WO2023085875A1 - Microorganism comprising class i type bira and biotin production method using same - Google Patents

Abstract

The present application relates to a microorganism or a biotin production method using same, the microorganism having reduced activity of class II type, intrinsic BirA and comprising class I type BirA.

Description

Microorganisms containing BirA of class I type and biotin production method using the same

The present application relates to a microorganism containing BirA of class I type or a biotin production method using the same.

Biotin belongs to 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 sulfur-containing ring structure 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 themselves, so it is classified as an essential vitamin and is a very useful substance, such as being 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 is increasing due to the expansion of antibiotic regulations, and the selling price is gradually rising. Most of the biotin sold on the market is being produced only through multi-step chemical methods, so the demand for biological production technology is increasing in the era of environmental protection. However, the biological production technology seems to have little advanced literature or patents since the 2000s, and the production efficiency is not high. Therefore, it is important to secure a number of biotin biological production technologies.

Under this background, research for increasing biotin production capacity is still required.

One object of the present application is to provide a microorganism of the genus Escherichia or Serratia sp., in which the activity of BirA of class II type is attenuated and the activity of BirA of class I type is attenuated, , The microorganism may have a biotin-producing ability.

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

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

A detailed description of this is 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 various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below. In addition, a number of papers and patent documents are referenced throughout this specification and their citations are indicated. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention belongs and the contents of the present invention.

In this application, the term "strain (or microorganism)" includes both wild-type microorganisms and naturally or artificially genetically modified microorganisms, and causes such as insertion of foreign genes or enhancement or inactivation of endogenous gene activity. As a microorganism whose specific mechanism is attenuated or enhanced due to, it may be a microorganism containing genetic modification for the production of a desired polypeptide, protein or product.

In the present application, "vector" is an expression encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to express the target polypeptide (eg, class I type BirA) in a suitable host. The expression control region may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control the termination of transcription and translation After transformation into a suitable host cell, the vector can replicate or function independently of the host genome, and can integrate into the genome itself.

Vectors used in the present application are not particularly limited, and any vectors known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. 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 , pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, pET-based, etc. can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.

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

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

In addition, the term "operably linked" in the above means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the object polynucleotide (eg, class I type BirA) of the present application. means there is

Hereinafter, this application will be described in detail.

One aspect of the present application for achieving the above object provides a microorganism of the genus Escherichia or Serratia sp., including BirA of class I type. The microorganism may be one in which the activity of class II type BirA is weakened.

In this application, “class II type BirA” refers to biotin protein ligase (BPL) that contains a DNA binding domain at the N-terminal position and thus includes transcriptional control functions of biotin biosynthesis genes and transport genes. can do. In this application, the class II type of BirA can obtain its sequence from NCBI's GenBank, a known database, for example, GenBank Accession No. AP009048.1, or NP418404.1 or the like. In one example, the class II type of BirA may be encoded by a birA gene derived from a microorganism of the genus Escherichia or the genus Serratia.

In the present application, "class I type BirA" may refer to a protein having only the function of biotin ligase without including a regulatory domain. In this application, the class I type of BirA can obtain its sequence from NCBI's GenBank, 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 of BirA may be encoded by a birA gene derived from a microorganism of the genus Mycobacterium or the genus Corynebacterium.

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

The attenuation is 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 gene expression of the polynucleotide encoding it, or translation into a polypeptide. When the overall level and/or concentration (expression level) of the polypeptide in the cell is lower than that of the native strain due to inhibition of translation, etc., when the polynucleotide is not expressed at all, and/or when the polynucleotide is expressed 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 transformation when the character is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activation before transformation”. "Inactivation, depletion, reduction, downregulation, reduction, attenuation" of the activity of a polypeptide compared to its intrinsic activity means that it is lower than the activity of a specific polypeptide originally possessed by the parent strain or non-transformed microorganism before transformation.

