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  • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)

Definitions

• the invention relates to a DNA regulating gene expression in coryneform bacteria.
• Corynebacterium glutamicu and the species closely related to it are c. mela ⁇ ecolae, Brevibacterium flavum and B. lactofermentum counted among the coryneform bacteria. Furthermore, the mentioned species to the 'glutamic acid bacteria' because they are able to excrete large amounts of glutamate into the medium under certain growth conditions. The microorganisms mentioned are of great industrial interest since they can be used for the production of amino acids, purines and proteins. For C. glutamicum, C. mela ⁇ secolae, B. flavum and B.
• lactofermentum growth on acetate or ethanol has already been demonstrated and it has been shown that they have a glyoxylate cycle, ie also the enzymes isocitrate lyase and malate synthase, (For an overview see: Kinoshita, Amino acids, in Biology of industrial organisms, 1985, pp. 115-142, Benjamin / cum ings Publishing).
• coryneform bacteria can be genetically modified for specific purposes.
• the genes to be cloned are cloned under the control of their own promoters onto vectors which are present in high copy numbers in coryneform bacteria. It was shown in several cases that a strong overexpression of individual genes had an adverse effect on the growth of coryneform bacteria and thus on the production of desired products. The reason for this was that the
• a desired gene can be integrated into the chromosome of coryneform bacteria in a single copy number. Since there is only one copy of this gene in the organism, there are usually no toxic effects from the corresponding gene product. A weakness of this method lies in the labor-intensive methodology to achieve the desired goal. In addition, the simple copy number of the inserted gene rarely produces a sufficient amount of a desired substance.
• An alternative to integrating a gene into the chromosome of coryneform bacteria is to clone a gene onto a low copy vector in coryneform bacteria. This has the advantage that the corresponding gene product is formed in a relatively small amount and is therefore usually not toxic to the cell. Allerdin ⁇ s is too in this case the relatively small amount of gene product for biotechnological applications is a disadvantage.
• each of the three promoters needs to induce a gene are relatively uninteresting for industrial purposes.
• the lac promoter requires the relatively expensive substance IPTG to induce a gene, which renders a large-scale application of this promoter unprofitable.
• IPTG IPTG
• Lambda P L is activated by heat. Heat not only damages the organism but could also have a harmful effect on the product formed, so that this promoter is of no industrial interest for coryneform bacteria 5 is.
• the trp promoter is activated by tryptophan deficiency. Since coryneform bacteria usually do not suffer from tryptophan deficiency, the use of this promoter would require the production of coryneform tryptophan deficiency mutants. Since the generation of such mutants is relatively complex, the trp promoter has not yet found its way into the biotechnological use of coryneform bacteria.
• the ideal case for a regulatable promoter was a coryneform promoter, which is regulated by an easily available, inexpensive substance.
• the only coryneform promoter described so far is that of the gene for isocitrate lyase (EP-OS 0 530 765).
• This promoter leads to the expression of genes as long as there is no sugar in the medium.
• sugar since sugar is used as a carbon source in most fermentation media, it would make sense to obtain a regulable promoter which also leads to the expression of a gene in the presence of sugars with an inexpensive inducer.
• the expression of the malate synthase gene in coryneform bacteria can be induced by the presence of inducers such as lactate, pyruvate and / or acetate.
• inducers such as lactate, pyruvate and / or acetate.
• This induction in particular through acetate, also occurs when there are other carbon sources in the medium. Even in the presence of sugars or in complex medium there is significant induction by acetate.
• Inducer such as acetate and regardless of the composition of the fermentation medium to express genes regulated in coryneform bacteria.
• the malate synthase gene of the present invention is a malate synthase gene of the malate synthase gene of the malate synthase gene of the malate synthase gene of the malate synthase gene of the malate synthase gene of the malate synthase gene of the malate synthase gene of the malate synthase gene of the malate synthase gene of the malate synthase gene of
• Coryjie ⁇ acter UJD glutamicum upstream and isolated from this DNA fragment provided; i.e. the gene for malate synthase (aceB) was isolated and sequenced from C-glutamicum together with the structures required for expression and regulation.
