KR102303662B1 - A microorganism producing compound and method for producing the compound using the same - Google Patents

Abstract

The present application relates to a microorganism producing a compound and a method for producing a compound using the same.

Description

A microorganism producing a compound and a method for producing a compound using the same {A microorganism producing compound and method for producing the compound using the same}

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 anticancer (Melo et al ., 2000, Ferreira et al., 2004, Kodach et al ., 2006, Duran et al., 2007), antiulcer (Antonisamy et al ., 2009), antibacterial (Duran et al ., 1983, Duran et al ., 2012), antifungal (Shirata et al ., 2000, Becker et al. ., 2009), antigenic animals (Leon et al ., 2001), and antiviral (Rettori et al ., 1998), etc., have various physiologically active effects. can also be used. 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). .

As such, the need for mass production of violacein or deoxybiolacein, which is a material having a number of industrially valuable physiological activities, is also increasing, and accordingly, the need for a method for producing violacein or deoxyviolacein with high efficiency increases and there is a situation.

Under this background, the present inventors 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.

[Formula 1]

In Formula 1, 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.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the present application described herein. Also, such equivalents are intended to be covered by this application.

One aspect of the present application is to provide a microorganism producing a compound of Formula 1, wherein Crr protein is inactivated.

[Formula 1]

In Formula 1, 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.

In one embodiment, in the compound, in Formula 1, 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).

[Formula 2]

The compound in which X is -OH in Formula 2 is referred to as "violacein" in the present application, and the compound in which X is -H in Formula 2 is referred to as "deoxyviolacein" in the present application.

Meanwhile, the compound of the present application also includes a derivative of the compound represented by Formula 1. For example, 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) may also be included in the scope of the present application, and 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.

As used herein, the term "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.

In addition, 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. In addition, 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. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting biological activity corresponding to the protein, it is apparent that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included in the scope of the present application.

In addition, as a 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 polypeptide, the sequence Polypeptides having the same activity as the protein consisting of the amino acid sequence of No. 18 may also be included without limitation.

That is, in the present application, '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 It is apparent that proteins having these deletions, modifications, substitutions, conservative substitutions or added amino acid sequences can also be used in the present application. For example, the amino acid sequence N-terminus and/or C-terminal addition of a sequence that does not alter the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof. .

The term “conservative 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. For example, 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, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan.

As used herein, the term "homology" or "identity" refers to the degree to which two given amino acid sequences or nucleotide sequences match and can be expressed as a percentage. The terms homology and identity can often be used interchangeably. In the present specification, 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.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the “FASTA” program. or, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine. (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215] : 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073) For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW, can be used to determine homology, similarity or identity.

Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. 48: 443 by comparing the sequence information using a GAP computer program. In summary, the 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. Thus, as used herein, 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".

In the present application, the term "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 may 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.

Specifically, 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.

In addition, it is obvious that 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. Also, a protein having the activity of a protein consisting of the amino acid sequence of SEQ ID NO: 18 by hydridation 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 the nucleotide sequence. It may be included without limitation as long as it encodes a sequence. The "stringent conditions" means conditions that allow specific hybridization between polynucleotides. Such conditions are described in, for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology. , John Wiley & Sons, Inc., New York). For example, 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 do not hybridize, or 60° C., 1ХSSC, 0.1% SDS, which are the washing conditions of normal Southern hybridization, specifically Lists conditions for washing once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to 60°C, 0.1ХSSC, 0.1% SDS, more specifically 68°C, 0.1ХSSC, 0.1% SDS. can do. 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.

Specifically, 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 above-described conditions. In addition, 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 appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

In the present application, the microorganism producing the compound may be one in which Crr protein is inactivated.

In the present application, the term "Crr protein is inactivated" means that the expression of the gene encoding the protein is not expressed at all compared to the parent strain or the strain in which the Crr protein is unmodified, or the activity of the protein is absent or reduced even if it is expressed. means that In this case, the decrease is a case in which the activity of the protein is reduced compared to the activity of the protein possessed by the original microorganism due to mutation or deletion of the gene encoding the protein, and inhibition of expression or translation inhibition of the gene encoding it. When the overall protein activity level in the cell is lower than that of the native strain or the strain before modification, it is a concept including a combination thereof.

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

In the present application, the inactivation may be achieved by applying various methods well known in the art. Examples of the method include: 1) a method of deleting all or part of the gene encoding the protein; 2) modification of the expression control sequence to reduce the expression of the gene encoding the protein, 3) modification of the gene sequence encoding the protein so that the activity of the protein is removed or weakened, 4) the gene encoding the protein introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of 5) By adding a sequence complementary to the Shine-Dalgarno sequence to the front end of the Shine-Dalgarno sequence of the gene encoding the protein, a secondary structure is formed to make attachment of the ribosome impossible Way; 6) There is a method of adding a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the polynucleotide sequence of the gene encoding the protein (Reverse transcription engineering, RTE), etc., and a combination thereof can also be achieved, but is not particularly limited thereto.

Specifically, in the method of deleting part or all of the gene encoding the protein, a polynucleotide encoding an endogenous target protein in a chromosome is converted into a polynucleotide or a marker gene in which some nucleotide sequences are deleted through a vector for chromosomal insertion in a microorganism. This can be done by replacing As an example of a method for deleting part or all of the polynucleotide, a method for deleting a polynucleotide by homologous recombination may be used, but the present invention is not limited thereto.

