene” and “coding region” are used synonymously as are the two terms “protein” and “polypeptide”.
“Functionally linked” means in this context the sequential arrangement of the polynucleotide having promoter activity according to the invention with a further oligo- or polynucleotide, resulting in transcription of said further polynucleotide.
If the further polynucleotide is a polynucleotide, referred to as second polynucleotide hereinbelow, which codes for a polypeptide/protein and consists of the coding region for a polypeptide, starting with a start codon, including the stop codon and, where appropriate, including a transcription termination sequence, “functionally linked” then means the sequential arrangement of the polynucleotide having promoter activity according to the invention with the second polynucleotide, resulting in transcription of said second polynucleotide and translation of the synthesized RNA.
The second polynucleotide codes for one or more polypeptide(s). A polynucleotide coding for a protein/polypeptide essentially consists of a start codon selected from the group consisting of ATG, GTG and TTG, preferably ATG or GTG, particularly preferably ATG, a protein-encoding sequence and one or more stop codon(s) selected from the group consisting of TAA, TAG and TGA.
If the second polynucleotide codes for a plurality of proteins/polypeptides, each gene may be preceded by a ribosome-binding site. Where appropriate, a termination sequence is located downstream of the last gene.
The second polynucleotide preferably codes for one or more polypeptides or proteins of the biosynthetic pathway of fine chemicals, preferably selected from the group of proteinogenic amino acids, non-proteinogenic amino acids, vitamins, nucleosides, nucleotides and organic acids. Particular preference is given to proteinogenic amino acids, non-proteinogenic amino acids, and organic acids.
The second polynucleotide preferably consists of one or more of the polynucleotides listed in Table 1 of EP 1 108 790 A2 which is hereby incorporated by reference.
The second polynucleotide preferably consists of one or more of the genes or polynucleotides coding for enzymes of the pentosephoshate pathway, selected from the group consisting of:
The second nucleotide further preferably consists of one or more of the genes or polynucleotides coding for enzymes of anaplerosis and gluconeogenesis, selected from the group consisting of:
In connection with the preparation of L-lysine, the second polynucleotide preferably consists of one or more of the genes or polynucleotides coding for enzymes of L-lysine biosynthesis, selected from the group consisting of:
In connection with the preparation of L-valine, L-isoleucine, α-ketoisovaleric acid, α-keto-β-methylvaleric acid or α-ketoisocaproic acid, the second polynucleotide preferably consists of one or more of the genes or polynucleotides coding for enzymes of the biosynthesis of L-valine, L-isoleucine, α-ketoisovaleric acid, α-keto-β-methylvaleric acid or α-ketoisocaproic acid, selected from the group consisting of:
In connection with the preparation of L-ornithine, the second polynucleotide preferably consists of one or more of the genes or polynucleotides coding for enzymes of L-ornithine biosynthesis, selected from the group consisting of:
The promoter according to the invention can thus be used in each case for (over) expressing the second polynucleotide in Corynebacterium glutamicum.
The second polynucleotide is positioned downstream of the promoter according to the invention, i.e. at the 3′ end, such that both polynucleotides are functionally linked to one another either directly or by means of a linker oligonucleotide or linker polynucleotide. Preference is given to both polynucleotides being functionally linked to one another by means of a linker oligonucleotide or linker polynucleotide.
Said linker oligonucleotide or linker polynucleotide consists of deoxyribonucleotides.
In this context, the expression “functionally linked to one another directly” means that the nucleotide via the adenosine nucleotide in position 461 at the 3′ end of SEQ ID NO:3 or SEQ ID NO:34 is linked directly to the first nucleotide of the start codon of a coding region. This results in “leaderless” mRNAs which start immediately with the 5′-terminal AUG start codon and therefore do not have any other translation initiation signals.
In this context, the expression “functionally linked to one another by means of a linker oligonucleotide” means that the nucleotide via the adenosine nucleotide in position 461 at the 3′ end of SEQ ID NO:3 or SEQ ID NO:34 is linked by an oligonucleotide of 1, 2, 3, 4 or 5 nucleotides in length to the first nucleotide of the start codon of a coding region.
In this context, the expression “functionally linked to one another by means of a linker polynucleotide” means that the nucleotide via the adenosine nucleotide in position 461 at the 3′ end of SEQ ID NO:3 or SEQ ID NO:34 is linked by a polynucleotide of from 6 to no more than (≦) 600 nucleotides in length to the first nucleotide of the start codon of a coding region.
In this context, the expression “functionally linked to one another” means that the second polynucleotide is bound to the polynucleotide having promoter activity according to the invention in such a way that transcription of the second polynucleotide and translation of the synthesized RNA are ensured.
Depending on the technical requirement, the linker polynucleotide is
6-600, 6-500, 6-400, 6-300, 6-200, 6-180, 6-160, 6-140, 6-120, 6-100, 6-80, 6-60, 6-50, 6-40, 6-30, 6-28, 6-27, 6-26, 6-25 or
8-600, 8-500, 8-400, 8-300, 8-200, 8-180, 8-160, 8-140, 8-120, 8-100, 8-80, 8-60, 8-50, 8-40, 8-30, 8-28, 8-27, 8-26, 8-25 or
10-600, 10-500, 10-400, 10-300, 10-200, 10-180, 10-160, 10-140, 10-120, 10-100, 10-80, 10-60, 10-50, 10-40, 10-30, 10-28, 10-27, 10-26, 10-25 or
12-600, 12-500, 12-400, 12-300, 12-200, 12-180, 12-160, 12-140, 12-120, 12-100, 12-80, 12-60, 12-50, 12-40, 12-30, 12-28, 12-27, 12-26, 12-25 or
14-600, 14-500, 14-400, 14-300, 14-200, 14-180, 14-160, 14-140, 14-120, 14-100, 14-80, 14-60, 14-50, 14-40, 14-30, 14-28, 14-27, 14-26, 14-25 or
16-600, 16-500, 16-400, 16-300, 16-200, 16-180, 16-160, 16-140, 16-120, 16-100, 16-80, 16-60, 16-50, 16-40, 16-30, 16-28, 16-27, 16-26, 16-25 or
18-600, 18-500, 18-400, 18-300, 18-200, 18-180, 18-160, 18-140, 18-120, 18-100, 18-80, 18-60, 18-50, 18-40, 18-30, 18-28, 18-27, 18-26, 18-25 or
20-600, 20-500, 20-400, 20-300, 20-200, 20-180, 20-160, 20-140, 20-120, 20-100, 20-80, 20-60, 20-50, 20-40, 20-30, 20-28, 20-27, 20-26, 20-25 nucleotides in length.
In particularly preferred embodiments, the linker polynucleotide is 20, 21, 22, 23, 24 or 25 nucleotides in length because this produces preferably functional constructs, as can be seen also in examples 3 to 5.
The linker polynucleotide is preferably a polynucleotide which comprises a nucleotide sequence which ensures translation of the synthesized RNA. Such a sequence is referred to in the art also as ribosome-binding site (RBS) or else Shine Dalgarno sequence.
The invention further relates accordingly to an isolated polynucleotide, essentially consisting of a polynucleotide of SEQ ID NO:3 or SEQ ID NO:34, which, via the adenosine nucleotide in position 461 at its 3′ end, is functionally linked, directly or by means of a linker polynucleotide which ensures translation of RNA, to a second polynucleotide which contains at its 5′ end an ATG or GTG start codon and codes for one or more polypeptide(s). Preference is given to both polynucleotides being functionally linked to one another by means of a linker polynucleotide.
The invention furthermore also relates to an isolated polynucleotide, essentially consisting of a polynucleotide of SEQ ID NO:3 or SEQ ID NO:34, which, via the adenosine nucleotide in position 461 at its 3′ end, is functionally linked to a linker oligonucleotide.
In addition, the invention furthermore relates to an isolated polynucleotide, essentially consisting of a polynucleotide of SEQ ID NO:3 or SEQ ID NO:34, which, via the adenosine nucleotide in position 461 at its 3′ end, is functionally linked to a linker polynucleotide which ensures translation of RNA.
In this context, the term “essentially” means that a polynucleotide of no more than (≦) 1000, no more than (≦) 800, no more than (≦) 700, no more than (≦) 600, no more than (≦) 500 or no more than (≦) 400 nucleotides in length has been added to the 5′ end of the polynucleotide of SEQ ID NO:3 or SEQ ID NO:34 and a polynucleotide of no more than (≦) 1000, no more than (≦) 800, no more than (≦) 700, no more than (≦) 600, no more than (≦) 500 or no more than (≦) 400 nucleotides in length has been added to the 3′ end of the second polynucleotide, or a polynucleotide of no more than (≦) 15000, no more than (≦) 10000, no more than (≦) 7500, no more than (≦) 5000, no more than (≦) 2500, no more than (≦) 1000, no more than (≦) 800, no more than (≦) 700, no more than (≦) 600, no more than (≦) 500 or no more than (≦) 400 nucleotides in length has been added to the 3′ end of the linker oligo- or polynucleotide.
Any useful combination of the features from the two lists is in accordance with the invention here.
“Useful combination” means, for example, a combination of features which results in an efficient recombination being carried out. The use of additions of the same length flanking a DNA region to be replaced facilitates the transfer of the region by homologous recombination in the experimental procedure. Relatively long flanking homologous regions are advantageous for efficient recombination between circular DNA molecules but cloning of the replacement vector is made more difficult with increasing length of the flanks (Wang et al., Molecular Biotechnology 32:43-53 (2006)).
Therefore preference is given to the specific combination of additions of in each case 600 to no more than (≦) 1000 nucleotides, with additions of in each case 750 to 850 nucleotides being particularly preferred.
