KR102688631B1 - Microorganism comprising class I type BirA and biotin production method using the same - Google Patents

Abstract

This application relates to a microorganism containing class I type BirA or a biotin production method using the same. In one example, the microorganism has the activity of the endogenous class II type BirA weakened and contains class I type BirA to produce biotin. The ability may be increased.

Description

Microorganism comprising class I type BirA and biotin production method using the same {Microorganism comprising class I type BirA and biotin production method using the same}

This application relates to a microorganism containing class I type BirA or a method for producing biotin using the same.

Biotin belongs to the vitamin B group (Vitamin B7), which plays an important role in cell growth, protein production, and activity, and is an essential nutrient for animals, plants, and microorganisms. Biotin, which has a structure in which one ring structure containing sulfur is connected to another ring structure, functions as a coenzyme for multi-carboxylase and plays an important role in glucose metabolism as well as amino acid and fatty acid metabolism.

Biotin can be biosynthesized by many microbial species, but most animals cannot synthesize it on their own, so it is classified as an essential vitamin and is very useful, as it is used as a food or feed additive, or as a raw material for the synthesis and production of other medicines. am. In particular, the demand for vitamins has recently increased due to expanded antibiotic regulations, and sales prices are gradually rising. However, most biotin sold on the market is produced only through multi-step chemical methods, and with the advent of the environmentally friendly era, the demand for biological production technology is increasing. However, biological production technology appears to have had little advanced literature or patents since the 2000s, and production efficiency is not high. Therefore, it is important to secure multiple biological production technologies for biotin.

Therefore, under this background, research to increase biotin production capacity is still needed.

One object of the present application is to provide a microorganism of Escherichia sp. or Serratia sp., which contains BirA of class I type and wherein the activity of BirA of class II type is attenuated. , the microorganism may have the ability to produce biotin.

Another object of the present application is to provide a composition for producing biotin containing the above microorganisms.

Another object of the present application is to provide a method for producing biotin, which includes culturing the microorganism in a medium.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may also 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. Additionally, the scope of the present application cannot be considered limited by the specific description described below. Additionally, numerous papers and patent documents are referenced and citations are indicated throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present invention and the level of technical field to which the present invention pertains.

In this application, the term "strain (or microorganism)" includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and can be caused by insertion of foreign genes or enhanced or inactivated activity of intrinsic genes. It is a microorganism whose specific mechanism is weakened or strengthened, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product.

In the present application, “vector” refers to a vector encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) to enable expression of the polypeptide of interest (e.g., class I type BirA) in a suitable host. The expression control region may include a DNA preparation containing a polynucleotide base sequence, a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, and a sequence encoding a suitable mRNA ribosome binding site. and sequences that regulate the termination of transcription and translation. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome and can be integrated into the genome itself.

The vector used in this application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, and pUC-based plasmid vectors can be used. , pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome may be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome has been inserted. The selection marker is used to select cells transformed with a vector, that is, to confirm the insertion of the target nucleic acid molecule, and to display selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides. Markers that provide may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other expression traits, so transformed cells can be selected.

In this application, the term “transformation” refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoding the polynucleotide can be expressed within the host cell. As long as the transformed polynucleotide can be expressed in the host cell, it can include both of these, regardless of whether it is inserted into the chromosome of the host cell or located outside the chromosome. Additionally, the polynucleotide includes DNA and/or RNA encoding the polypeptide of interest. The polynucleotide can be introduced in any form as long as it can be introduced and expressed into a host cell. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for self-expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term "operably linked" as used herein means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide of the subject of the present application (e.g., class I type BirA). It means that there is.

Hereinafter, this application will be described in detail.

One aspect of the present application for achieving the above object may provide a microorganism of the genus Escherichia ( Escherichia sp.) or the genus Serratia ( Serratia sp.) containing BirA of class I type. The microorganism may have a weakened activity of class II type BirA.

In this application, “class II type BirA” refers to biotin protein ligase (BPL), which contains a DNA binding domain at the N terminal and thus has the function of regulating the transcription of biotin biosynthetic genes and transport genes. can do. In the present application, the sequence of the class II type BirA can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be AP009048.1, or NP418404.1. In one example, the class II type BirA may be encoded by a birA gene derived from a microorganism of the genus Escherichia or Serratia.

In the present application, “BirA of class I type” may refer to a protein that does not contain a regulatory domain and has only the function of biotin ligase. In the present application, the sequence of BirA of the class I type can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be NZ_CP025534.1 or NP599941.1, etc. In one example, the class I type BirA may be encoded by a birA gene derived from a microorganism of the genus Mycobacterium or Corynebacterium.

In this application, the term “weakening” of a polypeptide is a concept that includes both reduced activity or no activity compared to the intrinsic activity. The attenuation may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

The weakening occurs when the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to mutation of the polynucleotide encoding the polypeptide, inhibition of expression of the gene of the polynucleotide encoding the polypeptide, or translation into a polypeptide. When the overall polypeptide activity level and/or concentration (expression amount) in the cell is lower than that of the natural strain due to (translation) inhibition, etc., when the polynucleotide is not expressed at all, and/or when the polynucleotide expression is low Even if there is no activity of the polypeptide, it may also be included. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain, wild type, or unmodified microorganism before the change in trait when the trait changes due to genetic mutation caused by natural or artificial factors. This can be used interchangeably with “activity before modification.” The fact that the activity of a polypeptide is “inactivated, depleted, reduced, down-regulated, lowered, or attenuated” compared to the intrinsic activity means that the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the transformation was lowered.

Attenuation of the activity of such a polypeptide may be performed by any method known in the art, but is not limited thereto, and may be achieved by applying various methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. 2014;15(2):2773-2793, Molecular Cloning 2012, etc.

Specifically, the attenuation of the polypeptide of the present application is

1) Deletion of all or part of the gene encoding the polypeptide;

2) Modification of the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide;

3) modification of the amino acid sequence constituting the polypeptide (e.g., deletion/substitution/addition of one or more amino acids on the amino acid sequence) such that the activity of the polypeptide is eliminated or weakened;

4) Modification of the gene sequence encoding the polypeptide to eliminate or weaken the activity of the polypeptide (e.g., one or more nucleotide bases on the nucleotide sequence of the polypeptide gene to encode a polypeptide modified to eliminate or weaken the activity of the polypeptide) deletion/substitution/addition of);

5) Modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide;

6) Introduction of an antisense oligonucleotide (eg, antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide;

7) Addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure to which ribosome attachment is impossible;

8) Addition of a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

9) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited.

for example,

1) Deletion of part or all of the gene encoding the polypeptide may be removal of the entire polynucleotide encoding the target polypeptide endogenous in the chromosome, replacement with a polynucleotide with some nucleotides deleted, or replacement with a marker gene.

In addition, the above 2) modification of the expression control region (or expression control sequence) is a mutation in the expression control region (or expression control sequence) caused by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or a weaker mutation. It may be replacement with an active sequence. 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 that regulates the termination of transcription and translation.

In addition, 3) the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another start codon with a lower polypeptide expression rate compared to the internal start codon. It may be a substitution by sequence, but is not limited thereto.

In addition, the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) includes deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide. Alternatively, a combination thereof may result in a mutation in the sequence, or a 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. For example, gene expression can be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon, but is not limited thereto.

6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide is described, for example, in Weintraub, H. et al., Antisense-RNA as a molecular tool. for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

7) In order to form a secondary structure to which ribosomes cannot attach, the addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide is carried out through mRNA translation. This may make it impossible or slow it down.

8) The addition of a promoter transcribed in the opposite direction (Reverse transcription engineering, RTE) to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide is an antisense complementary to the transcript of the gene encoding the polypeptide. It may be that the activity is weakened by creating nucleotides.

In one example, the microorganism may be one in which the endogenous birA gene has been deleted and the birA gene derived from a microorganism of the genus Mycobacterium or Corynebacterium has been introduced.

In one example, the microorganism is 7,8-diamino-pelargonic acid aminotransferase, biotin synthase, 8-amino-7-oxononanoate. One or more activities selected from the group consisting of synthase (8-amino-7-oxononanoate synthase), malonyl-ACP O-methyltransferase, and Dethiobiotin synthetase This may have been further strengthened. In one example, the bioABFCD operon (for example, the bioABFCD operon derived from E. coli) may be introduced. In one example, the 7,8-diamino-pelargonic acid aminotransferase, biotin synthase, 8-amino-7-oxononanoate synthase, malonyl-ACP O-methyltransferase, and dethiobiotin synthetase The other enzyme may be derived from a microorganism of the genus Serratia ( Serratia sp.; e.g., Serratia marcescens ) or Escherichia sp. (e.g., Escherichia coli ).

In this application, “biotin synthase (EC 2.8.1.6)” is an enzyme that catalyzes the conversion of dethiobiotin (DTB) to biotin, using a radical mechanism to convert dethiobiotin into thiol. It may refer to a SAM-dependent enzyme that thiolates and converts to biotin. The biotin synthase may be included regardless of its microbial origin as long as it is an enzyme having the above activity. The biotin synthase can be used interchangeably with BioB protein or BioB.

In this application, “7,8-diamino-pelargonic acid aminotransferase” refers to 8-amino-7-oxononanoate synthase (8-amino-7- By combining 8-amino-7-oxononanoate (7-Keto-8-aminopelargonic acid; KAPA) produced by the activity of oxononanoate synthase with an alpha-amino group obtained from a specific enzyme-specific substance with an amine group such as SAM or lysine, 7 , It may refer to a protein that produces 8-diamino-pelargonic acid (DAPA).

In this application, “8-amino-7-oxononanoate synthase” catalyzes the decarboxylation condensation reaction of 6-carboxyhexanoyl-CoA (Pimeloyl-coA/ACP) and alanine. It can refer to a protein that

In this application, “Malonyl-ACP O-methyltransferase (Malonyl-[acyl-carrier protein] O-methyltransferase)” converts carbon polymers in the fatty acid biosynthesis pathway into pimeloyl-coA/ACP. This enzyme can refer to a protein that methylates the carboxy group of malonyl thioester.