In addition, the method of deleting a part or all of the gene may be performed by inducing a mutation using light or a chemical such as ultraviolet light, and selecting a strain in which the target gene is deleted from the obtained mutant. The gene deletion method includes a method by DNA recombination technology. The DNA recombination technique may be accomplished by, for example, injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene into the microorganism to cause homologous recombination. In addition, the injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

In addition, the method of modifying the expression control sequence can be achieved by applying various methods well known in the art. As an example of the method, deletion, insertion, non-conservative or conservative substitution of the polynucleotide sequence or a combination thereof to further weaken the activity of the expression control sequence is performed by inducing mutations in the expression control sequence, or having a weaker activity by replacing it with a polynucleotide sequence. The expression control sequence 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.

In addition, the method of modifying the gene sequence is performed by inducing a mutation in the sequence by deleting, inserting, non-conservative or conservative substitution of the gene sequence, or a combination thereof to further weaken the activity of the enzyme, or to have weaker activity. It may be carried out by replacing the improved gene sequence or the modified gene sequence so as to have no activity, but is not limited thereto.

For example, by introducing a mutation in the gene sequence to form a stop codon, the expression of a gene can be inhibited or attenuated.

As used herein, the term "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 For example, 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. For example, 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.

In addition, the microorganism may have an enhanced ability to produce a target compound as compared to a microorganism that does not contain genetic mutations such as the aforementioned gene insertion or gene inactivation. However, it is not limited thereto.

Specifically, 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. More specifically, 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.

In the present application, 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), and vioE means a gene encoding VioE (protoviolaceinate synthase).

As used herein, the term "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.

In the vioABCDE, 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.

That is, 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 is Chromobacterium ( Chormobacterium ) genus, Xantinobacterium genus, Janthinobacterium genus, Duganella genus, Pseudoalteromonas genus, Colimonas genus, Iodobacter ) may be derived from strains such as genus, and specifically may be derived from the genus Chromobacterium , but is not limited thereto.

In one embodiment, the microorganism producing the compound may be a microorganism of the genus Escherichia (Escherichia sp.). Specifically, the microorganisms of the genus Escherichia are Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia blattae, Escherichia fergusonii, Escherichia hermanni ( Escherichia hermannii) or 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.

[Formula 1]

In one embodiment, the compound may be violacein or deoxyviolacein, but is not limited thereto.

In the present application, 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.

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. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used. In addition, 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.

During the culturing of the microorganism, 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. In addition, 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) may be injected into the culture to maintain aerobic conditions. 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. For the recovery method, methods such as centrifugation, filtration, ion exchange chromatography, and crystallization may be used. For example, 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. Alternatively, the desired compound may be recovered from the medium.

The recovery step may further include a separation process and/or a purification process.

Another aspect of the present application provides a composition for producing the compound of Formula 1 of the microorganism in which the Crr protein of the present application is inactivated.

The terms “Crr protein”, “compound of Formula 1”, and “inactivation” are the same as described above.

The composition may further include any suitable excipients commonly used in compositions for the production of the compound. Such 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 increasing the production of the 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.

The terms “Crr protein”, “compound of Formula 1”, and “inactivation” are the same as described above.

The microorganism of the present application can be usefully applied to various industrial fields using the high production efficiency of useful products having various physiological activities.

Hereinafter, the present application will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present application, and the scope of the present application is not limited to these Examples and Experimental Examples.

Example 1: Chromobacterium violaceum ( Chromobacterium violaceum ) Construction of ATCC 12472-derived violacein biosynthetic gene cluster overexpression vector

To construct a 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 and primer 2 (SEQ ID NO: 2) having a BamHI restriction enzyme site inserted at the 3' end were synthesized. Using this primer pair, a polymerase chain reaction (hereinafter abbreviated as PCR) was performed using the genomic DNA of Chromobacterium violaceum ATCC 12472 as a template to amplify the violacein biosynthesis gene cluster. For 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.

In addition, a trc promoter (SEQ ID NO: 3) in which a KpnI restriction enzyme site was inserted at the 5' end and an EcoRV restriction enzyme 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. After ligating this to the overexpression vector pECCG117 (Korean Patent Registration No. 1992-007401) having restriction enzymes KpnⅠ and BamHI terminus, it was transformed into E. coli DH5α 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.

Before selecting the colonies transformed with the vector into which the desired gene was inserted through PCR, it was confirmed that the color of the colonies of the already transformed E. coli DH5α was mostly purple. However, some indigo-colored colonies were found and marked separately (*). Plasmids were obtained from each transformed Escherichia coli DH5α 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. Unlike the wild-type VioD amino acid sequence (SEQ ID NO: 5), 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 (August PR et al ., 2000, J Mol Microbiol Biotechnol. 2(4):513-519) , Jiang PX et al ., 2012, Appl Microbiol Biotechnol. 94(6):1521-32, Rodrigues AL et al ., 2013, Metab Eng. 20:29-41), the present inventors separately deoxybiolacein biosynthesis gene The pECCG117-Ptrc-vioABCD*E prepared above was used for the production of deoxyviolacein without constructing an overexpression vector.

Example 2: Violacein biosynthesis gene cluster overexpression vector-introduced strain was prepared and violacein and deoxyviolacein production ability was confirmed

The strains produced by transforming the wild-type and mutant violacein biosynthetic gene cluster overexpression vectors pECCG117-Ptrc-vioABCDE and pECCG117-Ptrc-vioABCD*E prepared in Example 1 into E. coli W3110 strains, respectively, W3110/pECCG117- They were named Ptrc-vioABCDE and W3110/pECCG117-Ptrc-vioABCD*E. When the vector is transformed, it becomes resistant to kanamycin, so transformation was confirmed by growth in LB medium containing 25 mg/L of kanamycin.

