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WO2021125867A1 - Micro-organisme pour produire un composé et procédé de production de composé l'utilisant - Google Patents

Micro-organisme pour produire un composé et procédé de production de composé l'utilisant Download PDF

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Publication number
WO2021125867A1
WO2021125867A1 PCT/KR2020/018629 KR2020018629W WO2021125867A1 WO 2021125867 A1 WO2021125867 A1 WO 2021125867A1 KR 2020018629 W KR2020018629 W KR 2020018629W WO 2021125867 A1 WO2021125867 A1 WO 2021125867A1
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Prior art keywords
microorganism
protein
formula
compound
crr
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Korean (ko)
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배현애
전애지
류규리
장재우
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CJ CheilJedang Corp
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CJ CheilJedang Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom

Definitions

  • the present application relates to a microorganism producing a compound and a method for producing a compound using the same.
  • Violacein (violacein) is known as a purple dye, and deoxyviolacein (deoxyviolacein) is a compound with a structure similar to that of violacein, and is known to have a blue color than violacein in a solid state.
  • Violacein and deoxyviolacein are substances used as dyes for dyeing various fabrics such as cotton, nylon, rayon, and polyester, but have various physiologically active effects such as anticancer, antiulcer, antibacterial, antifungal, antibiotic, and antiviral. It can be used not only for use as a pigment, but also for use as various inhibitors, antibiotics, and the like. In particular, it is expected to play a role as a new antibiotic against multidrug-resistant bacteria due to its strong antibiotic action against Staphylococcus aureus and various other Gram-positive bacteria (Aruldass CA et al ., 2018, Environ Sci Pollut Res. 25:5164-5180). .
  • the present inventors have completed the present invention by confirming that, when the activity of the Crr protein is inactivated, the production capacity of violacein or deoxybiolacein is effectively increased.
  • the present application provides a microorganism producing the compound of Formula 1, wherein the Crr protein is inactivated.
  • X 1 is OH or H
  • X 2 is a ketone group or H
  • X 3 is H or OH
  • Dotted lines indicate double bonds or single bonds.
  • the present application provides a method for producing the compound of Formula 1 using the microorganism.
  • the present application provides a composition for producing a compound of Formula 1 containing the microorganism.
  • the present application provides a method for increasing the production of a compound of Formula 1, comprising the step of inactivating Crr protein.
  • microorganism of the present application can be usefully applied to various industrial fields using the high production efficiency of useful products.
  • One aspect of the present application is to provide a microorganism producing a compound of Formula 1, wherein Crr protein is inactivated.
  • X 1 is OH or H
  • X 2 is a ketone group or H
  • X 3 is H or OH
  • Dotted lines indicate double bonds or single bonds.
  • the compound of Formula 1 may be a compound named prodeoxyviolacein, proviolacein, oxyviolacein, deoxyviolacein, violacein, and the like, It is not limited thereto.
  • X 1 is -OH or -H
  • X 2 is a ketone group
  • X 3 may be a compound of -H, specifically, the compound may be represented by the following formula (2).
  • the compound of the present application also includes a derivative of the compound represented by Formula 1.
  • chromoviridan, oxychromoviridan, deoxychromoviridan, chromoazepinone pseudoviolacein, pseudodeoxyviola Sein (psuedodeoxyviolacein), protoviolaceinic acid, protodeoxyviolaceinic acid, chromopyrrolic acid (CPA), indigo (indigo; 2- (3-hydroxy-1 H -indol-2) Derivatives such as -yl)indol-3-one
  • substances obtainable in the route in which the microorganism of the present application biosynthesizes the derivative of the compound represented by Formula 1 is the compound of the present application may be included without limitation.
  • Crr protein or “Enzyme IIA Glc” protein refers to one of the proteins constituting the PTS system.
  • the Crr protein of the present application may be a protein comprising the amino acid sequence of SEQ ID NO: 18, and a protein consisting essentially of the amino acid sequence of SEQ ID NO: 18, a protein having the amino acid sequence of SEQ ID NO: 18, or the sequence It may be a protein consisting of the amino acid sequence of No. 18, but is not limited thereto.