In addition, the flank at the 3′ end of the linker oligo- or polynucleotide increases in length to no more than (≦) 15000 nucleotides when the 3′ end is functionally linked to a second polynucleotide which contains at its 5′ end an ATG or GTG start codon and codes for one or more polypeptide(s).
The ribosome-binding sites of the genus Corynebacterium, preferably of the species Corynebacterium glutamicum, are well-described. Von Amador et al. (Microbiology, 145, 915-924 (1999)) have determined the Anti-Shine Dalgarno sequence of the 16S rRNA of Corynebacterium glutamicum, which is depicted in SEQ ID NO:4. The sequence reported by Martin et al. (Journal of Biotechnology 104, 41-53 (2003)) is depicted in SEQ ID NO:5.
Other examples of suitable ribosome-binding sites are inter alia
the ribosome-binding site of the ppc gene of C. glutamicum ATCC13032 (O'Regan et al., Gene 77, 237-251 (1989)), depicted in SEQ ID NO:6;
the ribosome-binding site of the aceA gene of C. glutamicum ATCC13032 (Reinscheid et al., Journal of Bacteriology 176 (12), 3474-3483 (1994)), depicted in SEQ ID NO:7;
the ribosome-binding site of the thiX gene of C. glutamicum ATCC13032 (Reinscheid et al., Journal of Bacteriology 176 (12), 3474-3483 (1994)), depicted in SEQ ID NO:8;
the ribosome-binding site of the sod gene of C. glutamicum ATCC13032 (WO2005/059144), depicted in SEQ ID NO:9;
the ribosome-binding site of the tuf gene of C. glutamicum ATCC13032 (WO2005/059093), depicted in SEQ ID NO:10;
the ribosome-binding site of SEQ ID NO:44 of EP 1918378, depicted in SEQ ID NO:11;
the ribosome-binding site of the fbp gene of C. glutamicum ATCC13032, depicted in SEQ ID NO:12;
the ribosome-binding site of the gap gene of C. glutamicum ATCC13032 (Eikmanns, Journal of Bacteriology 174 (19), 6076-6086 (1992)), depicted in SEQ ID NO:13.
Preferably the linker polynucleotide contains the ribosome-binding site of the gap gene, depicted in SEQ ID NO:13, or of the fbp gene, depicted in SEQ ID NO:12.
Particularly preferred embodiments of the linker polynucleotide which contain the ribosome-binding site of the gap gene are depicted in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17.
A particularly preferred embodiment of the linker polynucleotide, which contains the ribosome-binding site of the fbp gene, is depicted in SEQ ID NO:18.
These particularly preferred embodiments of the linker polynucleotide ensure translation of RNA in an advantageous manner.
To facilitate chemical linking between the polynucleotide according to the invention having the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:34, the linker polynucleotide which ensures translation of RNA, and the second polynucleotide coding for one or more polypeptide(s), which has an ATG or GTG start codon at its 5′ end, functional nucleotide sequences required for cloning may be incorporated into said polynucleotides at their 5′ and 3′ ends and are at least partially retained even after said cloning.
The term “functional nucleotide sequence required for cloning” here represents any REII (type II restriction endonuclease) cleavage site present, whose sequence normally consists of from 4 to 8 nucleotides.
In addition, it should be mentioned here that site-specific mutagenesis by means of mutagenesis primers or a de novo gene synthesis (e.g. by GENEART AG (Regensburg, Germany)) of the nucleotide sequences to remove cleavage sites for restriction endonucleases may introduce silent mutations into the sequence in order to enable said cleavage sites to be used advantageously for subsequent cloning steps.
The polynucleotide resulting from the promoter according to the invention being functionally linked to the linker polynucleotide which ensures translation of RNA is also referred to as expression unit hereinbelow.
The invention furthermore relates to the use of the promoter according to the invention or of the expression unit according to the invention for expressing desired genes or polynucleotides in microorganisms. The promoter according to the invention or the expression unit according to the invention ensures transcription and translation of the synthesized RNA, preferably mRNA, into a polypeptide.
Expression is preferably carried out in microorganisms of the genus Corynebacterium. Preference is given to strains within the genus Corynebacterium which are based on the following species: Corynebacterium efficiens, with the deposited type strain being DSM44549, Corynebacterium glutamicum, with the deposited type strain being ATCC13032, and Corynebacterium ammoniagenes, with the deposited type strain being ATCC6871. Very particular preference is given to the species Corynebacterium glutamicum.
In this way it is possible to express polynucleotides that code for polypeptides having a property, preferably enzyme activity, which are not present or detectable in the corresponding host. Thus, for example, Yukawa et al. describe expression of Escherichia coli genes for utilizing D-xylose in Corynebacterium glutamicum R under the control of the constitutive Ptrc promoter (Applied Microbiology and Biotechnology 81, 691-699 (2008)).
The promoter according to the invention or the expression unit according to the invention is furthermore used for overexpressing desired genes or polynucleotides in microorganisms (overexpression).
Overexpression generally means an increase in the intracellular concentration or activity of a ribonucleic acid, a protein (polypeptide) or an enzyme in comparison with the starting strain (parent strain) or wild-type strain, if the latter is the starting strain. A starting strain (parent strain) means the strain that has been subjected to the measure resulting in overexpression.
For overexpression, preference is given to the methods of recombinant overexpression. These include all methods in which a microorganism is produced using a DNA molecule provided in vitro. Such DNA molecules comprise, for example, promoters, expression cassettes, genes, alleles, coding regions, etc. They are introduced into the desired microorganisms by methods of transformation, conjugation, transduction or similar methods.
In the case of the present invention, the promoters are preferably a polynucleotide of SEQ ID NO:3 or SEQ ID NO:34 and the expression cassettes are preferably a polynucleotide of SEQ ID NO:3 or SEQ ID NO:34 which, via the adenosine nucleotide in position 461 at its 3′ end, is functionally linked to a linker polynucleotide which ensures translation of RNA.
The measures of overexpression using the promoter according to the invention or the expression unit according to the invention increase the activity or concentration of the corresponding polypeptide usually by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, preferably by no more than 1000%, 2000%, 4000%, 10000% or 20000%, based on the activity or concentration of said polypeptide in the strain prior to the measure resulting in overexpression.
The extent of expression or overexpression may be established by measuring the amount of mRNA transcribed from the gene, by determining the amount of polypeptide and by determining enzyme activity.
The amount of mRNA may be determined inter alia by using the methods of “Northern Blotting” and of quantitative RT-PCR. Quantitative RT-PCR involves reverse transcription which precedes the polymerase chain reaction. For this, the LightCycler™ System from Roche Diagnostics (Boehringer Mannheim GmbH, Roche Molecular Biochemicals, Mannheim, Germany) may be used, as described in Jungwirth et al. (FEMS Microbiology Letters 281, 190-197 (2008)), for example. The concentration of the protein may be determined via 1- and 2-dimensional protein gel fractionation and subsequent optical identification of the protein concentration using appropriate evaluation software in the gel. A customary method of preparing protein gels for coryneform bacteria and of identifying said proteins is the procedure described by Hermann et al. (Electrophoresis, 22:1712-23 (2001)). The protein concentration may likewise be determined by Western-Blot hybridization using an antibody specific for the protein to be detected (Sambrook et al., Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and subsequent optical evaluation using appropriate software for concentration determination (Lohaus and Meyer (1998) Biospektrum 5:32-39; Lottspeich, Angewandte Chemie 321: 2630-2647 (1999)). The statistical significance of the data collected is determined by means of a T test (Gosset, Biometrika 6(1): 1-25 (1908)).
The measure of overexpressing desired genes using the promoter according to the invention may be combined in a suitable manner with further overexpression measures.
Overexpression is achieved by a multiplicity of methods available in the prior art. These include increasing the copy number in addition to modifying the nucleotide sequences which direct or control expression of the gene.
The copy number may be increased by means of plasmids which replicate in the cytoplasm of the microorganism. To this end, an abundance of plasmids are described in the prior art for very different groups of microorganisms, which plasmids can be used for setting the desired increase in the copy number of the gene. Plasmids suitable for the genus Corynebacterium are described, for example, in Tauch et al. (Journal of Biotechnology 104 (1-3), 27-40, (2003)), or in Stansen et al. (Applied and Environmental Microbiology 71, 5920-5928 (2005)).
The copy number may furthermore be increased by at least one (1) copy by introducing further copies into the chromosome of the microorganism. Methods suitable for the genus Corynebacterium are described, for example, in the patents WO 03/014330, WO 03/040373 and WO 04/069996.
Gene expression may furthermore be increased by positioning a plurality of promoters upstream of the desired gene or functionally linking them to the gene to be expressed and achieving increased expression in this way. Examples of this are described in the patent WO 2006/069711.
Transcription of a gene is controlled, where appropriate, by proteins which suppress (repressor proteins) or promote (activator proteins) transcription. Accordingly, overexpression can likewise be achieved by increasing the expression of activator proteins or reducing or switching off the expression of repressor proteins or else eliminating the binding sites of the repressor proteins.
The rate of elongation is influenced by the codon usage, it being possible to enhance translation by utilizing codons for t(transfer) RNAs which are frequent in the starting strain. Moreover, replacing a start codon with the ATG codon most frequent in many microorganisms (77% in Escherichia coli) may considerably improve translation, since, at the RNA level, the AUG codon is two to three times more effective than the codons GUG and UUG, for example (Khudyakov et al., FEBS Letters 232(2):369-71 (1988); Reddy et al., Proceedings of the National Academy of Sciences of the USA 82(17):5656-60 (1985)). It is also possible to optimize the sequences surrounding the start codon because synergistic effects between the start codon and the flanking regions have been described (Stenstrom et al., Gene 273(2):259-65 (2001); Hui et al., EMBO Journal 3(3):623-9 (1984)).