In order to compare the violacein and deoxyviolacein production ability of the prepared strains, 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. To quantify violacein and deoxyviolacein, 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) W3110 6.5 0 0 W3110/pECCG117-Ptrc-vioABCDE 8.2 78.6 11 W3110/pECCG117-Ptrc-vioABCD*E 7.9 0 82.9

As shown in [Table 1], when pECCG117-Ptrc-vioABCDE was transformed into E. coli W3110 strain, it was confirmed that violacein and deoxyviolacein were produced, and pECCG117-Ptrc-vioABCD encoding the variant VioD was * When transforming E, it was confirmed that only deoxyviolacein was produced. Hereinafter, they are collectively referred to as a violacein-producing strain and a deoxyviolacein-producing strain.

Example 3: Introduction of random mutations through UV irradiation of violacein-producing strains

In order to select strains with improved violacein-producing ability, 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.

In order to compare the violacein and deoxyviolacein production capacity of the 13 mutant strains subcultured, 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 was inoculated and incubated with shaking at 200 rpm for 48 hours at 37°C.

Potency medium composition

Glucose 40g/L, calcium carbonate 30g/L, ammonium sulfate 10g/L, potassium monophosphate 1g/L, magnesium sulfate 1.5g/L, yeast extract 4g/L

In order to quantify violacein and deoxyviolacein, 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) W3110 15.2 0 0 W3110/pECCG117-Ptrc-vioABCDE 30.5 446 42 C1 22.1 336 72 C2 43.1 711 68 C3 26.1 116 153 C4 32.1 400 82 C5 16.9 226 10 C6 39.2 125 422 C7 30.6 222 262 C8 10.6 241 22 C9 41.6 0 754 C10 12.8 156 29 C11 22.6 323 13 C12 30.5 426 43 C13 31.6 458 32

As shown in [Table 2], 13 mutants showed various production capacities and ODs. Among them, 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.

Example 4: Confirmation of mutations in biosynthetic genes and promoters of violacein mutants C2 and C9

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.

Nevertheless, since the C9 mutant has a higher deoxyviolacein-producing ability than the control, sequencing the genes on the genome of the C2 mutant as well as the C9 mutant to identify the mutant gene affecting the production of violacein or deoxyviolacein. .

Example 5: Confirmation of mutations through NGS analysis of violacein mutants C2 and C9

The genomes of the mutant strains C2 and C9 were sequenced and compared with the wild-type strain.

As a result, it was confirmed that the mutants C2 and C9 each contained a mutation in the gene region encoding Crr. Specifically, in 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 As a result, codon 84 was changed from Ser to Asn. (SEQ ID NO: 8), in 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).

Therefore, in the following Examples, it was attempted to confirm the effect of the mutation on the violacein and deoxybiolacein production ability of microorganisms of the genus Escherichia.

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) . As a marker for confirming the insertion into the gene, the chloramphenicol gene of pUCprmfmloxC was used (Korean Patent Application Laid-Open No. 2009-007554). Using primer 3 (SEQ ID NO: 10) and primer 4 (SEQ ID NO: 11) having a partial nucleotide sequence of the chloramphenicol resistance gene of the pUCprmfmloxC gene and a part of the two genes, pUCprmfmloxC as a template and at 94°C for 30 seconds 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.

According to the method developed by Datsenko KA et al. (Proc Natl Acad Sci USA., (2000) 97: 6640-6645), 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.

Example 7: Construction of crr mutation vectors of SEQ ID NOs: 8 and 9

In order to construct crr mutation vectors of SEQ ID NOs: 8 and 9, 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. For 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.

In addition, a trc promoter (SEQ ID NO: 17) in which a KpnI restriction enzyme site was inserted at the 5' end and an EcoRV restriction enzyme 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. After ligating this to pcc1BAC (Epicentre), a 1-copy vector having restriction enzymes KpnⅠ and BamHI terminus, it was transformed into E. coli DH5α 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 DH5α using a commonly known plasmid extraction method, and these plasmids were named pcc1BAC-Ptrc-crr (C2) and pcc1BAC-Ptrc-crr (C9).

Example 8: Confirmation of the influence of two crr mutants and crr deletion

In order to confirm the effect of crr mutations and deletions of SEQ ID NOs: 8 and 9 on violacein and deoxybiolacein production ability, each plasmid was transformed into W3110 and W3110Δcrr strains as shown in [Table 3] to prepare strains.

product strain name Antibiotic violacein W3110/pECCG117-Ptrc-vioABCDE kanamycin C2 kanamycin W3110Δcrr/pECCG117-Ptrc-vioABCDE kanamycin W3110Δcrr/pECCG117-Ptrc-vioABCDE/pcc1BAC-Ptrc-crr(C2) Kanamycin Chloroamphenicol W3110Δcrr/pECCG117-Ptrc-vioABCDE/pcc1BAC-Ptrc-crr(C9) Kanamycin Chloroamphenicol deoxyviolacein W3110/pECCG117-Ptrc-vioABCD*E kanamycin C9 kanamycin W3110Δcrr/pECCG117-Ptrc-vioABCD*E kanamycin W3110Δcrr/pECCG117-Ptrc-vioABCD*E/pcc1BAC-Ptrc-crr(C2) Kanamycin Chloroamphenicol W3110Δcrr/pECCG117-Ptrc-vioABCD*E/pcc1BAC-Ptrc-crr(C9) Kanamycin Chloroamphenicol

In order to confirm whether two crr mutations and crr deletion of SEQ ID NOs: 8 and 9 affect the production ability of violacein and deoxyviolacein, the strains of [Table 3] were transformed and produced (mutant strains C2, C9). Except), a total of 10 strains were subcultured on LB agar medium containing antibiotics for each strain.