In this application, “Dethiobiotin synthetase” may refer to an enzyme that converts dethiobiotin, a biotin production precursor, from 7,8-diamino-pelargonic acid (DAPA).

In this application, the term “enhancement” of polypeptide (protein) activity means that the activity of the polypeptide is increased compared to the intrinsic activity. The enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase may include showing an activity that it did not originally have, or showing improved activity compared to the intrinsic activity or activity before modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the change in trait when the trait changes due to genetic mutation caused by natural or artificial factors. This can be used interchangeably with “activity before modification.” “Enhanced,” “upregulated,” “overexpressed,” or “increased” in the activity of a polypeptide compared to its intrinsic activity means the activity and/or concentration (expression) of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the transformation. It means improvement compared to the amount).

The enhancement can be achieved by introducing an exogenous polypeptide or enhancing the activity and/or concentration (expression amount) of the endogenous polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed by increasing the activity level of the polypeptide, the expression level, or the amount of product released from the polypeptide.

Enhancement of the activity of the polypeptide can be done by applying various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to that of the microorganism before modification. Specifically, genetic engineering and/or protein engineering well known to those skilled in the art, which are routine methods of molecular biology, may be used, but are not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology 2010, Vol. 2. 1-16, Molecular Cloning 2012, etc.

Specifically, the reinforcement of the polypeptide of the present application is

1) Increased intracellular copy number of the polynucleotide encoding the polypeptide;

2) Replacement of the gene expression control region on the chromosome encoding the polypeptide with a highly active sequence;

3) Modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide;

4) modifying the amino acid sequence of the polypeptide to enhance its activity;

5) modification of the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide (e.g., modification of the polynucleotide sequence of the polypeptide gene to encode a polypeptide modified to enhance the activity of the polypeptide);

6) Introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

7) Codon optimization of the polynucleotide encoding the polypeptide;

8) Analyzing the tertiary structure of the polypeptide, select exposed sites and modify or chemically modify them; or

9) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited.

More specifically,

Above 1) the increase in the intracellular copy number of the polynucleotide encoding the polypeptide is achieved by the introduction into the host cell of a vector capable of replicating and functioning independently of the host to which the polynucleotide encoding the polypeptide is operably linked. It may be possible. Alternatively, this may be achieved by introducing one or two or more copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. The introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome of the host cell into the host cell, but is not limited to this. The vector is the same as described above.

2) Replacement of the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a highly active sequence, for example, by deletion, insertion, non-conservative or Sequence variation may occur due to conservative substitution or a combination thereof, or replacement may occur with a sequence with stronger activity. The expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but the method is not limited thereto.

Examples of known strong promoters include CJ1 to CJ7 promoters (US Patent US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13. (sm3) promoter (US Patent US 10584338 B2), O2 promoter (US Patent US 10273491 B2), tkt promoter, yccA promoter, etc., but is not limited thereto.

3) The base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base sequence encoding another start codon with a higher polypeptide expression rate than the internal start codon. It may be a substitution, but is not limited thereto.

The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) includes deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide. The combination of these may result in a mutation in the sequence, or a replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity, or an amino acid sequence or polynucleotide sequence improved to increase activity, but is not limited thereto. The replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may additionally include a selection marker to check whether chromosome insertion has occurred. The selection marker is as described above.

6) Introduction of a foreign polynucleotide showing the activity of the polypeptide may be introduction into the host cell of a foreign polynucleotide encoding a polypeptide showing the same/similar activity as the polypeptide. There are no restrictions on the origin or sequence of the foreign polynucleotide as long as it exhibits the same/similar activity as the polypeptide. The method used for the introduction can be performed by appropriately selecting a known transformation method by a person skilled in the art, and by expressing the introduced polynucleotide in the host cell, a polypeptide can be produced and its activity can be increased.

7) Codon optimization of the polynucleotide encoding the polypeptide is codon optimization of the native polynucleotide to increase transcription or translation within the host cell, or optimized transcription and translation of the foreign polynucleotide within the host cell. It may be that the codons have been optimized to allow this.

8) Analyzing the tertiary structure of a polypeptide and selecting exposed sites to modify or chemically modify the sequence information, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins to determine the degree of sequence similarity. Accordingly, a template protein candidate may be determined, the structure confirmed based on this, and an exposed site to be modified or chemically modified may be selected and modified or modified.

Such enhancement of polypeptide activity means that the activity or concentration of the corresponding polypeptide is increased based on the activity or concentration of the polypeptide expressed in the wild type or unmodified microbial strain, or the amount of the product produced from the polypeptide is increased. However, it is not limited to this.

Modification of part or all of the polynucleotide in the microorganism of the present application is (a) homologous recombination using a vector for chromosome insertion into the microorganism or genome editing using engineered nuclease (e.g., CRISPR-Cas9) and/or (b) It may be induced by, but is not limited to, light and/or chemical treatment, such as ultraviolet rays and radiation. The method of modifying part or all of the gene may include a method using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the gene of interest is injected into the microorganism to cause homologous recombination, thereby causing deletion of part or the entire gene. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

In one example, the strain of the present application may be a strain with biotin production ability.

The strain of the present application is a microorganism (parent strain) that naturally has the activity of class II type BirA, where class I type BirA or a polynucleotide encoding it (or a vector containing the polynucleotide) is introduced and/or class The activity of type II BirA is weakened, the biotin production ability is increased, and/or the microorganism may be endowed with biotin production ability, but is not limited thereto.

As an example, the microorganism (strain) of the present application is introduced (transformed) with a class I type BirA, a polynucleotide encoding it, and/or a vector containing the polynucleotide, and/or the activity of class II type BirA is introduced (transformed). It can contain all microorganisms that are weakened and capable of producing biotin. For example, the microorganism of the present application is a natural wild-type microorganism or a microorganism that produces biotin, where class I type BirA, a polynucleotide encoding it, and/or a vector containing the polynucleotide are introduced (transformed or expressed) ); And/or it may be a recombinant strain in which the activity of class II type BirA is weakened and biotin production ability is increased. The recombinant strain with increased biotin production ability may be a microorganism with increased biotin production ability compared to a natural wild-type microorganism or an unmodified microorganism (for example, a microorganism containing class II type BirA), but is not limited thereto. . For example, the unmodified microorganisms that are the target strains for comparing the increase in biotin production ability are wild-type E. coli (e.g., W3110 strain), E. coli overexpressing the E. coli-derived biotin operon, wild-type Serratia marcescens, and Serratia marce. It may be, but is not limited to, TA5027 (US 5374554 A; incorporated herein by reference in its entirety), a biotin-producing strain derived from Sense.

As an example, the recombinant strain with increased production capacity has a biotin production (or production capacity) of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more compared to the biotin production (or production capacity) of the parent strain or unmodified microorganism before mutation. 7% or more, about 8% or more, about 9% or more, about 10% or more, about 10.5% or more, about 11% or more, about 11.5% or more, about 12% or more, about 12.5% or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.5% or more, about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, about 17.5% or more, about 18% or more, about 18.5% or more, about 19% or more, about 19.5% or more, about 20% or more, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.5% or more, about 27% or more, about 27.5% or more, about 28% or more, about 28.5% or more, about 29% or more, about 29.5% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, or about 35% or more+ (the upper limit is a special limit) There is no, for example, about 500% or less, about 400% or less, about 300% or less, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, or may be about 35% or less). In another example, the recombinant strain with increased production (or production capacity) has a biotin production (or production capacity) of about 1.1 times more, about 1.12 times more, or about 1.13 times more than that of the parent strain or unmodified microorganism before mutation. , 1.15 times or more, 1.16 times or more, 1.17 times or more, 1.18 times or more, 1.19 times or more, about 1.2 times or more, 1.25 times or more, about 1.3 times or more, about 1.4 times or more, or about 1.5 times or more (the upper limit is a special limit) may be increased (for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less), but is not limited thereto. The term “about” is a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values in a range that are equivalent or similar to the value that appears after the term “about.” Not limited.

In this application, “unmodified microorganism” does not exclude strains containing mutations that may occur naturally in microorganisms, and is a wild-type strain or a natural strain itself, or whose characteristics change due to genetic mutation caused by natural or artificial factors. It may refer to a strain before becoming a strain. For example, the unmodified microorganism may have an attenuated class II type of BirA described herein and/or no class I type of BirA introduced, or a class II type of BirA attenuated and/or a class I type of BirA prior to introduction. It may mean a strain. The “non-modified microorganism” may be used interchangeably with “pre-transformed strain”, “pre-transformed microorganism”, “non-mutated strain”, “non-modified strain”, “non-mutated microorganism” or “reference microorganism”.

The microorganism may be a microorganism of the genus Escherichia ( Escherichia sp.), and the microorganism of the genus Escherichia includes Escherichia coli, Escherichia albertii, Escherichia faecalis, It may be one or more species selected from the group consisting of Escherichia fergusonii, Escherichia marmotae, Escherichia ruysiae, and Escherichia senegalensis. there is.

The microorganism may be a microorganism of the Serratia genus, and the microorganisms of the Serratia genus include Serratia marcescens , Serratia aquatilis, Serratia bockelmannii, and Serratia bozoiensis. (Serratia bozhouensis), Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia liquefaciens ), Serratia microhaemolytica, Serratia myotis, Serratia nematodiphila, Serratia oryzae, Serratia plymuthica It may be one or more types selected from the group consisting of etc.

The strains of the Corynebacterium genus include Corynebacterium glutamicum , Corynebacterium crudilactis , Corynebacterium deserti , and Corynebacterium eph. Corynebacterium efficiens , Corynebacterium callunae , Corynebacterium stationis , Corynebacterium singulare , Corynebacterium halotolerans ), Corynebacterium striatum , Corynebacterium ammoniagenes, Corynebacterium pollutisoli , Corynebacterium imitans , Corynebacterium It may be one or more species selected from the group consisting of Bacteria testudinoris ( Corynebacterium testudinoris ), Corynebacterium flavescens ( Corynebacterium flavescens ), etc.

