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  • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
  • C12Y103/01013—Prephenate dehydrogenase (NADP+) (1.3.1.13)

Definitions

• Another object of the present application is to provide an expression cassette comprising a gene encoding a trp operon regulatory region and a prephenate dehydratase operably linked thereto.
• one aspect of the present application includes a gene encoding a trp operon regulatory region and a prephenate dehydratase operably linked thereto, L-tyrosine. Provides the microorganisms that produce it.
• trp operon regulatory region refers to a site that is present upstream of a structural gene constituting the trp operon and can regulate the expression of a structural gene.
• Structural genes constituting the trp operon in microorganisms of the genus Corynebacterium may be composed of trpE, trpG, trpD, trpC, trpB, and trpA genes, and the structural genes constituting the trp operon in microorganisms of the genus Escherichia are It may be composed of trpE, trpD, trpC, trpB, and trpA genes.
• the trp operon regulatory region may be present upstream of trpE at the 5'position of the trp operon structural gene.
• a trp regulator trpR
• trp promoter a promoter
• trp operator an operator
• trp leader peptide trp L
• trp attenuation factor trp promoter
• trp promoter may include a promoter (trp promoter), an operator (trp operator), a trp leader peptide (trp L) and a trp attenuator (trp attenuator).
• trp operon regulatory region may be used interchangeably with the trp operon regulatory factor, trpE promoter, trp operon promoter, trpE regulatory region, trpE regulatory factor, and trpE regulatory sequence.
• the regulatory region is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with SEQ ID NO: 1 and/or SEQ ID NO: 1 ( It may be a nucleotide sequence having homology or identity. In addition, if it is a base sequence that has such homology or identity and exhibits a function corresponding to the control region, it is obvious that expression control sequences having a nucleotide sequence in which some sequences are deleted, modified, substituted or added are also included within the scope of the present application. .
• sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and a default gap penalty established by the program used can be used together.
• sequence homologous or identical sequences are generally at least about 50%, 60%, 70%, 80% of the sequence full or full-length in medium or high stringent conditions. Or it can hybridize to 90% or more. Hybridization is also contemplated for polynucleotides containing degenerate codons instead of codons in the polynucleotide.
• homology or identity to the polypeptide or polynucleotide sequence can be determined, for example, by the algorithm BLAST by literature [see Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)], or FASTA by Pearson (Methods Enzymol., 183, 63, 1990). Based on this algorithm BLAST, a program called BLASTN or BLASTX has been developed (see: http://www.ncbi.nlm.nih.gov).
• the microorganism genetically modified so that the pheA gene is regulated by the trp operon control region does not interfere with the growth of the cells, and phenylalanine It minimizes the accumulation of (phenylalanine) and is effective in improving tyrosine production.
• the microorganisms are, for example, Enterbacter genus, Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Corynebacterium ) And may be a microorganism belonging to the genus Brevibacterium , but is not limited thereto.
• Another aspect of the present application provides an expression cassette comprising a gene encoding the trp operon regulatory region and prephenate dehydratase operably linked thereto.
• expression cassette may be a gene construct unit comprising essential regulatory elements operably linked to the gene so that the introduced gene is expressed when present in the cell of an individual.
• the expression cassette may be, for example, in the form of an expression vector, but is not limited thereto, and all the gene constructs of the smallest unit capable of functioning to express the target gene to be introduced may be included.
• the expression cassette can be prepared and purified using standard recombinant DNA techniques.
• the type of expression cassette is not particularly limited as long as it has a function of expressing a desired gene in various host cells such as prokaryotic and eukaryotic cells and producing a desired protein.
• the expression cassette may include a promoter, an initiation codon, a gene encoding a protein of interest, or a stop codon, and in addition, a DNA encoding a signal peptide, an enhancer sequence, an untranslated region on the 5'side and 3'side of the gene of interest , A selection marker region, or a replicable unit may be appropriately included.