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. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, attenuation of the polypeptide of the present application

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 (eg, deletion/substitution/addition of one or more amino acids on the amino acid sequence) such that the activity of the polypeptide is eliminated or attenuated;

4) modification of the sequence of a gene encoding a polypeptide such that the activity of the polypeptide is eliminated or attenuated (e.g., one or more nucleotides on the nucleotide sequence of the polypeptide gene to encode a modified polypeptide such that the activity of the polypeptide is eliminated or attenuated) deletion / substitution / addition of);

5) modification of the nucleotide 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 complementarily to the transcript of the gene encoding the polypeptide;

7) addition of a sequence complementary to the Shine-Dalgarno sequence at the front end of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot be attached to ribosomes;

8) addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) 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), but is not particularly limited thereto.

for example,

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

In addition, the above 2) modification of the expression control region (or expression control sequence) is a deletion, insertion, non-conservative or conservative substitution, or a combination thereof, resulting in mutations in the expression control region (or expression control sequence), or weaker It may be a 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 controlling termination of transcription and translation.

In addition, the 3) modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another initiation codon with a lower polypeptide expression rate than the endogenous initiation codon. It may be substituted with a sequence, but is not limited thereto.

In addition, the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above can be a 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. Or a combination thereof may be a sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, expression of a gene may be inhibited or attenuated by introducing a mutation in a polynucleotide sequence to form a stop codon, but is not limited thereto.

6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) 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) Addition of a sequence complementary to the Shine-Dalgarno sequence to the front end of the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure to which ribosomes cannot attach is mRNA translation may be disabled or slowed down.

8) Addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) is antisense complementary to the transcript of the gene encoding the polypeptide. It could be making nucleotides and attenuating their activity.

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

In one example, the microorganism is 7,8-diamino-pelargonic acid aminotransferase (7,8-diamino-pelargonic acid aminotransferase), biotin synthase (Biotin synthase), 8-amino-7-oxononanoate At least one activity selected from the group consisting of synthase (8-amino-7-oxononanoate synthase), malonyl-ACP O-methyltransferase, and dethiobiotin synthetase This may be further enhanced. In one example, the bioABFCD operon (eg, the bioABFCD operon derived from Escherichia coli) may be introduced. In one embodiment, the 7,8-diamino-pelargonic acid aminotransferase, biotin synthase, 8-amino-7-oxononanoate synthase, malonyl-ACP O-methyltransferase, and dethiobiotin synthase The taje may be derived from a microorganism of the genus Serratia sp.; eg Serratia marcescens ( Serratia marcescens ) or Escherichia sp.; eg Escherichia coli .

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

In this application, “7,8-diamino-pelargonic acid aminotransferase” refers to 8-amino-7-oxononanoate synthase (8-amino-7- oxononanoate synthase) to 8-amino-7-oxononanoate (7-Keto-8-aminopelargonic acid; KAPA) produced by binding an alpha-amino group obtained from an enzyme-specific substance with an amine group such as SAM or lysine to form 7 It may mean a protein that generates ,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 mean a protein that

In this application, “malonyl-ACP O-methyltransferase (Malonyl-ACP O-methyltransferase or Malonyl-[acyl-carrier protein] O-methyltransferase)” converts carbon polymers in the fatty acid biosynthetic pathway to pimeloyl-coA/ACP It can mean a protein that methyl esterifies the carboxy group of malonyl thioester.

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

As used herein, 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. Herein, activation, enhancement, upregulation, overexpression, and increase may include those that exhibit an activity that was not originally possessed, or those that exhibit enhanced activity compared to 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 transformation when the character is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activation before transformation”. "Enhancement", "upregulation", "overexpression" or "increase" of the activity of a polypeptide compared to its intrinsic activity means the activity and/or concentration (expression amount) is improved.

The enhancement can be achieved by introducing a foreign polypeptide or by enhancing the activity and/or concentration (expression level) of an endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from an increase in the activity level, expression level, or amount of a product released from the corresponding polypeptide.