Instructions for handling DNA, digestion and ligation of DNA, transformation and selection of transformants can be found inter alia in the known manual by Sambrook et al. “Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989).
The invention also relates to vectors comprising the polynucleotide according to the invention.
Kirchner and Tauch (Journal of Biotechnology 104:287-299 (2003)) describe a selection of vectors to be used in Corynebacterium glutamicum.
Homologous recombination using the vectors according to the invention allows DNA segments on the chromosome to be replaced with polynucleotides according to the invention which are transported into the cell by the vector. For efficient recombination between the circular DNA molecule of the vector and the target DNA on the chromosome, the DNA region to be replaced with the polynucleotide according to the invention is provided at the ends with nucleotide sequences homologous to the target site which determine the site of integration of the vector and of replacement of the DNA.
Thus the polynucleotide having promoter activity according to the invention may
1. be replaced with the native promoter at the native gene locus of the second polynucleotide in the chromosome, or
2. be integrated with the second polynucleotide at the native gene locus of the latter or at another gene locus.
“Replacement of the native promoter at the native gene locus of the second polynucleotide” means the fact that the naturally occurring promoter of the gene which usually is naturally present by way of a single copy at its gene locus in the corresponding wild type or corresponding starting organism in the form of its nucleotide sequence is replaced.
“Another gene locus” means a gene locus whose nucleotide sequence is different from the sequence of the second polynucleotide. Said other gene locus or the nucleotide sequence at said other gene locus is preferably located within the chromosome and normally is not essential for growth and for production of the desired chemical compounds. It is furthermore possible to use intergenic regions within the chromosome, i.e. nucleotide sequences without coding function.
Expression or overexpression is preferably carried out in microorganisms of the genus Corynebacterium. Within the genus Corynebacterium, preference is given to strains based on the following species: Corynebacterium efficiens, with the deposited type strain being DSM44549, Corynebacterium glutamicum, with the deposited type strain being ATCC13032, and Corynebacterium ammoniagenes, with the deposited type strain being ATCC6871. Very particular preference is given to the species Corynebacterium glutamicum.
Some representatives of the species Corynebacterium glutamicum are also known in the prior art under different names. These include, for example: strain ATCC13870 referred to as Corynebacterium acetoacidophilum, strain DSM20137 referred to as Corynebacterium lilium, strain ATCC17965 referred to as Corynebacterium melassecola, strain ATCC14067 referred to as Brevibacterium flavum, strain ATCC13869 referred to as Brevibacterium lactofermentum, and strain ATCC14020 referred to as Brevibacterium divaricatum.
The term “Micrococcus glutamicus” has also been in use for Corynebacterium glutamicum. Some representatives of the species Corynebacterium efficiens have also been referred to as Corynebacterium thermoaminogenes in the prior art, such as the strain FERM BP-1539, for example.
The microorganisms or strains (starting strains) employed for the expression or overexpression measures according to the invention preferably already possess the ability to secrete a desired fine chemical into the surrounding nutrient medium and accumulate there. The expression “to produce” is also used for this hereinbelow. More specifically, the strains employed for the overexpression measures possess the ability to accumulate the desired fine chemical in concentrations of ≧ (at least) ≧0.10 g/l, 0.25 g/l, ≧0.5 g/l, ≧1.0 g/l, ≧1.5 g/l, ≧2.0 g/l, ≧4 g/l or ≧10 g/l in (no more than) 120 hours, ≦96 hours, ≦48 hours, ≦36 hours, ≦24 hours or ≦12 hours in the cell or in the nutrient medium. The starting strains are preferably strains prepared by mutagenesis and selection, by recombinant DNA technologies or by a combination of both methods.
A person skilled in the art understands that a microorganism suitable for the measures of the invention may also be obtained by firstly employing the promoter according to the invention of SEQ ID NO:3 or SEQ ID NO:34 for overexpression of the desired genes in a wild strain such as, for example, the Corynebacterium glutamicum type strain ATCC 13032 or the strain ATCC 14067, and then, by means of further genetic measures described in the prior art, causing the microorganism to produce the desired fine chemical(s).
The term “fine chemicals” means with regard to the measures of the invention amino acids, organic acids, vitamins, nucleosides and nucleotides. Particular preference is given to proteinogenic amino acids, non-proteinogenic amino acids, and organic acids.
“Proteinogenic amino acids” mean the amino acids which occur in natural proteins, i.e. in proteins of microorganisms, plants, animals and humans. They serve as structural units for proteins in which they are linked to one another via peptide bonds.
The terms protein and polypeptide are interchangeable.
The mentioning of proteinogenic L-amino acids hereinbelow refers to one or more of the amino acids, including their salts, selected from the group consisting of L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-arginine, L-proline, and, where appropriate, L-selenocystein and L-pyrrolysine. Particular preference is given to the L-amino acids L-lysine, L-tryptophan, L-methionine, L-valine, L-isoleucine and L-proline. Very particular preference is given to L-lysine and L-valine.
The mentioning of L-lysine or lysine hereinbelow refers to not only the bases but also the salts such as, for example, lysine monohydrochloride or lysine sulphate.
The non-proteinogenic amino acids include inter alia L-ornithine and L-homoserine.
The organic acids include inter alia α-keto acids, with particular preference being given to α-ketoisocaproic acid (KIC), α-ketovaleric acid (KIV) and α-keto-β-methylvaleric acid (KMV).
Examples of L-lysine-secreting or -producing strains of the species Corynebacterium glutamicum are:
An example of an L-lysine-secreting or -producing strain of the species Corynebacterium efficiens is:
L-lysine-producing microorganisms typically possess a “feedback”-resistant or densensitized aspartate kinase. “Feedback”-resistant aspartate kinases mean aspartate kinases (LysC) which, in comparison with the wild form (wild type), are less sensitive to inhibition by mixtures of lysine and threonine or mixtures of AEC (aminoethylcysteine) and threonine or lysine alone or AEC alone. The genes or alleles coding for these, compared to the wild type, desensitized aspartate kinases are also referred to as lysCFBR alleles. The wild type suitable in the case of the aspartate kinases of the species Corynebacterium glutamicum is the strain ATCC13032. The prior art describes numerous lysCFBR alleles which code for aspartate kinase variants which have amino acid substitutions in comparison with the wild-type protein. The lysC gene in bacteria of the genus Corynebacterium is also referred to as ask gene. The lysC gene-encoded aspartate kinase in Enterobacteriaceae is also referred to as aspartokinase III.
A detailed list with information about amino acid substitutions in the Corynebacterium glutamicum aspartate kinase protein that result in densensitization is included inter alia in WO2009141330A1 (page 10, line 21 to page 13, line 17) which is incorporated herein by reference. Preference is given to aspartate kinase variants carrying the following amino acid substitutions selected from the group consisting of: L-isoleucine for L-threonine in position 380 of the amino acid sequence and optionally L-phenylalanine for L-serine in position 381, L-isoleucine for L-threonine in position 311 and L-threonine for L-alanine in position 279.
An example of an L-methionine-secreting or -producing strain of the species Corynebacterium glutamicum is
Examples of known representatives of L-tryptophan-producing or -secreting strains of coryneform bacteria are:
Examples of known representatives of L-valine-producing or -secreting strains of coryneform bacteria are:
Corynebacterium glutamicum FERM BP-1764 described in U.S. Pat. No. 5,188,948.
L-valine-producing microorganisms typically possess a “feedback”-resistant or densensitized acetolactate synthase (AHAS, EC 4.1.3.18) which is the first enzyme of the parallel metabolic pathways for synthesizing isoleucine, valine and leucine (Umbarger, H. E. 1987. Biosynthesis of the branched-chain amino acids, p. 352-367. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, D.C.). The holoenzyme always consists of two large and two small subunits. The large AHAS subunit forms the catalytic domain and is encoded by ilvB, with the small subunit which acts as regulatory domain being encoded by ilvN. “Feedback”-resistant acetolactate synthases mean acetolactate synthases which, compared to the wild form (wild type), are less sensitive to inhibition by the branched-chain amino acids valine, leucine and isoleucine or mixtures thereof. The wild type suitable in the case of the acetolactate synthases of the species Corynebacterium glutamicum is the strain ATCC13032, and the wild type suitable within the species Brevibacterium flavum is the strain ATCC14067.
The ilvBN genes coding for acetolactate synthase in Corynebacterium glutamicum have been described, for example, by Keilhauer et al. (Journal of Bacteriology 175(17):5595-603 (1993)) or in EP1108790. Access number L09232 depicts the sequence of said genes.
AHAS variant enzymes which are no longer subject to feedback inhibition by the branched-chain amino acids (leucine, valine, isoleucine) are described, for example, in Mendel et al. (Journal of Molecular Biology 325, 275-284 (2003)), Elisakova et al. (Applied and Environmental Microbiology 71, 207-213 (2005)), Wada et al. (Bioscience Biotechnology & Biochemistry, 72 (11), 2959-2965, (2008)) and in EP1491634. Preference is given to variants of a “feedback”-resistant acetolactate synthase which carry one or more of the following amino acid substitutions in the ilvN-encoded small subunit, selected from the group consisting of: L-aspartic acid for glycine in position 20 of the amino acid sequence, L-aspartic acid for L-isoleucine in position 21 of the amino acid sequence, L-phenylalanine for L-isoleucine in position 22 of the amino acid sequence, any proteinogenic amino acid except L-alanine, preferably L-valine, L-isoleucine and L-leucine, particularly preferably L-valine in position 42 and optionally L-leucine in position 47 for L-histidine.