  • the Crr protein of the present application may be a protein consisting of the amino acid sequence of SEQ ID NO: 18, but includes without limitation a sequence having the same activity as the Crr protein, and a person skilled in the art can obtain sequence information from a known database, NCBI's GenBank, etc. have.
  • the protein comprising the amino acid sequence of SEQ ID NO: 18 of the present application is SEQ ID NO: 18 and at least 60%, 70%, 80%, 83%, 84%, 85%, 86%, 87% of SEQ ID NO: 18 , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% homology or identity.
  • polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a probe that can be prepared from a known gene sequence for example, a sequence complementary to all or part of a nucleotide sequence encoding the protein
  • sequence Polypeptides having the same activity as the protein consisting of the amino acid sequence of No. 18 may also be included without limitation.
  • 'protein or polypeptide comprising the amino acid sequence described in a specific SEQ ID NO:'
  • 'protein or polypeptide comprising the amino acid sequence described in the specific SEQ ID NO:'
  • 'essentially composed of the amino acid sequence described in the specific SEQ ID NO: Even if it is described as 'a protein or polypeptide having an amino acid sequence set forth in a specific SEQ ID NO:' or 'a protein or polypeptide having an amino acid sequence set forth in a specific sequence number,' if it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding SEQ ID NO:
  • proteins having these deletions, modifications, substitutions, conservative substitutions or added amino acid sequences can also be used in the present application.
  • amino acid substitution means substituting an amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues.
  • positively charged (basic) amino acids include arginine, lysine, and histidine
  • negatively charged (acidic) amino acids include glutamic acid and aspartic acid
  • Aromatic amino acids include phenylalanine, tryptophan and tyrosine
  • hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan.
  • homology refers to the degree to which two given amino acid sequences or nucleotide sequences match and may be expressed as a percentage.
  • homology and identity can often be used interchangeably.
  • a homologous sequence having the same or similar activity to a given amino acid sequence or polynucleotide sequence is expressed as "% homology”.
  • Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially, homologous or identical sequences are generally at least about 50%, 60%, 70%, 80% of the entire or full-length sequence under moderate or high stringent conditions. or more than 90% hybrid. Hybridization is also contemplated for polynucleotides containing degenerate codons instead of codons in the polynucleotides.
  • GAP program is defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids).
  • Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.
  • the term “homology” or “identity” refers to a relevance between sequences.
  • the polynucleotide encoding the Crr protein of the present application may be referred to as a "crr gene”.
  • polynucleotide has a meaning comprehensively encompassing DNA or RNA molecules, and nucleotides, which are basic structural units in polynucleotides, may include not only natural nucleotides, but also analogs in which sugar or base sites are modified ( Scheit, Nucleotide Analogs, John Wiley, New York (1980); see Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).
  • the polynucleotide sequence of the gene encoding the Crr protein can be obtained from a known database, for example, GenBank of NCBI, etc., but is not limited thereto.
  • the polynucleotide is 60%, 70%, 80%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the polynucleotide encoding the Crr protein of the present application or the protein of the present application. , 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% homology or identity.
  • the polynucleotide encoding the Crr protein of the present application is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO: 7 It may be a polynucleotide having homology or identity. However, as long as it is a polynucleotide sequence encoding a protein having an activity corresponding to the Crr protein, it is not limited thereto, and it is obvious that it is included in the scope of the present application.
  • a polynucleotide that can be translated into a protein having the same amino acid sequence or a protein having homology thereto may also be included due to the genetic code degeneracy of the codon.
  • a probe that can be prepared from a known gene sequence for example, a protein having the activity of a protein consisting of the amino acid sequence of SEQ ID NO: 18 by hybridizing under stringent conditions with a sequence complementary to all or part of the nucleotide sequence Any coding sequence may be included without limitation.
  • stringent conditions means conditions that allow specific hybridization between polynucleotides. Such conditions are described in, e.g., J.
  • genes with high homology are 40% or more, specifically 70% or more, 80% or more, 85% or more, 90% or more, more specifically 95% or more, more specifically 97% or more, in particular, the conditions in which genes having 99% or more homology are hybridized and genes having lower homology are not hybridized, or 60°C, 1 ⁇ SSC, 0.1% SDS, which are the washing conditions of normal Southern hybridization.
  • Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization.
