KR102712136B1 - Polypeptide variant having biotin synthase activity and biotin production method using the same - Google Patents

Abstract

The present application relates to a polypeptide variant having biotin synthase activity, a microorganism comprising the variant, and a method for producing biotin using the strain.

Description

Polypeptide variant having biotin synthase activity and biotin production method using the same {Polypeptide variant having biotin synthase activity and biotin production method using the same}

The present application relates to a polypeptide variant having biotin synthase activity or a method for producing biotin using the same.

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

Biotin is classified as an essential vitamin because many microorganisms can synthesize it, while most animals cannot, and it is used as a raw material for the synthesis and production of food or feed additives or other pharmaceuticals, making it a very useful substance. In particular, the demand for vitamins has been increasing due to the expansion of antibiotic regulations, and its sales price is gradually increasing. However, most biotin sold on the market is produced only through multi-step chemical processes, and now that the era of essential environmental technologies has arrived, the demand for biological production technologies is increasing. However, there appears to be almost no advanced literature or patents on biological production technologies since the 2000s, and their production efficiency is not high. Therefore, it is important to secure a large number of biological production technologies for biotin.

Therefore, under these circumstances, research to increase biotin production is still needed.

One object of the present application is to provide a polypeptide variant having biotin synthase activity.

Another object of the present application is to provide a polynucleotide encoding a variant of the present application.

Another object of the present application is to provide a recombinant vector comprising the polynucleotide.

Another object of the present application is to provide a microorganism producing biotin, comprising a polypeptide variant having biotin synthase activity.

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

Another object of the present application is to provide a method for producing biotin, which comprises a step of culturing a microorganism producing biotin.

In this application, we seek to provide a recombinant microorganism (strain) with excellent biotin production ability by searching for a biotin synthase or a variant thereof capable of improving biotin production ability and introducing the same into a microorganism.

One aspect of the present application for achieving the above object can provide a polypeptide variant having biotin synthase activity.

In the present application, “biotin synthase (EC 2.8.1.6)” may refer to an enzyme that catalyzes the conversion of dethiobiotin (DTB) into biotin, and may mean a SAM-dependent enzyme that thiolates dethiobiotin into biotin using a radical mechanism. The biotin synthase may be included regardless of the origin of the microorganism as long as it is an enzyme having the above activity, and may be, for example, derived from a microorganism such as Serratia sp. (for example, Serratia marcescens ) or Escherichia sp. (for example, Escherichia coli ). The biotin synthase may be used interchangeably with BioB protein or BioB. In one example, the biotin synthase may comprise or consist of the amino acid sequence of SEQ ID NO: 1, and if it has the same or corresponding conversion activity as biotin synthase, a polypeptide having an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added to the amino acid sequence of SEQ ID NO: 1 may also be included within the scope of the present application. In addition, regardless of the origin of the microorganism, a polypeptide having at least 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99%, 99.5% or 99.8% homology or identity to the amino acid sequence of SEQ ID NO: 1, and having the same or corresponding conversion activity as biotin synthase may also be included within the scope of the present application as the biotin synthase.

In the present application, even if it is described as 'a polypeptide (or protein) comprising an amino acid sequence described by a specific sequence number' or 'a polypeptide (or protein) composed of an amino acid sequence described by a specific sequence number', if it has the same or corresponding activity as the polypeptide (or protein) composed of the amino acid sequence of the corresponding sequence number, a polypeptide (or protein) having an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added can also be used as the protein or polypeptide that is the target of mutation of the present application. For example, a polypeptide (or protein) having a sequence in which a part of the amino acid sequence of sequence number 1 is deleted, modified or substituted or a sequence in which a part of the sequence is added to the amino acid sequence of sequence number 1 can belong to the 'polypeptide composed of the amino acid sequence of sequence number 1' if it has the same or corresponding activity as the 'polypeptide composed of the amino acid sequence of sequence number 1'.

The amino acid sequence of the above biotin synthase and the base sequence of the gene encoding biotin synthase can be easily obtained from databases known in the art, such as the National Center for Biotechnology Information (NCBI) and the DNA Data Bank of Japan (DDBJ), and may be, for example, GenBank Accession No. WP_000951213.1. The gene encoding the biotin synthase may be a bioB gene, and the biotin synthase may be encoded by the bioB gene.

In the present application, the phrase “a polynucleotide or a polypeptide comprises a specific nucleic acid sequence (base sequence) or amino acid sequence” may mean that the polynucleotide or the polypeptide consists of or essentially includes the specific nucleic acid sequence (base sequence) or amino acid sequence, and may be interpreted as including a sequence in which a mutation (deletion, substitution, modification, and/or addition) is added to the specific nucleic acid sequence (base sequence) or amino acid sequence within the scope of maintaining the original function and/or the desired function of the polynucleotide or the polypeptide (or not excluding the mutation). In one example, a polynucleotide or polypeptide "comprising a particular nucleic acid sequence (base sequence) or amino acid sequence" can mean that the polynucleotide or polypeptide (i) consists of or essentially comprises the particular nucleic acid sequence (base sequence) or amino acid sequence, or (ii) consists of or essentially comprises a nucleic acid sequence or amino acid sequence that has at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99.5%, or 99.9% homology to the particular nucleic acid sequence (base sequence) or amino acid sequence and retains its original function and/or desired function.

In this application, ‘homology’ or ‘identity’ means the degree of similarity between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be used together with a default gap penalty established by the program being used. In practice, homologous or identical sequences are generally capable of hybridizing with all or part of the sequence under moderate or high stringent conditions. It will be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account in the polynucleotide.

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

Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or, for example, Needleman et al. (1970), J Mol Biol. 48:443. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and (2) a matrix of 1-bit integers, as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), as disclosed in Gribskov et al(1986) Nucl. Acids Res. 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or gap opening penalty 10, gap extension penalty 0.5); and (3) no penalty for terminal gaps.

In one example, the polypeptide variant having biotin synthase activity may be an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity thereto, wherein the amino acid corresponding to the third residue of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid. In addition, in one example, the polypeptide variant of the present application may be a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with the amino acid sequence of SEQ ID NO: 1, as a polypeptide in which the amino acid corresponding to the 3rd residue of the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid. In addition, it is obvious that a variant having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted or added is also included in the variant of the present application, as long as it has such homology or identity and exhibits an effect corresponding to the variant of the present application. For example, in the case of having sequence additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions that do not alter the function of the variant of the present application at the N-terminus, C-terminus and/or within the amino acid sequence.

The above “conservative substitution” refers to the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

In this application, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and/or modified, thereby differing from the amino acid sequence of the variant before the mutation, but retaining functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. In addition, some variants may include variants in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted. Other variants may include variants in which portions are deleted from the N- and/or C-terminus of the mature protein. The above term “variant” may be used interchangeably with terms such as variant, modification, variant polypeptide, mutated protein, mutation, and variant (in English expressions, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited thereto if the term is used in the meaning of mutation. In one example, the variant may be a polypeptide including the amino acid sequence described in SEQ ID NO: 21, in which alanine, an amino acid corresponding to the third position of the amino acid sequence of SEQ ID NO: 1, is substituted with another amino acid (for example, an amino acid other than alanine).

Additionally, the variant may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be conjugated to a signal (or leader) sequence that is involved in co-translationally or post-translationally translocation of the protein. Additionally, the variant may be conjugated to other sequences or linkers so that it can be identified, purified, or synthesized.

In the present application, ‘the ~th amino acid sequence having sequence number ~’ can be used with the same meaning as ‘the ~th amino acid sequence from the N-terminus of a polypeptide consisting of (or made of) the amino acid sequence having sequence number ~’.

In this application, "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the polypeptide. Identifying an amino acid at a corresponding position can determine a particular amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position at which a modification, such as a substitution, insertion, or deletion, occurs, by comparing it to a query sequence (also referred to as a “reference sequence”).

For this alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) can be used, but is not limited thereto, and any sequence alignment program or pairwise sequence comparison algorithm known in the art can be appropriately used.

In one example, the polypeptide variant can comprise an amino acid sequence in which the amino acid corresponding to the third position of the amino acid sequence of SEQ ID NO: 1, alanine, is replaced with another amino acid, and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with the amino acid sequence of SEQ ID NO: 1.

In one example, the polypeptide variant having biotin synthase activity may have the third amino acid in the amino acid sequence of SEQ ID NO: 1 replaced with a different amino acid.

The above other amino acids may refer to other types of amino acid residues other than the amino acid (e.g., alanine) present at the position (the corresponding position in the amino acid sequence of SEQ ID NO: 1) before being substituted, and may be, for example, proline, arginine, leucine, isoleucine, valine, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, aspartic acid, and glutamic acid.

A polypeptide variant according to an example is:

Alanine, an amino acid corresponding to the third position of sequence number 1, may be substituted with an amino acid other than alanine. The amino acids other than alanine may include arginine, proline, leucine, isoleucine, valine, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, aspartic acid, and glutamic acid. A polypeptide variant according to one specific example may be one in which alanine corresponding to the third position is substituted with threonine.

A polypeptide variant according to an example may be one in which the third alanine in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine.

A polypeptide variant according to one example can comprise or consist essentially of the amino acid sequence of SEQ ID NO: 21.

A polypeptide variant according to an example may be one in which the amino acid corresponding to the third residue of the amino acid sequence of SEQ ID NO: 1 is replaced with a different amino acid in SEQ ID NO: 1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity thereto, such that the biotin producing activity is increased compared to the polypeptide before the replacement .

In this application, the term “enhancement” of polypeptide 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, up-regulation, overexpression, and increase may all include exhibiting an activity that was not originally present, or exhibiting an enhanced activity compared to the intrinsic activity or the activity before modification. The “intrinsic activity” means the activity of a specific polypeptide that a parent strain or an unmodified microorganism originally had before the trait change when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification.” “Enhanced,” “upregulated,” “overexpressed,” or “increased” activity of a polypeptide relative to its intrinsic activity means that the activity and/or concentration (expression level) of the specific polypeptide is enhanced compared to the activity and/or concentration (expression amount) that the parent strain or unmodified microorganism originally had prior to the transformation.

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

Enhancement of the activity of the above polypeptide can be achieved 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 the microorganism before modification. Specifically, it may be performed using genetic engineering and/or protein engineering, which are routine methods of molecular biology and are well known to those skilled in the art, 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, the enhancement of the polypeptide of the present application is

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

2) Replacing the gene expression control region on the chromosome that codes for a polypeptide with a highly active sequence;

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

4) Modification of the amino acid sequence of the polypeptide so as to enhance the polypeptide activity;

5) Modification of a polynucleotide sequence encoding said polypeptide such that the activity of said polypeptide is enhanced (e.g., modification of a polynucleotide sequence of said polypeptide gene such that the polynucleotide sequence of said polypeptide is encoded by a modified polypeptide such that the activity of said polypeptide is enhanced);

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

7) Codon optimization of polynucleotides encoding polypeptides;

8) Analyzing the tertiary structure of the polypeptide and selecting the exposed portion to modify or chemically modify; or

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

More specifically,

The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described above 1) may be achieved by 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. Alternatively, the polynucleotide encoding the polypeptide may be achieved by introduction of one copy or two or more copies into the chromosome of the host cell. The introduction into the chromosome may be performed by introduction into the host cell of a vector capable of inserting the polynucleotide into the chromosome of the host cell, but is not limited thereto. The vector is as described above.

The above 2) replacement of the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence having strong activity may be, for example, a mutation in the sequence such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the expression control region, or replacement with a sequence having stronger activity. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but is not limited thereto.

Examples of known strong promoters include, but are not limited to, cj1 to cj7 promoters (US Patent No. 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 No. US 10584338 B2), O2 promoter (US Patent No. US 10273491 B2), tkt promoter, yccA promoter, etc.

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

The modification of the amino acid sequence or polynucleotide sequence of the above 4) and 5) may be, but is not limited to, a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, 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 have increased activity. The replacement may be specifically performed by inserting the 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 confirm whether or not the chromosome has been inserted. The selection marker is as described above.

The introduction of the foreign polynucleotide exhibiting the activity of the above 6) polypeptide may be the introduction into the host cell of a foreign polynucleotide encoding a polypeptide exhibiting the same/similar activity as the above polypeptide. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same/similar activity as the above polypeptide. The method used for the above introduction can be performed by a person skilled in the art appropriately selecting a known transformation method, and the introduced polynucleotide is expressed in the host cell, thereby producing the polypeptide and increasing its activity.

The above 7) codon optimization of a polynucleotide encoding a polypeptide may be codon optimization of an endogenous polynucleotide to increase transcription or translation within a host cell, or codon optimization of a foreign polynucleotide to achieve optimized transcription or translation within a host cell.

The above 8) analyzing the tertiary structure of a polypeptide and selecting an exposed portion to modify or chemically modify may be done by, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins, determining a template protein candidate based on the degree of sequence similarity, confirming the structure based on this, and selecting an exposed portion to modify or chemically modify and modifying or modifying it.

Such enhancement of polypeptide activity may be, but is not limited to, an increase in the activity or concentration or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or unmodified microbial strain, or an increase in the amount of a product produced from the polypeptide.

In this application, the term “attenuation” of a polypeptide is a concept that includes both a decrease in 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 above weakening may also include cases where 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, etc., cases where the overall level of polypeptide activity and/or concentration (expression amount) within the cell is lower than that of the natural strain due to inhibition of expression of the gene of the polynucleotide encoding it or inhibition of translation into a polypeptide, cases where the polynucleotide is not expressed at all, and/or cases where the polynucleotide is expressed but there is no activity of the polypeptide. The above “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 is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification.” The term “inactivation, deficiency, reduction, down-regulation, reduction, attenuation” of a polypeptide activity relative to its intrinsic activity means that the activity of a particular polypeptide is lowered compared to the activity that the parent strain or unmodified microorganism originally had prior to the transformation.