The microorganisms in the Mycobacterium genus include Mycobacterium smegmatis , Mycobacterium albicans, Mycobacterium album, Mycobacterium alsense, and Mycobacterium albicans. Mycobacterium angelicum, Mycobacterium anthracenicu, Mycobacterium aquaticum, Mycobacterium aquiterrae, Mycobacterium attenuatum, Mycobacterium Mycobacterium arosiense, Mycobacterium leprae, Mycobacterium heckeshornense, Mycobacterium helveticum, Mycobacterium hyorhinis ), Mycobacterium innocens, Mycobacterium isoniacini, Mycobacterium jacuzzii, Mycobacterium kansasii, Mycobacterium marinum It may be one or more species selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium tuberculosis, etc.

Microorganisms according to one example include an exogenous class I type of BirA that does not contain a DNA binding site at the N-terminus and/or the activity of the endogenous class II type of BirA is weakened, or When cultured in biotin-free media, the growth rate is faster and/or biotin production ability (or production) is increased compared to the case containing class II type BirA with the N-terminal DNA bindig domain deleted. It could be.

Another aspect provides a composition for producing biotin comprising the microorganism.

In one example, the composition for production may further comprise any suitable excipients commonly used in compositions for biotin production, such as preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or It may be a topical topic, etc., but is not limited to this.

Another aspect includes attenuating class II type BirA and/or introducing (e.g., transforming) a class I type BirA, a polynucleotide encoding the same, and/or a recombinant vector containing the polynucleotide into a microorganism. It is possible to provide a method of increasing the biotin production ability of the microorganism or a method of imparting the biotin production ability to the microorganism, including.

Another aspect of the present application provides a method for producing biotin, comprising culturing the microorganism of the present application in a medium.

In the present application, “culturing” means growing the microorganism of the present application under appropriately controlled environmental conditions. The culture process of the present application can be carried out according to appropriate media and culture conditions known in the art. This culture process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culture may be batch, continuous, and/or fed-batch, but is not limited thereto.

In this application, “medium” refers to a material that is mainly mixed with nutrients necessary for cultivating the microorganisms of this application, and supplies nutrients and growth factors, including water, which are essential for survival and development. Specifically, the medium and other culture conditions used for cultivating the microorganisms of the present application can be any medium used for cultivating ordinary microorganisms without particular restrictions, but the microorganisms of the present application can be grown with an appropriate carbon source, nitrogen source, personnel, and inorganic substances. It can be cultured under aerobic conditions in a typical medium containing compounds, amino acids, and/or vitamins while controlling temperature, pH, etc. Specifically, culture media for microorganisms can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981)].

In the present application, the carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; Sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc.; Amino acids such as glutamic acid, methionine, lysine, etc. may be included. Additionally, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice bran, cassava, bagasse and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e. converted to reducing sugars). Carbohydrates such as molasses) can be used, and various other carbon sources in an appropriate amount can be used without limitation. These carbon sources may be used alone or in combination of two or more types, but are not limited thereto.

The nitrogen source includes inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used These nitrogen sources may be used individually or in combination of two or more types, but are not limited thereto.

The agent may include monopotassium phosphate, dipotassium phosphate, or the corresponding sodium-containing salt. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and/or appropriate precursors may be included. These components or precursors can be added to the medium batchwise or continuously. However, it is not limited to this.

In addition, during the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. Additionally, during culturing, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, to maintain the aerobic state of the medium, oxygen or oxygen-containing gas can be injected into the medium, or to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, and is limited thereto. That is not the case.

In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and culture can be performed for about 10 to 160 hours, but is not limited thereto.

Biotin produced by the culture of the present application may be secreted into the medium or remain within the cells.

The biotin production method of the present application includes preparing the microorganism of the present application, preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, cultivating the microorganism. It may be additionally included before the step.

The biotin production method of the present application may further include the step of recovering biotin from the culture medium (medium in which the culture was performed) or the microorganism. The recovering step may be additionally included after the culturing step.

The recovery may be to collect the desired biotin using a suitable method known in the art according to the microorganism culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity. Various chromatography such as chromatography, HPLC, or a combination of these methods can be used, and the desired biotin can be recovered from the medium or microorganism using a suitable method known in the art.

Additionally, the biotin production method of the present application may additionally include a purification step. The purification can be performed using a suitable method known in the art. In one example, when the biotin production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step are performed interchronously (or sequentially) regardless of the order, simultaneously, or integrated into one step. may be performed, but is not limited thereto.

In the method of the present application, the microorganisms, etc. of the present application are as described above.

In one example, the microorganism may have weakened activity of the endogenous class II type BirA and increased biotin production capacity, including class I type BirA.

Figure 1 shows the domains and origins of class I type and class II type BirA.
Figure 2 shows the results of measuring the growth rate of the control group and the strain of the present invention over time in M9 minimal medium without biotin or M9 medium with added biotin.

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

Example 1. Construction of E. coli birA deletion and foreign birA gene replacement vectors

In this example, the proteins encoded by birA from Mycobacterium smegmatis ( Mycobacterium smegmatis or M.sm) and Corynebacterium glutamicum (C.gl) (SEQ ID NOs: 3 and 4, respectively) We sought to determine the effect on the growth and biotin production of E. coli strains.

The birA gene (SEQ ID NO: 5) encoding the catalytic domain excluding the N-terminal DNA binding site (amino acid sequence 8 to 68) of E. coli BirA (SEQ ID NO: 1) and the foreign birA genes are respectively described. It was expressed using the known E. coli-derived cj1 promoter (Korean Patent Publication No. 10-2006-0068505; hereinafter referred to as Pcj1, SEQ ID NO: 9).

First, using the E. coli-derived cj1 promoter, an initiation codon (ATG) was inserted to express the remaining catalytic domain (Catalytic domain) lacking the N-terminal DNA binding site (N-terminal DNA bindig domain) in E. coli BirA. A vector was created to secure the birA gene. To construct the vector, a cj1 promoter fragment was obtained using E. coli W3110 as a template, birA upstream and downstream regions, and synthetic cj1 promoter DNA as a template. Specifically, through PCR, using E. coli W3110 chromosomal DNA as a template, primers VB7-1 (SEQ ID NO: 10) and VB7-2 (SEQ ID NO: 11) were used to purify about 0.5 kb of the upstream region (SEQ ID NO: 12). , a gene fragment of approximately 0.5 kb in the downstream region (SEQ ID NO: 15) was obtained using primers of VB7-3 (SEQ ID NO: 13) and VB7-4 (SEQ ID NO: 14), and the synthetic cj1 promoter DNA was used as a template. A cj1 promoter fragment (SEQ ID NO: 9) of about 0.3 kb was obtained using primers VB7-5 (SEQ ID NO: 16) and VB7-6 (SEQ ID NO: 17). SolgTM Pfu-X DNA polymerase was used as a polymerase for the PCR reaction, and the PCR amplification conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 60 seconds, and polymerization at 72°C for 30 seconds for 30 minutes. After repeating the process several times, polymerization was performed at 72°C for 5 minutes to obtain each fragment as a PCR product. The obtained PCR product was purified using QIAGEN's PCR Purification kit, and then a vector was constructed using the amplified upstream, downstream, and cj1 promoter fragments. According to the method described in PCT published patent WO2020032590A1, the R6K origin-based gene replacement vector pSKH containing the sacB gene was digested with EcoRⅤ restriction enzyme, and the prepared PCR product was subjected to Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method to create a vector pSKHΔbirADB(E.co)::Pci1_ATG capable of expressing the birA gene encoding BirA with a missing N-terminal DNA binding site. did. Cloning was performed by mixing Gibson assembly reagent and each gene fragment and storing them at 50°C for 1 hour.

In addition, in order to replace the E. coli birA gene with the foreign Class I birA genes described above, birA gene deletion and target gene insertion vectors were first constructed. To construct the vector, PCR was performed to obtain birA upstream and downstream regions using E. coli W3110 as a template. Specifically, using E. coli W3110 chromosomal DNA as a template, primers VB7_7 (SEQ ID NO: 18) and VB7_8 (SEQ ID NO: 19) were used to purify approximately 0.5 kb of the upstream region (SEQ ID NO: 20), VB7_9 (SEQ ID NO: 21), and VB7_10. A gene fragment of approximately 0.5 kb in the downstream region (SEQ ID NO: 23) was obtained through PCR using the primer (SEQ ID NO: 22). SolgTM Pfu-X DNA polymerase was used as the polymerase for the PCR reaction, and the PCR amplification conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 60 seconds, and polymerization at 72°C for 30 seconds for 30 minutes. After repeating the process several times, the polymerization reaction was performed at 72°C for 5 minutes to obtain the product. The obtained DNA product was purified using QIAGEN's PCR Purification kit, and then the amplified birA upstream and downstream fragments and the pSKH vector for chromosome transformation cut with EcoRⅤ restriction enzyme were used as Gibson assembly (DG Gibson et al., NATURE). Vector pSKHΔbirA (E.co) for birA gene deletion and target gene insertion was created by cloning using the (METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method. Next, based on the birA gene deletion vector pSKHΔbirA (E.co), a vector was constructed to insert the birA gene from Mycobacterium smegmatis and Corynebacterium glutamicum containing the known cj1 promoter. First, in order to secure the known cj1 promoter, a cj1 promoter fragment of about 0.3 kb was obtained through PCR using the synthetic cj1 promoter DNA as a template and primers VB7_11 (SEQ ID NO: 24) and VB7_12 (SEQ ID NO: 25). Using Mycobacterium smegmatis MC2155 chromosomal DNA as a template, a birA gene fragment of about 0.8 kb was obtained through PCR using primers VB7_13 (SEQ ID NO: 26) and VB7_14 (SEQ ID NO: 27) (SEQ ID NO: 7 ) Likewise, in order to secure the birA gene derived from Corynebacterium glutamicum containing the cj1 promoter, primers VB7_11 (SEQ ID NO: 24) and VB7_15 (SEQ ID NO: 28) were used using the synthetic cj1 promoter DNA as a template to produce about 0.3 kb of cj1. The promoter fragment was obtained through PCR, and a birA gene fragment of about 0.8 kb was generated using Corynebacterium glutamicum ATCC13032 chromosomal DNA as a template and primers VB7_16 (SEQ ID NO: 29) and VB7_17 (SEQ ID NO: 30). Obtained through PCR (SEQ ID NO: 8). The obtained DNA product was purified using QIAGEN's PCR Purification kit, and then recombined by cloning the cj1 promoter, each birA gene fragment, and the pSKHΔbirA (E.co) vector cut with ScaI restriction enzyme using the Gibson assembly method. Plasmids were obtained and named pSKHΔbirA(E.co)::Pcj1_birA(M.sm) and SKHΔbirA(E.co)::Pcj1_birA(C.gl), respectively.