In order to compare the violacein and deoxyviolacein production capacity of 10 subcultured strains, 1 platinum of each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of a titer medium containing an antibiotic suitable for each strain. and incubated with shaking at 200 rpm for 48 hours at 37 °C.

Potency medium composition

Glucose 40g/L, calcium carbonate 30g/L, ammonium sulfate 10g/L, potassium monophosphate 1g/L, magnesium sulfate 1.5g/L, yeast extract 4g/L

In order to quantify violacein and deoxyviolacein, the culture solution 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. Four strains with higher concentrations were finally selected, and the results are shown in [Table 4].

main product strain name OD
(600 nm absorption) violacein
(mg per liter of culture medium) deoxyviolacein
(mg per liter of culture medium) violacein W3110
/pECCG117-Ptrc-vioABCDE 32.2 436 52 C2 42.9 726 73 W3110Δcrr/pECCG117-Ptrc-vioABCDE 42.6 699 75 W3110Δcrr/pECCG117-Ptrc-vioABCDE
/pcc1BAC-Ptrc-crr(C2) 43.2 711 68 W3110Δcrr/pECCG117-Ptrc-vioABCDE
/pcc1BAC-Ptrc-crr(C9) 41.5 708 71 deoxyviolacein W3110
/pECCG117-Ptrc-vioABCD*E 32.4 0 471 C9 44.6 0 758 W3110Δcrr/pECCG117-Ptrc-vioABCD*E 43.2 0 782 W3110Δcrr/pECCG117-Ptrc-vioABCD*E
/pcc1BAC-Ptrc-crr(C2) 44.1 0 777 W3110Δcrr/pECCG117-Ptrc-vioABCD*E
/pcc1BAC-Ptrc-crr(C9) 43.3 0 761

As shown in [Table 4], in the case of the strains producing violacein, it was confirmed that all of the violacein-producing ability increased by about 1.6 times compared to the control W3110/pECCG117-Ptrc-vioABCDE, and crr of SEQ ID NOs: 8 and 9 It was confirmed that the violacein-producing strain containing two mutant strains and the strain completely deficient in crr showed the same fermentation pattern as the mutant C2.

In addition, in the case of the strains producing deoxyviolacein, as compared to the control group W3110/pECCG117-Ptrc-vioABCD*E, it was confirmed that the deoxyviolacein producing ability was increased by about 1.6 times as in the strains producing violacein. It was confirmed that the violacein-producing strain containing two crr mutants of SEQ ID NOs: 8 and 9 and the strain completely deficient in crr showed the same fermentation pattern as the mutant C9.

As a result, it was confirmed that the weakening or inactivation of crr is a factor that has an effective effect on the improvement of violacein or deoxyviolacein production capacity.

From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the claims described below, rather than the above detailed description, and equivalent concepts thereof, to be included in the scope of the present application.