  • the term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also include isolated nucleic acid fragments that are complementary to substantially similar nucleic acid sequences as well as the entire sequence.
  • polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the conditions described above.
  • the Tm value may be 60° C., 63° C. or 65° C., but is not limited thereto and may be appropriately adjusted by those skilled in the art depending on the purpose.
  • the microorganism producing the compound may be one in which Crr protein is inactivated.
  • the term "inactivation" of a protein is a concept that includes both reduced activity or no activity compared to intrinsic activity.
  • the inactivation may be used interchangeably with terms such as deficiency, down-regulation, decrease, reduce, attenuation, and weakening.
  • the inactivation is when the activity of the protein itself is reduced or eliminated compared to the activity of the protein possessed by the original microorganism due to mutation of the polynucleotide encoding the protein, etc.
  • the overall protein activity level and/or concentration (expression amount) in the cell is lower than that of the native strain due to translation inhibition, etc., when the expression of the polynucleotide is not made at all, and/or expression of the polynucleotide Even if this happens, it may also include a case where there is no protein activity.
  • the "intrinsic activity” refers to the activity of a specific protein originally possessed by the parent strain, wild-type or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification”. "Inactivation, deficiency, reduction, downregulation, reduction, attenuation” of the activity of a protein compared to the intrinsic activity means that it is lower than the activity of a specific protein originally possessed by the parent strain or unmodified microorganism before transformation.
  • This attenuation of protein activity may be performed by any method known in the art, but is not limited thereto, and may be achieved by application of various methods well known in the art (eg, Nakashima N et al., Bacterial). cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).
  • inactivation is
  • modification of the gene sequence encoding the protein to remove or attenuate the activity of the protein eg, one or more nucleotides on the nucleotide sequence of the protein gene to encode the modified protein to remove or attenuate the activity of the protein) deletion/replacement/addition of);
  • an antisense oligonucleotide eg, antisense RNA
  • an antisense oligonucleotide that complementarily binds to the transcript of the gene encoding the protein
  • deletion of a part or all of the gene encoding the protein may be the removal of the entire polynucleotide encoding the endogenous target protein in the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.
  • the expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.
  • the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the protein is, for example, a base encoding another start codon having a lower protein expression rate than the intrinsic start codon. It may be substituted with a sequence, but is not limited thereto.
  • the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above deletes, inserts, non-conservative or conservative substitution of the amino acid sequence of the protein or the polynucleotide sequence encoding the protein to weaken the activity of the protein. Or a combination thereof may result in sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto.
  • the expression of a gene may be inhibited or attenuated, but is not limited thereto.
  • antisense oligonucleotide eg, antisense RNA
  • antisense RNA an antisense oligonucleotide that complementarily binds to the transcript of the gene encoding the protein
  • RTE reverse transcription engineering
  • the inactivation of the Crr protein of the present application is the inactivation of the Crr transcriptional regulator, the inactivation of the protein comprising the amino acid sequence of SEQ ID NO: 18, or the protein encoded by the gene comprising the polynucleotide sequence of SEQ ID NO: 7 including inactivation of However, it is not limited thereto.
  • a microorganism producing a compound or "a microorganism having a compound-producing ability” refers to a microorganism having the ability to naturally produce the compound of the present application, or the parent strain without the ability to produce the compound of the present application. It refers to microorganisms that have been given the ability to produce compounds of
  • the microorganism may have a genetic mutation to produce a desired compound.
  • the genetic variation may be, for example, an insertion of a foreign gene. including inactivation of endogenous gene activity.
  • the genetic mutation includes mutations such as inactivation of the aforementioned Crr protein, introducing a part of the vioABCDE gene cluster or a mutated form thereof or enhancing its activity. These variations do not exclude the natural.
  • the microorganism may have an enhanced ability to produce a target compound as compared to a microorganism that does not include a genetic mutation such as the aforementioned gene insertion or gene inactivation.
  • a genetic mutation such as the aforementioned gene insertion or gene inactivation.
  • the microorganism producing the compound of the present application may be a microorganism having the ability to produce the compound of the present application by inactivating the Crr protein.
  • the protein comprising the amino acid sequence of SEQ ID NO: 18 may be an inactivated microorganism, and more specifically, the protein comprising the amino acid sequence of SEQ ID NO: 18 is inactivated, vioA, vioB, vioC and a microorganism into which the vioE gene is introduced or enriched, but is not limited thereto.