Example 2. E. coli birA Defects and outpatients birA Development of gene-introduced strains

pSKHΔbirADB(E.co)::Pcj1_ATG, pSKHΔbirA(E.co)::Pcj1_birA(M.sm), and pSKHΔbirA(E.co)::Pcj1_birA(C.gl) obtained in Example 1 were each incubated with E. coli wild type W3110. After transformation of electro-competent cells by electroporation, CV04-0003 (W3110 ΔbirADB(E.co)::Pcj1_ATG), CV04-0004 (W3110 ΔbirA(E.co): :Pcj1_birA(M.sm)) and CV04-0002 (W3110 ΔbirA(E.co)::Pcj1_birA(C.gl)) were obtained, respectively. In the CV04-0003 strain, the birA gene was modified to encode BirA with a disrupted N-terminal DNA binding site on the E. coli chromosome, and in the CV04-0004 and CV04-0002 strains, the birA gene on the E. coli chromosome was modified for Mycobacterium smegmatis and Mycobacterium smegmatis, respectively. It is a strain substituted with the birA gene from Corynebacterium glutamicum. Through PCR and genome sequencing using primers VB7_18 (SEQ ID NO: 31) and VB7_19 (SEQ ID NO: 32), which can amplify the external regions of the homologous recombination upstream and downstream regions where the corresponding gene is inserted in each recombinant strain, respectively. The genetic manipulation was confirmed.

Example 3. Confirmation of growth rate of E. coli recombinant strain

To determine changes in growth rate due to deletion and substitution of the birA gene in wild-type E. coli, the growth rates of the wild-type E. coli strain and the recombinant strain were compared. To measure the growth curve of E. coli cells growing in the exponential growth phase, they were inoculated into M9 minimal medium supplemented with 0.3% glucose as a carbon source and incubated for over 15 hours (overnight). ) Primary culture was performed at 37°C and 200rpm. Cultured cells were inoculated into 25 ml of M9 minimal medium without biotin or with 0.2 mg/L of biotin added so that the initial absorbance (Optical density 562 nm, OD562) was 0.1 and incubated at 37°C for more than 15 hours (overnight). , while culturing at 200 rpm, the absorbance was measured in OD562, and the results are shown in Figure 2.

As shown in Figure 2, the Escherichia coli birA gene is better than the strain (CV04-0003) in which the wild-type birA gene of E. coli is replaced with the birA gene encoding BirA with a defect in the N-terminal DNA binding site, Mycobacterium smegmatis or CV04-0004 and CV04-0002 strains substituted with the birA gene derived from Corynebacterium glutamicum showed fast growth rates even when biotin was not added, and when biotin was added, all strains showed equivalent growth rates. In particular, strain CV04-0002 showed a high growth rate even when biotin was not added.

Since the growth rate of E. coli strains in M9 minimal medium without biotin is related to the production of biotin, the above results show that E. coli in which the birA gene was replaced with the birA gene derived from Mycobacterium smegmatis or Corynebacterium glutamicum The result of the accelerated growth rate of the strain shows that the activity of the BirA protein from Mycobacterium or Corynebacterium is more advantageous for biotin production than the activity of the BirA protein with a missing N-terminal DNA binding site in E. coli.

Example 4. Production of biotin biosynthesis enhanced strain based on birA substitution strain and evaluation of biotin production ability

To determine the effect of E. coli birA gene deletion and foreign birA gene introduction on biotin production ability, three recombinant strains prepared in Example 3, CV04-0003 (W3110 ΔbirADB(E.co)::Pcj1_ATG) and CV04-0004 ( Overexpression vectors containing the E. coli biotin operon were introduced into W3110 ΔbirA(E.co)::Pcj1_birA(M.sm)) and CV04-00025 (W3110 ΔbirA(E.co)::Pcj1_birA(C.gl)), respectively.

For the purpose of high biotin production, an overexpression vector was created based on the pCL1920 vector (GenBank No AB236930) using the genes bioABFCD and the autologous promoter of bioABFCD, which contains all of the E. coli biotin operon. To construct the vector, the bioABFCD operon gene was obtained by PCR using the E. coli W3110 chromosome as a template and the primer pair VB7_20 (SEQ ID NO: 33) and VB7_21 (SEQ ID NO: 34). SolgTM Pfu-X DNA polymerase was used as the polymerase for the PCR reaction, and the PCR amplification conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 180 seconds. After repeating 30 times, polymerization reaction was performed at 72°C for 10 minutes. As a result, a 5020bp wild-type E. coli bioABFCD operon gene fragment bioABFCD (Eco) (SEQ ID NO: 46) was obtained. The obtained DNA product was purified using QIAGEN's PCR Purification kit, and then cloned into the pCL1920 vector treated with SmaI restriction enzyme using the Gibson assembly method to obtain a recombinant plasmid, which was named pCL1920-bioABFCD (Eco).

The above-constructed biotin operon overexpression vector (pCL1920-bioABFCD(Eco)) was used in the three recombinant strains prepared in Example 3, CV04-0003 (W3110 ΔbirADB(E.co)::Pcj1_ATG), CV04-0004 (W3110 ΔbirA(E) .co)::Pcj1_birA(M.sm)) and CV04-0002 (W3110 ΔbirA(E.co)::Pcj1_birA(C.gl)), respectively, using the heat shock introduction method. As a control, an operon overexpression vector (pCL1920-bioABFCD (Eco)) was introduced into W3110.

Each strain was plated on LB solid medium and cultured overnight in an incubator at 30°C. The strain cultured overnight on LB solid medium was inoculated into 25 mL of titer medium with the composition shown in Table 1 below, then cultured in an incubator at 30°C and 200 rpm for 40 hours, and the biotin concentration in the culture medium was measured by MS-MS method. These results are shown in Table 2 below. Cell growth was expressed as OD by measuring absorbance at 562 nm, and residual sugar was measured using a sugar analyzer (YSI 2900) and expressed as the difference from the initial input amount.

Furtherance Concentration (g/L) glucose 30 KH ₂ PO ₄ 0.3 K ₂ HPO ₄ 0.6 yeast extract 2.5 (NH ₄ ) ₂ SO ₄ 15 MgSO ₄ ,7H ₂ O One _{FeSO4,7H2O ​} 0.030 NaCl 2.5 calcium carbonate 40

Strain name OD562nm Sugar consumed (g/L) Biotin (mg/L) E.coli W3110/pCL1920-bioABFCD (Eco) 36.5 30 0.0 E.coli CV04-0003/pCL1920-bioABFCD (Eco) 25.1 22.9 0.52 E.coli CV04-0004/pCL1920-bioABFCD (Eco) 35.1 30 1.08 E.coli CV04-0002/pCL1920-bioABFCD (Eco) 33.1 30 1.30

As shown in Table 2, E. coli CV04-0004 /pCL1920-bioABFCD (Eco) and E. coli CV04 in which the E. coli birA gene was replaced with birA from Mycobacterium smegmatis or Corynebacterium glutamicum The biotin production of the -0002/pCL1920-bioABFCD (Eco) strain was significantly increased compared to E. coli W3110/pCL1920-bioABFCD (Eco) and E. coli CV04-0003/pCL1920-bioABFCD (Eco).

Example 5. Serratia marcescens ( Serratia marcescens ) Production of birA substitution strain based on biotin biosynthesis enhanced strain and evaluation of biotin production ability

To investigate the effect of Class II birA deletion and Class I birA gene substitution other than E. coli on biotin production ability, based on the biotin-producing strain TA5027 (US Patent US 5374554 A) derived from Serratia marcescens, birA gene deletion and Class I birA gene deletion were investigated. A recombinant strain into which the I birA gene was introduced was constructed.

To construct a birA gene deletion and foreign birA gene insertion vector, a birA gene deletion and target gene insertion vector was constructed using the chromosome of strain TA5027 as a template in the same manner as in Example 2. Specifically, using Serratia TA5027 chromosomal DNA as a template, primers VB7_22 (SEQ ID NO: 35) and VB7_23 (SEQ ID NO: 36) were used to clone approximately 0.5 kb of the upstream region (SEQ ID NO: 37), VB7_24 (SEQ ID NO: 38), and A gene fragment of approximately 0.5 kb in the downstream region (SEQ ID NO: 40) was obtained through PCR using primers of VB7_25 (SEQ ID NO: 39). SolgTM Pfu-X DNA polymerase was used as the polymerase for the PCR reaction, and the PCR amplification conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 60 seconds, and polymerization at 72°C for 30 seconds for 30 minutes. After repeating the process several times, the polymerization reaction was performed at 72°C for 5 minutes to obtain the product. The obtained DNA product was purified using QIAGEN's PCR Purification kit, and then the amplified birA upstream and downstream fragments and the pSKH vector for chromosome transformation cut with EcoRⅤ restriction enzyme were used as Gibson assembly (DG Gibson et al., NATURE). Vector pSKHΔbirA (S.ma) for birA gene deletion and target gene insertion was created by cloning using the (METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method.