<110> CJ CheilJedang Corporation <120> A microorganism producing compound and method for producing the compound using the same <130> KPA190157-KR <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggcgatatca tgaagcattc ttccgatat 29 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggcggatccc tagcgcttgg cggcgaaga 29 <210> 3 <211> 488 <212> DNA <213> Artificial Sequence <220> <223> trc <400> 3 ggtacccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc 60 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc 120 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 180 tgagcgcaac gcaattaatg taagttagcg cgaattgatc tggtttgaca gcttatcatc 240 gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct 300 gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata 360 atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca 420 attaatcatc cggctcgtat 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cgggctcaat gtcgacttcc ccgaggcgtg gagcggcgcg 780 cgctacggct cgctgccgct gttcaagggt ttcctcacct acggcgagcc atggtggctg 840 gactacaagc tggacgacca ggtgctgatc gtcgacaacc cgctgcgcaa gatctacttc 900 aagggcgaca agtacctgtt cttctacacc gacagcgaga tggccaatta ctggcgcggc 960 tgcgtggccg aaggagagga cggctacctg gagcagatcc gcacccatct ggccagcgcg 1020 ctgggcatcg ttcgcgagcg cattccccag cccctcgccc atgtgcacaa gtattgggcg 1080 catggcgtgg agttctgccg cgacagcgat atcgaccatc cgtccgcgct cagccaccgc 1140 gacagcggca tcatcgcctg ttcggacgcc tacaccgagc actgcggctg gatggagggc 1200 ggcctgctca gcgcccgcga agccagccgt ctgctgctgc agcgcatcgc cgcgtgaacg 1260 gtccggccgc cgcatcgcgt cgccgcccgg ttccgggcgg cgcttgtcag ccatgaccgt 1320 tcgggaaaca catgagcatt ctggattttc cacgcatcca tttccgcggc tgggcgcggg 1380 tcaacgcgcc caccgccaac cgcgatccgc acggccacat cgacatggcc agcaatacgg 1440 tggccatggc aggcgaaccg ttcgacctcg cgcgccatcc gaccgagttc caccgccacc 1500 tgcggtcgct ggggccgcgt ttcggcctgg acggccgggc tgacccggaa gggccgttca 1560 gcctggccga gggctacaac gcggccggca acaaccattt ctcctgggag agcgccaccg 1620 tcagccacgt gcagtgggat ggcggcgaag cggaccgcgg cgacggcctg gtcggcgcca 1680 ggctggcgct gtgggggcat tacaacgatt acctgcgcac caccttcaac cgcgcgcgct 1740 gggtggacag cgaccccacc cgccgcgacg cggcgcagat ctacgccggg cagttcacga 1800 tcagcccggc cggcgccgga ccgggcacgc cctggctgtt caccgccgac atcgacgaca 1860 gccacggcgc gcgctggacg cgcggcggcc acatcgccga gcgcggcggc catttcctgg 1920 acgaggagtt cggcctggcg cggctgttcc agttctcggt gcccaaagac catccgcact 1980 tcctgttcca cccggggcca ttcgattccg aagcctggcg caggctgcag ctggcgctgg 2040 aggacgacga cgtgctcggc ctgacggtgc agtacgcgct gttcaatatg tcgacgccgc 2100 cgcaacccaa ctcgccggtg ttccacgaca tggtcggcgt ggtcggcctg tggcggcgcg 2160 gcgaactggc cagctacccg gccggccggc tgctgcgtcc gcgccagccc gggctgggcg 2220 atctgacgct gcgcgtaagc ggcggccgcg tggcgctgaa tctggcctgc gccattccgt 2280 tctccacccg ggcggcgcag ccgtccgcgc cggacaggct gacgcccgat ctcggggcca 2340 agctgccgtt gggcgacctg ctgctgcgcg acgaggacgg cgcgttgctg gcgcgggtgc 2400 cgcaggcgct ttaccaggat tactggacga accacggcat cgtcgacctg ccgctgctgc 2460 gcgagcccag gggctcgctg acgctgtcca gcgagctggc cgaatggcgc gagcaggact 2520 gggtcacgca gtccgacgcc tccaatcttt atttggaagc gccggaccgc cgccacggcc 2580 gtttctttcc ggaaagcatc gcgctgcgca gctatttccg cggcgaggcc cgcgcgcgcc 2640 cggacattcc ccaccggatc gaggggatgg gtctggtcgg cgtggagtcg cgccaggacg 2700 gcgatgccgc cgaatggcgg ctgaccggcc tgcggcccgg cccggcgcgc atcgtgctcg 2760 acgacggcgc ggaggcgatc ccgctgcggg tgctgccgga cgactgggcg ttggacgacg 2820 cgacggtgga ggaggtcgat tacgccttcc tgtaccggca cgtgatggcc tattacgagc 2880 tggtctaccc gttcatgtcc gacaaggtgt tcagcctggc cgaccgctgc aagtgcgaga 2940 cctacgccag gctgatgtgg cagatgtgcg atccgcagaa ccggaacaag agctactaca 3000 tgcccagcac ccgcgagctg tcggcgccca aggccaggct gttcctcaaa tacctggccc 3060 atgtcgaggg ccaggccagg ctgcaggcgc cgccgccggc cgggccggcg cgcatcgaga 3120 gcaaggccca gctggcggcc gagctgcgca aggcggtgga tctggagttg tcggtgatgc 3180 tgcagtacct gtacgccgcc tattccattc ccaattacgc ccagggccag cagcgggtgc 3240 gcgacggcgc gtggacggcg gagcagctgc agctggcctg cggcagcggc gaccggcgcc 3300 gcgacggcgg catccgcgcc gcgctgctgg agatcgccca cgaggagatg atccattacc 3360 tggtggtcaa caacctgctg atggcgctgg gcgagccgtt ctacgccggc gtgccgctga 3420 tgggcgaggc ggcgcggcag gcgttcggcc tggacaccga