  • vioA is a gene encoding VioA (L-tryptophan oxidase)
  • vioB is VioB (N-[2-(carboxylatoamino)-1,2-bis(1H-indol-3-yl)ethyl]carbamate synthase)
  • vioC means a gene encoding VioC (violacein synthase)
  • vioE means a gene encoding VioE (protoviolaceinate synthase).
  • vioABCDE gene cluster refers to a gene cluster comprising 5 genes by adding the gene vioD encoding VioD (protoviolaceinate synthase) in addition to the vioA, vioB, vioC and vioE, and "vioABCDE” and can be used interchangeably.
  • the VioD polypeptide when the VioD polypeptide is inactivated because the VioD-encoding gene vioD exists in a mutated or deleted form, the production ability of deoxyviolasein may increase.
  • the vioD of the vioABCDE may be in an inactivated form.
  • the inactivation is as described above.
  • the microorganism of the present application is one in which Crr protein is inactivated and vioA, vioB, vioC and vioE genes are introduced, or Crr protein is inactivated and vioA, vioB, vioC, vioD and vioE genes are introduced, or The Crr protein may be inactivated, vioA, vioB, vioC, and vioE may be introduced, and a mutated vioD gene may be introduced such that activity is reduced or eliminated, but is not limited thereto.
  • the vioABCDE gene chromotherapy tumefaciens (Choromobacterium), A glass Martino tumefaciens (Janthinobacterium), A duga Nella (Duganella) genus, Pseudomonas Alteromonas (Psuedoalteromonas), An coli Pseudomonas (Collimonas), An iodo bakteo (Iodobacter ) may be derived from strains such as genus, and specifically may be derived from the genus Chromobacterium , but is not limited thereto.
  • the microorganism producing the compound may be a microorganism of the genus Escherichia sp .
  • the microorganisms of the genus Escherichia are Escherichia coli , Escherichia albertii , Escherichia blattae , Escherichia blattae , Escherichia fergusonii ), Escherichia hermanni ( Escherichia hermannii ) or Escherichia vulneris ( Escherichia vulneris ), but is not limited thereto. More specifically, it may be Escherichia coli , but is not limited thereto.
  • Another aspect of the present application provides a method for producing a compound of Formula 1 comprising the step of culturing the microorganism of the present application.
  • the compound may be violacein or deoxyviolacein, but is not limited thereto.
  • the term "cultivation” means growing microorganisms in an appropriately artificially controlled environmental condition.
  • the method of culturing the microorganism in the present application may be performed using a method widely known in the art. Specifically, the culture may be continuously cultured in a batch process, an injection batch or a repeated fed batch process, but is not limited thereto.
  • the step of culturing the microorganism is not particularly limited thereto, but may be performed by a known batch culture method, a continuous culture method, a fed-batch culture method, and the like.
  • the medium and other culture conditions used for culturing the microorganisms of the present application may be used without particular limitation as long as they are media used for culturing conventional microorganisms, but specifically, the microorganisms of the present application may be used with a suitable carbon source, nitrogen source, phosphorus, inorganic compound, It can be cultured while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing amino acids and/or vitamins.
  • Sugar sources that can be used in the medium include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, and stearic acid , fatty acids such as linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture, but are not limited thereto.
  • sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch and cellulose
  • oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, and stearic acid
  • fatty acids such as linoleic acid
  • alcohols such as glycerol and ethanol
  • organic acids such as acetic acid.
  • Nitrogen sources that can be used include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
  • the nitrogen source may also be used individually or as a mixture, but is not limited thereto.
  • the phosphorus that can be used may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or salts containing the corresponding sodium.
  • the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth.
  • essential growth substances such as amino acids and vitamins can be used.
  • precursors suitable for the culture medium may be used. The above-mentioned raw materials may be added batchwise or continuously by a method suitable for the culture during the culture process.
  • a basic compound such as sodium hydroxide, potassium hydroxide, ammonia, or an acid compound such as phosphoric acid or sulfuric acid may be used in an appropriate manner to adjust the pH of the culture.