• the DNA sequence and the amino acid sequence derived from it are shown in Table 2.
• the ribosome binding site of the aceB gene is underlined and marked with 'RBS'.
• the potential terminator of aceB transcription is shown by antiparallel arrows.
• the media used were 2xTY complete medium or CgC minimal medium (Eikmanns et al., Appl Microbiol Biotechnol 34 (1991) 617-622), each with 1% of glucose, acetate, pyruvate, lactate, citrate, succinate , Fumarate or glutamate used as a carbon source.
• the cultures were incubated again at 30 ° C and the OD 60Q was followed.
• the cells Upon reaching an OD of 60 o 8 ⁇ 10, the cells were harvested by centrifugation, washed once with buffer pH 7.6 (50 mM Tris / HCl), resuspended in 1 ml of the same buffer was added, and by sonication in a Branson Sonifier W250 (10 Minutes, pulsed with an interval of 20% and an output of 30 watts). To separate the cell debris, the homogenate was sigma at 13000 rpm for 30 minutes
• the enzyme test contained in a final volume of 1.0 ml, 50 mM
• the mixture was incubated at 30 ° C., the decrease in extinction at 232 nm was determined over a period of 2 minutes, which decrease results from the cleavage of the thioester bond of acetyl-CoA.
• the extinction coefficient of acetyl-CoA at 232 nm is 4.5 mM -1 cm -1 (Stadtman, Methods in Enzy ology, Vol. 3, 1957, New York: Academic Press).
• the protein content of the crude extracts was determined using the biuret method (Bradford, Anal Biochem 72 (1976) 248-254).
• the specific malate synthase activities obtained are listed in Table 1.
• Table 1 the activity of the MSY when growing on 2xTY complete medium and on CgC minimal medium with glucose, citrate, succinate, fumarate or glutamate as a carbon source is approximately 0.04 U / mg protein.
• CgC minimal medium with lactate or pyruvate as carbon sources the MSY activity increases to values of 0.173 U / mg protein or 0.192 U / mg protein. The highest MSY activity is observed with 2.212 U / mg protein when growing on CgC minimal medium with acetate.
• coli stam ED8654 (Murray et al. Mol Gen Genet 150 (1977) 53-61) was transfected therewith.
• the recombinant cosmids were packaged in the protein envelope of the phage lambda by a method by Sternberg et al. (Gene 1 (1979) 255-280), the transfection of E. coli ED8654 according to a method by Sambrook et al. (Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press).
• the corresponding cosmids were isolated from a total of 30 of the recombinant E.
• mutant DV21A05 (anderwinkel and De Vlieghere Eur J Biochem 5 (1968) 81-90) according to a known method (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press). Due to its MSY defect, mutant DV21A05 is no longer able to grow on acetate as the only carbon source. After transformation of the Cosmid gene bank into this mutant, a total of 1000 clones were obtained. Of these, three clones showed growth on M9 minimal medium (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press) with acetate as the only carbon source. After isolating the corresponding cosmids (Sambrook et al.,
• the plasmids generated in this way were used to generate deletion constructs according to the method of Henikoff (Gene 28 (1984) 351-359), which were subsequently used by the chain termination sequencing method (Sanger et al., Proc Natl Acad Sei USA, 74 (1977) 5463-5467) were sequenced.
• the entire sequence of the 3 kb Bfrl-Pvul fragment obtained in this way is shown in Table 2.
• the protein sequence derived from the aceB gene for the MSY from C. glutamicum, the ribosome binding site located in front of the gene and the termination structure for the transcription behind the gene are shown in Table 2.