Next, based on the birA gene deletion vector pSKHΔbirA (S.ma), a vector was constructed to insert the birA gene derived from Mycobacterium smegmatis or Corynebacterium glutamicum containing the known cj1 promoter. In order to secure the known cj1 promoter, a cj1 promoter fragment of about 0.3 kb was obtained through PCR using the synthetic cj1 promoter DNA as a template and primers VB7_26 (SEQ ID NO: 41) and VB7_12 (SEQ ID NO: 25). Using Bacterium smegmatis MC ² 155 (http://www.lgcpromochem.com/atcc/) chromosomal DNA as a template, primers VB7_13 (SEQ ID NO: 26) and VB7_27 (SEQ ID NO: 42) were used to produce about 0.8 kb of DNA. The birA gene fragment was obtained through PCR. Similarly, in order to secure the birA gene derived from Corynebacterium glutamicum containing the cj1 promoter, primers VB7_26 (SEQ ID NO: 41) and VB7_15 (SEQ ID NO: 28) were used as a template using the synthetic cj1 promoter DNA, and about 0.3 kb of the cj1 promoter was generated. The fragment was obtained through PCR. Using Corynebacterium glutamicum ATCC13032 chromosomal DNA as a template, the birA gene fragment of about 0.8 kb was PCR-processed using primers VB7_16 (SEQ ID NO: 29) and VB7_28 (SEQ ID NO: 43). Obtained through practice. The obtained DNA product was purified using QIAGEN's PCR Purification kit, and then the cj1 promoter, each birA gene fragment, and the pSKHΔbirA (S.ma) vector cut with ScaI restriction enzyme were cloned using the Gibson assembly method to create a recombinant plasmid. were obtained, and were named pSKHΔbirA(S.ma)::Pcj1_birA(M.sm) and pSKHΔbirA(S.ma)::Pcj1_birA(C.gl), respectively.

The vector produced above was transformed into competent cells of the Serratia marcescens TA5027 strain using electroporation using the same method as in Example 3, and then the birA gene was transferred to the chromosome of the TA5027 strain through a second crossover process. Strains substituted with the birA gene from Mycobacterium smegmatis or Corynebacterium glutamicum were obtained, and these were designated CV04-9991 (TA5027 ΔbirA(S.ma)::Pcj1_birA(M.sm)), CV04-, respectively. It was named 9992 (TA5027 ΔbirA(S.ma)::Pcj1_birA(C.gl)). The genetic manipulation was performed through PCR and genome sequencing using primers VB7_29 (SEQ ID NO: 44) and VB7_30 (SEQ ID NO: 45), which can amplify the external regions of the homologous recombination upstream and downstream regions where the gene was inserted, respectively. Confirmed.

The two strains prepared above, CV04-9991 and CV04-9992, and the TA5027 strain to be used as a control were each plated on LB solid medium and cultured in an incubator at 30°C overnight. The strain cultured overnight on LB solid medium was inoculated into 25 ml of titer medium with the composition shown in Table 3 below, and then cultured for 40 hours in an incubator at 30°C and 200 rpm, and the biotin concentration in the culture medium was analyzed by MS-MS method. The results are shown in Table 4 below. Cell growth was expressed as OD by measuring absorbance at 562 nm, and residual sugar was measured using a sugar analyzer (YSI 2900) and expressed as the difference from the initial input amount.

Furtherance Concentration (g/L) glucose 30 KH ₂ PO ₄ 0.3 K ₂ HPO ₄ 0.6 yeast extract 2.5 (NH ₄ ) ₂ SO ₄ 15 MgSO ₄ ,7H ₂ O One FeSO ₄ ,7H ₂ O 0.030 NaCl 2.5 calcium carbonate 40

Strain name OD562nm Sugar consumed (g/L) Biotin (mg/L) TA5027 37.3 30.0 32.5 CV04-9991 25.1 22.9 35.8 CV04-9992 25.1 22.9 39.7

As shown in Table 4 above, the biotin production of CV04-9991 and CV04-9992 strains in which the birA gene of Serratia marcescens was replaced with the birA gene from Mycobacterium smegmatis or Corynebacterium glutamicum, respectively, was It was confirmed that it was significantly higher than the strain TA5027.