Examples of known representatives of L-isoleucine-producing or -secreting strains of coryneform bacteria are:
Examples of known representatives of L-homoserine-producing or -secreting strains of coryneform bacteria are:
Examples of known representatives of L-ornithine-secreting or -producing strains of the species Corynebacterium glutamicum are:
α-keto acid-secreting or -producing strains are based on, for example:
The present invention provides a microorganism which produces a fine chemical, said microorganism having increased expression of one or more genes in comparison to the particular starting strain by using the promoter according to the invention of SEQ ID NO:3 or SEQ ID NO:34.
The present invention furthermore provides a process for fermentative preparation of a fine chemical, comprising the steps of:
Preference is given here to obtaining from the fine chemical-containing fermentation broth the fine chemical or a liquid or solid fine chemical-containing product.
The microorganisms produced may be cultured continuously—as described, for example, in WO 05/021772—or discontinuously in a batch process (batch cultivation) or in a fed-batch or repeated fed-batch process for the purpose of producing the desired organic-chemical compound. A summary of a general nature about known cultivation methods is available in the textbook by Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium or fermentation medium to be used must in a suitable manner satisfy the demands of the respective strains. Descriptions of culture media for various microorganisms are present in the “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 interchangeable.
It is possible to use, as carbon source, sugars and carbohydrates such as, for example, 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, for example, soybean oil, sunflower oil, groundnut oil and coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerol, methanol and ethanol, and organic acids such as, for example, acetic acid or lactic acid.
It is possible to use, as nitrogen source, organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used individually or as a mixture.
It is possible to use, as phosphorus source, phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts.
The culture medium must additionally comprise salts, for example in the form of chlorides or sulphates of metals such as, for example, sodium, potassium, magnesium, calcium and iron, such as, for example, magnesium sulphate or iron sulphate, which are necessary for growth. Finally, essential growth factors such as amino acids, for example homoserine and vitamins, for example thiamine, biotin or pantothenic acid, may be employed in addition to the above-mentioned substances.
Said starting materials may be added to the culture in the form of a single batch or be fed in during the cultivation in a suitable manner.
The pH of the culture can be controlled by employing basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds such as phosphoric acid or sulphuric acid in a suitable manner. The pH is generally adjusted to a value of from 6.0 to 8.5, preferably 6.5 to 8. To control foaming, it is possible to employ antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids, it is possible to add to the medium suitable selective substances such as, for example, antibiotics. The fermentation is preferably carried out under aerobic conditions. In order to maintain these conditions, oxygen or oxygen-containing gas mixtures such as, for example, air are introduced into the culture. It is likewise possible to use liquids enriched with hydrogen peroxide. The fermentation is carried out, where appropriate, at elevated pressure, for example at an elevated pressure of from 0.03 to 0.2 MPa. The temperature of the culture is normally from 20° C. to 45° C. and preferably from 25° C. to 40° C., particularly preferably from 30° C. to 37° C. In batch or fed-batch processes, the cultivation is preferably continued until an amount of the desired organic-chemical compound sufficient for being recovered has formed. This aim is normally achieved within 10 hours to 160 hours. In continuous processes, longer cultivation times are possible. The activity of the microorganisms results in a concentration (accumulation) of the organic-chemical compound in the fermentation medium and/or in the cells of said microorganisms.
Examples of suitable fermentation media can be found inter alia in the U.S. Pat. Nos. 5,770,409, 5,990,350, 5,275,940, WO 2007/012078, U.S. Pat. No. 5,827,698, WO 2009/043803, U.S. Pat. No. 5,756,345 and U.S. Pat. No. 7,138,266.
Analysis of L-amino acids to determine the concentration at one or more time(s) during the fermentation can take place by separating the L-amino acids by means of ion exchange chromatography, preferably cation exchange chromatography, with subsequent post-column derivatization using ninhydrin, as described in Spackman et al. (Analytical Chemistry 30: 1190-1206 (1958)). It is also possible to employ ortho-phthaldialdehyde rather than ninhydrin for post-column derivatization. An overview article on ion exchange chromatography can be found in Pickering (LC•GC (Magazine of Chromatographic Science) 7(6), 484-487 (1989)).
It is likewise possible to carry out a pre-column derivatization, for example using ortho-phthadialdehyde or phenyl isothiocyanate, and to fractionate the resulting amino acid derivates by reversed-phase chromatography (RP), preferably in the form of high-performance liquid chromatography (HPLC). A method of this type is described, for example, in Lindroth et al. (Analytical Chemistry 51: 1167-1174 (1979)).
Detection is carried out photometrically (absorption, fluorescence).
A review regarding amino acid analysis can be found inter alia in the textbook “Bioanalytik” from Lottspeich and Zorbas (Spektrum Akademischer Verlag, Heidelberg, Germany 1998).
The analysis of determining the concentration of α-keto acids at one or more point(s) in the course of the fermentation may be carried out by separating the keto acids and other secreted products by means of ion exchange chromatography, preferably cation exchange chromatography, on a sulphonated styrene-divinylbenzene polymer in the H+ form, for example by means of 0.025 M sulphuric acid with subsequent UV detection at 215 nm (alternatively also at 230 or 275 nm). Preferably, a REZEK RFQ—Fast Fruit H+ column (Phenomenex) may be employed, but other suppliers for the separating phase (e.g. Aminex from BioRad) are feasible. Similar separations are described in corresponding application examples by the suppliers.
The performance of the processes or fermentation processes containing the promoter variant according to the invention, in terms of one or more of the parameters selected from the group of concentration (compound formed per unit volume), yield (compound formed per unit carbon source consumed), formation (compound formed per unit volume and time) and specific formation (compound formed per unit dry cell matter or dry biomass and time or compound formed per unit cellular protein and time) or else other process parameters and combinations thereof, is increased by at least 0.5%, at least 1%, at least 1.5% or at least 2%, based on processes or fermentation processes using microorganisms not containing the promoter variant according to the invention. This is considered to be very worthwhile in terms of a large-scale industrial process.
The fermentation measures result in a fermentation broth which contains the desired fine chemical, preferably amino acid or organic acid.
A product containing the fine chemical is then provided or produced or recovered in liquid or solid form.
A fermentation broth means a fermentation medium or nutrient medium in which a microorganism has been cultivated for a certain time and at a certain temperature. The fermentation medium or the media employed during fermentation comprise(s) all the substances or components which ensure production of the desired compound and typically propagation and viability.
When the fermentation is complete, the resulting fermentation broth accordingly comprises
The organic byproducts include substances which are produced by the microorganisms employed in the fermentation in addition to the particular desired compound and are optionally secreted.
The fermentation broth is removed from the culture vessel or fermentation tank, collected where appropriate, and used for providing a product containing the fine chemical in liquid or solid form. The expression “recovering the fine chemical-containing product” is also used for this. In the simplest case, the fine chemical-containing fermentation broth itself, which has been removed from the fermentation tank, constitutes the recovered product.
One or more of the measures selected from the group consisting of
The partial (>0% to <80%) to complete (100%) or virtually complete (≧80% to <100%) removal of the water (measure a)) is also referred to as drying.
In one variant of the process, complete or virtually complete removal of the water, of the biomass, of the organic byproducts and of the unconsumed constituents of the fermentation medium employed results in pure (≧80% by weight, ≧90% by weight) or high-purity (≧95% by weight, ≧97% by weight, ≧99% by weight) product forms of the desired organic-chemical compound, preferably L-amino acids. An abundance of technical instructions for measures a), b), c) and d) are available in the prior art.
In the case of the amino acid L-lysine, essentially four different product forms are known in the prior art.
One group of L-lysine-containing products includes concentrated aqueous alkaline solutions of purified L-lysine (EP-B-0534865). A further group, as described for example in U.S. Pat. No. 6,340,486 and U.S. Pat. No. 6,465,025, includes aqueous acidic biomass-containing concentrates of L-lysine-containing fermentation broths. The best-known group of solid products includes pulverulent or crystalline forms of purified or pure L-lysine, which is typically in the form of a salt such as, for example, L-lysine monohydrochloride. A further group of solid product forms is described for example in EP-B-0533039. The product form described therein comprises besides L-lysine most of the starting materials used during the fermentative production and not consumed and, where appropriate, the biomass of the microorganism employed with a proportion of >0%-100%.
A wide variety of processes appropriate for the various product forms are known for producing the L-lysine-containing product or the purified L-lysine from the fermentation broth.
The methods essentially used to produce pure solid L-lysine are those of ion exchange chromatography, where appropriate with use of activated carbon, and methods of crystallization. The corresponding base or a corresponding salt such as, for example, the monohydrochloride (Lys-HCl) or the lysine sulphate (Lys2-H2SO4) is obtained in this way.
EP-B-0534865 describes a process for producing aqueous basic L-lysine-containing solutions from fermentation broths. In the process described therein, the biomass is separated from the fermentation broth and discarded. A base such as, for example, sodium hydroxide, potassium hydroxide or ammonium hydroxide is used to set a pH of between 9 to 11. The mineral constituents (inorganic salts) are removed from the broth by crystallization after concentration and cooling and are either used as fertilizer or discarded.
In processes for producing lysine by using bacteria of the genus Corynebacterium, preferred processes are those resulting in products which comprise constituents of the fermentation broth. These are used in particular as animal feed additives.
Depending on requirements, the biomass can be removed wholly or partly from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decantation or a combination thereof, or be left completely therein. Where appropriate, the biomass or the biomass-containing fermentation broth is inactivated during a suitable process step, for example by thermal treatment (heating) or by addition of acid.
In one procedure, the biomass is completely or virtually completely removed so that no (0%) or at most 30%, at most 20%, at most 10%, at most 5%, at most 1% or at most 0.1% biomass remains in the prepared product. In a further procedure, the biomass is not removed, or is removed only in small proportions, so that all (100%) or more than 70%, 80%, 90%, 95%, 99% or 99.9% biomass remains in the product prepared. In one process according to the invention, accordingly, the biomass is removed in proportions of from ≧0% to ≦100%.