Attenuation of the activity of such polypeptides can be accomplished by any method known in the art, including but not limited to, and can be achieved by application of 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, the weakening of the polypeptide of the present application is

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

2) Modification of the expression control region (or expression control sequence) so as to decrease 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 in the amino acid sequence) so as to eliminate or weaken the activity of the polypeptide;

4) Modification of the gene sequence encoding the polypeptide such that the activity of the polypeptide is eliminated or weakened (e.g., deletion/substitution/addition of one or more nucleotides in the nucleotide sequence of the polypeptide gene such that the polypeptide is modified such that the activity of the polypeptide is eliminated or weakened);

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

6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to a 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 a gene encoding a polypeptide to form a secondary structure to which ribosome attachment is impossible;

8) Addition of a promoter that is 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 of the above 1) to 8), but is not particularly limited thereto.

For example,

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

In addition, the above 2) modification of the expression control region (or expression control sequence) may be a mutation in the expression control region (or expression control sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence having weaker activity. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

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

In addition, the modification of the amino acid sequence or polynucleotide sequence of the above 4) and 5) may be, but is not limited to, a mutation in the sequence such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence improved to have a weaker activity, or an amino acid sequence or polynucleotide sequence improved to have no activity. For example, the expression of the gene may be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon, but is not limited thereto.

The introduction of an antisense oligonucleotide (e.g., antisense RNA) that complementarily binds to a transcript of the gene encoding the polypeptide described above 6) can be described, for example, with reference to the literature [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 in front of the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure to which ribosome attachment is impossible may render mRNA translation impossible or slow it down.

The 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 above 8) polypeptide (Reverse transcription engineering, RTE) may weaken the activity by creating an antisense nucleotide complementary to the transcript of the gene encoding the above polypeptide.

Another aspect may be to provide a polynucleotide encoding the above polypeptide variant.

In this application, "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, a DNA or RNA strand of a certain length or longer, and more specifically, a polynucleotide fragment encoding the variant.

A polynucleotide encoding a variant of the present application may comprise a base sequence encoding an amino acid sequence set forth in SEQ ID NO: 21. As an example of the present application, the polynucleotide of the present application may have or comprise the sequence of SEQ ID NO: 22. Additionally, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 22.

The polynucleotide of the present application may have various modifications made to the coding region within a range that does not change the amino acid sequence of the variant of the present application, taking into account the degeneracy of the codon or the codon preferred in an organism that is intended to express the variant of the present application. Specifically, the polynucleotide of the present application has or includes a base sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less than 100% homology or identity with the sequence of SEQ ID NO: 2, or may consist of or consist essentially of a base sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less than 100% homology or identity with the sequence of SEQ ID NO: 2, but is not limited thereto. At this time, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 220th position of SEQ ID NO: 1 may be one of the codons encoding cysteine.

In addition, the polynucleotide of the present application may include, without limitation, a probe that can be prepared from a known genetic sequence, for example, a sequence that can hybridize under stringent conditions with a complementary sequence to all or a part of the polynucleotide sequence of the present application. The “stringent conditions” above mean conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, the conditions under which polynucleotides having high homology or identity hybridize with each other, or polynucleotides having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridize, and polynucleotides having lower homology or identity do not hybridize, or the conditions of washing once, specifically twice to three times, at a salt concentration and temperature corresponding to the washing conditions of conventional southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS.

Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term “complementary” is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60° C., 63° C., or 65° C. and can be appropriately adjusted by a person skilled in the art depending on the purpose.

The appropriate stringency to hybridize the polynucleotides will depend on the length and degree of complementarity of the polynucleotides and variables are well known in the art (see, e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition; F.M. Ausubel et al., Current Protocols in Molecular Biology, 9.50-9.51, 11.7-11.8).

Another aspect may provide a vector comprising the polynucleotide. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

In the present application, a “vector” may include a DNA construct comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences controlling termination of transcription and translation. The vector may be capable of replicating or functioning independently of the host genome after being transformed into a suitable host cell, and may be integrated into the genome itself.

The vector used in the present 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 series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors can be used.

For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for confirming whether the chromosome has been inserted can be additionally included. The selection marker is for selecting cells transformed with the vector, i.e., confirming whether the target nucleic acid molecule has been inserted, and markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, resistance to cytotoxic agents, or expression of a surface polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, so that the 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. The transformed polynucleotide may include both a position inserted into the chromosome of the host cell or an extrachromosomal position, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and/or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own 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 above 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.

Additionally, the term "operably linked" as used herein means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target variant of the present application is functionally linked to the polynucleotide sequence.

Another aspect can be provided by providing a microorganism producing biotin having enhanced biotin synthase activity. In one example, the biotin synthase can be from a microorganism of the genus Serratia (e.g., Serratia marcescens ) or the genus Escherichia (e.g., Escherichia coli ). The enhancement of polypeptide (e.g., biotin synthase) activity is as described above.

In one example, the microorganism may comprise at least one selected from the group consisting of a polypeptide comprising an amino acid sequence of sequence number 1; a polypeptide variant of the present application; a polynucleotide encoding the polypeptide or the polypeptide variant; and a vector comprising the polynucleotide.

In one example, the microorganism may further have an enhanced activity of at least one selected from the group consisting of 7,8-diamino-pelargonic acid aminotransferase, 8-amino-7-oxononanoate synthase, malonyl-ACP O-methyltransferase, and dethiobiotin synthetase, for example, a bioABFCD operon including a bioB gene mutated to encode the variant of the present application may be introduced. The enhancement of the polypeptide activity is as described above.

In the present application, “7,8-diamino-pelargonic acid aminotransferase” may mean a protein that generates 7,8-diamino-pelargonic acid (DAPA) by bonding an alpha-amino group obtained from an enzyme-specific specific substance having an amine group such as SAM or lysine to 8-amino-7-oxononanoate (7-Keto-8-aminopelargonic acid; KAPA) produced by the activity of 8-amino-7-oxononanoate synthase.

In the present application, “8-amino-7-oxononanoate synthase” may mean a protein that catalyzes the decarboxylative condensation reaction of 6-carboxyhexanoyl-CoA (Pimeloyl-coA/ACP) and alanine.

In the present application, “malonyl-ACP O-methyltransferase (or malonyl-[acyl-carrier protein] O-methyltransferase)” may mean a protein that methyl esterifies the carboxyl group of malonyl thioester by converting a carbon polymer in the fatty acid biosynthetic pathway into pimeloyl-coA/ACP.

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

In this application, the term "strain (or microorganism)" includes both wild-type microorganisms and microorganisms that have undergone genetic modification naturally or artificially, and may be microorganisms that have a specific mechanism weakened or strengthened due to causes such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene, and may be microorganisms that include genetic modification for the production of a desired polypeptide, protein or product.

The strain of the present application may be, but is not limited to, a strain comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 1; a strain comprising at least one of a variant of the present application, a polynucleotide of the present application, and a vector comprising a polynucleotide of the present application; a strain modified to express the variant of the present application or the polynucleotide of the present application; a strain (e.g., a recombinant strain) expressing the variant of the present application or the polynucleotide of the present application; or a strain (e.g., a recombinant strain) having the activity of the variant of the present application.

The strain of the present application may be a strain having biotin production ability.

The strain of the present application may be, but is not limited to, a microorganism naturally having biotin synthase or biotin production ability, or a parent strain lacking biotin synthase or biotin production ability, into which the variant of the present application or a polynucleotide encoding the same (or a vector including the polynucleotide) is introduced and/or biotin production ability is imparted.

For example, the strain of the present application is a cell or microorganism that is transformed with a vector including a polynucleotide encoding the polynucleotide of the present application or a variant of the present application, and expresses the variant of the present application. For the purpose of the present application, the strain of the present application may include all microorganisms capable of producing biotin, including the variant of the present application. For example, the strain of the present application may be a recombinant strain in which a variant having biotin synthase activity is expressed by introducing a polynucleotide encoding the variant of the present application into a natural wild-type microorganism or a microorganism that produces biotin, thereby increasing biotin production ability. The recombinant strain in which biotin production ability is increased may be a microorganism in which biotin production ability is increased compared to a natural wild-type microorganism or a biotin synthase-unmodified microorganism (i.e., a microorganism expressing wild-type biotin synthase), but is not limited thereto. For example, the target strain for comparing the increase in biotin production ability, a biotin synthase non-modified microorganism, may be, but is not limited to, a wild-type E. coli strain, E. coli W3110, E. coli CV04-0002 strain, and/or E. coli CV04-0104 strain (a strain into which an E. coli biosynthetic operon based on the CV04-0002 strain is additionally introduced).

For example, the recombinant strain with increased productivity has a biotin production (or productivity) of 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, or about 20% 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 not particularly limited, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% Below, about 40% or less, or about 35% or less) may be increased. In another example, the recombinant strain with increased production may have an increased biotin production (or productivity) of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more, about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.2 times or more, about 1.25 times or more, or about 1.3 times or more (the upper limit is not particularly limited, and may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less), compared to the parent strain before mutation or the unmodified microorganism, but is not limited thereto. The above term “about” includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values in a range equal to or similar to the numerical value following the term “about,” but is not limited thereto.

In the present application, the term "unmodified microorganism" does not exclude a strain containing a mutation that may occur naturally in a microorganism, and may refer to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed by genetic mutation due to natural or artificial factors. For example, the unmodified microorganism may refer to a strain into which the biotin synthase variant described herein is not introduced or before it is introduced. The term "unmodified microorganism" may be used interchangeably with "pre-modified strain", "pre-modified microorganism", "unmutated strain", "unmodified microorganism", or "reference microorganism".

The above microorganism may be Escherichia sp., and the Escherichia sp. microorganism may be Escherichia coli .

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

Another aspect can provide a composition for producing biotin, comprising at least one selected from the group consisting of a polypeptide variant having the biotin synthase activity, a polynucleotide encoding the variant, a vector comprising the polynucleotide, and the microorganism (or strain, recombinant cell). The variant, the polynucleotide, the vector, and the microorganism, etc. are as described above.

In one example, the production composition may further comprise any suitable excipient commonly used in compositions for biotin production, including but not limited to preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers, or isotonic agents.

Another aspect may provide a method for increasing biotin production ability of a microorganism or a method for imparting biotin production ability to a microorganism, the method comprising a step of introducing (e.g., transforming) a polypeptide variant having the biotin synthase activity, a polynucleotide encoding the same, and/or a recombinant vector comprising the polynucleotide into the microorganism.

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

In this application, "cultivation" means growing the microorganism of this application under appropriately controlled environmental conditions. The culturing process of this application can be performed according to appropriate media and culturing 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 culturing may be batch, continuous, and/or fed-batch, but is not limited thereto.

In this application, "medium" means a material containing nutrients as main components necessary for culturing the microorganism of the present application, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of the present application may be any medium used for culturing general microorganisms without particular limitation, but the microorganism of the present application may be cultured in a general medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid, and/or vitamin, etc., under aerobic conditions 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)], etc.

In the present application, the carbon source may include 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. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane residue, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugar) may be used, and other appropriate amounts of carbon sources 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.

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

The above-mentioned personnel may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. The 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 may be added to the medium in batch or continuous manner. However, the present invention is not limited thereto.

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. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or in order to maintain the anaerobic and microaerobic state, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

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

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

The method for producing biotin of the present application may additionally include a step of preparing a microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

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

The above recovery may be to collect the target biotin using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination of these methods may be used, and the target biotin can be recovered from the 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 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 may be performed in a sequential manner (or sequentially) regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

In the method of the present application, the variant, polynucleotide, vector, strain, etc. are as described above.

When culturing a microorganism comprising a polypeptide variant having biotin synthase activity of the present invention, biotin can be produced at a higher yield compared to a microorganism having an existing unmodified polypeptide.

The present invention will be described in more detail with reference to the following examples, but the scope of the invention is not intended to be limited by the following examples.

Example 1. S . marcescens Biotin synthase mutant derived

Example 1-1. S. marcescens Construction of expression vectors of derived biotin synthase mutants and construction of mutant strains

Serratia marcescens ( Serratia marcescens or S.mar ) To determine the effect of a mutant (A3T, S.mar) in which the third amino acid residue, alanine, was substituted with threonine in the derived biotin synthase protein on biotin production, an overexpression vector containing the above-mentioned amino acid mutation was constructed.

S. marcescens based on the pCL1920 vector (GenBank No AB236930) containing the pSC101 origin Two overexpression vectors capable of expressing only the protein encoded by the bioB gene were constructed using an autonomous promoter of the derived bioB gene. To construct the vectors, the chromosome of the S. mar wild-type strain (KTCT NO. 2354) was used as a template, and the primer pairs of SEQ ID NO: 3 and SEQ ID NO: 4 were used to perform PCR to obtain a wild-type bioB gene without mutation, and the bioB gene encoding the mutant was obtained by PCR using the primer pairs of SEQ ID NO: 3 and SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 4. The primer sequences used are listed in Table 1. PfuUltra ^TM high-fidelity DNA polymerase (Stratagene) was used as a polymerase for the PCR reaction. PCR was performed under the following conditions: denaturation at 95°C for 30 sec; annealing at 55°C for 30 sec; and polymerization at 72°C for 2 min, repeated 25 times, and then polymerization at 72°C for 5 min. As a result, DNA fragments of the wild-type 1166 bp bioB (S.mar Wt) (SEQ ID NO: 7) and the mutant fragments of the 132 bp bioB (g7a) 1 (SEQ ID NO: 8) and the 1075 bp bioB (g7a) 2 (SEQ ID NO: 9) were obtained. The obtained DNA products were purified using a PCR Purification kit from QIAGEN and then cloned using the pCL1920 vector treated with the SmaI restriction enzyme and the Infusion Cloning Kit from TaKaRa, thereby constructing two types of wild-type biotin synthase overexpression vectors, pCL1920-Pn-bioB (S.mar) and mutant biotin synthase overexpression vectors, pCL1920-Pn-bioB (A3T, S.mar).