<110> CJ CheilJedang Corporation <120> Microorganism comprising class I type BirA and biotin production method using the same <130> DPP20210313KR <160> 46 <170> koPatentIn 3.0 <210> 1 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> BirA of Escherichia coli (255aa) <400> 1 Met Lys Asp Asn Thr Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu Phe His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser 20 25 30 Arg Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val 35 40 45 Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile 50 55 60 Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly Gly Ser 65 70 75 80 Val Ala Val Leu Pro Val Ile Asp Ser Thr Asn Gln Tyr Leu Leu Asp 85 90 95 Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala Cys Ile Ala Glu Tyr Gln 100 105 110 Gln Ala Gly Arg Gly Arg Arg Gly Arg Lys Trp Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu Ser Met Phe Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala Ile Gly Leu Ser Leu Val Ile Gly Ile Val Met Ala Glu Val 145 150 155 160 Leu Arg Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp 165 170 175 Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu Thr 180 185 190 Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly Ile Asn 195 200 205 Met Ala Met Arg Arg Val Glu Glu Ser Val Val Asn Gln Gly Trp Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp Arg Asn Thr Leu Ala Ala 225 230 235 240 Met Leu Ile Arg Glu Leu Arg Ala Ala Leu Glu Leu Phe Glu Gln 245 250 255 <210> 2 <211> 160 <212> PRT <213> Artificial Sequence <220> <223> BirA of Serratia marcescens (160aa) <400> 2 Met Arg Asp Thr Lys Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asp 1 5 10 15 Gly Glu Phe His Ser Gly Glu Gln Leu Gly Glu Ser Leu Gly Met Ser 20 25 30 Arg Ala Ala Ile Asn Lys His Ile Gln Thr Val Arg Glu Trp Gly Leu 35 40 45 Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Ala Pro Met 50 55 60 Gln Leu Leu Glu Ala Glu Arg Ile Leu Arg Gly Leu Asp Asp Lys Arg 65 70 75 80 Val Thr Val Leu Pro Val Val Asp Ser Thr Asn Gln Tyr Leu Leu Asp 85 90 95 Arg Ile Glu Thr Leu Gln Ser Gly Asp Ala Cys Ile Ala Glu Tyr Gln 100 105 110 Leu Ala Gly Arg Gly Arg Arg Gly Arg Gln Trp Ile Ser Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu Ser Met Phe Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala Met Gly Leu Ser Leu Val Ile Gly Met Val Met Ala Glu Val 145 150 155 160 <210> 3 <211> 286 <212> PRT <213> Artificial Sequence <220> <223> BirA of Corynebacterium glutamicum (286aa) <400> 3 Met Ser Pro Leu Lys Arg Ala Phe Arg Arg Asp Pro Thr Thr Leu Ala 1 5 10 15 Ser Met Asn Val Asp Ile Ser Arg Ser Arg Glu Pro Leu Asn Val Glu 20 25 30 Leu Leu Lys Glu Lys Leu Leu Gln Asn Gly Asp Phe Gly Gln Val Ile 35 40 45 Tyr Glu Lys Val Thr Gly Ser Thr Asn Ala Asp Leu Leu Ala Leu Ala 50 55 60 Gly Ser Gly Ala Pro Asn Trp Thr Val Lys Thr Val Glu Phe Gln Asp 65 70 75 80 His Ala Arg Gly Arg Leu Gly Arg Pro Trp Ser Ala Pro Glu Gly Ser 85 90 95 Gln Thr Ile Val Ser Val Leu Val Gln Leu Ser Ile Asp Gln Val Asp 100 105 110 Arg Ile Gly Thr Ile Pro Leu Ala Ala Gly Leu Ala Val Met Asp Ala 115 120 125 Leu Asn Asp Leu Gly Val Glu Gly Ala Gly Leu Lys Trp Pro Asn Asp 130 135 140 Val Gln Ile His Gly Lys Lys Leu Cys Gly Ile Leu Val Glu Ala Thr 145 150 155 160 Gly Phe Asp Ser Thr Pro Thr Val Val Ile Gly Trp Gly Thr Asn Ile 165 170 175 Ser Leu Thr Lys Glu Glu Leu Pro Val Pro His Ala Thr Ser Leu Ala 180 185 190 Leu Glu Gly Val Glu Val Asp Arg Thr Thr Phe Leu Ile Asn Met Leu 195 200 205 Thr His Leu His Thr Arg Leu Asp Gln Trp Gln Gly Pro Ser Val Asp 210 215 220 Trp Leu Asp Asp Tyr Arg Ala Val Cys Ser Ser Ile Gly Gln Asp Val 225 230 235 240 Arg Val Leu Leu Pro Gly Asp Lys Glu Leu Leu Gly Glu Ala Ile Gly 245 250 255 Val Ala Thr Gly Gly Glu Ile Arg Val Arg Asp Ala Ser Gly Thr Val 260 265 270 His Thr Leu Asn Ala Gly Glu Ile Thr His Leu Arg Leu Gln 275 280 285 <210> 4 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> BirA of Mycobacterium smegmatis (270aa) <400> 4 Met Asn Ser Asp Thr Glu Arg Pro Ala Leu Asp Ala Asp Ala Ile Arg 1 5 10 15 Ser Ala Val Val Arg Pro Arg Gly Ser Trp Arg Ser Phe Asp Val Val 20 25 30 Ala Glu Thr Gly Ser Thr Asn Ala Asp Leu Leu Ala Arg Ala Arg Ser 35 40 45 Gly Thr Asp Ile Asn Gly Ala Val Leu Ala Ala Glu His Gln Thr Ala 50 55 60 Gly Arg Gly Arg Asn Gly Arg Gln Trp Thr Thr Pro Pro Arg Ser Gln 65 70 75 80 Ile Ala Val Ser Val Gly Ile Asp Thr Thr Gly Val Pro Ser Thr Ala 85 90 95 Trp Gly Leu Leu Pro Leu Ala Thr Gly Val Ala Val Val Val Asp Ala Ile 100 105 110 Ser Ala Val Thr Gly Val Glu Ala Lys Leu Lys Trp Pro Asn Asp Val 115 120 125 Leu Val Asp Thr Gly Lys Leu Ala Gly Ile Leu Ala Glu Val Ala Ser 130 135 140 Pro Ala Pro Thr Val Val Ile Gly Thr Gly Leu Asn Val Ser Val Met 145 150 155 160 Pro Asp Asp Val Pro Asp Ser Val Ala Thr Ser Leu Ala Met Leu Thr 165 170 175 Asp Ala Pro Val Asp Arg Ser Ala Leu Leu Thr Glu Phe Leu Thr Arg 180 185 190 Leu Ala Asp Arg Val Glu Ser Trp Arg Ala Ala Gly Gly Ala Asp Asp 195 200 205 Arg Leu Leu Asp Asp Tyr Arg Gln Cys Ser Gly Thr Leu Gly Thr Ala 210 215 220 Val Arg Val Leu Leu Pro Gly Asp Arg Gln Leu Leu Gly Asp Ala Val 225 230 235 240 Asp Ile Asp Glu Ser Gly Arg Leu Leu Ile Asp Ser Glu Gly Glu Arg 245 250 255 Ile Thr Val Ala Ala Gly Asp Val Thr His Leu Arg Pro Ala 260 265 270 <210> 5 <211> 966 <212> DNA <213> Artificial Sequence <220> <223> birA gene of Escherichia coli (966bp) <400> 5 atgaaggata acaccgtgcc actgaaattg attgccctgt tagcgaacgg tgaatttcac 60 tctggcgagc agttgggtga aacgctggga atgagccggg cggctattaa taaacacatt 120 cagacactgc gtgactgggg cgttgatgtc tttaccgttc cgggtaaagg atacagcctg 180 cctgagcc ta tccagttact taatgctaaa cagatattgg gtcagctgga tggcggtagt 240 gtagccgtgc tgccagtgat tgactccacg aatcagtacc ttcttgatcg tatcggagag 300 cttaaatcgg gcgatgcttg cattgcagaa taccagcagg ctggccgtgg tcgccggggt 360 cggaa atggt tttcgccttt tggcgcaaac ttatatttgt cgatgttctg gcgtctggaa 420 caaggcccgg cggcggcgat tggtttaagt ctggttatcg gtatcgtgat ggcggaagta 480 ttacgcaagc tgggtgcaga taaagttcgt gttaaatggc ctaatgacct ctatctgcag 540 gatcgcaagc tggcaggcat tctggtggag ctgactggca aaactggcga tgcggcgcaa 600 atagtcat tg gagccgggat caacatggca atgcgccgtg ttgaagagag tgtcgttaat 660 caggggtgga tcacgctgca ggaagcgggg atcaatctcg atcgtaatac gttggcggcc 720 atgctaatac gtgaattacg tgctgcgttg gaactcttcg aacaagaagg attggcacct 780 ta tctgtcgc gctgggaaaa gctggataat tttattaatc gcccagtgaa acttatcatt 840 ggtgataaag aaatatttgg catttcacgc ggaatagaca aacagggggc tttattactt 900 gagcaggatg gaataataaa accctggatg ggcggtgaaa tatccctgcg tagtgcagaa 960 aaataa 966 <210> 6 <211> 998 <212> DNA <213> Artificial Sequence <220> <223> birA gene of Serratia marcescen s (wheren the CDS is 1~963bp) <400> 6 atgagagata ccaaggtccc gttaaaactg atcgcgctat tggccgacgg tgaattccat 60 tccggggaac agctcggtga gtcattgggc atgagccgtg ccgcgatcaa taagcatatt 120 caaaccgttc gcgagtgggg gctggatgtc tttactgtac cggggaaag atac agcttg 180 cctgctccca tgcagctatt ggaggcggaa cgcattctta gagggctgga cgacaagcga 240 gttaccgtgc tgccggtcgt agactctacc aatcaatatt tgctggatcg tatcgaaacg 300 ctgcaatctg gcgacgcctg catcgctgag taccaactgg t cgtcgtggg 360 cgtcagtgga tttcgccgtt tggcgccaat ttatacctgt cgatgttttg gcgattggaa 420 caggggccgg ctgcggcgat ggggctgagc ctggtgatcg gcatggtgat ggcggaggtc 480 ttgcagcgtc ttggcgctaa agacgtgcga gtcaaatggc ccaacgatct ttacctgaac 540 gatcgcaaat gat cttggtcgag ttgacgggga aaaccggcga tgcggctcag 600 ttggtcatcg gcgccggcat taatttggcg atgcgggata ccaatgcgag tgggatcaat 660 caaggttgga tcaacctgca agaagccggt attaacatcg atcgtaatga gctcaccgcc 720 actctgctta atga attgcg gcagtcatta aaacaattcg agatagatgg gctggcgcca 780 ttcattggcc gttggcgcac gctggataat tttatcgaca gaccggtcaa actgctgatt 840 ggcgagcgac agattgtggg tatcgcccgg ggtatcgacg cgcaaggtgc actcctgctg 900 gaacaagaag gggaaatcaa accctttatc ggcggagaga tatcgctgcg cagcgcggaa 960 taggctgcag gaattcgatt tcgctattgt agcc gtag 998 <210> 7 <211> 813 <212> DNA <213> Artificial Sequence <220> <223> birA gene of Mycobacterium smegmatis ( 813bp) <400> 7 atgaacagcg ataccgagcg cccggcgctc gatgccgacg ccatccgttc cgcagtggtg 60 cgaccgcgcg ggtcgtggcg cagtttcgat gtcgtcgcgg agaccgggtc gacgaatgcc 120 gacctgctgg cacgcgcgag atcgggtacc gacatcaacg gtgcggtgtt ggccgccgaa 180 caccagaccg cgggccgcgg tcgcaacggc agacagtgga ccacgccgcc gcgatcgcag 240 atcgcggtct cggtggggat cgacacgacc ggtgttccgt cgacagcgtg gggcctgctc 300 ccgctcgcga ccggcgtggc ggtggtcgac gccatctcgg cggtcaccgg ggtcgaagcg 360 aaactcaagt ggcccaacga tgttctggtc gacaccggca agctggccgg catcctggcc 420 gaggtcgcct cgcccgcacc gaccgtggtg atcggcaccg gcctgaacgt ctctgtcatg 480 ccggacgacg tgcccgattc ggtcgcgacg tcactggcga tgctgaccga cgcccccgtg 540 g atcgttcgg cactgctgac ggagttcctg acccgactgg ccgaccgggt cgaaagctgg 600 cgtgcggcag gcggagccga cgacaggctg ctcgacgact accggcagtg cagcggcacg 660 ctgggcaccg cggtccgggt gctgcttccg ggagatcggc aactgctggg c gacgcggtg 720 gacatcgacg aatccggaag actgctgatc gactccgagg gtgagcgcat caccgtggcc 780 gcaggcgacg tgacgcatct gcgccccgcg tga 813 <210> 8 <211> 861 <212> DNA <213> Artificial Sequence <220> <223> birA gene of Corynebacterium glutamicum (861bp) <400> 8 atgtcgccct taaagcgcgc ttttcgacgc gaccccacta cattggcttc catgaacgtt 60 gacattt cac gatccagaga gccgctaaac gttgagctcc tgaaggaaaa attgctccaa 120 aacggtgact ttggccaggt catttacgaa aaagtgacag gctccactaa tgctgacttg 180 ctggcacttg caggttctgg cgctccaaac tggacggtga aaactgtcga gtttcaagat 240 catgcgcgtg ggcgactcgg ccgcccgtgg tctgcccctg agggtt ccca aacaatcgtg 300 tctgtgctcg ttcaactatc tattgatcaa gtggaccgga ttggcactat tccactcgcg 360 gcgggactcg ctgtcatgga tgcgttgaat gacctcggtg tggaaggtgc cggactgaaa 420 tggcccaacg atgttcaaat ccacggcaag aa actctgcg gcatcctggt ggaagccacc 480 ggctttgatt ccaccccaac agttgtcatc ggttggggca ctaatatcag cctgactaaa 540 gaggagcttc ctgttcctca tgcaacttcc ctcgcattgg aaggtgttga agtcgacaga 600 accacattcc ttattaatat gctcacacat ctgcatactc gactggacca gtggcagggt 660 ccaagtgtgg attggctcga tgattaccgt gcggtatgtt ccagtattgg ccaagatgtt 720 cgagtgcttc tacctgggga taaagaactc ttaggtgaag cgatcggtgt cgcgactggc 780 ggagaaattc gtgttcgcga tgcttcgggc accgttcaca ccctcaacgc cggtgaaatt 840 acgcaccttc gcctgcagta a 861 <210> 9 <21 1> 304 <212> DNA <213> Artificial Sequence <220> <223> cj1 promoter <400> 9 caccgcgggc ttattccatt acatggaatg accaggaatg gcagggaatg cgacgaaatt 60 gactgtgtcg ggagcttctg atccgatgct gccaaccagg agagaaaata atgacatgtg 120 caggcacgct ggtgagctgg agatttatga tctcaagtac cttttttctt gcactcgagg 18 0 gggctgagtg ccagaatggt tgctgacacc aggttgaggt tggtacacac tcaccaatcc 240 tgccgtcgcg ggcgcctgcg tggaacataa accttgagtg aaacccaatc taggagatta 300 agat 304 <210> 10 <211> 35 <212> DNA < 213> Artificial Sequence <220> <223> primer_VB7_1 <400> 10 gctgcaggaa ttcgatttcg ctattgtagc cgtag 35 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_2 <400> 11 agccc gcggtgtggc acggtgttat ccttc 35 <210> 12 <211> 536 <212> DNA <213> Artificial Sequence <220> <223> birA upstream fragment <400> 12 ttcgctattg tagccgtagg tctgcgtctg ccaaaagagt ggcaacctgt actaacgtat 60 ggtgacttaa ct cgtctgga tcctacaaca gtaacgccac agcaagtatt taatgcggtg 120 tgtcatatgc gcaccaaccaa actccctgat ccaaaagtga atggcaatgc cggtagtttc 180 ttcaaaaacc ctgttgtatc tgccgaaacg gctaaagcat tactgtcaca atttccaaca 240 gcaccaaatt acccccaggc ggatggttca gtaaaactgg cagcaggttg gcttatcgat 300 cagtgccagc taaaagggat ggt ggggctgcgg tgcaccgtca acaggcgtta 360 gttctcatta atgaagacaa tgcaaaaagc gaagatgttg tacagctggc gcatcatgta 420 agacagaaag ttggtgaaaa atttaatgtc tggcttgagc ctgaagtccg ctttattggt 480 gcatcaggtg aagtgagcg c agtggagaca atttcatgaa ggataacacc gtgcca 536 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_3 <400> 13 tgaaggataa caccgtgcca caccgcgggc ttatt 35 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <220> < 223> primer_VB7_4 <400> 14 cattaagtaa ctggatcata tcttaatctc ctagat 36 <210> 15 <211> 560 <212> DNA <213> Artificial Sequence <220> <223> birA fragment downstream <400> 15 atccagttac ttaatgctaa acagatattg ggtcagctgg atggcggtag tgtagccgtg 60 ctgccagtga ttgactccac gaatcagtac cttcttgatc gtatcggaga gcttaaatcg 120 ggcgatgctt gcattgcaga ataccagcag gctggccgtg gtcgccgggg tcggaaatgg 180 ttttcgcctt ttggcgcaaa cttatatttg tcgatgttct ggcgtctgga cccg 240 gcggcggcga ttggtttaag tctggttatc ggtatcgtga tggcggaagt attacgcaag 300 ctgggtgcag ataaagttcg tgttaaatgg cctaatgacc