Enhancement of the activity of the polypeptide can be applied by 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 the microorganism before transformation. Specifically, it may be using genetic engineering and / or protein engineering, which is well known to those skilled in the art, which is a routine method of molecular biology, but is not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, enrichment of the polypeptides of the present application

1) increase in intracellular copy number of a polynucleotide encoding a polypeptide;

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

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

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

5) modification of the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide (eg, modification of the polynucleotide sequence of the polypeptide gene to encode the modified polypeptide to enhance the activity of the polypeptide);

6) introduction of a foreign polypeptide that exhibits the activity of the polypeptide or a foreign polynucleotide encoding the same;

7) codon optimization of polynucleotides encoding the polypeptide;

8) Analysis of the tertiary structure of the polypeptide to 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), but is not particularly limited thereto.

More specifically,

1) The increase in the intracellular copy number of the polynucleotide encoding the polypeptide is achieved by introducing into the host cell a vector capable of replicating and functioning independently of the host, to which the polynucleotide encoding the corresponding polypeptide is operably linked. it may be Alternatively, it may be achieved by introducing one copy or two or more copies of a polynucleotide encoding the corresponding 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 thereto. The vector is as described above.

2) Replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a highly active sequence, for example, deletion, insertion, non-conservative or Conservative substitutions or combinations thereof may result in mutations in the sequence, or replacement with sequences having stronger activity. The expression control region, but is not particularly limited thereto, may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating termination of transcription and translation. For example, the original promoter may be replaced with a strong promoter, but is not limited thereto.

Examples of known strong promoters include the CJ1 to CJ7 promoter (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 are not limited thereto.

3) Modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a nucleotide sequence encoding another initiation codon with a higher polypeptide expression rate than the endogenous initiation codon. It may be substituted, but is not limited thereto.

Modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may include 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 thereof may be a sequence mutation, or 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 further include a selection marker for checking whether the chromosome is inserted. The selectable marker is as described above.

6) Introduction of a foreign polynucleotide exhibiting the activity of the polypeptide may be introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same/similar activity as the polypeptide into a host cell. The foreign polynucleotide is not limited in origin or sequence 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 expression of the introduced polynucleotide in a host cell can generate a polypeptide and increase its activity.

7) The codon optimization of the polynucleotide encoding the polypeptide is codon optimization of the endogenous polynucleotide to increase transcription or translation in the host cell, or optimization of the transcription or translation of the foreign polynucleotide in the host cell. It may be that the codons of this have been optimized.

8) Analyzing the tertiary structure of the polypeptide to select and modify or chemically modify the exposed site, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, depending on the degree of sequence similarity. It may be to determine a template protein candidate according to the method, confirm the structure based on this, and modify or modify an exposed portion to be chemically modified to be modified or modified.

Such enhancement of polypeptide activity is an increase in the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or unmodified microbial strain, or an increase in the amount of the product produced from the corresponding polypeptide. It may be, but is not limited thereto.

Modification of some or all of the polynucleotides in the microorganism of the present application is (a) genome editing using homologous recombination or genetic scissors (engineered nuclease, e.g., CRISPR-Cas9) using a vector for chromosomal insertion into the microorganism and / or (b) It may be induced by light and/or chemical treatment, such as ultraviolet light and radiation, but is not limited thereto. A method of modifying part or all of the gene may include a method using DNA recombination technology. For example, a part or all of a gene may be deleted by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene into the microorganism to cause homologous recombination. The injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

According to one embodiment, the strain of the present application may be a strain having biotin production ability.

In the strain of the present application, class I type BirA or a polynucleotide encoding the same (or a vector containing the polynucleotide) is introduced into a microorganism (parent strain) naturally having class II type BirA activity and/or a class The activity of type II BirA is attenuated, and the biotin-producing ability may be increased and/or the microorganism may be endowed with the biotin-producing ability, but is not limited thereto.