attcgcgctg gagccgttct 3480 ccgagtcgac gctggcgcgc ttcgtccggc tggaatggcc gcacttcatc cctgcgccgg 3540 gcaaatccat cgccgactgc tacgccgcca tccgccaggc ctttctcgat ctgcccgacc 3600 tgttcggcgg cgaggccggc aagcgcggcg gcgagcacca cttgttcctc aacgagctga 3660 ccaaccgcgc ccatcccggc taccagctgg aggtgttcga tcgcgacagc gcgctgttcg 3720 gcatcgcctt cgtcaccgac cagggcgagg gcggggcgct ggactcgccg cattacgagc 3780 attcgcattt ccagcggctg cgggagatgt cggccaggat catggcgcag tccgcgccgt 3840 tcgagccggc gttgccggcg ctgcgcaacc cggtgctgga cgagtcgccg ggctgccagc 3900 gcgtggcgga cggacgggcg cgcgcgctga tggcgctgta ccagggcgtg tacgagctga 3960 tgttcgcgat gatggcgcag cacttcgcgg tcaagccgct gggcagcctc aggcgctcgc 4020 ggctgatgaa cgcggcgatc gacctgatga ccggcctgct caggccgctg tcctgcgcgc 4080 tgatgaacct gccgtcgggc atcgccggac gcaccgccgg gccgccgctg ccggggccgg 4140 tggatacccg cagctacgac gactacgcgc tgggctgccg gatgctggcg cggcgctgcg 4200 agcgcctgct ggagcaggcg tcgatgctgg agccgggctg gctgcccgac gcgcaaatgg 4260 aactgctgga tttctaccgc cggcagatgc tggatttggc ttgtggaaag ctttctagag 4320 aggcctgaaa tgaaaagagc aatcatagtc ggaggcgggc tcgccggcgg gctgaccgcc 4380 atctacctgg cgaagcgcgg ctacgaggtc cacgtggtgg aaaagcgcgg cgacccgctg 4440 cgggacctgt cttcctacgt ggatgtggtc agctcgcggg cgataggcgt cagcatgacc 4500 gtgcgcggca tcaagtcggt gctggcggcc ggcattccgc gcgcggagct ggacgcctgc 4560 ggcgaaccca tcgtggcgat ggcgttttcc gtcggcggcc agtaccggat gcgggagctc 4620 aagccgctgg aggatttccg cccgctgtcg ctgaaccgcg cggcgtttca gaagctgctg 4680 aacaagtacg ccaacctggc cggcgtccgc tactacttcg agcacaagtg cctggacgtg 4740 gatctggacg gcaagtcggt gctgatccag ggcaaggacg gccagccgca gcgcttgcag 4800 ggcgatatga tcatcggcgc cgacggcgcg cactcggcgg tgcggcaggc gatgcagagc 4860 gggttgcgcc gcttcgaatt ccagcagact ttcttccgcc acggctacaa gacgctggtg 4920 ctgccggacg cgcaggcgct gggctaccgc aaggacacgc tgtatttctt cggcatggac 4980 tccggcggcc tgttcgccgg ccgcgccgcc accatcccgg acggcagcgt cagcatcgcg 5040 gtctgcctgc cgtacagcgg cagccccagc ctgaccacca ccgacgagcc gacgatgcgc 5100 gcctttttcg accgttactt cggcggcctg ccgcgggacg cgcgcgacga gatgctgcgc 5160 cagttcctgg ccaagcccag caacgacctg atcaacgtcc gttccagcac cttccactac 5220 aagggcaatg tgctgctgct gggcgacgcc gccccacgcca ccgcgccttt cctcggccag 5280 ggcatgaaca tggcgctgga ggacgcgcgc accttcgtcg agctgctgga ccgccaccag 5340 ggcgaccagg acaaggcctt tcccgagttc accgagctgc gcaaggtgca ggccgacgcg 5400 atgcaggaca tggcgcgcgc caactacgac gtgctcagct gctccaatcc catcttcttc 5460 atgcgggccc gctacacccg ctacatgcat agcaagtttc ccggccttta cccgccggac 5520 atggcggaga agctgtactt cacgtccgag ccgtacgaca gactgcagca gatccagaga 5580 aaacagaacg tttggtacaa gatagggagg gtcaactgat gaagattctg gtcatcggcg 5640 cggggccggc cggcctggtg ttcgccagcc aactgaaaca ggcgcgtccg ctgtgggcga 5700 tagacatcgt cgaaaagaac gacgagcagg aagtgctggg ctggggcgtg gtgctgcccg 5760 gccggcccgg ccagcatccg gccaatccgc tgtcctacct ggacgcgccg gagaggctga 5820 atccgcagtt cctggaagac ttcaagctgg tccaccacaa cgagcccagc ctgatgagca 5880 ccggcgtgct gctgtgcggc gtggagcgcc gcggcctggt gcacgccttg cgcgacaagt 5940 gccgctcgca gggcatcgcc atccgcttcg aatcgccgct gctggagcat ggcgagctgc 6000 cgctggccga ctacgacctg gtggtgctgg ccaacggcgt caatcacaag accgcccact 6060 tcaccgaggc gctggtgccg caggtggact acggccgcaa caagtacatc tggtacggca 6120 ccagccagct gttcgaccag atgaacctgg tgttccgcac ccacggcaag gacattttca 6180 tcgcccacgc ctacaagtac tcggacacga tgagcacctt catcgtcgag tgcagcgagg 6240 agacctatgc ccgcgcccgc ctgggcgaga tgtcggaaga ggcgtcggcc gaatacgtcg 6300 ccaaggtgtt ccaggccgag ctgggcggcc acggcctggt gagccagccc ggcctcggct 6360 ggcgcaactt catgaccctg agccacgacc gctgccacga cggcaagctg gtgctgctgg 6420 gcgacgcgct gcagtccggc cacttctcca tcggccacgg caccacgatg gcggtggtgg 6480 tggcgcagct gctggtgaag gcgctgtgca ccgaggacgg cgtgccggcc gcgctgaagc 6540 gcttcgagga gcgcgcgctg ccgctggtcc agctgttccg cggccatgcc gacaacagcc 6600 gggtctggtt cgagacggtg gaggagcgca tgcacctgtc cagcgccgag ttcgtgcaga 6660 gcttcgacgc gcgccgcaag tcgctgccgc cgatgccgga agcgctggcg cagaacctgc 6720 gctacgcgct gcaacgctga ggaggccgca tggaaaaccg ggaaccgccg ctgctgccgg 6780 cgcgctggag cagcgcctat gtgtcgtact ggatccgat gctgccggat gaccagctga 6840 cgtccggcta ctgctggttc gactacgagc gcgacatctg tcggatagac ggcctgttca 6900 atccctggtc ggagcgcgac accggctacc ggctgtggat gtccgaggtc