Name Sequence (5'->3') Sequence number VB07_0001 GACTCTAGAGGATCCCCgggTGTTGTAAACCAAATTGGAT Sequence number 3 VB07_0002 ATTCGAGCTCGGGTACCCgggTCACGGCGCCGCGTTGTAAA Sequence number 4 VB07_0003 TGTCCAGTGAATGCGGTCGGTCATCATGGCGTCTCCAAAAC Sequence number 5 VB07_0004 GTTTTGGAGACGCCATGATGACCGACCGCATTCACTGGACA Sequence number 6

To confirm whether biotin production ability was increased, the two types of bioB gene overexpression vectors constructed above (wild-type biotin synthase overexpression vector pCL1920-Pn-bioB (S.mar) and mutant biotin synthase overexpression vector pCL1920-Pn-bioB (A3T, S.mar)) were introduced into an E. coli strain (CV04-0002) that produces a small amount of biotin by heat shock transformation. As a control, the pCL1920 vector was introduced into the CV04-0002 strain.

The above CV04-0002 strain is a strain (W3110 ΔbirA(E.co)::Pcj1_birA(C.gl)) in which the E. coli (W3110) birA gene has been deleted and the Corynebacterium glutamicum-derived birA gene has been inserted, and was obtained by the following method:

Using E. coli W3110 chromosomal DNA as a template, a gene fragment of about 0.5 kb in the upstream region (SEQ ID NO: 34; upstream fragment) was obtained using the primers VB7_7 (SEQ ID NO: 32) and VB7_8 (SEQ ID NO: 33) (SEQ ID NO: 34; upstream fragment) and a gene fragment of about 0.5 kb in the downstream region (SEQ ID NO: 37; downstream fragment) was obtained by PCR using the primers VB7_9 (SEQ ID NO: 35) and VB7_10 (SEQ ID NO: 36). SolgTM Pfu-X DNA polymerase was used as the polymerase for the PCR reaction, and the PCR amplification conditions were as follows: denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 60 seconds, and polymerization at 72°C for 30 seconds, and then polymerization at 72°C for 5 minutes. The obtained DNA product was purified using a PCR Purification kit from QIAGEN, and the amplified birA upstream and downstream fragments and the pSKH vector for chromosomal transformation digested with EcoRⅤ restriction enzyme (see WO2020032590A1) were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to produce a vector pSKHΔbirA(E.co) for birA gene deletion and target gene insertion. Next, a vector inserting the birA gene derived from Corynebacterium glutamicum including the disclosed cj1 promoter was produced based on the birA gene deletion vector pSKHΔbirA(E.co). First, using the synthetic cj1 promoter DNA (SEQ ID NO: 38) as a template and the primers VB7_11 (SEQ ID NO: 39) and VB7_15 (SEQ ID NO: 40), a cj1 promoter fragment of about 0.3 kb was obtained through PCR, and using the Corynebacterium glutamicum ATCC13032 chromosomal DNA as a template and the primers VB7_16 (SEQ ID NO: 41) and VB7_17 (SEQ ID NO: 42), a birA gene fragment of about 0.8 kb was obtained through PCR (SEQ ID NO: 43). The obtained DNA product was purified using a PCR Purification kit from QIAGEN, and the cj1 promoter, each birA gene fragment, and the pSKHΔbirA(E.co) vector digested with ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named SKHΔbirA(E.co)::Pcj1_birA(C.gl). The obtained pSKHΔbirA(E.co)::Pcj1_birA(C.gl) was transformed into a wild-type E. coli W3110 electro-competent cell by electroporation, and CV04-0002 (W3110 ΔbirA(E.co)::Pcj1_birA(C.gl)) was obtained through a second crossing over process.

Each strain was spread on LB solid medium and cultured overnight in an incubator at 30°C. The strains cultured overnight on the LB solid medium were inoculated into 25 ml of a titer medium having the composition shown in Table 2 below, and cultured for 40 hours in an incubator at 30°C and 200 rpm, and the biotin production of each strain was measured using the MS-MS technique, and the results are shown in Table 3.

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 1 FeSO ₄ ,7H ₂ O 0.030 NaCl 2.5 Calcium carbonate 40

Strain name OD562nm Consumption (g/L) Biotin (mg/L) E. coli CV04-0002/pCL1920 35.3 30 0.17 E.coli CV04-0002/pCL1920-Pn-bioB(S.mar) 34.1 30 0.35 E.coli CV04-0002/pCL1920-Pn-bioB(A3T, S.mar) 34.3 30 0.49

As can be seen in Table 3 above, S. marcescens In the strain where the bioB gene from the strain was overexpressed, biotin production increased, and the strain (CV04-0002/pCL1920-Pn-bioB(A3T, S.mar)) into which the biotin synthase mutant (A3T, S.mar) from S. marcescens was introduced also showed increased biotin production compared to the parent strain without the mutant and the strain into which the wild-type biotin synthase was introduced.

Example 1-2. S. marcescens Construction of expression vectors of biotin operons containing derived biotin synthase mutants and construction of mutant strains

In this example, we aimed to determine whether the mutant form of the bioB gene could have a greater effect on increasing biotin production in a biotin biosynthesis-enhanced system that includes the entire biotin operon. Based on the pCL1920 vector, S. marcescens origin Two overexpression vectors capable of expressing proteins encoded by the bioABFCD gene were constructed using the self-promoter of the biotin operon bioABFCD gene. To construct the vectors, the chromosome of the S. mar wild-type (KTCT NO: 2354) strain was used as a template, and the primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11 were used to perform PCR to obtain the wild-type bioABFCD operon gene without mutation, and the bioABFCD operon gene including the bioB gene that can encode the mutant (A3T, S. ma) was obtained by PCR using the primer pairs of SEQ ID NO: 10 and SEQ ID NO: 5 and SEQ ID NO: 11 and SEQ ID NO: 6, respectively. The sequences of the primers used are listed in Tables 1 and 4. PfuUltra ^TM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were denaturation, 95°C, 30 sec; After repeating 25 cycles of annealing at 55°C for 30 seconds and polymerization at 72°C for 6 minutes, polymerization was performed at 72°C for 10 minutes. As a result, DNA fragments of the wild-type fragment bioABFCD (Wt) (SEQ ID NO: 12) of 5016 bp and the mutant fragments bioAB (g7a)FCD 1 (SEQ ID NO: 13) of 1407 bp and bioAB (g7a)FCD 2 (SEQ ID NO: 14) of 3650 bp were obtained. The obtained DNA product was purified using QIAGEN's PCR Purification kit and then cloned using pCL1920 vector treated with SmaI restriction enzyme and TaKaRa's Infusion Cloning Kit to produce two wild-type overexpression vectors, pCL1920-Pn-bioABFCD(S.mar) and mutant overexpression vector pCL1920-Pn-bioAB(A3T, S.mar)FCD.

designation Sequence (5'->3') Sequence number VB07_0005 GACTCTAGAGGATCCCCgggTTAACGCGCGGCGTCGGCCA Sequence number 10 VB07_0006 ATTCGAGCTCGGTACCCgggTCACTGCGCCAGCAAGCTGA Sequence number 11

To confirm whether biotin production ability increases, the two types of bioABFCD gene overexpression vectors constructed above (wild-type overexpression vector pCL1920-Pn-bioABFCD(S.ma) and mutant overexpression vector pCL1920-Pn-bioAB(A3T, S.ma)FCD) were introduced into an E. coli strain (CV04-0002) that produces a small amount of biotin by the heat shock method. As a control, the pCL1920 vector was introduced into the CV04-0002 strain.

Each strain was spread on LB solid medium and cultured overnight in an incubator at 30°C. The strain cultured overnight on the LB solid medium was inoculated into 25 ml of a titer medium having the composition of Table 2, which was then cultured for 48 hours in an incubator at 30°C and 200 rpm, and the biotin production of each strain was measured using the MS-MS technique, and the results are shown in Table 5.

Strain name OD562nm Consumption (g/L) Biotin (mg/L) E. coli CV04-0002/pCL1920 36.1 30 0.2 E.coli CV04-0002/ pCL1920-Pn- bioABFCD (S.mar) 35.6 30 1.6 E.coli CV04-0002/ pCL1920-Pn- bioAB (A3T, S.mar) FCD 35.8 30 2.7

As can be seen in Table 5 above, the biotin production of the strain overexpressing the biotin operon derived from the Serratia strain increased and could be confirmed, and the biotin production of the pCL1920-Pn-bioAB(A3T, S.ma)FCD strain, into which the biotin operon including the A3T mutation in the protein (biotin synthase) encoded by the bioB gene was introduced, increased compared to the parent strain without the mutation and the strain into which the wild-type biotin operon was introduced.

Example 2. Biotin synthase derived from E. coli

Example 2-1. Production of E. coli-derived biotin synthase expression vector and production of mutant strains

In this example, S. marcescens To compare the effects of E. coli-derived biotin synthase and E. coli-derived biotin synthase on biotin production, an overexpression vector of the E. coli-derived bioB gene was constructed.

Based on the pCL1920 vector, an overexpression vector capable of expressing only the protein encoded by the bioB gene was constructed using the autonomous promoter of the bioB gene derived from E. coli. To construct the vector, the chromosome of the E. coli W3110 strain was used as a template and the primer pair of SEQ ID NO: 15 and SEQ ID NO: 16 was used to perform PCR to obtain a wild-type bioB gene without mutation. The primer sequences used are listed in Table 6. PfuUltra ^TM high-fidelity DNA polymerase (Stratagene) was used as a polymerase for the PCR reaction, and the PCR conditions were as follows: denaturation at 95°C for 30 sec; annealing at 55°C for 30 sec; and polymerization at 72°C for 2 min, repeated 25 times, and then polymerization was performed at 72°C for 5 min. As a result, a wild-type fragment of 1167 bp, bioB (Wt, Eco) (SEQ ID NO: 17) DNA fragment was obtained. The obtained DNA product was purified using QIAGEN's PCR Purification kit and then cloned using pCL1920 vector treated with SmaI restriction enzyme and TaKaRa's Infusion Cloning Kit to produce two types of bioB coding protein wild-type biotin synthase overexpression vector pCL1920-Pn-bioB (Eco, WT).

Name Sequence (5'->3') Sequence number VB07_0007 GACTCTAGAGGATCCCCgggAATCGACTTGTAAACCAAAT Sequence number 15 VB07_0008 ATTCGAGCTCGGTACCCgggTCATAATGCTGCCGCGTTGT Sequence number 16

To confirm whether biotin production ability increased, the bioB gene overexpression vector constructed above (wild-type overexpression vector pCL1920-Pn-bioB (Eco)) was introduced into an E. coli strain (CV04-0002) (see Example 1-1) that produces a small amount of biotin by the heat shock method. As a control, the pCL1920 vector was also introduced into the CV04-0002 strain.

Each strain was spread on LB solid medium and cultured overnight in an incubator at 30°C. The strain cultured overnight on the LB solid medium was inoculated into 25 ml of a titer medium having the composition of Table 2, which was then cultured in an incubator at 30°C and 200 rpm for 40 hours, and the biotin production of each strain was measured using the MS-MS technique, and the results are shown in Table 7.

Strain name OD562nm Consumption (g/L) Biotin (mg/L) E. coli CV04-0002/pCL1920 35.1 30 0.16 E.coli CV04-0002/pCL1920-Pn-bioB(Eco) 34.4 30 0.23

As shown in Table 7 above, the strain introduced with a vector capable of expressing wild-type biotin synthase (E. coli CV04-0002/pCL1920-Pn-bioB(Eco)) showed a slight increase in biotin production compared to the strain introduced with the empty vector.

Example 2-2. Production of expression vector of biotin operon containing E. coli-derived biotin synthase and production of mutant strain

To determine whether mutation of the bioB gene can have a greater effect on increasing biotin production in a biotin biosynthesis enhanced vector containing the entire biotin operon, an overexpression vector was constructed using the autologous promoter of the bioABFCD gene of the E. coli biotin operon based on the pCL1920 vector (GenBank No AB236930) to express only the protein encoded by the bioABFCD bioB gene. To construct the vector, the chromosome of E. coli W3110 strain was used as a template and PCR was performed using the primer pair of SEQ ID NO: 18 and SEQ ID NO: 19 to obtain the wild-type bioABFCD operon gene without mutation. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as a polymerase for the PCR reaction, and the PCR conditions were as follows: denaturation, 95°C, 30 sec; annealing, 55°C, 30 sec; And after repeating the polymerization reaction at 72℃ for 6 minutes 25 times, the polymerization reaction was performed at 72℃ for 10 minutes. As a result, a wild-type fragment of 5020 bp bioABFCD (Eco WT) (SEQ ID NO: 20) DNA fragment was obtained. The obtained DNA product was purified using QIAGEN's PCR Purification kit and then cloned using pCL1920 vector treated with SmaI restriction enzyme and TaKaRa's Infusion Cloning Kit, thereby constructing two types of bioABFCD coding protein wild-type overexpression vectors pCL1920-Pn-bioABFCD (Eco).