Finally, the fermentation broth obtained after the fermentation can be adjusted, before or after the complete or partial removal of the biomass, to an acidic pH with an inorganic acid such as, for example, hydrochloric acid, sulphuric acid or phosphoric acid, or organic acid such as, for example, propionic acid, so as to improve the handling properties of the final product (GB 1,439,728 or EP 1 331 220). It is likewise possible to acidify the fermentation broth with the complete content of biomass. Finally, the broth can also be stabilized by adding sodium bisulphite (NaHSO3, GB 1,439,728) or another salt, for example ammonium, alkali metal or alkaline earth metal salt of sulphurous acid.
During the removal of the biomass, any organic or inorganic solids present in the fermentation broth are partially or completely removed. The organic byproducts dissolved in the fermentation broth, and the dissolved unconsumed constituents of the fermentation medium (starting materials), remain at least partly (>0%), preferably to an extent of at least 25%, particularly preferably to an extent of at least 50% and very particularly preferably to an extent of at least 75% in the product. Where appropriate, they also remain completely (100%) or virtually completely, meaning >95% or >98% or greater than 99%, in the product. If a product in this sense comprises at least part of the constituents of the fermentation broth, this is also described by the term “product based on fermentation broth”.
Subsequently, water is removed from the broth, or said broth is thickened or concentrated, by known methods such as, for example, using a rotary evaporator, thin-film evaporator, falling-film evaporator, by reverse osmosis or by nanofiltration. This concentrated fermentation broth can then be worked up to free-flowing products, in particular to a fine powder or preferably coarse granules, by methods of freeze drying, spray drying, spray granulation or by other processes such as in the circulating fluidized bed, as described for example according to PCT/EP2004/006655. A desired product is isolated where appropriate from the resulting granules by screening or dust removal.
It is likewise possible to dry the fermentation broth directly, i.e. without previous concentration by spray drying or spray granulation.
“Free-flowing” means powders which, from a series of glass orifice vessels with orifices of different sizes, flow unimpeded at least out of the vessel with a 5 mm (millimeters) orifice (Klein: Seifen, Öle, Fette, Wachse 94, 12 (1968)).
“Fine” means a powder predominantly (>50%) having a particle size of diameter from 20 to 200 μm.
“Coarse” means a product predominantly (>50%) of a particle size of diameter from 200 to 2000 μm.
The particle size determination can be carried out by methods of laser diffraction spectrometry. Corresponding methods are described in the textbook on “Teilchengröβenmessung in der Laborpraxis” by R. H. Müller and R. Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) or in the textbook “Introduction to Particle Technology” by M. Rhodes, published by Wiley & Sons (1998).
The free-flowing, fine powder can in turn be converted by suitable compaction or granulation processes into a coarse, very free-flowing, storable and substantially dust-free product.
The term “dust-free” means that the product comprises only small proportions (<5%) of particle sizes below 100 μm in diameter.
“Storable” in the sense of this invention means a product which can be stored for at least one (1) year or longer, preferably at least 1.5 years or longer, particularly preferably two (2) years or longer, in a dry and cool environment without any substantial loss of the respective amino acid occurring. “Substantial loss” means a loss of >5%.
The invention further relates to a process described in principle in WO 2007/042363 A1. To this end, a process is carried out which uses the fermentation broth obtained according to the invention, from which the biomass has been removed completely or partially, where appropriate, and which comprises the following steps:
where one or both of the following measures is/are carried out where appropriate before step a):
Where appropriate, also before step b), a salt of sulphurous acid, preferably alkali metal bisulphite, particularly preferably sodium bisulphite, is added in a concentration of from 0.01 to 0.5% by weight, preferably 0.1 to 0.3% by weight, particularly preferably 0.1 to 0.2% by weight, based on the fermentation broth.
Preferred sulphate-containing compounds which should be mentioned in the context of the abovementioned process steps are in particular ammonium sulphate and/or ammonium bisulphate or appropriate mixtures of ammonia and sulphuric acid and sulphuric acid itself.
The molar sulphate/L-lysine ratio V is calculated by the formula: V=2x[SO42−]/[L-lysine]. This formula takes account of the fact that the SO42− anion and sulphuric acid are divalent. A ratio of V=1 means that a stoichiometric composition Lys2−(H2SO4) is present, whereas the finding with a ratio of V=0.9 is a 10% sulphate deficit and with a ratio of V=1.1 is a 10% sulphate excess.
It is advantageous to employ during the granulation or compaction the usual organic or inorganic auxiliaries or carriers such as starch, gelatine, cellulose derivatives or similar substances, as normally used in the processing of food products or feeds as binders, gelling agents or thickeners, or further substances such as, for example, silicas, silicates (EP0743016A) and stearates.
It is further advantageous to treat the surface of the resulting granules with oils or fats as described in WO 04/054381. Oils which can be used are mineral oils, vegetable oils or mixtures of vegetable oils. Examples of such oils are soybean oil, olive oil, soybean oil/lecithin mixtures. In the same way, silicone oils, polyethylene glycols or hydroxyethylcellulose are also suitable. Treatment of the surfaces with said oils achieves an increased abrasion resistance of the product and a reduction in the dust content. The oil content in the product is 0.02 to 2.0% by weight, preferably 0.02 to 1.0% by weight, and very particularly preferably 0.2 to 1.0% by weight, based on the total amount of the feed additive.
Preferred products have a proportion of ≧97% by weight with a particle size of from 100 to 1800 μm or a proportion of ≧95% by weight with a particle size of diameter 300 to 1800 μm. The proportion of dust, i.e. particles with a particle size<100 μm, is preferably >0 to 1% by weight, particularly preferably not exceeding 0.5% by weight.
However, alternatively, the product may also be absorbed on an organic or inorganic carrier known and customary in the processing of feeds, such as, for example, silicas, silicates, meals, brans, flours, starches, sugars or others, and/or be mixed and stabilized with customary thickeners or binders. Examples of use and processes therefor are described in the literature (Die Mühle+Mischfuttertechnik 132 (1995) 49, page 817).
Finally, the product can also be brought, by coating processes with film-formers such as, for example, metal carbonates, silicas, silicates, alginates, stearates, starches, gums and cellulose ethers, as described in DE-C-4100920, into a state which is stable to digestion by animal stomachs, especially the stomach of ruminants.
To establish a desired L-lysine concentration in the product it is possible, depending on requirements, to add the L-lysine during the process in the form of a concentrate or, where appropriate, of a substantially pure substance or its salt in liquid or solid form. These can be added singly or as mixtures to the resulting or concentrated fermentation broth, or else during the drying or granulation process.
The invention further relates to a combination with a process for preparing a solid lysine-containing product, which process is described in principle in US 20050220933. This involves carrying out a process which uses the fermentation broth obtained according to the invention and which comprises the following steps:
The coating agents preferably used for the coating in step d) are selected from the group consisting of
L-lysine content of <1% by weight, preferably <0.5% by weight, preferably selected from the group of starch, carrageenan, agar, silicas, silicates, meals, brans and flours, and
The L-lysine content is adjusted to a desired value by the measures corresponding to steps d1) to d4), in particular d1) to d3).
In the production of L-lysine-containing products, the ratio of the ions is preferably adjusted so that the molar ion ratio corresponding to the following formula
2x[SO42−]+[Cl−]−[NH4+]−[Na+]−[K+]−2x[Mg2+]−2x[Ca2+]/[L-Lys]
gives 0.68 to 0.95, preferably 0.68 to 0.90, particularly preferably 0.68 to 0.86, as described by Kushiki et al. in US 20030152633.
In the case of L-lysine, the solid product produced in this way has, based on the fermentation broth, a lysine content (as lysine base) of from 10% by weight to 70% by weight or 20% by weight to 70% by weight, preferably 30% by weight to 70% by weight and very particularly preferably from 40% by weight to 70% by weight, based on the dry matter of the product. Maximum lysine base contents of 71% by weight, 72% by weight, 73% by weight are likewise possible.
The water content of the L-lysine-containing solid product is up to 5% by weight, preferably up to 4% by weight, and particularly preferably less than 3% by weight.
The Corynebacterium glutamicum strain DM1547 was prepared by multiple, non-directed mutagenesis, selection and mutant choice from C. glutamicum ATCC13032. The strain is resistant to the lysine analogue S-(2-aminoethyl)-L-cysteine.
A pure culture of the DM1547 strain was deposited with the Deutsche Sammlung für Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany) as DSM 13994 in accordance with the Budapest Treaty on 16 Jan. 2001. The strain DSM 13994 is listed in EP 1 239 040.
The nucleotide sequence of the Corynebacterium glutamicum genome ATCC13032 is described in Ikeda and Nakagawa (Applied Microbiology and Biotechnology 62, 99-109 (2003)), in EP 1 108 790 and in Kalinowski et al. (Journal of Biotechnology 104(1-3), (2003)). The nucleotide sequence of the Corynebacterium glutamicum R genome is described in Yukawa et al. (Microbiology 153(4):1042-1058 (2007)).
The nucleotide sequence of the Corynebacterium efficiens genome is described in Nishio et al (Genome Research. 13 (7), 1572-1579 (2003)).
The nucleotide sequences of the Corynebacterium glutamicum and Corynebacterium efficiens genomes are likewise available in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA), in the DNA Data Bank of Japan (DDBJ, Mishima, Japan), or in the nucleotide sequence database of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany, and Cambridge, UK).