• vector refers to a DNA preparation containing the nucleotide sequence of a polynucleotide encoding the protein of interest operably linked to a suitable regulatory sequence so that the protein of interest can be expressed in a suitable host.
• the regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling termination of transcription and translation.
• Vectors can be transformed into a suitable host cell and then replicated or function independently of the host genome, and can be integrated into the genome itself.
• pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. may be used, but are not limited thereto.
• operably linked in the above means that the polynucleotide sequence is functionally linked to a promoter sequence or regulatory region that initiates and mediates transcription of the polynucleotide encoding the protein of interest in the present application.
• the operable linkage may be prepared using a gene recombination technique known in the art, and site-specific DNA cleavage and linkage may be prepared using a cleavage and linkage enzyme in the art, but is not limited thereto.
• compositions for producing L-tyrosine comprising a gene encoding the trp operon regulatory region and prephenate dehydratase operably linked thereto.
• the upstream and downstream regions to which the amplified variant aroG will be additionally inserted, and the vector pDZ for chromosome transformation digested with SmaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, and named pDZ- ⁇ BBD29_14470. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated mole number and then storing at 50° C. for 1 hour.
• pheA a first-stage gene branching from Prephenic acid
• the upstream and downstream regions of the pheA gene were obtained. Specifically, using the chromosomal DNA of Corynebacterium glutamicum ATCC13869 as a template, the pheA upstream region using the primers of SEQ ID NO: 31 and SEQ ID NO: 32, and the downstream region using the primers of SEQ ID NO: 33 and SEQ ID NO: 34 The (Downsteam) region of the gene fragment was obtained through PCR.
• Example 4 Evaluation of the production ability of the pheA gene-deficient strain of the L-tyrosine producing strain
• the strain was cultured using the method and medium composition described in Example 2.
• upstream of the region to be inserted and the region downstream of the region to be inserted and the region to be inserted were obtained.
• the upstream region of the region to be inserted using the primers of SEQ ID NO: 37 and SEQ ID NO: 38 using the Corynebacterium glutamicum ATCC13869 chromosome DNA as a template, and the primers of SEQ ID NO: 39 and SEQ ID NO: 40
• a gene fragment of a downstream region of the region to be inserted was obtained through PCR.
• Solg TM Pfu-X DNA polymerase was used, and PCR amplification conditions were after denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization for 30 seconds at 72°C. , Polymerization was performed at 72° C. for 5 minutes.
• Recombinant plasmids were obtained by cloning the amplified upstream and downstream regions of the region to be inserted, the trpE regulatory region, and the vector pDZ for chromosome transformation digested with SmaI restriction enzyme using the Gibson assembly method, and pDZ- ⁇ PpheA::PtrpE It was named as. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated mole number and then storing at 50° C. for 1 hour.
• Solg TM Pfu-X DNA polymerase was used, and PCR amplification conditions were after denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization for 30 seconds at 72°C. , Polymerization was performed at 72° C. for 5 minutes.
• Recombinant plasmids were obtained by cloning the amplified upstream and downstream regions of the region to be inserted, the ilvB promoter region, and the vector pDZ for chromosome transformation digested with SmaI restriction enzyme using the Gibson assembly method, and pDZ- ⁇ PpheA::PilvB It was named as. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated mole number and then storing at 50° C. for 1 hour.
• an upstream region of the region to be inserted and a promoter region of leuC and a downstream region of the region to be inserted were obtained.
• the primers of SEQ ID NO: 37 and SEQ ID NO: 51 using the Corynebacterium glutamicum ATCC13869 chromosome DNA as a template, the upstream region of the region to be inserted, and the primers of SEQ ID NO: 52 and SEQ ID NO: 53
• a gene fragment of the Downsteam region of the region to be inserted was obtained through PCR.
• the genetic manipulation was confirmed by PCR method and genome sequencing using primers of SEQ ID NO: 55 and SEQ ID NO: 56 that can amplify the regions outside the homologous recombination upstream region and downstream region into which the corresponding regulatory region or promoter is inserted, respectively.