Name Sequence (5'->3') Sequence number VB07_0009 GACTCTAGAGGATCCCCgggTTATTGGCAAAAAAAATGTTT Sequence number 18 VB07_0010 ATTCGAGCTCGGGTACCCgggCTACAACAAGGCAAGGTTTA Sequence number 19

To confirm whether biotin production ability increased, the two types of bioABFCD bioB gene overexpression vectors produced above (wild-type overexpression vector pCL1920-Pn-bioABFCD (Eco)) were introduced into E. coli strain CV04-0002 (Example 1-1) that produces small amounts of biotin. As a control, the pCL1920 vector was introduced into the CV04-0002 strain.

Each strain was spread on LB solid medium and cultured overnight in an incubator at 30°C. The strain cultured overnight on the LB solid medium was inoculated into 25 ml of a titer medium having the composition of Table 2, which was then cultured for 48 hours in an incubator at 30°C and 200 rpm, and the amount of biotin produced in the culture medium was measured by the MS-MS method, and the results are shown in Table 9.

Strain name OD562nm Consumption (g/L) Biotin (mg/L) E. coli CV04-0002/pCL1920 32.8 30 0.2 E.coli CV04-0002/pCL1920-Pn-bioABFCD(Eco) 33.1 30 1.3.

As shown in Table 9 above, the strain overexpressing the wild-type biotin operon (E. coli CV04-0002/pCL1920-Pn-bioABFCD(Eco)) showed increased biotin production compared to the control group (E. coli CV04-0002/pCL1920).

Example 3. Production of expression vector of biotin operon including biotin synthase mutant based on high-producing biotin strain and production of mutant strain

The three biotin biosynthesis operon-enhanced vectors pCL1920-Pn-bioABFCD(S.mar), pCL1920-Pn-bioAB(A3T, S.mar)FCD, and pCL1920-Pn-bioABFCD(Eco) constructed in Examples 1 and 2 were introduced into the CV04-0104 strain, a high-producing biotin strain with an additional introduction of the E. coli biotin biosynthesis operon. The CV04-0104 strain, a high-producing biotin strain, is a strain with an additional insertion of one copy of the E. coli biotin biosynthesis operon into the insAB gene locus based on the CV04-0002 strain, and was constructed through the following process.

To construct a vector for producing a high-producing biotin strain, E. coli W3110 was used as a template to obtain the insA upstream and downstream regions and a biotin operon fragment. Specifically, through PCR, using E. coli W3110 chromosomal DNA as a template, a gene fragment of about 0.5 kb (SEQ ID NO: 25) in the upstream region was obtained using the primers VB07_0011 (SEQ ID NO: 23) and VB07_0012 (SEQ ID NO: 24), a gene fragment of about 5 kb (SEQ ID NO: 28) in the biotin operon region was obtained using the primers VB07_0013 (SEQ ID NO: 26) and VB07_0014 (SEQ ID NO: 27), and a gene fragment of about 0.5 kb (SEQ ID NO: 31) in the downstream region was obtained using the primers VB07_0015 (SEQ ID NO: 29) and VB07_0016 (SEQ ID NO: 30). SolgTM Pfu-X DNA polymerase was used as the polymerase for PCR reaction, and the PCR amplification conditions were as follows: denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 60 seconds, and polymerization at 72°C for 30 seconds, and then polymerization at 72°C for 5 minutes to obtain each fragment as a PCR product. The obtained PCR products were purified using QIAGEN's PCR Purification kit, and then vectors were constructed using the upstream and downstream of the amplified insAB gene and the biotin operon fragment. According to the method described in PCT registered patent WO2020032590A1, the gene replacement vector pSKH was used to cut the PCR product with EcoRⅤ restriction enzyme, and the prepared PCR product was cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to produce a biotin operon 1 copy addition vector pSKHΔinsAB::Pn-bioABFCD. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment and then storing it at 50°C for 1 hour.

The vector pSKHΔinsAB::Pn-bioABFCD constructed above was introduced into the CV04-0002 strain (Example 1-1) via electroporation to construct the CV04-0104 strain, a biotin biosynthesis-enhanced strain with an added biotin operon.

The primer sequences used are shown in Table 10 below:

Name Sequence (5'->3') Sequence number VB07_0011 gggcTGCAGGAATTCGATCCAGATACGCTTTTTCCA Sequence number 23 VB07_0012 AAACATTTTTTTGCCAATAACACGCAAGCACCTTAAAATCAC Sequence number 24 VB07_0013 GTGATTTTAAGGTGCTTGCGTGTTATTGGCAAAAAAAATGTTT Sequence number 26 VB07_0014 TCAGATAATGCCCGATGACCCTACAACAAGGCAAGGTTTAT Sequence number 27 VB07_0015 ATAAACCTTGCCTTGTTGTAGGGGTCATCGGGCATTATCTGA Sequence number 29 VB07_0016 GCTAGGTCGACTAGCGTGATGCCTTTTTTGTCGTGGGT Sequence number 30

The three biotin biosynthesis operon-enhanced vectors pCL1920-Pn-bioABFCD(S.mar), pCL1920-Pn-bioAB(A3T, S.mar)FCD, and pCL1920-Pn-bioABFCD(Eco) constructed in Examples 1 and 2 were introduced into the prepared CV04-0104 strain using the heat shock introduction method, respectively, to construct E. coli CV04-0104/pCL1920-Pn-bioABFCD(S.mar), E. coli CV04-0104/pCL1920-Pn-bioAB(A3T, S.mar)FCD, and E. coli CV04-0104/pCL1920-Pn-bioABFCD(Eco), respectively. As a control, E. coli CV04-0104/pCL1920, in which pCL1920 was introduced into the CV04-0104 strain, was prepared.

Each strain was spread on LB solid medium and cultured overnight in an incubator at 30°C. The strain cultured overnight on the LB solid medium was inoculated into 25 ml of a titer medium having the composition of Table 2, which was then cultured in an incubator at 30°C and 200 rpm for 40 hours, and the biotin production of each strain was measured using the MS-MS technique, and the results are shown in Table 11.

Strain name OD562nm Consumption (g/L) Biotin (mg/L) E. coli CV04-0104/pCL1920 36.7 30 1.5 E.coli CV04-0104/pCL1920-Pn- bioABFCD (S.mar) 36.1 30 4.5 E.coli CV04-0104/pCL1920-Pn- bioAB(A3T, S.mar)FCD 35.4 30 7.1 E.coli CV04-0104/pCL1920-Pn- bioABFCD(Eco) 36.5 30 4.3

As can be seen in Table 11 above, the biotin concentrations of all strains introduced with the biotin biosynthesis operon derived from Serratia marcescens or E. coli were significantly improved compared to the control group (E. coli CV04-0104/pCL1920).

In particular, the biotin production of E. coli CV04-0104/ pCL1920-Pn-bioAB(A3T, S.mar)FCD, in which an A3T mutation was introduced into biotin synthase from Serratia marcescens, increased compared to that of the E. coli CV04-0104/ pCL1920-Pn-bioABFCD(S.mar) strain in which the mutation was not introduced, and this was improved compared to the biotin production of the E. coli CV04-0104/pCL1920-Pn-bioABFCD(Eco) strain in which the biotin biosynthesis operon from E. coli was enhanced.