  • an antifoaming agent such as a fatty acid polyglycol ester may be used to inhibit the formation of bubbles.
  • Oxygen or oxygen-containing gas eg, air
  • the temperature of the culture may be usually 20 °C to 45 °C, specifically 25 °C to 40 °C.
  • the incubation time may be continued until the desired compound production amount is obtained, but specifically, it may be 10 to 160 hours.
  • the compound produced by the culturing may be secreted into the medium or may remain in the cell.
  • the production method of the present application may further include the step of recovering the compound of the present application from the microorganism or medium after the culturing step.
  • the compound may be recovered by a conventional method known in the art.
  • methods such as centrifugation, filtration, ion exchange chromatography, and crystallization may be used.
  • the supernatant obtained by removing the biomass by centrifuging the culture at low speed may be separated through ion exchange chromatography, but is not limited thereto, and microorganisms cultured using a suitable method known in the art.
  • the desired compound may be recovered from the medium.
  • the recovery step may further include a separation process and/or a purification process.
  • compositions for producing a compound of Formula 1, comprising a microorganism in which the Crr protein of the present application is inactivated.
  • composition may further include any suitable excipients commonly used in compositions for the production of the compound.
  • excipients may include, but are not limited to, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents and isotonic agents, and the like.
  • Another aspect of the present application provides the use of a microorganism in which the Crr protein of the present application is inactivated, for producing a compound of Formula 1.
  • Another aspect of the present application provides a method for increasing the production of the compound of Formula 1, comprising the step of inactivating the Crr protein of the present application in a microorganism.
  • Example 1 Chromobacterium violaceum ( Chromobacterium violaceum ) Construction of ATCC 12472-derived violacein biosynthetic gene cluster overexpression vector
  • the violacein biosynthetic gene cluster vioABCDE was amplified using the genomic DNA of Chromobacterium violaceum ATCC 12472.
  • Primer 1 SEQ ID NO: 1 having an EcoRV restriction enzyme site inserted at the 5' end
  • primer 2 SEQ ID NO: 2 having a BamHI restriction enzyme site inserted at the 3' end were synthesized.
  • PCR polymerase chain reaction
  • trc promoter (SEQ ID NO: 3) with a KpnI restriction enzyme site inserted at the 5' end and an EcoRV restriction enzyme site inserted at the 3' end was synthesized.
  • the PCR-amplified gene cluster fragment was treated with restriction enzymes EcoRV and BamHI, and the synthesized trc promoter fragment was treated with restriction enzymes KpnI and EcoRV to obtain each DNA fragment.
  • the overexpression vector pECCG117 (Korean Patent Registration No. 1992-007401) having restriction enzymes KpnI and BamHI terminus, it was transformed into E. coli DH5 ⁇ and LB (Luria Bertani, tryptone per liter) containing kanamycin at 25 mg/l 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0) was smeared on a solid medium.
  • Plasmids were obtained from each transformed Escherichia coli DH5 ⁇ using a commonly known plasmid extraction method, and these plasmids were named pECCG117-Ptrc-vioABCDE and pECCG117-Ptrc-vioABCD*E.
  • the plasmid pECCG117-Ptrc-vioABCD*E found above was compared with the nucleotide sequence (SEQ ID NO: 4) of the wild-type violacein biosynthesis gene cluster through nucleotide sequence analysis, thereby confirming the amino acid sequence of the mutated VioD protein.
  • the mutant VioD amino acid sequence (SEQ ID NO: 6) can be expected to not perform the role of wild-type VioD properly due to the stop codon generated by the mutation, and accordingly, the plasmid pECCG117
  • the biosynthetic gene cluster of -Ptrc-vioABCD*E can be predicted as a gene cluster capable of biosynthesis only with deoxyviolacein, not violacein. Since it is already known that only deoxybiolacein can be produced using the vioD biosynthetic gene cluster in a mutated or deleted form (March PR et al ., 2000, J Mol Microbiol Biotechnol.
  • Example 2 Violacein biosynthesis gene cluster overexpression vector-introduced strain was prepared and violacein and deoxyviolacein production ability was confirmed
  • the vector When the vector is transformed, it becomes resistant to kanamycin, so transformation was confirmed through growth in LB medium containing 25 mg/L of kanamycin.