Chromosomal DNA was isolated from the strain DM1547 bp the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). A DNA section containing the upstream region of the gap gene was amplified with the aid of the polymerase chain reaction. Owing to the known sequence of the C. glutamicum gap gene, the following oligonucleotides were used as primers:
Pgap3-1 (SEQ ID NO:31):
5′ cgtttggggtcaatgtccat 3′
Pgap3-2 (SEQ ID NO:32):
5′ cccagtccaggttctttg 3′
The primers shown were synthesized by MWG Biotech (Ebersberg, Germany). They enable a 529 bp DNA section to be amplified. The Pgap3-2 primer binds to the region corresponding to positions 742 to 759 of the strand complementary to SEQ ID NO:30 (and SEQ ID NO:33). The Pgap3-1 primer binds to the region corresponding to positions 231 to 250 of the strand of SEQ ID NO:30 (and SEQ ID NO:33).
The PCR reaction was carried out using the Phusion High Fidelity DNA polymerase (New England Biolabs, Frankfurt, Germany). The reaction mixture was prepared according to the manufacturer's information and contained in a total volume of 50 μl 10 μl of the supplied 5× Phusion HF Buffer, deoxynucleoside triphosphates at a concentration of 200 μm each, primers at a concentration of 0.5 μm, approximately 50 ng of template DNA and two units of Phusion Polymerase. The volume was adjusted to 50 μl by adding H2O.
The PCR mixture was first subjected to an initial denaturation at 98° C. for 30 seconds. This was followed by 35 repeats of a denaturation step at 98° C. for 20 seconds, a step for binding the primers to the DNA presented at 60° C. for 20 seconds, and the extension step to extend the primers at 72° C. for 60 seconds. After the final extension step at 72° C. for 5 minutes, the PCR mixture was subjected to an agarose gel electrophoresis (0.8% agarose). A DNA fragment of approx. 0.53 kb in length was identified, isolated from the gel and purified using the QIAquick Gel Extraction kit from Qiagen (Hilden, Germany).
The nucleotide sequence of the amplified DNA fragment or PCR product was determined by Agowa (Berlin, Germany).
The nucleotide sequence of the promoter region of the strain DM1547 gap gene contains the nucleobase adenine in position 379 (see SEQ ID NO:33). The promoter region of the wild type (see SEQ ID NO:30) contains the nucleobase guanine in this position. This mutation is referred to as Pgap3 hereinbelow.
To characterize the mutation according to the invention, an upstream region of the gap gene, including the promoter, is used for enhancing a synthetic operon. The synthetic operon consists of a densensitized Corynebacterium glutamicum aspartate kinase encoded by an allele of the lysC gene, in which the wild-type nucleobase threonine has been replaced with the nucleobase isoleucine in position 311 of the encoded aspartate kinase protein (see WO 00/63388 and U.S. Pat. No. 6,893,848), and a Corynebacterium glutamicum aspartate-semialdehyde dehydrogenase encoded by the asd gene. Said synthetic operon is integrated via homologous recombination into an intergenic region within the Corynebacterium glutamicum chromosome in such a way that the insertion results in an increase in the copy number of the lysC and asd genes. A homologous DNA sequence which enables the synthetic operon to be incorporated was synthesized by Geneart AG (Regensburg, Germany). This DNA sequence is depicted in SEQ ID NO:35 and is denoted IBcg0054. The DNA sequence contains a 383 bp intergenic region flanked by a 595 bp region (cg0055 gene coding for a putative membrane protein and part of the cg0057 gene coding for a serine/threonine protein kinase) and a 663 bp region (part of the cg0054 gene coding for an iron uptake protein).
In addition to the restriction cleavage sites of the enzymes AvrII and NsiI, in a central position within the intergenic region for the insertion of various DNA sequences, flanking cleavage sites for the restriction endonucleases MunI and HindIII were generated for procedures of subcloning the entire fragment.
The 1660 bp Geneart fragment (SEQ ID NO:35) was digested with the restriction enzymes MunI and HindIII and then subcloned into the mobilizable vector pK18mobsacB described by Schafer et al. (Gene, 145, 69-73 (1994)). For this, pK18mobsacB was digested with the restriction enzymes EcoRI and HindIII. The vector prepared in this way was mixed with the IBcg0054 fragment, and the mixture was treated with the Ready-To-Go T4 DNA Ligase Kit (Amersham-Pharmacia, Freiburg, Germany).
Subsequently, the E. coli strain S17-1 (Simon et al., Bio/Technologie 1: 784-791, 1993) was transformed with the ligation mixture (Hanahan, in DNA cloning. A practical approach. Vol. 1. ILR-Press, Cold Spring Harbor, N.Y., 1989). Plasmid-carrying cells were selected by plating the transformation mixture on LB agar (Sambrock et al., Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor, N.Y., 1989) which had been supplemented with 50 mg/l kanamycin.
Plasmid DNA was isolated from a transformant with the aid of the Qiagen QIAprep Spin Miniprep Kit and checked by restriction cleavage with the enzymes EcoRI and HindIII and subsequent agarose gel elektrophoresis. The plasmid has the name pK18mobsacB_IBcg0054.
pK18mobsacB_PgapWT_lysCT311I_asd,
pK18mobsacB_Pgap3_lysCT311I_asd and
pK18mobsacB_Pg3N3_lysCT311I_asd
The mutation in the upstream region of the gap gene is characterized on the basis of a DNA cassette containing a synthetic operon of the lysC and asd genes under the control of the respective promoters PgapWT, Pgap3 and Pg3N3, which is inserted into the intergenic region described in example 2. The synthetic operons were synthesized by Geneart AG (Regensburg, Germany) and are named PgapWT_lysCT311I_asd, Pgap3_lysCT311I_asd and Pg3N3_lysCT311I_asd, respectively. The DNA sequences are depicted in Seq ID No:36, for PgapWT_lysCT311I_asd, in Seq ID No:37, for Pgap3_lysCT311I_asd, and in Seq ID No:38, for Pg3N3_lysCT311I_asd, respectively.
The promoter region of the gap gene comprises positions 9 to 469, the sequence of the constructs in this region corresponding in each case to the sequences depicted in Seq ID No:2 (for PgapWT), in Seq ID No:3 (for Pgap3) or Seq ID No:34 (for Pg3N3). This is followed downstream by the coding sequence of a desensitized Corynebacterium glutamicum aspartate kinase encoded by an allele of the lysC gene, in which the wild-type nucleobase threonine has been replaced with the nucleobase isoleucine in position 311 of the encoded aspartate kinase protein (see WO 00/63388 and U.S. Pat. No. 6,893,848). In addition, the nucleobase guanine was replaced with the nucleobase adenine within the lysC start codon. This is followed again downstream by the coding sequence of Corynebacterium glutamicum aspartate-semialdehyde dehydrogenase, encoding the asd gene. The ribosome-binding site of the gap gene is located upstream of the two genes lysC and asd in each case (upstream of lysC in the form of Seq ID No:16, upstream of asd in the form of Seq ID No:26), and the termination sequence of the gap gene is located downstream of asd.
Restriction cleavage sites of the enzymes AvrII and NsiI flanking the synthetic operon were generated for subcloning procedures.
The fragments were cleaved with the restriction enzymes AvrII and NsiI and then subcloned into the mobilizable vector pK18mobsacB_IBcg0054 described in example 2. For this, pK18mobsacB_IBcg0054 was digested with the restriction enzymes AvrII and NsiI. The vector prepared in this way was mixed with the PgapWT_lysCT311I_asd, Pgap3_lysCT311I_asd or Pg3N3_lysCT311I_asd fragments, and the mixture was treated with the Ready-To-Go T4 DNA Ligase Kit (Amersham-Pharmacia, Freiburg, Germany).
Subsequently, the E. coli strain S17-1 (Simon et al., Bio/Technologie 1: 784-791, 1993) was transformed with the ligation mixture (Hanahan, In. DNA cloning. A practical approach. Vol. 1. ILR-Press, Cold Spring Harbor, N.Y., 1989). Plasmid-carrying cells were selected by plating the transformation mixture on LB agar (Sambrock et al., Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor, N.Y., 1989) which had been supplemented with 50 mg/l kanamycin.
Plasmid DNA was isolated from a transformant with the aid of the Qiagen QIAprep Spin Miniprep Kit and checked by restriction cleavage with the enzymes AvrII and NsiI and subsequent agarose gel electrophoresis. Depending on the gap-promoter allele, the plasmids are named
pK18mobsacB_PgapWT_lysCT311I_asd,
pK18mobsacB_Pgap3_lysCT311I_asd and
pK18mobsacB_Pg3N3_lysCT311I_asd, respectively.
pK18mobsacB_Pgap3_lysCT311I_asd is depicted in
The mutations PgapWT_lysCT311I_asd, Pgap3_lysCT311I_asd and Pg3N3_lysCT311I_asd are intended to be introduced into the strain Corynebacterium glutamicum DM1729. The strain DM1729 is an aminoethylcysteine-resistant mutant of Corynebacterium glutamicum ATCC13032 and is described in the patent application EP 1 717 616 A2 and in Georgi et al. (Metabolic Engineering 7, 291-301 (2005)). It has been deposited under the name DSM17576 with the Deutsche Sammlung für Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany).
The vectors described in example 3,
pK18mobsacB_PgapWT_lysCT311I_asd,
pK18mobsacB_Pgap3_lysCT311I_asd and
pK18mobsacB_Pg3N3_lysCT311I_asd, were transferred by conjugation into the C. glutamicum strain DM1729 according to the protocol by Schäfer et al. (Journal of Microbiology 172: 1663-1666 (1990)). The vector cannot self-replicate in DM1729 and is retained in the cell only if it has been integrated into the chromosome as the result of a recombination event. Transconjugants, i.e. clones with integrated pK18mobsacB_PgapWT_lysCT311I_asd, pK18mobsacB_Pgap3_lysCT311I_asd or pK18mobsacB_Pg3N3_lysCT311I_asd, were selected by plating the conjugation mixture on LB agar which had been supplemented with 25 mg/l kanamycin and 50 mg/l nalidixic acid. Kanamycin-resistant transconjugants were then straightened out on kanamycin (25 mg/1)-supplemented LB agar plates and incubated at 33° C. for 24 hours. Mutants in which the plasmid had been excised due to a second recombination event were selected by culturing the clones non-selectively in LB liquid medium for 30 hours, followed by straightening them out on LB agar which had been supplemented with 10% sucrose, and incubating them at 33° C. for 24 hours.