<110> CJ CheilJedang Corporation <120> Polypeptide variant having biotin synthase activity and biotin production method using the same <130> DPP20205278KR <160> 43 <170> koPatentIn 3.0 <210> 1 <211> 346 <212> PRT <213> Artificial Sequence <220> <223> wild-type biotin synthase (BioB) of Serratia marcescens <400> 1 Met Met Ala Asp Arg Ile His Trp Thr Val Gly Gln Ala Gln Ala Leu 1 5 10 15 Phe Asp Lys Pro Leu Leu Glu Leu Leu Phe Glu Ala Gln Thr Val His 20 25 30 Arg Gln His Phe Asp Pro Arg Gln Val Gln Val Ser Thr Leu Leu Ser 35 40 45 Ile Lys Thr Gly Ala Cys Pro Glu Asp Cys Lys Tyr Cys Pro Gln Ser 50 55 60 Ser Arg Tyr Lys Thr Gly Leu Glu Ser Glu Arg Leu Met Gln Val Glu 65 70 75 80 Gln Val Leu Glu Ser Ala Arg Lys Ala Lys Ala Asn Gly Ser Thr Arg 85 90 95 Phe Cys Met Gly Ala Ala Trp Lys Asn Pro His Glu Arg Asp Met Pro 100 105 110 Tyr Leu Gln Gln Met Val Gln Gly Val Lys Ala Met Gly Met Glu Thr 115 120 125 Cys Met Thr Leu Gly Thr Leu Asp Gly Thr Gln Ala Glu Arg Leu Ala 130 135 140 Glu Ala Gly Leu Asp Tyr Tyr Asn His Asn Leu Asp Thr Ser Pro Glu 145 150 155 160 Phe Tyr Gly Ser Ile Ile Thr Thr Arg Ser Tyr Gln Glu Arg Leu Asp 165 170 175 Thr Leu Asp Lys Val Arg Asp Ala Gly Ile Lys Val Cys Ser Gly Gly 180 185 190 Ile Val Gly Leu Gly Glu Thr Val Arg Asp Arg Ala Gly Leu Leu Val 195 200 205 Gln Leu Ala Asn Leu Pro Lys Pro Pro Glu Ser Val Pro Ile Asn Met 210 215 220 Leu Val Lys Val Lys Gly Thr Pro Leu Ala Asp Asn Asp Asp Val Asp 225 230 235 240 Pro Phe Asp Phe Ile Arg Thr Ile Ala Val Ala Arg Ile Met Met Pro 245 250 255 Ser Ser Tyr Val Arg Leu Ser Ala Gly Arg Glu Gln Met Asn Glu Gln 260 265 270 Thr Gln Ala Met Cys Phe Met Ala Gly Ala Asn Ser Ile Phe Tyr Gly 275 280 285 Cys Lys Leu Leu Thr Thr Pro Asn Pro Glu Glu Asp Lys Asp Leu Gln 290 295 300 Leu Phe Arg Lys Leu Gly Leu Asn Pro Gln Gln Thr Ala Thr Glu His 305 310 315 320 Gly Asp Asn Gln Gln Gln Gln Val Leu Ala Lys Gln Leu Leu Asn Ala 325 330 335 Asp Thr Ala Glu Phe Tyr Asn Ala Ala Leu 340 345 <210> 2 <211> 1041 <212> DNA <213> Artificial Sequence <220> <223> gene encoding wild-type biotin synthase (BioB) of Serratia marcescens <400> 2 atgatggccg accgcattca ctggacagta gggcaagccc aggccctgtt tgataaaccg 60 ctgctggaac tgctgttcga agcgcaaacc gtacaccgcc agcacttcga cccgcgtcag 120 gtgcaggtca gcacgctgct gtcgatcaag accggcgctt g cccggaaga ctgcaaatac 180 tgcccgcaga gctcacgcta caagaccggc ctggagtcgg agcggctgat gcaggtcgag 240 caggtgctgg aatcggcacg caaggccaag gcgaacggtt cgacccgttt ttgcatgggc 300 gcggcgtgga agaacccgca cgagcgcgat atgccttatc tgcagcaaat ggtgcagg gc 360 gtgaaagcga tgggcatgga aacctgcatg acgctgggca cattggatgg cacccaggcc 420 gagcggctgg cggaggccgg gctggattac tacaaccata acctcgacac ctcgccggag 480 ttctacggca gcatcatcac cacccgcagc taccaggagc gcctggatac gctcgacaag 540 gtgcgcgacg ccggcatcaa agtgtgctcc ggcggcatcg tcgggctggg tgaaacggtg 600 cgcgatcgcg ccgggctgct ggtgcagctg gccaacctgc caaaaccacc ggagagcgtg 660 ccgatcaaca tgttggtgaa ggt gaaaggc acgccgctgg cggataacga tgacgtcgat 720 ccgtttgatt tcatccgcac catcgcggtg gcgcgcatca tgatgccatc ttcttatgtc 780 cgtctctccg caggccgcga acagatgaac gaacagacgc aggcgatgtg cttcatggcc 840 ggcgccaact cgatcttcta cggttgcaag ctgctgacca cgccgaatcc ggaagaagac 900 aaagacctgc agctgttccg caagctgggg ctcaacccgc agcagaccgc aaccgaacac 960 ggcgacaacc agcaacagca ggtgctggcc aagcaactgc tgaacgccga taccgccga g 1020 ttttacaacg cggcgccgtg a 1041 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0001 < 400> 3 gactctagag gatccccggg tgttgtaaac caaattggat 40 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0002 <400> 4 attcgagctc ggtacccggg tcacggcgcc gcgttgtaaa 40 <210> 5 <211> 41 <212> DNA <213> Artificial Sequence <220> < 223> primer_VB07_0003 <400> 5 tgtccagtga atgcggtcgg tcatcatggc gtctccaaaa c 41 <210> 6 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0004 <400> 6 gttttggaga cgccat gatg accgaccgca ttcactggac a 41 <210> 7 <211> 1166 <212> DNA <213> Artificial Sequence <220> <223> wild-type bioB gene fragment of Serratia marcescens (1166bp), wherein atg at positions 106-108 is a start codon for encoding SEQ ID NO: 1 <400> 7 gactctagag gatccccggg tgttgtaaac caaattggat taaaattggt tgacagtata 60 tccacaatat ttaaactggc gacacttttt cgttttggag acgccatgat ggccgaccgc 120 attcactgga cagtagggca agcccaggcc ctgtt tgata aaccgctgct ggaactgctg 180 ttcgaagcgc aaaccgtaca ccgccagcac ttcgacccgc gtcaggtgca ggtcagcacg 240 ctgctgtcga tcaagaccgg cgcttgcccg gaagactgca aatactgccc gcagagctca 300 cgctacaaga ccggcctgga gtcggagcgg ctgatgcagg ggt gctggaatcg 360 gcacgcaagg ccaaggcgaa cggttcgacc cgtttttgca tgggcgcggc gtggaagaac 420 ccgcacgagc gcgatatgcc ttatctgcag caaatggtgc agggcgtgaa agcgatgggc 480 atggaaacct gcatgacgct gggcacattg gatggcaccc aggccgagcg gctggcggag 540 gccgggctgg attactacaa ccataacctc gacacctcgc cggagttcta cggcagcatc 600 atcaccaccc gcagctacca ggagcgcctg gatacgctcg acaaggtgcg cgacgccggc 660 atcaaag tgt gctccggcgg catcgtcggg ctgggtgaaa cggtgcgcga tcgcgccggg 720 ctgctggtgc agctggccaa cctgccaaaa ccaccggaga gcgtgccgat caacatgttg 780 gtgaaggtga aaggcacgcc gctggcggat aacgatgacg tcgatccgtt tgatttcatc 840 cgcaccatcg cggtggcgcg catcatgatg ccatcttctt atgtccgtct ctccgcaggc 900 cgcgaacaga tgaacgaaca gacgcaggcg atgtgcttca tggccggcgc caactcgatc 960 ttctacggtt gcaagctgct gaccacgccg aatccggaag a agacaaaga cctgcagctg 1020 ttccgcaagc tggggctcaa cccgcagcag accgcaaccg aacacggcga caaccagcaa 1080 cagcaggtgc tggccaagca actgctgaac gccgataccg ccgagtttta caacgcggcg 1140 ccgtgacccg ggtaccgagc tcgaat 1166 <210> 8 <211> 132 <212> DNA <213> Artificial Sequence <220> <223> bioB gene mutant (g7a) fragment of Serratia marcescens (132bp), wherein atg at positions 106-108 corresponds to the start codon of SEQ ID NO: 7 <400> 8 gactctagag gatccccggg tgttgtaaac caaattggat taaaattggt tgacagtata 60 tccacaatat ttaaactggc gacacttttt cgttttggag acgccatgat gaccgaccgc 120 attcactgga ca 132 <210> 9 <211 > 1075 <212> DNA <213> Artificial Sequence <220> <223> bioB gene mutant (g7a) fragment of Serratia marcescens (1075bp), wherein atg at positions 15-17 corresponds to the start codon of SEQ ID NO: 7 <400> 9 gttttggaga cgccatgatg accgaccgca ttcactggac agtagggcaa gcccaggccc 60 tgtttgataa accgctgctg gaactgctgt tcgaagcgca aaccgtacac cgccagcact 120 tcgacccgcg tcaggtgcag gtcagcacgc tgctgtcgat caagaccggc gcttgcccgg 180 aagactgcaa atactgcccg cagagctcac gctacaagac cggcctggag tcggagcggc 240 tgatgcaggt cgagcaggtg ctggaatcgg cacgcaaggc caaggcgaac ggttcgaccc 300 gtttttgcat gggcgcggcg tggaagaacc cgcacgagcg cgatatgcct tatctgcagc 360 aaatggtgca gggcgtgaaa gcgatgggca tggaaac ctg catgacgctg ggcacattgg 420 atggcaccca ggccgagcgg ctggcggagg ccgggctgga ttactacaac cataacctcg 480 acacctcgcc ggagttctac ggcagcatca tcaccacccg cagctaccag gagcgcctgg 540 atacgctcga caaggtgcgc gacgccggca tcaaagtgtg ctccggcggc atcgtcgggc 600 tgggtgaaac ggtgcgcgat cgcgccgggc tgctggtgca gctggccaac ctgccaaaac 660 caccggagag cgtgccgatc aacatgttgg tgaaggtgaa aggcacgccg ctggcggata 720 acgatgacgt cgatccgttt gatttcatcc gcaccatcgc ggtggcgcgc atcatgatgc 780 catcttctta tgtccgtctc tccgcaggcc gcgaacagat gaacgaacag acgcaggcga 840 tgtgcttcat ggccggcgcc aactcgatct tctacggttg caagctgctg accacgccga 900 atccggaaga agacaaagac ctgcagctgt tccgcaagct ggggctcaac ccgcagcaga 960 ccgcaaccga acacggcgac aaccagcaac agcaggtgct ggccaagcaa ctgctgaacg 1020 ccgataccgc cgagttttac aacgcggcgc acccgg gtaccgagct cgaat 1075 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0005 <400> 10 gactctagag gatccccggg ttaacgcgcg gcgtcggcca 40 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0006 <400> 11 attcgagctc ggtacccggg tcactgcgcc agcaagctga 40 <210> 12 <211> 5016 <212> DNA <213> Artificial Sequence <220> <223> FCD( Wt) fragment of Serratia marcescens (5016bp) <400> 12 gactctagag gatccccggg ttaacgcgcg gcgtcggcca cggcggcggt caggcggctg 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 tcgatggcgc tcacctgcgc ctg ccagcga 300 ttttccgcca gcagcgccag actggcgtcc gccacggcgc aggccagcgg attgcccata 360 aaggtcggcc cgtgcataaa gcagccggcc gcgccgttgc tgatggtctc cgccacgtgg 420 cgggtagtca aggtggcgga aagggtcata tagccgccgg tcagcgcctt gcccagacag 480 agaatgtccg gcaccacctg cgcgtgctcg caggcgaaca gcttgccggt gcggccgaaa 540 ccggtggcga tctcgtcggc gatcagcagc acctgatggc gatcgcacag ctcgcgcacc 600 cgcttgagat aggtcgg atg gtagatgcgc atgccgccgg cgccttgcac caccggttcc 660 agaatcaccg ccgccacttc accggcgtgc tgctccagca gcgcggcgaa cggcgcgata 720 tcctcttcac gccattcctc gtcgaagcgg cactgcggcg cggtggcgaa caggtgcggc 780 gccagatacc cctgatagag gctgtgcatc gagttgtccg gatcgcagac cgacatcgcg 840 ccgaaggtat cgccgtgata gccgtggcgc agcgtcagga tgcgctgccg gcgctcgccg 900 cgcgcctgcc agtactgcag cgccattttc agcgac actt ctaccgccac cgaaccagag 960 tccgccagga acacgcactg cagtgcttcc ggcgtcattt ccaccagccg acggcacaac 1020 gagatggcgg ccggatgggt aatgccgccg aacatcacgt gcgacatctt ctccaactgc 1080 tggctggcgg cctgattcag acgcggat gg ttgtaaccgt ggatcgccgc ccaccaggag 1140 gacatgccgt ccaccagact ccggccgtcc gccagctgca gttcgacgcc gctggccgat 1200 tcgatcgggt aacagggtaa cgggcggctc atggaggtgt aggggtgcca gatatggcgt 1260 tggtcaaac g ccaggtcgga agcggtgaca gacattgttg taaaccaaat tggattaaaa 1320 ttggttgaca gtatatccac aatatttaaa ctggcgacac tttttcgttt tggagacgcc 1380 atgatggccg accgcattca ctggacagta gggcaagccc aggccctgtt tgataaaccg 144 0 ctgctggaac tgctgttcga agcgcaaacc gtacaccgcc agcacttcga cccgcgtcag 1500 gtgcaggtca gcacgctgct gtcgatcaag accggcgctt gcccggaaga ctgcaaatac 1560 tgcccgcaga gctcacgcta caagaccggc ctggagtcgg agc ggctgat gcaggtcgag 1620 caggtgctgg aatcggcacg caaggccaag gcgaacggtt cgacccgttt ttgcatgggc 1680 gcggcgtgga agaacccgca cgagcgcgat atgccttatc 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 cgcgatcgcg 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 cgatctt cta cggttgcaag ctgctgacca cgccgaatcc ggaagaagac 2280 aaagacctgc agctgttccg caagctgggg ctcaacccgc agcagaccgc aaccgaacac 2340 ggcgacaacc agcaacagca ggtgctggcc aagcaactgc tgaacgccga taccgccgag 2400 ttttacaacg cggcgccgtg atgagctggc agcaacgcat cgagcaggcg ctggctgagc 2460 ggcgcctgaa cgccgcctac cgccggcgac agaccaccga gggcggcaac ggccgccaga 2520 tccggctcgg