  • each strain was inoculated with 1 platinum in a 250 ml corner-baffle flask containing 25 ml of LB medium containing kanamycin at a concentration of 25 mg/L, Incubated at 37° C. for 24 hours with shaking at 200 rpm.
  • the culture medium was diluted 10-fold in ethanol, extracted with shaking for 1 hour, centrifuged at 13,000 rpm for 5 minutes, and the supernatant was analyzed by HPLC. The results are shown in [Table 1].
  • strain name OD 600 nm absorption
  • violacein mg per liter of culture medium
  • deoxyviolacein mg per liter of culture medium
  • the violacein-producing strain W3110/pECCG117-Ptrc-vioABCDE prepared in the above Example was plated on LB agar medium containing 25 mg/L of kanamycin and then UV irradiated. Random mutations within the strain were induced. The UV-irradiated plate was incubated at 37°C for 24 hours. Among the colonies formed, 13 mutant strains with a deeper purple color than the W3110/pECCG117-Ptrc-vioABCDE colony were selected (each mutant was named C1 ⁇ C13) and kanamycin 25 It was subcultured on LB agar medium containing mg/L.
  • each strain was placed in a 250 ml corner-baffle flask containing 25 ml of titer medium containing kanamycin at a concentration of 25 mg/l, 1 platinum. This inoculation was carried out at 37 DEG C for 48 hours and shaking at 200 rpm.
  • Glucose 40g/L calcium carbonate 30g/L, ammonium sulfate 10g/L, potassium monophosphate 1g/L, magnesium sulfate 1.5g/L, yeast extract 4g/L
  • the culture medium was diluted 10-fold in ethanol, extracted with shaking for 1 hour, centrifuged at 13,000 rpm for 5 minutes, and the supernatant was analyzed by HPLC. The results are shown in [Table 2]. indicated.
  • strain name OD 600 nm absorption
  • violacein (mg per liter of culture medium) deoxyviolacein (mg per liter of culture medium)
  • 13 mutants showed various production capacities and ODs.
  • the C2 mutant strain showed a 1.6-fold higher violacein production capacity compared to the control W3110/pECCG117-Ptrc-vioABCDE, and a pattern in which the ratio of deoxyviolacein was maintained similar to that of the control group was confirmed. is not produced, but it was confirmed that the production capacity of deoxyviolacein was increased by 1.5 times compared to the total production of deoxyviolacein and violacein of the control group.
  • Plasmids were obtained from C2 and C9 mutants with improved violacein or deoxyviolacein production ability by the UV irradiation, and it was first confirmed whether there was a mutation in vioABCDE or the promoter region.
  • the plasmid obtained from C2 had no mutations in the biosynthetic operon and promotor regions, but in the plasmid obtained from C9, the 610th nucleotide G of vioD was changed to T, and accordingly, the 204th Glu coding codon had an amber displacement. mutation) was introduced and it was confirmed that it was changed to a stop codon. That is, it is judged that VioD was inactivated by the amber displacement introduced into vioD when the C9 candidate group failed to produce violacein and produced only deoxyviolacein.
  • the genomes of the mutant strains C2 and C9 were sequenced and compared with the wild-type strain.
  • mutants C2 and C9 contained mutations in the gene region encoding Crr, respectively.
  • mutant C2 compared to wild-type crr (SEQ ID NO: 7), amber mutation was introduced as the 43rd nucleotide was replaced with A, so that the 14th codon was changed from Lys to the stop codon, and the 251th nucleotide G was substituted with A
  • codon 84 was changed from Ser to Asn.
  • mutant C9 a frame shift occurred as nucleotide A was inserted at the 12th position of the crr gene, and a stop codon was generated as the 6th codon was introduced with an ochre mutation. was confirmed (SEQ ID NO: 9).
  • Example 6 W3110 wild-type strain-based crr-deficient strain production
  • the gene encoding Crr (SEQ ID NO: 7) inherent in microorganisms of the genus Escherichia was deleted by homologous recombination.
  • a one-step inactivation method a mutagenesis technique using lambda red recombinase developed by Datsenko KA et al., was used (Proc Natl Acad Sci USA., (2000) 97: 6640 -6645) .
  • the chloramphenicol gene of pUCprmfmloxC was used (Korean Patent Application Laid-Open No. 2009-007554).