Like the starting plasmid pK18mobsacB, the plasmids
pK18mobsacB_PgapWT_lysCT311I_asd,
pK18mobsacB_Pgap3_lysCT311I_asd and
pK18mobsacB_Pg3N3_lysCT311I_asd include, in addition to the kanamycin-resistance gene, a copy of the sacB gene coding for Bacillus subtilis levansucrase. Sucrose-inducible expression of the sacB gene results in the formation of levansucrase which catalyses the synthesis of the product levan which is toxic to C. glutamicum. Consequently, only those clones in which the integrated
pK18mobsacB_PgapWT_lysCT311I_asd,
pK18mobsacB_Pgap3_lysCT311I_asd or
pK18mobsacB_ Pg3N3_ lysCT311I_asd has been excised due to a second recombination event grow on sucrose-supplemented LB agar. Depending on the position of the second recombination event with respect to the site of mutation, excision comprises allele replacement or incorporation of the mutation, or the original copy is retained in the chromosome of the host.
Screening was then carried out in each case for a clone in which the desired replacement, i.e. incorporation of the cassette PgapWT_lysCT311I_asd, Pgap3_lysCT311I_asd or Pg3N3_lysCT311I_asd into the intergenic region, had taken place.
To this end, in each case 20 clones with the phenotype “Growth in the presence of sucrose” and “No growth in the presence of kanamycin” were checked for integration of the cassette PgapWT_lysCT311I_asd, Pgap3_lysCT311I_asd or Pg3N3_lysCT311I_asd using the polymerase chain reaction (PCR).
For this, the following synthetic oligonucleotides (primers) were used:
Primer cg0054—9.p (SEQ ID No.39):
5′ CCACCCATCACCCTCACTTC 3′
Primer cg0054—10.p (SEQ ID No.40):
5′ GCACTCTCGTTTGGCAGTTC 3′
Primer QlysC_WT_P2 (SEQ ID No.41):
5′ AAGTGCCGGGATCATTACTA 3′
The primers shown were synthesized by MWG Biotech (Ebersberg, Germany). The primers cg0054—9.p and cg0054—10.p enable a 1032 bp DNA fragment to be amplified if the wild-type arrangement is present in the intergenic region. Using a further primer (QlysC_WT_P2) in the same reaction mixture, which is capable of annealing to the lysC gene, enables a 1330 bp DNA fragment to be amplified, whereby integration of the synthetic operon PgapWT_lysCT311I_asd, Pgap3_lysCT311I_asd or Pg3N3_lysCT311I_asd into the intergenic region IBcg0054 is specifically detected. Under the conditions chosen for the PCR reaction, the combination of primers cg0054—9.p and cg0054—10.p does not result in an amplicon in the case of integration into the intergenic region. In addition, PCR reactions which have not yielded an amplicon for technical reasons are identified with the aid of the detection reactions described.
The PCR reactions were carried out using the Taq PCR Core Kit from Qiagen (Hilden, Germany), containing the Thermus aquaticus Taq DNA polymerase, in a mastercycler from Eppendorf (Hamburg, Germany). The conditions in the reaction mixture were adjusted according to the manufacturer's information. The PCR mixture was first subjected to an initial denaturation at 94° C. for two minutes. This was followed by 35 repeats of a denaturation step at 94° C. for 30 seconds, a step of binding the primers to the DNA at 57° C. for 30 seconds, and the extension step for extending the primers at 72° C. for 60 s. After the final extension step at 72° C. for 5 min, the products amplified in this way were checked by electrophoresis in an agarose gel.
In this manner, mutants were identified, in which the cassette PgapWT_lysCT311I_asd, Pgap3_lysCT311I_asd or Pg3N3_lysCT311I_asd has been integrated, with one of the strains C. glutamicum obtained in each case being named DM1729_PgapWT_lysCT311I_asd, DM1729_Pgap3_lysCT311I_asd and DM1729_Pg3N3_lysCT311I_asd.
The C. glutamicum strains obtained in example 4, DM1729_PgapWT_lysCT311I_asd, DM1729_Pgap3_lysCT311I_asd and DM1729_ Pg3N3_ lysCT311I_asd, and the starting strain DM1729 were cultured in a nutrient medium suitable for producing lysine, and the lysine content in the culture supernatent was determined.
For this purpose, the clones were first propagated on brain-heart agar plates (Merck, Darmstadt, Germany) at 33° C. for 24 hours. Starting from these agar plate cultures, in each case a preculture was inoculated (10 ml medium in a 100 ml conical flask). The medium used for the preculture was MM medium. The preculture was incubated at 33° C. on a shaker at 240 rpm for 24 hours. A main culture was inoculated from this preculture such that the starting OD (660 nm) of the main culture was 0.1 OD. MM medium was also used for the main culture.
CSL (corn steep liquor), MOPS (morpholinopropanesulphonic acid) and the salt solution were adjusted to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions and the dry-autoclaved CaCO3 were then added.
Culturing was carried out in volumes of 10 ml in 100 ml conical flasks with baffles. The temperature was 33° C., with 250 revolutions per minute and a humidity of 80%.
After 24 hours the optical density (OD) was measured at a wavelength of 660 nm using a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of lysine formed was determined using an amino acid analyser from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection.
The result of the experiment is depicted in Table 1.
All values are averages of three independent experiments with the strains listed
The result indicates the positive effect on the formation of the desired product (L-lysine) due to using the promoter variants according to the invention, Pgap3 and Pg3N3, with respect to the starting strains mentioned above.
Starting from the genomic sequence of Corynebacterium glutamicum ATCC13032, a 2722 bp DNA fragment was synthesized at GeneArt (Regensburg, Germany) (Seq ID: 29), which carries the Pgap3 promoter and the genes argJ (coding for a glutamate N-acetyltransferase) and argB (coding for an acetylglutamate kinase). The promoter region of the gap gene comprises positions 10 to 470, with the sequence of the construct in this region corresponding to the sequence depicted in Seq ID No:3 (for Pgap3). This is followed downstream by the coding sequences for a glutamate N-acetyltransferase and an acetylglutamate kinase from Corynebacterium glutamicum. The fragment was cut by SphI and BlnI cleavage via the terminally introduced cleavage sites SphI and BlnI and then cloned into the vector pK18mobsacB_IBcg0054 (from example 2) which had been cut likewise with SphI and BlnI. The plasmid is named pK18mobsacB_IBcg0054_Pg3_argJB.
The vector mentioned in example 6, pK18mobsacB_IBcg0054_Pg3_argJB, was transferred into the Corynebacterium glutamicum strain ATCC13032_DargFRGH (from patent application DE102010003419.3) by means of conjugation according to a protocol by Schäfer et al. (Journal of Microbiology 172: 1663-1666 (1990)), analogous to example 4.
For this purpose, the vector was first transformed into the E. coli S17-1 strain (Simon et al., Biotechnology 1:784-791). The vector pK18mobsacB or pK18mobsacB_IBcg0054_Pg3_argJB cannot self-replicate in C. glutamicum ATCC13032 and is retained in the cell only if it has integrated into the chromosome as the result of a recombination event. Clones with integrated pK18mobsacB_IBcg0054_Pg3_argJB are selected by plating the conjugation mixture on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y., 1989) supplemented with 15 mg/l kanamycin and 50 mg/ml nalidixic acid. 15 established clones are straightened out on LB agar plates containing 25 mg/1 kanamycin and incubated at 33° C. for 16 hours. To select mutants in which the plasmid has been excised due to a second recombination event, the clones are cultured non-selectively in LB liquid medium for 20 hours, then straightened out on LB agar containing 10% sucrose and incubated for 24 hours. Like the starting plasmid pK18mobsacB, the pK18mobsacB_IBcg0054_Pg3_argJB plasmid includes, in addition to the kanamycin-resistance gene, a copy of the sacB gene coding for Bacillus subtilis levansucrase. Sucrose-inducible expression results in the formation of levansucrase which catalyses the synthesis of the product levan which is toxic to C. glutamicum. Consequently, only those clones in which the integrated pK18mobsacB_IBcg0054_Pg3_argJB has again been excised grow on LB agar containing sucrose. Either the complete cassette IBcg0054_Pg3_argJB or the chromosomal intergenic region IBcg0054 may be excised together with the plasmid. Approximately 40 to 50 colonies were tested for the phenotype “Growth in the presence of sucrose” and “No growth in the presence of kanamycin”. In order to establish that the cassette IBcg0054_Pg3_argJB has remained in the chromosome, approximately 20 colonies which have the phenotype 5 “Growth in the presence of sucrose” and “No growth in the presence of kanamycin” were investigated with the aid of the polymerase chain reaction by the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press). This involved amplifying from the chromosomal DNA of the colonies a DNA fragment which carries the surrounding region of the intergenic region IBcg0054 and part of the inserted arg gene. The following primer oligonucleotides were selected for the PCR.
argA-E1: TGTCGCGGAAGTGCTGCAAC
cg0054—10.p: GCACTCTCGTTTGGCAGTTC
The correct clones were detected by generating a 1949 bp DNA fragment which was detected in an agarose gel.
To test their ability to produce L-ornithine, in each case three clones of the strain C. glutamicum ATCC13032_DargFRGH_Pg3_argJB and three clones of the strain ATCC 13032_Delta_argFRGH were precultured in in each case 10 ml of test medium at 33° C. for 16 h. For the production assay, in each case 10 ml of test medium were inoculated with the preculture obtained such that the starting OD600 (optical density at 600 nm) was 0.1. Each clone was tested in three shaker flasks, so that each strain is represented by a total of nine shaker flasks.