For example, the microorganism (strain) of the present application is a class I type of BirA, a polynucleotide encoding the same, and/or a vector containing the polynucleotide is introduced (transformed) and/or the activity of class II type BirA is It can include 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 biotin-producing microorganism in which class I type BirA, a polynucleotide encoding the same, and/or a vector containing the polynucleotide are introduced (transformed, or expressed). ); and/or a recombinant strain in which the activity of class II type BirA is attenuated and the biotin-producing ability is increased. The recombinant strain with increased biotin-producing ability may be a microorganism with increased biotin-producing ability compared to natural wild-type microorganisms or unmodified microorganisms (eg, class II type BirA-containing microorganisms), but is not limited thereto. . For example, non-transformed microorganisms, which are strains to be compared for an increase in the biotin-producing ability, are wild-type E. coli (e.g., W3110 strain), E. coli overexpressing the biotin operon derived from E. coli, wild-type Serratia marcescens, and Serratia marse It may be TA5027 (US 5374554 A; the entire document is incorporated herein by reference), a biotin-producing strain derived from sense, but is not limited thereto.

For example, the recombinant strain with increased production capacity is about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 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+ 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 about 35% or less) may be increased. In another example, the recombinant strain having increased production (or production capacity) has biotin production (or production capacity) about 1.1 times, about 1.12 times or more, or about 1.13 times or more, compared to the parent strain before mutation or unmodified microorganisms. , 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 There is no, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) may be increased, but is not limited thereto. The term “about” includes all ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all ranges equivalent to or similar to the ranges following the term “about”. Not limited.

In this application, "non-modified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, and is either a wild-type strain or a wild-type strain itself, or a change in character due to genetic mutation caused by natural or artificial factors. It may mean a strain before becoming. For example, the unmodified microorganism is attenuated class II type BirA and/or class I type BirA is not introduced or class II type BirA is attenuated and/or class I type BirA is introduced. It may mean the previous strain. The "unmodified microorganism" may be used interchangeably with "strain before transformation", "microorganism before transformation", "non-transformation strain", "non-transformation strain", "non-transformation microorganism" or "reference microorganism".

The microorganism may be a microorganism of the genus Escherichia sp., and the microorganism of the genus Escherichia may include Escherichia coli, Escherichia albertii , Escherichia faecalis , Escherichia Fergusonii ( Escherichia fergusonii ), Escherichia marmotae ( Escherichia marmotae ), Escherichia Louisiae ( Escherichia ruysiae ), Escherichia Senegalensis ( Escherichia senegalensis ), and the like. there is.

The microorganism may be a microorganism of the genus Serratia, and the microorganism of the genus Serratia includes Serratia marcescens , Serratia aquatilis , Serratia bockelmannii , and Serratia bozoensis. ( Serratia bozhouensis ), Serratia entomophila ( Serratia entomophila ), Serratia picaria ( Serratia ficaria ), Serratia fonticola ( Serratia fonticola ), Serratia grimesii ( Serratia grimesii ), Serratia liquefaciens ( Serratia liquefaciens ), Serratia microhaemolytica , Serratia myotis ( Serratia myotis ), Serratia nematodiphilia ( Serratia nematodiphila ), Serratia duckjae ( Serratia oryzae ), Serratia plymuthica ( Serratia plymuthica ) It may be one or more selected from the group consisting of and the like.

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

Microorganisms of the Mycobacterium genus include Mycobacterium smegmatis, Mycobacterium albicans , Mycobacterium album , Mycobacterium alsense , Mycobacterium Angelicum ( Mycobacterium angelicum ), Mycobacterium anthracenicu ( Mycobacterium anthracenicu ), Mycobacterium aquaticum ( Mycobacterium aquaticum ), Mycobacterium aquiterrae ( Mycobacterium aquiterrae ), Mycobacterium attenuatum ( Mycobacterium attenuatum ), Myco Bacterium arosiense, Mycobacterium leprae, Mycobacterium hekesonense, Mycobacterium helveticum , Mycobacterium hyorhinis ), Mycobacterium innocens ( Mycobacterium innocens ), Mycobacterium isoniacini ( Mycobacterium isoniacini ), Mycobacterium jacuzzi ( Mycobacterium jacuzzii ), Mycobacterium kansasii ( Mycobacterium kansasii ), Mycobacterium marinum ( Mycobacterium marinum ) , Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) It may be one or more selected from the group consisting of the like.