cgatcgtctc tatctgaact tctcgggcaa cgactacctg gg cttgagcc 2580 aggatgcgcg 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 cagcacaat c 2880 aaccgcaggc cttggcggat ctgctggcca aaccctgcga cgggcagcgg ctggcggtca 2940 ccgaaggggt gttcagcatg gatggcgacg gcgcgccgtt ggcggagctg catcgcttaa 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 ggcggcgctg gcccgtattc gagagggtga tgatctgcga gcccggctgc 3300 aggacaacat tcggcgtttc cgtcagggcg cggcgccgtt ggcgctgacc ctgacggatt 3360 ccgacaccgc catccagccg ctgctggtgg gggataatca gcgcgcgctc gatctggcga 3420 cccgcctgcg cgagtgcggc ctgtgggtga gcgccatccg tccgccgacg gtgccccgg 3480 gcggcgcgcg gcgcatt accctgacgg cggcgcatca gtcgcaggat atcgatcgcc 3540 tgctggaggt gctgaatgac gtcagccaat gacacagtga acaaacaggc ggtcgcctcg 3600 gccttcagcc gcgcggccgg cagctacgat gccgccgccg cgctgcagcg c 3660 gagcgcttac tggggatggg gagttcccac ccgggcgaac agctgctgga cgccggctgc 3720 ggcaccggct atttcagccg tatgtggcgt gagcgcggca aacgggtgac cgcgctcgat 3780 ttggcgccgg gcatgctgga cgtcgcccgc caacggcagg cggcgcatca ttatctgctg 3840 ggcgatatcg aacaggtgcc 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 agttgcgc ac cctgaactac 4140 ccggacgtga tgacgctgat gcgttcgctc aagggcatcg gggcgacgca tttgcatcag 4200 gggcgtgagg gcggcctgat gtcgcgtggc cgcctcgccg cgctgcaggc ggcttacccg 4260 tgccggcagg gctcagctat catctggctt atggagtgat ttatcgtgag 4320 taaacgttgg ttcgtgaccg gcaccgacac cgaagtgggg aaaaccgtcg ccagcagcgc 4380 cttgctgcag gccgccaacc gggcgggcta ccgcagcgcc ggctataagc cggtggcctc 44 40 cggcagcgag atgaccgccg aggggctgcg caacggcgac gcgctggcgc tgcaggccaa 4500 cagcggtgtg gcgctggatt acgacgaagt gaacccttac gtatttgccg aaccgacctc 4560 gccgcatatc gtcagcgccg atgaaggccg gccgatcgac gcggcgcggc tgtccgacgg 4620 cctgcgccgg ctggagcagc gcgccgactg ggtgctggtc gagggggccg gcgggtggtt 4680 taccccgttg tcggcggagt acaccttcgc cgactgggtg cggcaagagc aactgccggt 4740 gatcctggtg gcatca 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 4980 cttgctggcg cagtgacccg ggtaccgagc tcgaat 5016 <210> 13 <211> 1407 <212> DNA <220> Artificial Sequence <220> 223> bioAB(g7a)FCD mutant fragment of Serratia marcescens (1407bp) < 400> 13 gactctagag gatccccggg ttaacgcgcg gcgtcggcca cggcggcggt caggcggctg 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 tcgatggcgc cctgcgc ctgccagcga 300 ttttccgcca gcagcgccag actggcgtcc gccacggcgc aggccagcgg attgcccata 360 aaggtcggcc cgtgcataaa gcagccggcc gcgccgttgc tgatggtctc cgccacgtgg 420 cgggtagtca aggtggcgga aagggtcata tagccgccgg tcagcgcctt gcccagacag 480 agaatgtccg gcaccacctg cgcgtgctcg caggcgaaca gcttgccggt gcggccgaaa 540 ccggtggcga tctcgtcggc gatcagcagc acctgatggc gatcgcacag ctcgcgcacc 600 cg cttgagat aggtcggatg gtagatgcgc atgccgccgg cgccttgcac caccggttcc 660 agaatcaccg ccgccacttc accggcgtgc tgctccagca gcgcggcgaa cggcgcgata 720 tcctcttcac gccattcctc gtcgaagcgg cactgcggcg cggtggcgaa caggtgcggc 780 gccagatacc cctgatagag gctgtgcatc gagttgtccg gatcgcagac cgacatcgcg 840 ccgaaggtat cgccgtgata gccgtggcgc agcgtcagga tgcgctgccg gcgctcgccg 900 cgcgcctgcc agtactgcag cgccattt tc agcgacactt ctaccgccac cgaaccagag 960 tccgccagga acacgcactg cagtgcttcc ggcgtcattt ccaccagccg acggcacaac 1020 gagatggcgg ccggatgggt aatgccgccg aacatcacgt gcgacatctt ctccaactgc 1080 tggctggcgg g acgcggatgg ttgtaaccgt ggatcgccgc ccaccaggag 1140 gacatgccgt ccaccagact ccggccgtcc gccagctgca gttcgacgcc gctggccgat 1200 tcgatcgggt aacagggtaa cgggcggctc atggaggtgt aggggtgcca gatatggcgt 126 0 tggtcaaacg ccaggtcgga agcggtgaca gacattgttg taaaccaaat tggattaaaa 1320 ttggttgaca gtatatccac aatatttaaa ctggcgacac tttttcgttt tggagacgcc 1380 atgatgaccg accgcattca ctggaca 1407 <210> 14 <211 > 3650 <212> DNA <213> Artificial Sequence <220> <223> bioAB(g7a)FCD mutant fragment of Serratia marcescens (3650bp) <400> 14 gttttggaga cgccatgatg accgaccgca ttcactggac agtagggcaa gcccaggccc 60 tgtttgataa accgctgctg gaactgctgt tcgaagcgca aaccgtacac cgccagcact 120 tcgacccgcg tcaggtgcag gtcagcacgc tgctgtcgat caagaccggc gcttgcccgg 180 aagactgcaa atactgcccg cagagctcac gctacaagac cggcctggag tcggagcggc 240 tgatgcaggt cgagcaggtg ctggaatcgg cacgcaaggc caaggcgaac ggttcgaccc 300 gtttttgcat gggcgcggcg tggaagaacc cgcacgagcg cgatatgcct tatctgcagc 360 aaatggtgca gggcgtgaaa gcgatgggca tggaaacctg catgacgctg ggcacattgg 420 atggcaccca ggccgagcgg ctggcggagg ccgggctgga ttactacaac cataacctcg 480 acacctcgcc ggagttctac ggcagcatca tcaccacccg cagctaccag gagcgcctgg 540 atacgctcga caaggtgcgc gacgccggca tcaaagtgtg ctccggcggc atcgtcgggc 6 00 tgggtgaaac ggtgcgcgat cgcgccgggc tgctggtgca gctggccaac ctgccaaaac 660 caccggagag cgtgccgatc aacatgttgg tgaaggtgaa aggcacgccg ctggcggata 720 acgatgacgt cgatccgttt gatttcatcc gcaccatcgc ggtggcgcgc atcatgatgc 780 catcttctta tgtccgtctc tccgcaggcc gcgaacagat gaacgaacag acgcaggcga 840 tgtgcttcat ggccggcgcc aactcgatct tctacggttg caagctgctg accacgccga 900 atccggaaga agacaaagac ctgcagctgt tccgcaagct ggggctcaac ccgcagcaga 960 ccgcaaccga acacggcgac aaccagcaac agcaggtgct ggccaagcaa ctgctgaacg 1020 ccgataccgc cgagttttac aacgcggcgc cgtgatgagc tggcagcaac gcatcgagca 1080 ggcgctggct gagcggcgcc tgaacgccgc ctaccgccgg cgacagacca ccgagggcgg 1140 caacggccgc cagatccggc tcggcgatcg tctctatctg aacttctcgg gcaacgacta 1200 cctgggcttg agccagggatg cgcgggtgat cgccgcctgg cagcagggcg c gcagcgtta 1260 cggcgtcggc agcggcggtt cgggccacgt gaccggtttt agcgcggcgc atcaggcgct 1320 ggaagagcaa ctggcggctt ggctcggcta tccgcgcgcg ctgctgttca tctccggcta 1380 cgccgccaac caggcggtgc tggcggcgtt gatgcaaaag ggcgatcgca ttttggccga 1440 tcgtctcagc catgcctcgc tgctggaggc ggcggcgcag tcgccggccg agctgcgccg 1500 gttccagcac aatcaaccgc aggccttggc ggatctgctg gccaaac cct gcgacgggca 1560 gcggctggcg gtcaccgaag gggtgttcag catggatggc gacggcgcgc cgttggcgga 1620 gctgcatcgc ttaacccgtg cggcgggcgc ctggctgatg gtggatgacg cccacggcat 1680 cggcgtgcgc ggcgaacaag gccgcggcag ttgctggcag cagggcgtgc gccctgaact 1740 gctggtggcg accttcggca aggcgttcgg cgtcagcggc gcggcggtgc tgtgcgatga 1800 ggcgaccgcc gagtatctgc tgcagttcgc ccgccatctg atctacagca ccgcgat gcc 1860 gccggcgcag gcctgcgcgc tgcaggcggc gctggcccgt attcgagagg gtgatgatct 1920 gcgagcccgg ctgcaggaca acattcggcg tttccgtcag ggcgcggcgc cgttggcgct 1980 gaccctgacg gattccgaca ccgccatc ca gccgctgctg gtgggggata atcagcgcgc 2040 gctcgatctg gcgacccgcc tgcgcgagtg cggcctgtgg gtgagcgcca tccgtccgcc 2100 gacggtgccc ccgggcggcg cgcggctgcg cattaccctg acggcggcgc atcagtcgca 216 0 ggatatcgat cgcctgctgg aggtgctgaa tgacgtcagc caatgacaca gtgaacaaac 2220 aggcggtcgc ctcggccttc agccgcgcgg ccggcagcta cgatgccgcc gccgcgctgc 2280 agcgtgacgt tggcgagcgc ttactgggga tggggagttc ccacccgggc gaacagctgc 2340 tggacgccgg ctgcggcacc ggctatttca gccgtatgtg gcgtgagcgc ggcaaacggg 2400 tgaccgcgct cgatttggcg ccgggcatgc tggacgtcgc ccgccaacgg caggcggcgc 2460 at cattatct gctgggcgat atcgaacagg tgccgctgcc cgatgcggcg atggacatct 2520 gtttcagcag cctggtggtg cagtggtgca gcgatctgcc tgccgcgctg gcagagctgt 2580 atcgcgtgac ccgtcccggc ggcgtgatcc tgttttccac gctggcggcg ggctcgctgc 2640 aggaattggg ggacgcctgg caacaggtgg acggcgaacg tcacgtgaac gcctttttgc 2700 cgttgacgca gatccgcacc gcctgcgccg cctatcgaca cgagctggtg acggagttgc 2760 gcaccctgaa ct acccggac gtgatgacgc tgatgcgttc gctcaagggc atcggggcga 2820 cgcatttgca tcaggggcgt gagggcggcc tgatgtcgcg tggccgcctc gccgcgctgc 2880 aggcggctta cccgtgccgg caggggcagt tcccgctcag ctatcatctg gcttatggag 2940 tgatttatcg tgagtaaacg ttggttcgtg accggcaccg acaccgaagt ggggaaaacc 3000 gtcgccagca gcgccttgct gcaggccgcc aaccgggcgg gctaccgcag cgccggctat 3060 aagccggtgg cctccggcag cgagatgacc gcc gaggggc tgcgcaacgg cgacgcgctg 3120 gcgctgcagg ccaacagcgg tgtggcgctg gattacgacg aagtgaaccc ttacgtattt 3180 gccgaaccga cctcgccgca tatcgtcagc gccgatgaag gccggccgat cgacgcggcg 3240 cggctgtccg acggcctgcg ccggctggag cagcgcgccg actgggtgct ggtcgagggg 3300 gccggcgggt ggtttacccc gttgtcggcg gagtacacct tcgccgactg ggtgcggcaa 3360 gagcaactgc cggtgatcct ggtggtgggc atcaagctgg gctgcatcaa ccacgc ggtg 3420 ctgacggccc aggcggtgca acaggccggg ctgacgctgg ccggttggat cgccaacgac 3480 gtggcgccgc cggggcggcg gcatcaggaa tacctggcta cgctgcgccg tatgctgccc 3540 gcgccgctgc tgggcgaaat cccgcacctg ccgcaggccg aacgggcgcc gctggggcag 3600 tatctggata tcagcttgct ggcgcagtga cccgggtacc gagctcgaat 3650 <210> 15 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0007 < 400> 15 gactctagg gatccccggg aatcgacttg taaaccaaat 40 <210> 16 <211 > 40 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0008 <400> 16 attcgagctc ggtacccggg tcataatgct gccgcgttgt 40 <210> 17 <211> 1167 <212> DNA <213> Artificial Sequence <220> <223> wild-type bioB fragment of E. coli (1167bp) <400> 17 gactctagag gatccccggg aatcgacttg taaaccaaat tgaaaagatt taggtttaca 60 agtctacacc gaattaacaa caaa aaacac gttttggaga agccccatgg ctcaccgccc 120 acgctggaca ttgtcgcaag tcacagaatt atttgaaaaa ccgttgctgg atctgctgtt 180 tgaagcgcag caggtgcatc gccagcattt cgatcctcgt caggtgcagg tcagcacgtt 240 gctgtcgatt aagaccggag cttgtccgga agattgcaaa tactgcccgc aaagctcgcg 300 ctacaaaacc gggctggaag ccgagcggtt gatggaagtt gaacaggtgc tggagtcggc 360 gcgcaaagcg aaagcggcag gatcgacgcg cttctgtatg ggcgcggcgt ggaagaatcc 420 ccacgaacgc gatatgccgt acctggaaca aatggtgcag ggggtaaaag cgatggggct 480 ggaggcgtgt atgacgctgg gcacgttgag tgaatctcag gcgcagcgcc tcgcgaacgc 540 cgggctggat tactacaacc acaacctgg a cacctcgccg gagttttacg gcaatatcat 600 caccacacgc acttatcagg aacgcctcga tacgctggaa aaagtgcgcg atgccgggat 660 caaagtctgt tctggcggca ttgtgggctt aggcgaaacg gtaaaagatc gcgccggatt 720 attgctgcaa ctggcaaacc t gccgacgcc gccggaaagc gtgccaatca acatgctggt 780 gaaggtgaaa ggcacgccgc ttgccgataa cgatgatgtc gatgcctttg attttattcg 840 caccattgcg gtcgcgcgga tcatgatgcc aacctcttac gtgcgccttt ctgccggacg 900 cgagcagatg aacgaacaga ctcaggcgat gtgctttatg gcaggcgcaa actcgatttt 960 ctacggttgc aaactgctga ccacgccgaa tccggaagaa gataaagacc tgcaactgtt 1020 ccgcaaactg gggctaaatc cgcagcaaac tgccgt gctg gcaggggata acgaacaaca 1080 gcaacgtctt gaacaggcgc tgatgacccc ggacaccgac gaatattaca acgcggcagc 1140 attatgaccc gggtaccgag ctcgaat 1167 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0009 <400> 18 gactctagag gatccccggg ttattggcaa aaaaatgttt 40 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0010 <400> 19 gctc ggtacccggg ctacaacaag gcaaggttta 40 < 210> 20 <211> 5020 <212> DNA <213> Artificial Sequence <220> <223> wold-type bioABFCD(Eco) fragment of E. coli (5020bp) <400> 20 gactctagag gatccccggg ttattggcaa aaaaatgttt catcctgtac cgcgcggtta 60 accgctgcgg tcagacgctg caactgttgc gggagaataa tatagggcgg catcaggtaa 120 atcagtttgc caaaaggccg gatccagaca ccctgttcga caaagaattt ttgcagcgcc 180 gccatattca ccggatgagt ggtttcgacc acgccaatgg cccccagtac gcgcacatcg 240 gcaaccattt cggcatcacg ggcgggggca agttgctcgc gcagctgtac ttcaatatcc 300 gccacctgtt gctgccagtc gccagattcg agaatcgcca ggctggcgtt tgctgccgcg 360 caggccagcg gattgcccat aaaagttggc ccatgcataa agcaaccggc ttcaccgtta 420 ctgatggttt ctgcaacctc gcgcgtggtg agtgtggcgg aaagggtcat tgtgccgccg 480 gttaaggctt taccgaggca caaaatgtcc ggcgcgattt ctgcatgttc acaggcaaac 540 a gtttcccgg tacgaccaaa tccagtggcg atctcgtcgg caatcagcaa gataccttcg 600 cgatcgcata ttttgcggat tcgttttaac cattccggat ggtacatgcg catcccgcct 660 gcgccctgga caatcggctc aatgatcacc gccgcgattt catgacgatg cgccgccatc 720 aggcgggcaa agcccaccat atcgcgctca tcccattcgc catccatgcg gctttgcggg 780 gcgggagcaa acaggttttc tggcaggtag cctttccaca gactgtgcat tgagttatcc 840 ggatcgcaca ccgacatcgc gccaaaggta tcgc catgat aaccattgcg gaaggtcaga 900 aaacgctggc gcgcttcgcc tttggcttgc cagtactgca acgccatttt catcgccact 960 tccaccgcta cggaaccgga gtccgcgaga aaaacgcact ccagcggttg cggcgtcatc 1020 gccaccagtt tgcggcacag ctcaatggct ggcgcatggg tgataccgcc aaacatcaca 1080 tgcgacatgg catcaatttg cgacttcatc gccgcattaa gctgcgggtg attgtagccg 1140 tggatcgccg cccaccagga cgacataccg tcaaccaggc gtctgccgtc agacaaaatc 1200 agctc gcaac cttcggcgct caccaccgga taaaccggca gaggggaggt catggatgtg 1260 tatgggtgcc agatatggcg ttggtcaaag gcaagatcgt ccgttgtcat aatcgacttg 1320 taaaccaaat tgaaaagatt taggtttaca agtctacacc gaattaacaa caaaaaacac 1380 gttttggaga agccccatgg ctcaccgccc acgctggaca ttgtcgcaag tcacagaatt 1440 atttgaaaaa ccgttgctgg atctgctgtt tgaagcgcag caggtgcatc gccagcattt 1500 cgatcctcgt caggtgcagg tcagcacgtt g ctgtcgatt aagaccggag cttgtccgga 1560 agattgcaaa tactgcccgc aaagctcgcg ctacaaaacc gggctggaag ccgagcggtt 1620 gatggaagtt gaacaggtgc tggagtcggc gcgcaaagcg aaagcggcag gatcgacgcg 1680 cttctgtatg ggcgcggcgt ggaagaatcc ccacgaacgc gatatgccgt acctggaaca 1740 aatggtgcag ggggtaaaag cgatggggct ggaggcgtgt atgacgctgg gcacgttgag 1800 tgaatctcag gcgcagcgcc tcgcgaacgc cgggctggat tact acaacc acaacctgga 1860 cacctcgccg gagttttacg gcaatatcat caccacacgc acttatcagg aacgcctcga 1920 tacgctggaa aaagtgcgcg atgccgggat caaagtctgt tctggcggca ttgtgggctt 1980 aggcgaaacg gtaaaagatc gcgccggatt attgctgcaa ctggcaaacc tgccgacgcc 2040 gccggaaagc gtgccaatca acatgctggt gaaggtgaaa ggcacgccgc ttgccgataa 2100 cgatgatgtc gatgcctttg attttattcg caccattgcg gtcgcgcgga tcatgatgcc 2160 aacct cttac gtgcgccttt ctgccggacg cgagcagatg aacgaacaga ctcaggcgat 2220 gtgctttatg gcaggcgcaa actcgatttt ctacggttgc aaactgctga ccacgccgaa 2280 tccggaagaa gataaagacc tgcaactgtt ccgcaaactg gggctaaatc cgcagcaaac 2340 tgccgtgctg gcaggggata acgaacaaca gcaacgtctt gaacaggcgc tgatgacccc 2400 ggacaccgac gaatattaca acgcggcagc attatgagct ggcaggagaa aatcaacgcg 2460 gcgctcgatg cgcggcgtgc tgccgatgcc ctgcgtcgcc gttatccggt ggcgcaagga 2520 gccggacgct ggctggtggc ggatgatcgc cagtatctga acttttccag taacgattat 2580 ttaggtttaa gccatcatcc gcaaattatc cgtgcctggc agcagggggc ggagcaattt 2640 ggcatcggta gcggcggctc cggtcacgtc agcggttata gcgtggtgca tcaggcactg 2700 gaagaagagc tggccgagtg gcttggctat tcgcgggcac tgctgtttat ctctggtttc 2760 gccgctaatc aggcagttat tgccgcgatg atggcgaaag aggac cgtat tgctgccgac 2820 cggcttagcc atgcctcatt gctggaagct gccagtttaa gcccgtcgca gcttcgccgt 2880 tttgctcata acgatgtcac tcatttggcg cgattgcttg cttccccctg tccggggcag 2940 caaatggtgg tgacagaagg cgtgttcagc atggacggcg atagtgcgcc actggcggaa 3000 atccagcagg taacgcaaca gcacaatggc tggttgatgg tcgatgatgc ccacggcacg 3060 ggcgttatcg gggagcaggg gcgcggcagc tgctggctgc aaaaggtaaa accagaattg 3120 ct ggtagtga cttttggcaa aggatttggc gtcagcgggg cagcggtgct ttgctccagt 3180 acggtggcgg attatctgct gcaattcgcc cgccacctta tctacagcac cagtatgccg 3240 cccgctcagg cgcaggcatt acgtgcgtcg ctggcggtca ttcgcagtga tgagggtgat 3300 gcacggcgcg aaaaactggc ggcactcatt acgcgttttc gtgccggagt acaggatttg 3360 ccgtttacgc ttgctgattc atgcagcgcc atccagccat tgattgtcgg tgataacagc 3420 cgtgcgttac aact ggcaga aaaactgcgt cagcaaggct gctgggtcac ggcgattcgc 3480 ccgccaaccg tacccgctgg tactgcgcga ctgcgcttaa cgctaaccgc tgcgcatgaa 3540 atgcaggata tcgaccgtct gctggaggtg ctgcatggca acggttaata aacaagccat 3600 tgcagcggca tttggtcggg cagccgcaca ctatgagcaa catgcagatc tacagcgcca 3660 gagtgctgac gccttactgg caatgcttcc acagcgtaaa tacacccacg tactggacgc 3720 gggttgtgga cctggctgga tgagccgcca ctggcgggaa cgt cacgcgc aggtgacggc 3780 cttagatctc tcgccgccaa tgcttgttca ggcacgccag aaggatgccg cagaccatta 3840 tctggcggga gatatcgaat ccctgccgtt agcgactgcg acgttcgatc ttgcatggag 3900 caatctcgca gtgcagtggt gcggtaattt atccacggca ctccgcgagc tgtatcgggt 3960 ggtgcgcccc aaaggcgtgg tcgcgtttac cacgctggtg cagggatcgt tacccgaact 4020 gcatcaggcg tggcaggcgg tggacgagcg tccgcatgct aatcgct ttt taccgccaga 4080 tgaaatcgaa cagtcgctga acggcgtgca ttatcaacat catattcagc ccatcacgct 4140 gtggtttgat gatgcgctca gtgccatgcg ttcgctgaaa ggcatcggtg ccacgcatct 4200 tcatgaaggg cgcgacccgc gaatattaac gcgttcgcag ttgcagcgat tgcaactggc 4260 ctggccgcaa cagcaggggc gatatcctct gacgtatcat ctttttttgg gagtgattgc 4320 tcgtgagtaa acgttatttt gtcaccggaa cggataccga agtggggaaa actgt cgcca 4380 gttgtgcact tttacaagcc gcaaaggcag caggctaccg gacggcaggt tataaaccgg 4440 tcgcctctgg cagcgaaaag accccggaag gtttacgcaa tagcgacgcg ctggcgttac 4500 agcgcaacag cagcctgcag ctggattacg caacagtaaa tccttacacc ttcgcagaac 4560 ccacttcgcc gcacatcatc agcgcgcaag agggcagacc gatagaatca ttggtaatga 4620 gcgccggatt acgcgcgctt gaacaacagg ctgactgggt gttagtggaa ggtgctggcg 4680 gctggtttac gccgctttct gacactt tca cttttgcaga ttgggtaaca caggaacaac 4740 tgccggtgat actggtagtt ggtgtgaaac tcggctgtat taatcacgcg atgttgactg 4800 cacaggtaat acaacacgcc ggactgactc tggcgggttg ggtggcgaac gatgttacgc 4860 ctccgggaaa acgtcacgct gaatatatga ccacgctcac ccgcatgatt cccgcgccgc 4920 tgctgggaga gatcccctgg cttgcagaaa atccagaaaa tgcggcaacc ggaaagtaca 4980 taaaccttgc cttgttgtag cccgggtacc gagctcgaat 502 0 <210> 21 <211> 346 <212> PRT <213> Artificial Sequence <220> <223> mutant biotin synthase (BioB) of Serratia marcescens <400> 21 Met Met Thr Asp Arg Ile His Trp Thr Val Gly Gln Ala Gln Ala Leu 1 5 10 15 Phe Asp Lys Pro Leu Leu Glu Leu Leu Phe Glu Ala Gln Thr Val His 20 25 30 Arg Gln His Phe Asp Pro Arg Gln Val Gln Val Ser Thr Leu Leu Ser 35 40 45 Ile Lys Thr Gly Ala Cys Pro Glu Asp Cys Lys Tyr Cys Pro Gln Ser 50 55 60 Ser Arg Tyr Lys Thr Gly Leu Glu Ser Glu Arg Leu Met Gln Val Glu 65 70 75 80 Gln Val Leu Glu Ser Ala Arg Lys Ala Lys Ala Asn Gly Ser Thr Arg 85 90 95 Phe Cys Met Gly Ala Ala Trp Lys Asn Pro His Glu Arg Asp Met Pro 100 105 110 Tyr Leu Gln Gln Met Val Gln Gly Val Lys Ala Met Gly Met Glu Thr 115 120 125 Cys Met Thr Leu Gly Thr Leu Asp Gly Thr Gln Ala Glu Arg Leu Ala 130 135 140 Glu Ala Gly Leu Asp Tyr Tyr Asn His Asn Leu Asp Thr Ser Pro Glu 145 150 155 160 Phe Tyr Gly Ser Ile Ile Thr Thr Arg Ser Tyr Gln Glu Arg Leu Asp 165 170 175 Thr Leu Asp Lys Val Arg Asp Ala Gly Ile Lys Val Cys Ser Gly Gly 180 185 190 Ile Val Gly Leu Gly Glu Thr Val Arg Asp Arg Ala Gly Leu Leu Val 195 200 205 Gln Leu Ala Asn Leu Pro Lys Pro Pro Glu Ser Val Pro Ile Asn Met 210 215 220 Leu Val Lys Val Lys Gly Thr Pro Leu Ala Asp Asn Asp Asp Val Asp 225 230 235 240 Pro Phe Asp Phe Ile Arg Thr Ile Ala Val Ala Arg Ile Met Met Pro 245 250 255 Ser Ser Tyr Val Arg Leu Ser Ala Gly Arg Glu Gln Met Asn Glu Gln 260 265 270 Thr Gln Ala Met Cys Phe Met Ala Gly Ala Asn Ser Ile Phe Tyr Gly 275 280 285 Cys Lys Leu Leu Thr Thr Pro Asn Pro Glu Glu Asp Lys Asp Leu Gln 290 295 300 Leu Phe Arg Lys Leu Gly Leu Asn Pro Gln Gln Thr Ala Thr Glu His 305 310 315 320 Gly Asp Asn Gln Gln Gln Gln Val Leu Ala Lys Gln Leu Leu Asn Ala 325 330 335 Asp Thr Ala Glu Phe Tyr Asn Ala Ala Leu 340 345 <210> 22 <211> 1041 <212> DNA <213> Artificial Sequence <220> <223> mutant bioB of Serratia marcescens encoding mutant biotin synthase of SEQ ID NO: 21 <400> 22 atgatgaccg accgcattca ctggacagta gggcaagccc aggccctgtt tgataaaccg 60 ctgctggaac tgctgttcga agcgcaaacc gtacaccgcc agcacttcga gtcag 120 gtgcaggtca gcacgctgct gtcgatcaag accggcgctt gcccggaaga ctgcaaatac 180 tgcccgcaga gctcacgcta caagaccggc ctggagtcgg agcggctgat gcaggtcgag 240 caggtgctgg aatcggcacg caaggccaag gcgaacggtt cgacccgttt ttgcatgggc 300 gcggcgtgga agaacccgca cgagcgcgat atgccttatc tgcagcaaat ggtgcagggc 360 gtgaaagcga tgggcatgga aacctgcatg acgctgggca cattggatgg cacccaggcc 420 gagcggctgg cggaggccgg gctggattac tacaaccata acctcgacac ctcgccggag 480 ttctacggca gcatcatcac cacccgcagc taccaggagc gcctggatac gctcgacaag 540 gtgcgcgacg ccggcatcaa agtgtgctcc ggcggcatcg tcgggctggg tgaaacggtg 600 cgcgatcgcg ccgggct gct ggtgcagctg gccaacctgc caaaaccacc ggagagcgtg 660 ccgatcaaca tgttggtgaa ggtgaaaggc acgccgctgg cggataacga tgacgtcgat 720 ccgtttgatt tcatccgcac catcgcggtg gcgcgcatca tgatgccatc ttcttatgtc 780 cgtctctccg caggccgcga acagatgaac gaacagacgc aggcgatgtg cttcatggcc 840 ggcgccaact cgatcttcta cggttgcaag ctgctgacca cgccgaatcc ggaagaagac 900 aaagacctgc agctgttccg caagctgggg ctcaaccc gc agcagaccgc aaccgaacac 960 ggcgacaacc agcaacagca ggtgctggcc aagcaactgc tgaacgccga taccgccgag 1020 ttttacaacg cggcgccgtg a 1041 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0011 <400> 23 gggctgcagg aattcgatcc agatacgctt tttcca 36 <210> 24 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0012 <400> 24 aaacattttt ccaataa cacgcaagca ccttaaaatc ac 42 <210> 25 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> insAB upstream fragment (594bp) <400> 25 gggctgcagg aattcgatcc agatacgctt tttccagcgc gtttcggcga aacaggtcca 60 ccatcacgcg tttggcgata gtgcagagga aggagcgagg atcgcggatc gtcgagagcg 120 tttcgctgac cattacccgc aaaaaagtgt cctgggcaat gtcatctgca tcaaaagcag 180 actggagttt gcgcgtcagc cagcttttca accagccgtg atgtgtgcca