  • primer 3 SEQ ID NO: 10
  • primer 4 SEQ ID NO: 11
  • the gene fragment of about 1300 base pairs was amplified by the polymerase chain reaction method (hereinafter 'PCR' method) in which the conditions of denaturation, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute were repeated 30 times.
  • the DNA fragment obtained through the above process was eluted after 0.8% agarose gel electrophoresis and used for recombination.
  • the target E. coli transformed with pKD46 was prepared in a competent state, and the 1300 base pair size obtained by PCR method was transformed by introducing a gene fragment of The obtained strain was selected from LB having chloramphenicol resistance. It was confirmed that the genes were deleted by confirming that the amplified size was about 1440 base pairs, respectively, through PCR using primers 5 (SEQ ID NO: 12) and primer 6 (SEQ ID NO: 13).
  • the primary recombinant strain with chloramphenicol resistance removes pKD46 and then introduces pJW168 to remove the chloramphenicol marker gene from the cells (Gene, (2000) 247,255-264).
  • the cells finally obtained were about 340 pairs of amplification products obtained through PCR using primers 5 (SEQ ID NO: 12) and primer 6 (SEQ ID NO: 13), and it was confirmed that each gene deletion was made as intended.
  • the crr genes of SEQ ID NO: 8 and SEQ ID NO: 9 containing mutations were amplified using the genomic DNAs of mutants C2 and C9, respectively.
  • Primer 7 (SEQ ID NO: 14) and primer 8 (SEQ ID NO: 15) having an EcoRV restriction site inserted at the 5' end and primer 9 (SEQ ID NO: 16) having a BamHI restriction site inserted at the 3' end were synthesized.
  • Violacein biosynthesis gene by performing a polymerase chain reaction (hereinafter abbreviated as PCR) using each genomic DNA of mutant strain C2 as a template using primers 7 and 9 and mutant strain C9 using primers 8 and 9
  • the cluster was amplified.
  • PCR conditions denaturation at 94 °C for 5 minutes, denaturation at 94 °C for 30 seconds, annealing at 60 °C for 30 seconds, and polymerization at 72 °C for 10 minutes were repeated 30 times, followed by polymerization at 72 °C for 7 minutes.
  • trc promoter (SEQ ID NO: 17) in which a KpnI restriction site was inserted at the 5' end and an EcoRV restriction site was inserted at the 3' end was synthesized.
  • the PCR-amplified gene cluster fragment was treated with restriction enzymes EcoRV and BamHI, and the synthesized trc promoter fragment was treated with restriction enzymes KpnI and EcoRV to obtain each DNA fragment.
  • pcc1BAC Epicentre
  • a 1-copy vector having restriction enzymes KpnI and BamHI terminus it was transformed into E. coli DH5 ⁇ and LB (Luria Bertani, 10 g of tryptone per liter, containing 25 mg/l of chloroamphenicol, Yeast extract 5 g, NaCl 10 g, pH 7.0) was spread on a solid medium.
  • Plasmids were obtained from each transformed Escherichia coli DH5 ⁇ using a commonly known plasmid extraction method, and these plasmids were named pcc1BAC-Ptrc-crr (C2) and pcc1BAC-Ptrc-crr (C9).
  • each plasmid was transformed into W3110 and W3110 ⁇ crr strains as shown in [Table 3] to prepare strains.
  • Glucose 40g/L calcium carbonate 30g/L, ammonium sulfate 10g/L, potassium monophosphate 1g/L, magnesium sulfate 1.5g/L, yeast extract 4g/L
  • the culture medium was diluted 10-fold in ethanol, extracted with shaking for 1 hour, centrifuged at 13,000 rpm for 5 minutes, and the supernatant was analyzed by HPLC and then the control group W3110/pECCG117-Ptrc-vioABCDE Four strains with higher concentrations were finally selected, and the results are shown in [Table 4].

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Abstract

La présente invention concerne un micro-organisme ayant une capacité de production d'un composé, et un procédé de production de composé l'utilisant.
PCT/KR2020/018629 2019-12-20 2020-12-18 Micro-organisme pour produire un composé et procédé de production de composé l'utilisant Ceased WO2021125867A1 (fr)

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