The microorganism according to an embodiment in which the activity of the endogenous class II type BirA is attenuated and/or contains exogenous class I type BirA that does not contain a DNA binding site at the N-terminus inherently contains class II type BirA, or When cultured in a non-biotin-added medium, the growth rate is faster and/or the biotin-producing ability (or production) is increased than in the case of containing Class II type BirA in which the N-terminal DNA bindig domain is deleted. it could be

Another aspect provides a composition for producing biotin, including the microorganism.

In one example, the composition for production may further include any suitable excipient commonly used in compositions for biotin production, and such excipients may include, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or It may be an isotonic agent or the like, but is not limited thereto.

Another aspect is introducing (eg, transforming) a recombinant vector comprising attenuated class II type BirA and/or class I type BirA, a polynucleotide encoding the same, and/or the polynucleotide into a microorganism It provides a method for increasing the biotin-producing ability of the microorganism or a method for imparting the biotin-producing ability to the microorganism.

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

In the present application, "cultivation" means growing the microorganism of the present application under appropriately controlled environmental conditions. The culture process of the present application may be performed according to suitable media and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culture may be batch, continuous and/or fed-batch, but is not limited thereto.

In this application, "medium" means a material in which nutrients necessary for culturing the microorganisms of the present application are mixed as main components, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganisms of the present application can be any medium without particular limitation as long as it is a medium used for culturing ordinary microorganisms, but the microorganisms of the present application are suitable as carbon sources, nitrogen sources, personnel, and inorganic materials. It can be cultured while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing compounds, amino acids, and/or vitamins. 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)] and the like.

Examples of the carbon source in the present application include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sorghum pomace and corn steep liquor can be used, specifically glucose and sterilized pretreated molasses (i.e. converted to reducing sugar). Carbohydrates such as molasses) may be used, and other carbon sources in an appropriate amount may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, etc., organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

The number of persons may include monopotassium phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and amino acids, vitamins, and/or appropriate precursors may be included. These components or precursors may be added to the medium either batchwise or continuously. However, it is not limited thereto.

In addition, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid and the like to the medium in an appropriate manner during the cultivation of the microorganism of the present application. In addition, during cultivation, the formation of bubbles can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without gas injection or nitrogen, hydrogen or carbon dioxide gas may be injected to maintain the anaerobic and non-aerobic state. It is not.

In the culture of the present application, the culture temperature may be maintained at 20 to 45 ° C, specifically 25 to 40 ° C, and may be cultured 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 in 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, the culture Prior to the step of doing, it may be further included.

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

The recovery may be to collect desired biotin using a suitable method known in the art according to the microorganism culture method of the present application, for example, a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a precipitating agent for crystallized proteins (salting out method), extraction, sonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity Various types of chromatography such as doe chromatography, HPLC, or a combination thereof may be used, and desired biotin may be recovered from a medium or microorganism using a suitable method known in the art.

In addition, the biotin production method of the present application may additionally include a purification step. The purification may be performed using suitable methods 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 simultaneously (or sequentially) regardless of order, or simultaneously or integrated into one step. It can be performed, but is not limited thereto.

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

Another aspect of the present application provides a use of the microorganism of the present application for biotin production. The microorganisms and the like are as described above.

In the microorganism according to one embodiment, the activity of the endogenous class II type BirA is weakened, and the biotin production ability is increased by including the class I type BirA, so that a high yield of biotin can be produced.

Figure 1 shows the domain and origin of class I type and class II type BirA.

Figure 2 shows the results of measuring the growth rates of the control and strains of the present invention according to time in M9 minimal medium without biotin or M9 medium supplemented with biotin.

Hereinafter, the present application will be described in more detail through examples. However, these examples are intended to illustrate the present application by way of example, and the scope of the present application is not limited to these examples.