• the strain in which the trpE regulatory region was inserted in front of the gene of pheA was CM06-0005
• the strain in which the promoter of ilvB was inserted was CM06-0006
• the strain in which the promoter of leuA was inserted was CM06-0007
• the promoter of leuC was inserted.
• the strain was named CM06-0008.
• Example 6 Evaluation of the production ability of the pheA gene promoter replacement strain of the L-tyrosine producing strain
• the strain was cultured using the method and medium composition described in Example 2.
• CM06-0005 strain produced L-tyrosine in a 5.37% yield because the production capacity was improved than that of the CM06-0004 strain.
• the production of L-tyrosine of CM06-0005 increased by 283% compared to the parent strain CM06-0003 and increased by 144% compared to CM06-0004.
• Solg TM Pfu-X DNA polymerase As the polymerase, Solg TM Pfu-X DNA polymerase was used, and PCR amplification conditions were after denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds. , Polymerization was performed at 72° C. for 5 minutes.
• the amplified ptsG upstream and downstream gene fragments, the glf gene fragment containing the cj7 promoter, and the vector pDZ for chromosome transformation digested with the ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, pDZ- ⁇ ptsG Named as ::pcj7-glf. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated mole number and then storing at 50° C. for 1 hour.
• the genetic manipulation was confirmed by PCR method and genome sequencing using primers of SEQ ID NO: 55 and SEQ ID NO: 56 that can amplify the regions outside the homologous recombination upstream region and downstream region into which the corresponding regulatory region or promoter is inserted, respectively.
• the strain in which the trpE regulatory region was inserted in front of the gene of pheA was CM06-0010
• the strain in which the promoter of ilvB was inserted was CM06-0012
• the strain in which the promoter of leuA was inserted was CM06-0013
• the promoter of leuC was inserted.
• the strain was named CM06-0014.
• Example 8 pheA gene deletion of non-PTS L-tyrosine-producing strain and evaluation of production ability of promoter replacement strain
• NTG N-methyl-N'-nitro-N-nitrosoguanidine
• ATCC 13869 strain was cultured in a seed medium for 18 hours, inoculated into 4 ml of a seed medium, and cultured until an OD660 reached about 1.0.
• the culture medium was centrifuged to recover the cells, washed twice with 50 mM Tris-malate buffer (pH6.5), and suspended in the final 4 ml of the same buffer solution.
• NTG solution (2 mg/ml in 0.05M Tris-malate buffer (pH6.5) was added to the cell suspension and allowed to stand for 20 minutes at room temperature, and then the cells were centrifuged.
• L-tyrosine production ability was confirmed with respect to the L-tyrosine hydroxamate resistant strain of 100 strains obtained in Example 9.
• culture was performed with shaking at 30° C. for 20 hours at 200 rpm.
• 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium and cultured with shaking at 200 rpm for 48 hours at 30°C.
• the pheA genes of ATCC 13869 YAR-6 and YAR-8 strains selected in Example 10 were regulated in the regulatory region of the trp operon.
• pDZ- ⁇ PpheA::PtrpE prepared in Example 5 was transformed into ATCC 13869 YAR-6 and YAR-8 strains by electroporation, respectively, and then through a second cross-over process, the pheA gene was the regulatory region of trpE A strain made to be controlled was obtained.
• the genetic manipulation was confirmed by PCR method and genome sequencing using primers of SEQ ID NO: 55 and SEQ ID NO: 56 that can amplify the external regions of the homologous recombination upstream region and downstream region into which the regulatory region is inserted, respectively.
• ATCC 13869 YAR-6 and YAR-8 strains, which inserted the trpE regulatory region in front of the pheA gene, were named YAR-6P and YAR-8P, respectively.
• the production amount of L-tyrosine can be significantly increased to an unexpected level without slowing the sugar consumption rate, and the combination of the regulatory region of the trp operon and the pheA gene is L-tyrosine. It was confirmed that the synergy effect was great for production.

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