ggcaacgccg 6960 ccagcggccg cacctggaag cagaaggtgg cctatggccg cgagcggacc gccctgggcg 7020 agcagctgtg cgagcggccg ctggacgacg agaccggccc gttcgccgag ctgttcctgc 7080 cgcgcgacgt gctgcgccgg ctgggcgccc gccatatcgg ccgccgcgtg gtgctgggca 7140 gggaagccga cggctggcgc taccagcgtc cgggcaaggg gccgtccacg ttgtacctgg 7200 acgccgccag cggtacgccg ctgaggatgg tgaccgggga cgaggcgtcg cgcgcgtcgc 7260 tgcgcgattt ccccaacgtc agcgaggccg agattcccga cgccgtcttc gccgccaagc 7320 gctag 7325 <210> 5 <211> 373 <212> PRT <213> Chromobacterium violaceum <400> 5 Met Lys Ile Leu Val Ile Gly Ala Gly Pro Ala Gly Leu Val Phe Ala 1 5 10 15 Ser Gln Leu Lys Gln Ala Arg Pro Leu Trp Ala Ile Asp Ile Val Glu 20 25 30 Lys Asn Asp Glu Gln Glu Val Leu Gly Trp Gly Val Val Leu Pro Gly 35 40 45 Arg Pro Gly Gln His Pro Ala Asn Pro Leu Ser Tyr Leu Asp Ala Pro 50 55 60 Glu Arg Leu Asn Pro Gln Phe Leu Glu Asp Phe Lys Leu Val His His 65 70 75 80 Asn Glu Pro Ser Leu Met Ser Thr Gly Val Leu Leu Cys Gly Val Glu 85 90 95 Arg Arg Gly Leu Val His Ala Leu Arg Asp Lys Cys Arg Ser Gln Gly 100 105 110 Ile Ala Ile Arg Phe Glu Ser Pro Leu Leu Glu His Gly Glu Leu Pro 115 120 125 Leu Ala Asp Tyr Asp Leu Val Val Leu Ala Asn Gly Val Asn His Lys 130 135 140 Thr Ala His Phe Thr Glu Ala Leu Val Pro Gln Val Asp Tyr Gly Arg 145 150 155 160 Asn Lys Tyr Ile Trp Tyr Gly Thr Ser Gln Leu Phe Asp Gln Met Asn 165 170 175 Leu Val Phe Arg Thr His Gly Lys Asp Ile Phe Ile Ala His Ala Tyr 180 185 190 Lys Tyr Ser Asp Thr Met Ser Thr Phe Ile Val Glu Cys Ser Glu Glu 195 200 205 Thr Tyr Ala Arg Ala Arg Leu Gly Glu Met Ser Glu Glu Ala Ser Ala 210 215 220 Glu Tyr Val Ala Lys Val Phe Gln Ala Glu Leu Gly Gly His Gly Leu 225 230 235 240 Val Ser Gln Pro Gly Leu Gly Trp Arg Asn Phe Met Thr Leu Ser His 245 250 255 Asp Arg Cys His Asp Gly Lys Leu Val Leu Leu Gly Asp Ala Leu Gln 260 265 270 Ser Gly His Phe Ser Ile Gly His Gly Thr Thr Met Ala Val Val Val 275 280 285 Ala Gln Leu Leu Val Lys Ala Leu Cys Thr Glu Asp Gly Val Pro Ala 290 295 300 Ala Leu Lys Arg Phe Glu Glu Arg Ala Leu Pro Leu Val Gln Leu Phe 305 310 315 320 Arg Gly His Ala Asp Asn Ser Arg Val Trp Phe Glu Thr Val Glu Glu 325 330 335 Arg Met His Leu Ser Ser Ala Glu Phe Val Gln Ser Phe Asp Ala Arg 340 345 350 Arg Lys Ser Leu Pro Pro Met Pro Glu Ala Leu Ala Gln Asn Leu Arg 355 360 365 Tyr Ala Leu Gln Arg 370 <210> 6 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> VioD_Variant <400> 6 Met Lys Ile Leu Val Ile Gly Ala Gly Pro Ala Gly Leu Val Phe Ala 1 5 10 15 Ser Gln Leu Lys Gln Ala Arg Pro Leu Trp Ala Ile Asp Ile Val Glu 20 25 30 Lys Asn Asp Glu Gln Glu Val Leu Gly Trp Gly Val Val Leu Pro Gly 35 40 45 Arg Pro Gly Gln His Pro Ala Asn Pro Leu Ser Tyr Leu Asp Ala Pro 50 55 60 Glu Arg Leu Asn Pro Gln Phe Leu Glu Asp Phe Lys Leu Val His His 65 70 75 80 Asn Glu Pro Ser Leu Met Ser Thr Gly Val Leu Leu Cys Gly Val Glu 85 90 95 Arg Arg Gly Leu Val His Ala Leu Arg Asp Lys Cys Arg Ser Gln Gly 100 105 110 Ile Ala Ile Arg Phe Glu Ser Pro Leu Leu Glu His Gly Glu Leu Pro 115 120 125 Leu Ala Asp 130 <210> 7 <211> 510 <212> DNA <213> Escherichia coli <400> 7 atgggtttgt tcgataaact gaaatctctg gtttccgacg acaagaagga taccggaact 60 attgagatca ttgctccgct ctctggcgag atcgtcaata tcgaagacgt gccggatgtc 120 gtttttgcgg aaaaaatcgt tggtgatggt attgctatca aaccaacggg taacaaaatg 180 gtcgcgccag tagacggcac cattggtaaa atctttgaaa ccaaccacgc attctctatc 240 gaatctgata gcggcgttga actgttcgtc cacttcggta tcgacaccgt tgaactgaaa 300 ggcgaaggct tcaagcgtat tgctgaagaa ggtcagcgcg tgaaagttgg cgatactgtc 360 attgaatttg atctgccgct gctggaagag aaagccaagt ctaccctgac tccggttgtt 420 atctccaaca tggacgaaat caaagaactg atcaaactgt ccggtagcgt aaccgtgggt 480 gaaaccccgg ttatccgcat caagaagtaa 510 <210> 8 <211> 510 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli_C2_crr <400> 8 atgggtttgt tcgataaact gaaatctctg gtttccgacg actagaagga taccggaact 60 attgagatca ttgctccgct ctctggcgag atcgtcaata tcgaagacgt gccggatgtc 120 gtttttgcgg aaaaaatcgt tggtgatggt attgctatca aaccaacggg taacaaaatg 180 gtcgcgccag tagacggcac cattggtaaa atctttgaaa ccaaccacgc attctctatc 240 gaatctgata acggcgttga actgttcgtc cacttcggta tcgacaccgt tgaactgaaa 300 ggcgaaggct tcaagcgtat tgctgaagaa ggtcagcgcg