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

In this Example, the proteins (SEQ ID NOs: 3 and 4, respectively) encoded by birA derived from Mycobacterium smegmatis (M.sm) and Corynebacterium glutamicum (C.gl) The purpose of this study was to examine the effects 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 Escherichia coli BirA (SEQ ID NO: 1) and the foreign birA gene were prepared separately. It was expressed with the known E. coli-derived cj1 promoter (Korean Patent Publication No. 10-2006-0068505; hereinafter, Pcj1, SEQ ID NO: 9).

First, by using the cj1 promoter derived from E. coli, for the expression of the catalytic domain in which the N-terminal DNA binding domain is missing in E. coli BirA, an initiation codon (ATG) is inserted. A vector for securing the birA gene was constructed. 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 the primers of VB7-1 (SEQ ID NO: 10) and VB7-2 (SEQ ID NO: 11) using E. coli W3110 chromosomal DNA as a template, the upstream region of about 0.5 kb (SEQ ID NO: 12) , Using the primers of VB7-3 (SEQ ID NO: 13) and VB7-4 (SEQ ID NO: 14), a gene fragment of about 0.5 kb (SEQ ID NO: 15) was obtained in the downstream region, 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 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, polymerization at 72 ° C for 30 seconds, and polymerization for 30 seconds. After repeating this twice, 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 and downstream and cj1 promoter fragments. According to the method described in PCT Publication 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) to construct 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 preserving at 50° C. for 1 hour.

In addition, in order to replace the E. coli birA gene with the foreign-derived Class I birA genes described above, first, a vector with birA gene deletion and target gene insertion was constructed. To construct the vector, PCR was performed to obtain birA upstream and downstream regions using E. coli W3110 as a template. Specifically, using primers of VB7_7 (SEQ ID NO: 18) and VB7_8 (SEQ ID NO: 19) using E. coli W3110 chromosomal DNA as a template, upstream regions of about 0.5 kb (SEQ ID NO: 20), VB7_9 (SEQ ID NO: 21) and VB7_10 A gene fragment of about 0.5 kb (SEQ ID NO: 23) in the downstream region was obtained through PCR using the primer of (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, polymerization at 72 ° C for 30 seconds, and polymerization for 30 seconds. After repeating this twice, a polymerization reaction was performed at 72° C. for 5 minutes to obtain. After purifying the obtained DNA product using QIAGEN's PCR Purification kit, the amplified birA upstream and downstream fragments and the pSKH vector, a vector for chromosomal transformation cut with EcoRV restriction enzyme, were used for Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method was used to construct a vector pSKHΔbirA (E.co) for birA gene deletion and target gene insertion. Next, based on the birA gene-defective vector pSKHΔbirA (E.co), vectors into which birA genes derived from Mycobacterium smegmatis and Corynebacterium glutamicum were inserted including the previously known cj1 promoter were constructed. First, in order to secure the known cj1 promoter, a cj1 promoter fragment of about 0.3 kb was obtained through PCR using synthetic cj1 promoter DNA as a template and primers VB7_11 (SEQ ID NO: 24) and VB7_12 (SEQ ID NO: 25), 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) using Mycobacterium smegmatis MC2155 chromosomal DNA as a template (SEQ ID NO: 7 ) Likewise, in order to secure the birA gene derived from Corynebacterium glutamicum, including the cj1 promoter, about 0.3 kb of cj1 was obtained using primers VB7_11 (SEQ ID NO: 24) and VB7_15 (SEQ ID NO: 28) using synthetic cj1 promoter DNA as a template. The promoter fragment was obtained through PCR, and a birA gene fragment of about 0.8 kb was obtained using Corynebacterium glutamicum ATCC13032 chromosomal DNA as a template and primers of VB7_16 (SEQ ID NO: 29) and VB7_17 (SEQ ID NO: 30). It was obtained through a PCR run (SEQ ID NO: 8). The obtained DNA product was purified using QIAGEN's PCR Purification kit, and then the cj1 promoter, each birA gene fragment, and the pSKHΔbirA (E.co) vector cut with ScaI restriction enzyme were cloned using the Gibson assembly method to recombine Plasmids were obtained and named as pSKHΔbirA(E.co)::Pcj1_birA(M.sm) and SKHΔbirA(E.co)::Pcj1_birA(C.gl), respectively.