• Electroporation (Liebl et al., FEMS Microbiol Lett 65 (1989) 299-304) was used to isolate the plasmids pEKBla and pEKBlb in C. introduced glutamicum and the resulting strains as WT (pEKBla) and WT (pEKBlb) called.
• WT pEKBla
• WT pEKBlb
• the newly constructed C. glutamicum strains were grown on CgC minimal medium with glucose, glucose / acetate or acetate as carbon sources up to an OD 60 o of 8-10, crude extracts were prepared and the specific MSY activity was determined in them .
• the measured MSY activities are shown in Table 3.
• This result proves that on the 3 kb Bfrl-Pvul fragment the aceB gene from C. Functionally available glutamicum.
• aceB gene required structures, ie the promoter and regulatory sequences, are located. These structures are in front of the aceB gene. Since the cloned fragment still bears 584 bp before the actual aceB structural gene (see Table 2), the structures for expression and regulation must be located in this DNA region. 5. Studies on the regulation and expression of the aceB gene from C glutamicum.
• MSY accounts for approximately 20% of the total cell protein in this strain.
• the result shows that the structures necessary for the expression and regulation of aceB induce the re-synthesis of large amounts of protein under induced conditions.
• the result shows that the observed increase in MSY activity after growth on acetate is due to the new synthesis of the MSY protein.
• the DNA region in front of the aceB gene was isolated according to known methods as a 574 bp Bfrl-DraJ fragment, the overhanging ends were filled in to blunt ends with Klenow polymerase and into the Sall interface of the vector pEKplCm (Eikmanns.) Filled in with Klenow polymerase et al., Gene 102 (1991) 93-98).
• This plasmid carries the chloramphenicol acetyltransferase gene (cat) behind the insertion site, but without its own promoter, ie the cat gene in C. glutamicum cannot be read from the plasmid pEKplCm.
• the strains to be investigated were cultivated according to a known method on the above-mentioned media up to an OD 60 o 8 to 10, crude extracts were prepared and in these the specific CAT activity according to the method of Shaw (Meth Enzymol 43 (1975) 737-755).
• the test contained in a final volume of 1.0 ml 100 mM Tris / HCl pH 7.8, 1 mM acetyl-coenzyme A, 1 mM 5,5-dithiobis (2-nitrobenzoic acid) and crude extract and was treated with 2.5 mM chloramphenicol started. The mixture was incubated at 30 ° C. The increase in absorbance at 412 nm was determined over a period of 2 minutes.
• the protein content of the crude extracts was determined using the biuret method (Bradford, Anal Biochem 72 (1976) 248-254).
• the specific CAT activities obtained are listed in Table 4. While there was no CAT activity detected in the C. glutamicum WT under any of the conditions tested, the recombinant strain C. glutamicum WT (pIWI) showed CAT activity for all carbon sources. However, the CAT activity after growth on CgC glucose was about 20 times lower than after growth on CgC glucose / acetate and even 50 times lower than after growth on CgC acetate. This result confirms that the isolated 574 bp Bfrl-Dral fragment allows the regulated gene expression of foreign genes. The foreign gene is induced by acetate, even in the presence of sugar.
• the supernatant was chromatographed on an FPLC system with an HR5 / 5 MonoQ anion exchange column (Pharmacia LKB, Freiburg Germany).
• the MSY was eluted with a 0.1 M to 0.4 M NaCl gradient in 50 mM MES / NaOH pH 6.
• the buffer of the partially purified MSY was changed from 50 mM MES / NaOH pH 6 to 50 mM Tris / HCl pH 8 by means of ultrafiltration.
• MSY malate synthase
• Table 4 Specific activity of chloramphenicol acetyltransferase (CAT) in crude extracts of the C. glutamicum wild type (WT) and the recombinant C. glutamicum strain WT (pIWI) after growth on CgC minimal medium with glucose, glucose / acetate or acetate as Carbon sources.
• CAT chloramphenicol acetyltransferase

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