tgaaagttgg cgatactgtc 360 attgaatttg atctgccgct gctggaagag aaagccaagt ctaccctgac tccggttgtt 420 atctccaaca tggacgaaat caaagaactg atcaaactgt ccggtagcgt aaccgtgggt 480 gaaaccccgg ttatccgcat caagaagtaa 510 <210> 9 <211> 511 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli_C9_crr <400> 9 atgggtttgt tacgataaac tgaaatctct ggtttccgac gacaagaagg ataccggaac 60 tattgagatc attgctccgc tctctggcga gatcgtcaat atcgaagacg tgccggatgt 120 cgtttttgcg gaaaaaatcg ttggtgatgg tattgctatc aaaccaacgg gtaacaaaat 180 ggtcgcgcca gtagacggca ccattggtaa aatctttgaa accaaccacg cattctctat 240 cgaatctgat agcggcgttg aactgttcgt ccacttcggt atcgacaccg ttgaactgaa 300 aggcgaaggc ttcaagcgta ttgctgaaga aggtcagcgc gtgaaagttg gcgatactgt 360 cattgaattt gatctgccgc tgctggaaga gaaagccaag tctaccctga ctccggttgt 420 tatctccaac atggacgaaa tcaaagaact gatcaaactg tccggtagcg taaccgtggg 480 tgaaaccccg gttatccgca tcaagaagta a 511 <210> 10 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 aatctgctaa tccacgagat gcggcccaat ttactgctta ggagaagatc taggtgacac 60 tatagaacgc g 71 <210> 11 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 aagcataaaa aaatggcgcc gatgggcgcc atttttcact gcggcaagaa tagtggatct 60 gatgggtacc 70 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ttctcatgct gaaggcaa 18 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tgttttgtgc tcagctca 18 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 caggatatca tgggtttgtt cgata 25 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 caggatatca tgggtttgtt acgata 26 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gaaggatcct tacttcttga tgcggat 27 <210> 17 <211> 494 <212> DNA <213> Artificial Sequence <220> <223> trc <400> 17 ctgggtaccc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg 60 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc 120 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg 180 cagtgagcgc aacgcaatta atgtaagtta gcgcgaattg atctggtttg acagcttatc 240 atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg 300 gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg 360 ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg 420 acaattaatc atccggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca 480 ggaaagatat cata 494 <210> 18 <211> 170 <212> PRT <213> Escherichia coli <400> 18 Met Gly Leu Phe Asp Lys Leu Lys Ser Leu Val Ser Asp Asp Lys Lys 1 5 10 15 Asp Thr Gly Thr Ile Glu Ile Ile Ala Pro Leu Ser Gly Glu Ile Val 20 25 30 Asn Ile Glu Asp Val Pro Asp Val Val Phe Ala Glu Lys Ile Val Gly 35 40 45 Asp Gly Ile Ala Ile Lys Pro Thr Gly Asn Lys Met Val Ala Pro Val 50 55 60 Asp Gly Thr Ile Gly Lys Ile Phe Glu Thr Asn His Ala Phe Ser Ile 65 70 75 80 Glu Ser Asp Ser Gly Val Glu Leu Phe Val His Phe Gly Ile Asp Thr 85 90 95 Val Glu Leu Lys Gly Glu Gly Phe Lys Arg Ile Ala Glu Glu Gly Gln 100 105 110 Arg Val Lys Val Gly Asp Thr Val Ile Glu Phe Asp Leu Pro Leu Leu 115 120 125 Glu Glu Lys Ala Lys Ser Thr Leu Thr Pro Val Val Ile Ser Asn Met 130 135 140 Asp Glu Ile Lys Glu Leu Ile Lys Leu Ser Gly Ser Val Thr Val Gly 145 150 155 160 Glu Thr Pro Val Ile Arg Ile Lys Lys *** 165 170

Claims (11)

delete A microorganism producing a compound represented by the following formula (2), wherein the microorganism is
(i) the Crr protein is inactivated,
(ii) vioA, vioB, vioC and vioE; or Escherichia coli into which vioA, vioB, vioC, vioD and vioE are introduced, microorganisms:
[Formula 2]

In Formula 2, X is H or OH.
The microorganism according to claim 2, wherein the Crr protein consists of SEQ ID NO: 18.
delete delete delete delete A method for producing a compound of Formula 2, comprising the step of culturing the microorganism of claim 2 or 3 in a medium:
[Formula 2]

In Formula 2, X is H or OH.
The method of claim 8 , wherein the method further comprises recovering the compound from the medium or microorganism.
in E. coli,
inactivating Crr protein; and
(i) vioA, vioB, vioC and vioE; or (ii) introducing vioA, vioB, vioC, vioD and vioE.
[Formula 2]

In Formula 2, X is H or OH.
A composition for producing a compound of Formula 2, comprising the microorganism of claim 2 or 3 or a culture thereof:
[Formula 2]

In Formula 2, X is H or OH.