Example 2. Escherichia coli birA missing and outpatient birA Development of transgenic 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 converted to E. coli wild type W3110. After transformation of electro-competent cells by electroporation, CV04-0003 (W3110 ΔbirADB(E.co)::Pcj1_ATG) and CV04-0004 (W3110 ΔbirA(E.co): :Pcj1_birA(M.sm)) and CV04-0002 (W3110 ΔbirA(E.co)::Pcj1_birA(C.gl)), respectively. In the CV04-0003 strain, the birA gene was modified to encode BirA in which the N-terminal DNA binding site was disrupted on the E. coli chromosome, and in the CV04-0004 and CV04-0002 strains, the birA gene on the E. coli chromosome was Mycobacterium smegmatis and It is a strain substituted with the birA gene derived from Corynebacterium glutamicum. In each recombinant strain, a PCR method using primers of VB7_18 (SEQ ID NO: 31) and VB7_19 (SEQ ID NO: 32) capable of amplifying the external regions of the upstream and downstream regions of the homologous recombination into which the gene was inserted, respectively, and genome sequencing were performed. Corresponding genetic manipulations were confirmed.

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

In order to confirm the change in growth rate due to the 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. In order to measure the growth curve of E. coli cells growing in the exponential phase, they were inoculated into M9 minimal medium supplemented with 0.3% glucose as a carbon source and cultured for 15 hours or more (overnight). ) 37 ℃, 200rpm conditions were the primary culture. The cultured cells were inoculated in a 25 ml volume of M9 minimal medium without biotin or with 0.2 mg/L of biotin added so that the initial absorbance (Optical density 562 nm, O.D562) was 0.1 and kept at 37°C for 15 hours or more (overnight). , while culturing at 200 rpm, absorbance was measured in O.D562, and the results are shown in FIG. 2 .

As shown in FIG. 2, the strain (CV04-0003) in which the wild-type birA gene of E. coli is replaced with the birA gene encoding BirA having a missing N-terminal DNA binding site has a higher E. coli birA gene than Mycobacterium smegmatis or The CV04-0004 and CV04-0002 strains substituted with the birA gene derived from Corynebacterium glutamicum exhibited rapid growth rates even when biotin was not added, and all strains exhibited the same growth rate when biotin was added. In particular, the CV04-0002 strain showed a high level of growth rate even when biotin was not added.

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

Example 4. Construction of biotin biosynthesis-enhanced strain based on birA substituted strain and evaluation of biotin production ability

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

Three recombinant strains prepared in Example 3 using the biotin operon overexpression vector (pCL1920-bioABFCD (Eco)) prepared above were 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 respectively introduced using the thermal 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 then cultured overnight in a 30° C. incubator. The strain cultured overnight in LB solid medium was inoculated into 25 mL of a titer medium having 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 the consumption sugar was measured using a sugar analyzer (YSI 2900) to measure residual sugar 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	Consumed sugar (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 derived from Mycobacterium smegmatis or Corynebacterium glutamicum. The -0002/pCL1920-bioABFCD (Eco) strain showed significantly higher biotin production than E. coli W3110/pCL1920-bioABFCD (Eco) and E. coli CV04-0003/pCL1920-bioABFCD (Eco).

Example 5. Serratia marcescens ( Serratia marcescens ) Production of biotin biosynthesis-enhanced strain-based birA-substituted strain and evaluation of biotin production capacity

In order to investigate the effect of Class II birA deficiency and introduction substitution of Class I birA genes other than Escherichia coli on biotin production capacity, based on the biotin-producing strain TA5027 (US patent US 5374554 A) derived from Serratia marcescens, birA gene deletion and class A recombinant strain into which I birA gene was introduced was prepared.