tctatctgca ggatcgcaag 360 ctggcaggca ttctggtgga gctgactggc aaaactggcg cgca aatagtcatt 420 ggagccggga tcaacatggc aatgcgccgt gttgaagaga gtgtcgttaa tcaggggtgg 480 atcacgctgc aggaagcggg gatcaatctc gatcgtaata cgttggcggc catgctaata 540 cgtgaattac gtgctgcgtt 560 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_5 <400> 16 tgaaggataa caccgtgcca caccgc gggc ttatt 35 <210> 17 <211> 36 < 212> DNA <213> Artificial Sequence <220> <223> primer_VB7_6 <400> 17 cattaagtaa ctggatcata tcttaatctc ctagat 36 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_7 <400 > 18 ctgcaggaat tcgatttcgc tattgtagcc gtagg 35 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_8 <400> 19 ctgcactacg cagggtggca cggtgttatc cttca 35 <210> <211> 515 <212 > DNA <213> Artificial Sequence <220> <223> birA upstream fragment for exogenous insertion <400> 20 ttcgctattg tagccgtagg tctgcgtctg ccaaaagagt ggcaacctgt actaacgtat 60 ggtgacttaa ctcgtctgga tcctacaaca gtaacgccac agcaagtatt taatgcgg tg 120 tgtcatatgc gcaccaccaa actccctgat ccaaaagtga atggcaatgc cggtagtttc 180 ttcaaaaacc ctgttgtatc tgccgaaacg gctaaagcat tactgtcaca atttccaaca 240 gcaccaaatt acccccaggc ggatggttca gtaaaactgg cagcaggttg gcttatcgat 300 cagtgccagc taaaagggat gcaaataggt ggggctgcgg tgcaccgtca acaggcgtta 360 gttctcatta atgaagacaa tgcaaaaagc gaagatgttg tacagctggc gcatcatg ta 420 agacagaaag ttggtgaaaa atttaatgtc tggcttgagc ctgaagtccg ctttatggt 480 gcatcaggtg aagtgagcgc agtggagaca atttc 515 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_9 <400> 21 gataacaccg tgccaccctg cgtagtgcag aaaaa 35 <210> 22 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_10 <400> 22 gtcgactagc tatgca tcgcctggtg aagtt 35 <210> 23 <211> 512 <212> DNA <213> Artificial Sequence <220> <223> birA downstream fragment for exogenous insertion <400> 23 gaaaggggag tattcgctcc cctgcaaatt atttgcgtag tctgacctct tctaccgcat 60 gattagcact tttcgtcagg attaaactgg cg cgctcacg agtaggtaga atattttgct 120 ttaagttcag ccagttgatc tctttccaca atgtcatggc agtcttaatc gcttcttctt 180 tagttaattt cgcgtagtta tgaaaatagg aatccgggtc ggtaaaagcc ccttcgcgga 240 atttcagaaa acggttgata taccatgtct gaagtaagtc ttccggtgca tcaacatata 300 tcgaaaaatc gacaaaatca gaaac aaata catgatgtgg atcgtgtgga taatccatcc 360 cgctctgtaa gacatttaac ccttcaagaa ttaaaatatc aggctgaaca accgttttat 420 ctccatccgg gatcacatca taaataagat gtgagtaaac aggtgctgta acgtttggca 480 cgccggattt gagatcggaa ca cc 512 <210> 24 <211 > 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_11 <400> 24 gtcgactagc gtgatatgca tcgcctggtg aagtt 35 <210> 25 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_12 <400> 25 tcggtatcgc tgttcatatc ttaatctcct agat 34 <210> 26 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_13 <400> 26 atctaggaga ttaagatatg aacagcgata ccga 34 <210> 27 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_14 <400> 27 atctgcgccc tgcgtagtgc aga 33 < 210> 28 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_15 <400> 28 ctttaagggc gacatatctt aatctcctag attgg 35 <210> 29 <211> 35 <212> DNA <213> Artificial Sequence < 220> <223> primer_VB7_16 <400> 29 aatctaggag attaagatat gtcgccctta aagcg 35 <210> 30 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_17 <400> 30 tctgcactac gcagggttac t gcaggcgaa ggtgc 35 <210 > 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_18 <400> 31 gttgattctg ttgaactggc 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> < 223> primer_VB7_19 <400> 32 ggaacatcgt cgtgttgaac 20 <210> 33 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_20 <400> 33 gactctagag gatccccggg ttattggcaa aaaaatgttt 40 <210> 34 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_21 <400> 34 attcgagctc ggtacccggg ctacaacaag gcaaggttta 40 <210> 35 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7 _22 <400> 35 ggctgcagga attcgatctg ggtctgcgtc tgag 34 <210> 36 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_23 <400> 36 gtcttttctc aagtactgac agcaccacca cagcg 35 < 210> 37 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> birA upstream fragment of Serratia marcescens <400> 37 ctgggtctgc gtctgagcaa gcagtggcaa cccaagttga gctatggcga tctggccaag 60 ctggatcccg ccacggtgac gccacttcag gtattcgagt ccg tatgtgc catgcgccgc 120 agcaagctgc cggacccgcg cgaaaccggt aatgccggca gtttcttcaa gaatccgctg 180 gtgaacgcgg ggaaagccgc agaactcatc acacaatatc ccggcatgcc gcattatccg 240 cagcaggatg gtcaggtgaa gctggccgct ggctggctga tcgatcagtg cgaactgaag 300 gggtatcgca tcggtggcgc agccgtacac cgccagcagg cgctggtgct ggtgaatatc 360 gataatgcgc atagtcagga ct ctggcgcgcc atgtccgtaa gactgtggcc 420 gataaatttg gcgtatggct ggaacctgag gtgcgtttca ttggcgcaac tggcgaattg 480 aacgctgtgg tggtgctgtc 500 <210> 38 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_24 <400> 38 cgctgtggtg gtgctgtcag tacttgagaa aagac 35 <210> 39 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_25 <400> 39 aggtcgacta gcgtgatcca aacgtgtcac cgccc 35 <210> 40 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> birA downstream fragment of Serratia marcescens <400> 40 tgagaaaaga caaggggacg gcagcgtccc cttgatcgtt cggtaggctt attttcttaa 60 acgcacgctt tcaacggcat ggttggcgct tttggtcata atcaggctcg cgcgttcgcg 120 ggtaggcagt atgttctgct gcaaattcaa tccattgatt tcattccata attgcgtggc 180 gatattaatc gcctccggtt ccggcaattt tgaataatta tggaaataag aatcaggatt 240 ggaaaatgcg ccctgacgga atttcaggaa acggttgata taccagcttt gcaatagcgt 300 ctccggcgca tcgacatata tagagaagtc agtcg gaaacgaata cgcgatgcgg 360 atcgtgagga taatccatac cgctttgcaa tacgttaagc ccttccagga taagaatgtc 420 cggctgttcg atgactttat taccttcggg cacaacgtca tagatcaaat gggaataaac 480 cggggcggtg acacgtttgg 500 <210> 41 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_26 <400> 41 gtggtggtgc tgtcagtcac cgcgggctta ttcca 35 <210> 42 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_2 7 < 400> 42 ccttgtcttt tctcaagttc acgcggggcg cagat 35 <210> 43 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_28 <400> 43 cttgtctttt ctcaagttta ctgcaggcga aggtg 35 <210> 44 <211> 20 < 212> DNA <213> Artificial Sequence <220> <223> primer_VB7_29 <400> 44 atgggtactg aacgttatgt 20 <210> 45 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_30 <400> 45 tttcccaacc tgcgttaa 18 <210> 46 <211> 5016 <212> DNA <213> Artificial Sequence <220> <223> biotin operon of Escherichia coli (5016bp) <400> 46 gactctagag gatccccggg ttaacgcgcg gcgtcggcca cggcggcggt cagg cggctg 60 agctgttccg cttcgatgat gtaaggcggc atcaggtaaa tcagcttgcc gaacggccgg 120 atccataccc cgcgctcgac gaacccgcgc tgcagctcgg cgacatccac cggttcgcgc 180 atctccacca ccccaatcgc gcccagcacc cgcacgtcgg ccaccttcgg cagcgccgcc 240 aacggcaaca gttcccgctt caactgggtt ggcgc tcacctgcgc ctgccagcga 300 ttttccgcca gcagcgccag actggcgtcc gccacggcgc aggccagcgg attgcccata 360 aaggtcggcc cgtgcataaa gcagccggcc gcgccgttgc tgatggtctc cgccacgtgg 420 cgggtagtca a aagggtcata tagccgccgg tcagcgcctt gcccagacag 480 agaatgtccg gcaccacctg cgcgtgctcg caggcgaaca gcttgccggt gcggccgaaa 540 ccggtggcga tctcgtcggc gatcagcagc acctgatggc gatcgcacag ctcgcgcacc 600 cgcttgagat aggtcggatg gtagatgcgc atgccgccgg cgccttgcac caccggttcc 660 agaatcaccg ccgccacttc accggcgtgc tgctccag ca gcgcggcgaa cggcgcgata 720 tcctcttcac gccattcctc gtcgaagcgg cactgcggcg cggtggcgaa caggtgcggc 780 gccagatacc cctgatagag gctgtgcatc gagttgtccg gatcgcagac cgacatcgcg 840 ccgaaggtat cgccgtgata gccg tggcgc agcgtcagga tgcgctgccg gcgctcgccg 900 cgcgcctgcc agtactgcag cgccattttc agcgacactt ctaccgccac cgaaccagag 960 tccgccagga acacgcactg cagtgcttcc ggcgtcattt ccaccagccg acggcacaac 1020 gagatggcgg ccggatgggt aatgccgccg aacatcacgt gcgacatctt ctccaactgc 1080 tggctggcgg cctgattcag acgcggatgg ttgtaaccgt ggatcgccg c ccaccaggag 1140 gacatgccgt ccaccagact ccggccgtcc gccagctgca gttcgacgcc gctggccgat 1200 tcgatcgggt aacagggtaa cgggcggctc atggaggtgt aggggtgcca gatatggcgt 1260 tggtcaaacg ccaggtcgga agcggtgaca g acattgttg taaaccaaat tggattaaaa 1320 ttggttgaca gtatatccac aatatttaaa ctggcgacac tttttcgttt tggagacgcc 1380 atgatggccg accgcattca ctggacagta gggcaagccc aggccctgtt tgataaaccg 1440 ctgctggaac tgctgttcga agcgcaaacc gtacaccgcc agcacttcga cccgcgtcag 1500 gtgcaggtca gcacgctgct gtcgatcaag accggcgctt gcccggaaga ctgcaa atac 1560 tgcccgcaga gctcacgcta caagaccggc ctggagtcgg agcggctgat gcaggtcgag 1620 caggtgctgg aatcggcacg caaggccaag gcgaacggtt cgacccgttt ttgcatgggc 1680 gcggcgtgga agaacccgca cgagcgcgat atgcctta tc tgcagcaaat ggtgcagggc 1740 gtgaaagcga tgggcatgga aacctgcatg acgctgggca cattggatgg cacccaggcc 1800 gagcggctgg cggaggccgg gctggattac tacaaccata acctcgacac ctcgccggag 1860 ttctacggca gcatcatcac cacccgcagc taccaggagc gcctggatac gctcgacaag 1920 gtgcgcgacg ccggcatcaa agtgtgctcc ggcggcatcg tcgggctggg tgaaacggtg 1980 cgcgatc gcg ccgggctgct ggtgcagctg gccaacctgc caaaaccacc ggagagcgtg 2040 ccgatcaaca tgttggtgaa ggtgaaaggc acgccgctgg cggataacga tgacgtcgat 2100 ccgtttgatt tcatccgcac catcgcggtg gcgcgcatca tgatgccatc ttcttatgtc 2160 cgtctctccg caggccgcga acagatgaac gaacagacgc aggcgatgtg cttcatggcc 2220 ggcgccaact cgatcttcta cggttgcaag ctgctgacca cgccgaatcc ggaagaagac 2280 aaagacctgc agctgttccg caagctgggg ctcaacccgc agcagaccgc aaccgaacac 2340 ggcgacaacc agcaacagca ggtgctggcc aagcaactgc tgaacgccga taccgccgag 2400 ttttacaacg cggcgccgt g atgagctggc agcaacgcat cgagcaggcg ctggctgagc 2460 ggcgcctgaa cgccgcctac cgccggcgac agaccaccga gggcggcaac ggccgccaga 2520 tccggctcgg cgatcgtctc tatctgaact tctcgggcaa cgactacctg ggcttgagcc 2580 aggatg cgcg ggtgatcgcc gcctggcagc agggcgcgca gcgttacggc gtcggcagcg 2640 gcggttcggg ccacgtgacc ggttttagcg cggcgcatca ggcgctggaa gagcaactgg 2700 cggcttggct cggctatccg cgcgcgctgc tgttcatctc cggctacgcc gccaaccagg 2760 cggtgctggc ggcgttgatg caaaagggcg atcgcatttt ggccgatcgt ctcagccatg 2820 cctcgctgct ggaggcggcg gcgcagtcgc cggccgagct gcgccggttc cagcacaatc 2880 aaccgcaggc cttggcggat ctgctggcca aaccctgcga cgggcagcgg ctggcggtca 2940 ccgaaggggt gttcagcatg gatggcgacg gcgcgccgtt ggccatcgcttaa 3000 cccgtgcggc gggcgcctgg ctgatggtgg atgacgccca cggcatcggc gtgcgcggcg 3060 aacaaggccg cggcagttgc tggcagcagg gcgtgcgccc tgaactgctg gtggcgacct 3120 tcggcaaggc gttcggcgtc agcggcgcgg cggtgctgtg cgatgaggcg accgccgagt 3180 atctgctgca gttcgcccgc catctgatct acagcaccgc gatgccgccg gcgcaggcct 3240 gcgcgctgca ggcggcg ctg gcccgtattc gagagggtga tgatctgcga gcccggctgc 3300 aggacaacat tcggcgtttc cgtcagggcg cggcgccgtt ggcgctgacc ctgacggatt 3360 ccgacaccgc catccagccg ctgctggtgg gggataatca gcgcgcgctc gatctggcga 342 0 cccgcctgcg cgagtgcggc ctgtgggtga gcgccatccg tccgccgacg gtgcccccgg 3480 gcggcgcgcg gctgcgcatt accctgacgg cggcgcatca gtcgcaggat atcgatcgcc 3540 tgctggaggt gctgaatgac gtcagccaat gacacagtga acaaacaggc ggtcgcctcg 3600 gccttcagcc gcgcggccgg cagctacgat gccgccgccg cgctgcagcg tgacgttggc 3660 gagcgcttac tggggatggg gagttcccac ccgggcga ac agctgctgga cgccggctgc 3720 ggcaccggct atttcagccg tatgtggcgt gagcgcggca aacgggtgac cgcgctcgat 3780 ttggcgccgg gcatgctgga cgtcgcccgc caacggcagg cggcgcatca ttatctgctg 3840 ggcgatatcg tgcc gctgcccgat gcggcgatgg acatctgttt cagcagcctg 3900 gtggtgcagt ggtgcagcga tctgcctgcc gcgctggcag agctgtatcg cgtgacccgt 3960 cccggcggcg tgatcctgtt ttccacgctg gcggcgggct cgctgcagga attgggggac 4020 gcctggcaac aggtggacgg cgaacgtcac gtgaacgcct ttttgccgtt gacgcagatc 4080 cgcaccgcct gcgccgccta tcgacacgag ctggtgacgg a gttgcgcac cctgaactac 4140 ccggacgtga tgacgctgat gcgttcgctc aagggcatcg gggcgacgca tttgcatcag 4200 gggcgtgagg gcggcctgat gtcgcgtggc cgcctcgccg cgctgcaggc ggcttacccg 4260 tgccggcagg cagttccc gctcagctat catctggctt atggagtgat ttatcgtgag 4320 taaacgttgg ttcgtgaccg gcaccgacac cgaagtgggg aaaaccgtcg ccagcagcgc 4380 cttgctgcag gccgccaacc gggcgggcta ccgcagcgcc ggctataagc cggtggcctc 4440 cggcagcgag atgaccgccg aggggctgcg caacggcgac gcgctggcgc tgcaggccaa 4500 cagcggtgtg gcgctggatt acgacgaagt gaacccttac gtatttgccg aaccgacc tc 4560 gccgcatatc gtcagcgccg atgaaggccg gccgatcgac gcggcgcggc tgtccgacgg 4620 cctgcgccgg ctggagcagc gcgccgactg ggtgctggtc gagggggccg gcgggtggtt 4680 taccccgttg tcggcggagt acaccttcgc c gactgggtg cggcaagagc aactgccggt 4740 gatcctggtg gtgggcatca agctgggctg catcaaccac gcggtgctga cggcccaggc 4800 ggtgcaacag gccgggctga cgctggccgg ttggatcgcc aacgacgtgg cgccgccggg 4860 gcggcggcat caggaatacc tggctacgct gcgccgtatg ctgcccgcgc cgctgctggg 4920 cgaaatcccg cacctgccgc aggccgaacg ggcgccgctg gggcagtatc tggatatcag 498 0cttgctggcg cagtgacccg ggtaccgagc tcgaat 5016