The test medium was identical to the CgXII medium described in Keilhauer et al. (Journal of Bacteriology (1993) 175: 5593-5603) but additionally contained 7.5 g/l yeast extract (Difco), 25 μg/ml kanamycin, 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside) and 40 g/l sucrose instead of glucose. For reasons of simplicity, the composition of the test medium is summarized in Table 2 below.
Culturing was carried out in 100 ml shaker flasks at 33° C. and 200 rpm. The deflection of the shaker was 5 cm. Samples were taken from the cultures after 24 and 48 hours, and the optical density, the sucrose content and the L-ornithine content were determined, and the cells were removed by brief centrifugation (table-top centrifuge type 5415D (Eppendorf) at 13000 rpm, 10 min, room temperature).
The optical density was determined at a wavelength of 660 nm, using a GENios microtitre plate photometer (Tecan, Reading UK). The samples were diluted 1:100 with demineralized water prior to the measurement.
Sucrose was determined using a test system (Cat. No. 10 716 251 035) from R-Biopharm AG (Darmstadt, Germany). This involves inversion of sucrose and the glucose formed being detected using a coupled enzyme assay (hexokinase/glucose-6-phosphate dehydrogenase) via NADH formation.
Quantitative determination of the extracellular amino acid concentrations from the culture supernatent was carried out by means of reversed-phase HPLC (Lindroth et al., Analytical chemistry (1979) 51: 1167-1174), using an HP1100 series HPLC instrument (Hewlett-Packard, Waldbronn, Germany) with connected fluorescence detector (G1321A); system control and data evaluation were carried out using an HP Chem Station (Hewlett-Packard). 1 μl of the amino acid solution to be analysed was mixed in an automated pre-column derivatization with 20 μl of ready-to-use ortho-phthalaldehyde/2-mercaptoethanol reagent (Pierce Europe BV, Oud-Beijerland, Netherlands). The resulting fluorescent, thio-substituted isoindoles (Jones et al., Journal of Chromatography (1983) 266: 471-482) were fractionated on a pre-column (40×4 mm Hypersil ODS 5) and main-column (Hypersil ODS 5) combination (both columns from CS-Chromatographie Service GmbH, Langerwehe, Germany) using a gradient program with an increasingly non-polar phase (methanol). The polar eluent was sodium acetate (0.1 M; pH 7.2); the flow rate was 0.8 ml per minute. Fluorescence of the derivatized amino acids was detected at an excitation wavelength of 230 nm and an emission wavelength of 450 nm. The L-ornithine and/or L-ornithine hydrochloride concentrations were calculated by way of comparison with an external standard and L-asparagine as additional internal standard.
The molecular weight of L-ornithine hydrochloride is 168.6 g×mol−1 and that of L-ornithine is 132.1 g×mol−1.
The yield was calculated by dividing the amount of L-ornithine formed (measured as L-ornithine hydrochloride) by the amount of sucrose consumed.
The results are depicted in Table 3.
Table 3: L-ornithine formation after 24 hours (Table 3A) and 48 hours (Table 3B) of incubation. Abbreviations: Orn-HCl: L-ornithine hydrochloride.
Construction of the Replacement Vector pK18mobsacB_homUP_Pg3_hom
Starting from the genomic sequence of Corynebacterium glutamicum ATCC13032, a 2549 bp DNA fragment was synthesized at GeneArt (Regensburg, Germany) (Seq ID No. 42), which carries a part of the gene region upstream of the hom gene, the Pgap3 promoter and a part of the hom gene. The fragment was cut by BamHI and XbaI cleavage via the terminally introduced cleavage sites BamHI and XbaI and then cloned into the vector pK18mobsacB which had been cut likewise with BamHI and XbaI. The plasmid is named pK18mobsacB_homUP_Pg3_hom.
Construction of the Replacement Vector pK18mobsacB_ilvAUP_Pg3_ilvA
Similarly, a 2442 bp DNA fragment was synthesized at GeneArt (Seq ID No. 43), which carries a part of the gene region upstream of the ilvA gene, the Pgap3 promoter and a part of the ilvA gene. The fragment was cut by BamHI and XbaI cleavage via the terminally introduced cleavage sites BamHI and XbaI and then cloned into the vector pK18mobsacB which had been cut likewise with BamHI and XbaI. The plasmid is named pK18mobsacB_ilvAUP_Pg3_ilvA.
Construction of the Replacement Vector pK18mobsacB_pycUP_Pg3_pyc
Similarly, a 2072 bp DNA fragment was synthesized at GeneArt (Seq ID No. 44), which carries a part of the gene region upstream of the pyc gene, the Pgap3 promoter and a part of the pyc gene. The fragment was cut by BamHI and HindIII cleavage via the terminally introduced cleavage sites BamHI and HindIII and then cloned into the vector pK18mobsacB which had been cut likewise with BamHI and HindIII. The plasmid is named pK18mobsacB_pycUP_Pg3_pyc.
The vectors mentioned in example 9, pK18mobsacB_homUP_Pg3_hom, pK18mobsacB_ilvAUP_Pg3_ilvA and pK18mobsacB_pycUP_Pg3_pyc, were transferred in each case individually by conjugation into the Corynebacterium glutamicum strain C. glutamicum ATCC14310 (similarly to example 4) by means of conjugation according to a protocol by Schäfer et al. (Journal of Microbiology 172: 1663-1666 (1990)).
The following primer oligonucleotides were used for detection PCR of the strains.
Primer for Detecting homUP_Pg3_hom in ATCC14310:
Hom_Xl-A1
LC-hom2
The correct clones were detected by generation of a 1502 bp DNA fragment which was detected in an agarose gel and then confirmed by sequencing.
Primer for detecting ilvAUP_Pg3_ilvA in ATCC14310:
Pgap3—1.p
ilvA-int-rev2
The correct clones were detected by generation of a 712 bp DNA fragment which was detected in an agarose gel and then confirmed by sequencing.
Primer for Detecting pycUP_Pg3_pyc in ATCC14310:
pycUP—3.p
2xpyc-1
The correct clones were detected by generation of a 1004 bp DNA fragment which was detected in an agarose gel and then confirmed by sequencing.
In each case, 10 ml of Caso broth containing 0.5% glucose were inoculated with 100 μl of a bacterial culture from example 10 (ATCC 14310 derivatives) in a conical flask with baffles (volume: 100 ml) and shaken at 200 rpm by way of a preculture at 33° C. for 16 h (amplitude: 5 cm). From the preculture, 10 ml of main culture in each of three 100 ml conical flasks with baffles were inoculated with an inoculation volume of 1%. A medium containing 40 g of glucose, 20 g of ammonium sulphate, 0.5 g of potassium dihydrogen phosphate, 0.5 g of dipotassium hydrogen phosphate, 20 g of calcium carbonate, 0.25 g of magnesium sulphate (heptahydrate), 10 mg of iron sulphate (heptahydrate), 10 mg of manganese sulphate (×4H2O), 1 mg of zinc sulphate (heptahydrate), 0.2 mg of copper sulphate and 300 mg/l of L-leucine was used for the main culture.
The main culture was incubated at 33° C. and a shaker speed of 200 rpm (amplitude: 5 cm) for 48 h. The culture fluid obtained was removed by centrifugation and the concentration of isoleucine was subsequently determined in the supernatant. Table 4 depicts the results.
The abbreviations and names used have the following meaning.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2011 118019 | Jun 2011 | DE | national |
This application is a divisional of U.S. application Ser. No. 13/534,359 filed Jun. 27, 2012, U.S. Pat. No. 8,912,313, and claims the benefit DE 10 2011 118019.6 filed Jun. 28, 2011 and U.S. provisional application Ser. No. 61/502,675 filed Jun. 29, 2011, each of which are incorporated herein by reference in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20040043458 | Bathe et al. | Mar 2004 | A1 |
| 20070026505 | Madden et al. | Feb 2007 | A1 |
| 20070259408 | Bathe et al. | Nov 2007 | A1 |
| 20080050786 | Bathe et al. | Feb 2008 | A1 |
| 20080171371 | Yukawa et al. | Jul 2008 | A1 |
| 20100173368 | Nakanishi et al. | Jul 2010 | A1 |
| 20100255544 | Bathe et al. | Oct 2010 | A1 |
| Number | Date | Country |
|---|---|---|
| 1 790 721 | May 2007 | EP |
| WO 03014330 | Feb 2003 | WO |
| WO 03014330 | Feb 2003 | WO |
| WO 2006138689 | Dec 2006 | WO |
| WO 2006138689 | Dec 2006 | WO |
| Entry |
|---|
| International Search Report issued Sep. 21, 2012, in International Patent Application No. PCT/EP2012/062046. |
| Database Gensenq [Online], “DNA fragment containing a pomoter region SEQ ID No. 32.”, XP002682941, Database accession No. AEG41826, May 18, 2006, 1 page. |
| Miroslav Patek, et al., “Promoters of Corynebacterium glutamicum”, Journal of Biotechnology 104, Elsevier Science, XP002323247, Sep. 4, 2003, pp. 311-323. |
| Miroslav Patek, et al., “Function of Corynebacterium glutamicum promoters in Escherichia coli, Streptomyces lividans, and Bacillus subtilis”, Journal of Biotechnology 104, Elsevier Science, XP002323103, Jan. 1, 20003, pp. 325-334. |
| Number | Date | Country | |
|---|---|---|---|
| 20150056666 A1 | Feb 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61502675 | Jun 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13534359 | Jun 2012 | US |
| Child | 14532259 | US |