In order to construct the birA gene-defective and foreign birA gene transduction vector, the birA gene-defective and target gene-inserted vectors were constructed using the chromosome of strain TA5027 as a template in the same manner as in Example 2 above. Specifically, using primers of VB7_22 (SEQ ID NO: 35) and VB7_23 (SEQ ID NO: 36) using Serratia TA5027 chromosomal DNA as a template, upstream regions of about 0.5 kb (SEQ ID NO: 37), VB7_24 (SEQ ID NO: 38) and A gene fragment (SEQ ID NO: 40) of about 0.5 kb in the downstream region 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, polymerization at 72 ° C for 30 seconds, and polymerization for 30 seconds. After repeating this twice, a polymerization reaction was performed at 72° C. for 5 minutes to obtain. After purifying the obtained DNA product using QIAGEN's PCR Purification kit, the amplified birA upstream and downstream fragments and the pSKH vector, a vector for chromosomal transformation cut with EcoRV restriction enzyme, were used for Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method was used to construct a vector pSKHΔbirA (S.ma) for birA gene deletion and target gene insertion.

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

After transforming the prepared vector into competent cells of the Serratia marcescens TA5027 strain by electroporation in the same manner as in Example 3, the birA gene on the chromosome of the TA5027 strain through a secondary crossover process. Strains substituted with the birA gene derived from Mycobacterium smegmatis or Corynebacterium glutamicum were obtained, respectively, CV04-9991 (TA5027 ΔbirA(S.ma)::Pcj1_birA(M.sm)), CV04- 9992 (TA5027 ΔbirA(S.ma)::Pcj1_birA(C.gl)). The genetic manipulation was performed through PCR and genome sequencing using primers of VB7_29 (SEQ ID NO: 44) and VB7_30 (SEQ ID NO: 45) capable of amplifying the external regions of the upstream and downstream regions of the homologous recombination into which 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 plated on LB solid medium, and then cultured overnight in an incubator at 30 ° C. The strain cultured overnight in LB solid medium was inoculated into 25 ml of titer medium having 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 the consumption sugar was measured using a sugar analyzer (YSI 2900) to measure residual sugar 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	Consumed sugar (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, the biotin production of strains CV04-9991 and CV04-9992 in which the birA gene of Serratia marcescens was substituted with the birA gene derived from Mycobacterium smegmatis or Corynebacterium glutamicum, respectively, was It was confirmed that it was significantly higher than that of the strain TA5027.

Claims (11)

1. A microorganism of the genus Escherichia or Serratia sp., wherein the activity of endogenous BirA of class II type is attenuated, and the class I type of BirA is contained.
2. The microorganism according to claim 1, wherein the class I type of BirA is encoded by a birA gene derived from a microorganism of the genus Mycobacterium or the genus Corynebacterium.
3. The microorganism according to claim 1, wherein the endogenous birA gene is deleted and the birA gene derived from a microorganism of the genus Mycobacterium or the genus Corynebacterium is introduced.
4. According to claim 1, 7,8-diamino-pelargonic acid aminotransferase (7,8-diamino-pelargonic acid aminotransferase), biotin synthase (Biotin synthase), 8-amino-7-oxononanoate synthase At least one activity selected from the group consisting of 8-amino-7-oxononanoate synthase, malonyl-ACP O-methyltransferase, and dethiobiotin synthetase Further enhanced, microbes.
5. The microorganism according to claim 1, having a biotin-producing ability.
6. The microorganism according to claim 1, wherein the microorganism of the genus Escherichia is Escherichia coli.
7. The microorganism according to claim 1, wherein the microorganism of the genus Serratia is Serratia marcescens.
8. A composition for producing biotin, comprising a microorganism of the genus Escherichia or Serratia sp., in which the activity of class II-type endogenous BirA is attenuated, and the class I-type BirA is contained.
9. Biotin production, comprising culturing a microorganism of the genus Escherichia or Serratia sp., in which the activity of endogenous BirA of class II type is attenuated, and containing BirA of class I type method.
10. The method for producing biotin according to claim 9, further comprising the step of recovering biotin from the cultured medium or microorganism.
11. Use of a microorganism of the genus Escherichia or Serratia sp., in which the activity of endogenous BirA of class II type is attenuated and containing BirA of class I type, for biotin production.

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