Claims (12)

The activity of the class II type of intrinsic BirA is attenuated and includes the class I type of BirA,
BirA of the class I type is encoded by the birA gene derived from a microorganism of the genus Mycobacterium or Corynebacterium,
Escherichia sp. Microorganisms. The microorganism according to claim 1, wherein the endogenous birA gene is deleted and a birA gene derived from a microorganism of the genus Mycobacterium or Corynebacterium is introduced. The method of claim 1, wherein 7,8-diamino-pelargonic acid aminotransferase, biotin synthase, 8-amino-7-oxononanoate syntha At least one activity selected from the group consisting of 8-amino-7-oxononanoate synthase, malonyl-ACP O-methyltransferase, and Dethiobiotin synthetase Additional enhanced microorganisms. The microorganism according to claim 1, wherein the Escherichia genus microorganism is Escherichia coli. The microorganism according to claim 1, which has the ability to produce biotin. The activity of class II type endogenous BirA is attenuated, including class I type BirA,
BirA of the class I type is encoded by the birA gene derived from a microorganism of the genus Mycobacterium or Corynebacterium,
Serratia sp. Microorganisms. The microorganism according to claim 6, wherein the endogenous birA gene is deleted and a birA gene derived from a microorganism of the genus Mycobacterium or Corynebacterium is introduced. The method of claim 6, wherein 7,8-diamino-pelargonic acid aminotransferase, biotin synthase, 8-amino-7-oxononanoate syntha At least one activity selected from the group consisting of 8-amino-7-oxononanoate synthase, malonyl-ACP O-methyltransferase, and Dethiobiotin synthetase Additional enhanced microorganisms. The microorganism according to claim 6, wherein the microorganism of the Serratia genus is Serratia marcescens. The microorganism according to claim 6, which has the ability to produce biotin. A composition for producing biotin, comprising the microorganism of any one of claims 1 to 10. A method for producing biotin, comprising culturing the microorganism of any one of claims 1 to 10.