taaagcgact 240 cgaacgttaa ggaagctgtg gtagtggcgc ggtcagacat gcggagtgca tcaaaagtta 300 attatcacgt agtcatatta atatgagaat ggttatcatt acaattggaa ataaaattgt 360 ttccaataga catttttaac atgttgtttt tctaagtgtt ataaggtagg tataaaatgg 420 gatggagcct ctgcttctgg catgtgtcgg tcagaatgac tcatgatgtg gtctgctatt 480 attgacatcc tcactgccct aaaggatggg gatttcggta atgctgccaa cttactgatt 540 tagtgtatga tggtgatttt aaggtgcttg cgtgttattg gcaaaaaaat gttt 594 <210> 26 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0013 <400> 26 gtgattttaa ggtgcttgcg tgttattggc aaaaaaatgt tt 42 <210> 27 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0014 <400> 27 tcagataatg cccgatgacc ctacaacaag gcaaggttta t 41 <210> 28 <211> 5022 <212> DNA <213> Artificial Sequence <220> <223> biotin operon fragment (10044bp) <400> 28 gtgattttaa ggtgcttgcg tgttattggc aaaaaaaatgt ttcatcctgt accgcgcggt 60 taaccgctgc ggtcagacgc tgcaactgtt gcggggagaat aatatagggc ggcatcaggt 120 aaatcagttt gccaaaaggc cggatccaga caccctgtt c gacaaagaat ttttgcagcg 180 ccgccatatt caccggatga gtggtttcga ccacgccaat ggcccccagt acgcgcacat 240 cggcaaccat ttcggcatca cgggcggggg caagttgctc gcgcagctgt acttcaatat 300 ccgccacctg ttgctgccag tcgccagatt cgagaatcgc caggctggcg tttgctgccg 360 cgcaggccag cggattgccc ataaaagttg gcccatgcat aaagcaaccg gcttcaccgt 420 tactgatggt ttctgcaacc tcgcgcgtgg tgagtgtggc ggaaagggtc attgtgccgc 480 c ggttaaggc tttaccgagg cacaaaatgt ccggcgcgat ttctgcatgt tcacaggcaa 540 acagtttccc ggtacgacca aatccagtgg cgatctcgtc ggcaatcagc aagatacctt 600 cgcgatcgca tattttgcgg attcgtttta accattccgg atggtacatg cgcatcccgc 660 ctgcgccctg gacaatcggc tcaatgatca ccgccgcgat ttcatgacga tgcgccgcca 720 tcaggcgggc aaagcccacc atatcgcgct catcccattc gccatccatg cggctttgcg 780 gggcgggagc aaacaggttt tctggcaggt ag cctttcca cagactgtgc attgagttat 840 ccggatcgca caccgacatc gcgccaaagg tatcgccatg ataaccattg cggaaggtca 900 gaaaacgctg gcgcgcttcg cctttggctt gccagtactg caacgccatt ttcatcgcca 960 cttccaccgc tacggaaccg gagtccgcga gaaaaaacgca ctccagcggt tgcggcgtca 1020 tcgccaccag tttgcggcac agctcaatgg ctggcgcatg ggtgataccg ccaaacatca 1080 catgcgacat ggcatcaatt tgcgacttca tcgccgcatt aagctgcggg t gattgtagc 1140 cgtggatcgc cgcccaccag gacgacatac cgtcaaccag gcgtctgccg tcagacaaaa 1200 tcagctcgca accttcggcg ctcaccaccg gataaaccgg cagaggggag gtcatggatg 1260 tgtatgggtg ccagatatgg cgttggtcaa aggcaagatc gtccgttgtc ataatcgact 1320 tgtaaaccaa attgaaaaga tttaggttta caagtctaca ccgaattaac aacaaaaaac 1380 acgttttgga gaagccccat ggctcaccgc ccacgctgga cattgtcgca agtcacagaa 1440 ttattgaaa aaccgt tgct ggatctgctg tttgaagcgc agcaggtgca tcgccagcat 1500 ttcgatcctc gtcaggtgca ggtcagcacg ttgctgtcga ttaagaccgg agcttgtccg 1560 gaagattgca aatactgccc gcaaagctcg cgctacaaaa ccgggctgga agccgagcgg 1620 ttgatggaag ttgaacaggt gctggagtcg gcgcgcaaag cgaaagcggc aggatcgacg 1680 cgcttctgta tgggcgcggc gtggaagaat ccccacgaac gcgatatgcc gtacctggaa 1740 caaatggtgc agggggtaaa agcgat gggg ctggaggcgt gtatgacgct gggcacgttg 1800 agtgaatctc aggcgcagcg cctcgcgaac gccgggctgg attactacaa ccacaacctg 1860 gacacctcgc cggagtttta cggcaatatc atcaccacac gcacttatca ggaacgcctc 1920 aaaaagtgcg cgatgccggg atcaaagtct gttctggcgg cattgtgggc 1980 ttaggcgaaa cggtaaaaga tcgcgccgga ttattgctgc aactggcaaa cctgccgacg 2040 ccgccggaaa gcgtgccaat caacatgctg gtgaaggtga aaggcacgcc gcttgcc gat 2100 aacgatgatg tcgatgcctt tgattttatt cgcaccattg cggtcgcgcg gatcatgatg 2160 ccaacctctt acgtgcgcct ttctgccgga cgcgagcaga tgaacgaaca gactcaggcg 2220 atgtgcttta tggcaggcgc aaactcgatt ttctacggtt gcaaactgct gaccacgccg 2280 aatccggaag aagataaaga cctgcaactg ttccgcaaac tggggctaaa tccgcagcaa 2340 actgccgtgc tggcagggga taacgaacaa cagcaacgtc ttgaacaggc gctgatgacc 2400 ccggacaccg acgaatatta caac gcggca gcattatgag ctggcaggag aaaatcaacg 2460 cggcgctcga tgcgcggcgt gctgccgatg ccctgcgtcg ccgttatccg gtggcgcaag 2520 gagccggacg ctggctggtg gcggatgatc gccagtatct gaacttttcc agtaacgatt 2580 atttaggttt aagccatcat ccgcaaatta tccgtgcctg gcagcagggg gcggagcaat 2640 ttggcatcgg tagcggcggc tccggtcacg tcagcggtta tagcgtggtg catcaggcac 2700 tggaagaaga gctggccgag tggcttggct attcgcgggc actgctgttt atct ctggtt 2760 tcgccgctaa tcaggcagtt attgccgcga tgatggcgaa agaggaccgt attgctgccg 2820 accggcttag ccatgcctca ttgctggaag ctgccagttt aagcccgtcg cagcttcgcc 2880 gttttgctca taacgatgtc actcatttgg cgcgattgct tgcttccccc tgtccggggc 2940 agcaaatggt ggtgacagaa ggcgtgttca gcatggacgg cgatagtgcg ccactggcgg 3000 aaatccagca ggtaacgcaa cagcacaatg gctggttgat ggtcgatgat gcccacggca 3060 c gggcgttat cggggagcag gggcgcggca gctgctggct gcaaaaggta aaaccagaat 3120 tgctggtagt gacttttggc aaaggatttg gcgtcagcgg ggcagcggtg ctttgctcca 3180 gtacggtggc ggattatctg ctgcaattcg cccgccacct tatctacagc accagtatgc 3240 cgcccgctca ggcgcaggca ttacgtgcgt cgctggcggt cattcgcagt gatgagggtg 3300 atgcacggcg cgaaaaactg gcggcactca ttacgcgttt tcgtgccgga gtacaggatt 3360 tgccgtttac gcttgctgat tcat gcagcg ccatccagcc attgattgtc ggtgataaca 3420 gccgtgcgtt acaactggca gaaaaactgc gtcagcaagg ctgctgggtc acggcgattc 3480 gcccgccaac cgtacccgct ggtactgcgc gactgcgctt aacgctaacc gctgcgcatg 3540 aaatgcagga tatcgaccgt ctgctggagg tgctgcatgg caacggttaa taaacaagcc 3600 attgcagcgg catttggtcg ggcagccgca cactatgagc aacatgcaga tctacagcgc 3660 cagagtgctg acgccttact ggcaatgctt ccacagcgta aatacaccca cgtactggac 3720 gcgggttgtg gacctggctg gatgagccgc cactggcggg aacgtcacgc gcaggtgacg 3780 gccttagatc tctcgccgcc aatgcttgtt caggcacgcc agaaggatgc cgcagaccat 3840 tatctggcgg gagatatcga atccctgccg ttagcgactg cgacgttcga tcttgcatgg 3900 agcaatctcg cagtgcagtg gtgcggtaat ttatccacgg cactccgcga gctgtatcgg 3960 gtggtgcgcc ccaaaggcgt ggtcgcgttt accacgctgg tgcagggatc gttacccgaa 4020 ctgcatcagg ggcaggc ggtggacgag cgtccgcatg ctaatcgctt tttaccgcca 4080 gatgaaatcg aacagtcgct gaacggcgtg cattatcaac atcatattca gcccatcacg 4140 ctgtggtttg atgatgcgct cagtgccatg cgttcgctga aaggcatcgg tgccacgcat 42 00 cttcatgaag ggcgcgaccc gcgaatatta acgcgttcgc agttgcagcg attgcaactg 4260 gcctggccgc aacagcaggg gcgatatcct ctgacgtatc atcttttttt gggagtgatt 4320 gctcgtgagt aaacgttatt ttgtcaccgg aacggatacc gaagtgggga aaactgtcgc 4380 cagttgtgca cttttacaag ccgcaaaggc agcaggctac cggacggcag gttataaacc 4440 ggtcgcctct ggcagcgaaa agaccccgga aggtttacgc aatagcgacg cgctggcgtt 4500 acagcgcaac agcagcct gc agctggatta cgcaacagta aatccttaca ccttcgcaga 4560 acccacttcg ccgcacatca tcagcgcgca agagggcaga ccgatagaat cattggtaat 4620 gagcgccgga ttacgcgcgc ttgaacaaca ggctgactgg gtgttagtgg aaggtgctgg 4680 cggctggtt t acgccgcttt ctgacacttt cacttttgca gattgggtaa cacaggaaca 4740 actgccggtg atactggtag ttggtgtgaa actcggctgt attaatcacg cgatgttgac 4800 tgcacaggta atacaacacg ccggactgac tctggcgggt tgggtggcga acgatgttac 4860 gcctccggga aaacgtcacg ctgaatatat gaccacgctc acccgcatga ttcccgcgcc 4920 gctgctggga gagatcccct ggcttgcaga aaatccagaa aatgcggcaa ccggaaagta 4980 cataaacctt gccttgttgt agggtcatcg ggcat tatct ga 5022 <210> 29 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0015 <400> 29 ataaaccttg ccttgttgta gggtcatcgg gcattatctg a 41 <210> 30 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer_VB07_0016 <400> 30 gctaggtcga ctagcgtgat gccttttttg tcgtgggt 38 <210> 31 <211> 536 <212> DNA <213> Artificial Sequence <220> <223> insAB downstream fragment ( 536bp) <400> 31 ataaaccttg ccttgttgta gggtcatcgg gcattatctg aacataaaac actatcagta 60 agttggagtc attaccgacc atgtttattt catacattgt gggtattgtt cttattatcg 120 ccgctaatca ataaaatcct gccccatatc tacatggggc agttgttcat tcttttagtg 180 tggtaattca cacgccagca aaaactctgc cgttccttca tcaacaatca ggtccgtgac 240 atatcctccc agcagggcac ccaacgttgc gtcatagcct ctttccccac cagcaaggaa 300 gatcttccgt tcaatctgcc ttagctgagc cagactgatt c ccagaatac gctggtcaac 360 atcagccacg acgggcatcc cttccttgtc atagaagcga ccacaaatga cacctactgc 420 gcctaaatcc cgatacgtct gcatttcctt cttattcagc acgccccacc gaatcagggg 480 attttcatca agcgcgttac ccacgacaaa aaaggcatca cgctagtcga cctagc 536 <210> 32 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_7 <400> 32 ctgcaggaat tcgatttcgc tattgtagcc gtagg 35 <210> 33 <211> 35 2> DNA < 213> Artificial Sequence <220> <223> primer_VB7_8 <400> 33 ctgcactacg cagggtggca cggtgttatc cttca 35 <210> 34 <211> 515 <212> DNA <213> Artificial Sequence <220> <223> birA upstream fragment for exogenous insertion < 400> 34 ttcgctattg tagccgtagg tctgcgtctg ccaaaagagt ggcaacctgt actaacgtat 60 ggtgacttaa ctcgtctgga tcctacaaca gtaacgccac agcaagtatt taatgcggtg 120 tgtcatatgc gcaccaccaa actccctgat ccaaaagtga atggcaatgc cggtagtt tc 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 gcatcatgta 420 agacagaaag ttggtgaaaa atttaatgtc tggcttgagc ctgaagtccg ctttattggt 480 gcatcaggtg aagtgagcgc agtggagaca atttc 515 <210> 35 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_9 <400> 35 gataacaccg tgccaccctg cgtagtgcag aaaaa 35 <210> 36 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_10 <400> 36 gtcgactagc gtgatatgca tcgcctggtg aagtt 35 <210> 37 <211> 512 <212> DNA <213> Artificial Sequence <220> <223> birA downstream fragment for exogenous insertion <400> 37 gaaaggggag ctcc cctgcaaatt atttgcgtag tctgacctct tctaccgcat 60 gattagcact tttcgtcagg attaaactgg cgcgctcacg 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 gaaacaaata catgatgtgg atcgtgtgga taatccatcc 360 cgct ctgtaa gacatttaac ccttcaagaa ttaaaatatc aggctgaaca accgttttat 420 ctccatccgg gatcacatca taaataagat gtgagtaaac aggtgctgta acgtttggca 480 cgccggattt gagatcggaa acaaacttca cc 512 <210> 38 <211> 304 <212> DNA <213> Artificial Sequence <220> <223> cj1 promoter <400> 38 caccgcgggc ttattccatt acatggaatg accaggaatg gcagggaatg cgacgaaatt 60 gactgtgtcg ggagcttctg atccgatgct gccaaccagg agagaaaata atgacatgtg 120 caggcac gct ggtgagctgg agatttatga tctcaagtac cttttttctt gcactcgagg 180 gggctgagtg ccagaatggt tgctgacacc aggttgaggt tggtacacac tcaccaatcc 240 tgccgtcgcg ggcgcctgcg tggaacataa accttgagtg aaacccaatc taggagatta 300 agat 304 <210> 39 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_11 <400> 39 gtcgactagc gtgatatgca tcgcctggtg aagtt 35 <210> 40 <211> 35 > DNA < 213> Artificial Sequence <220> <223> primer_VB7_15 <400> 40 ctttaagggc gacatatctt aatctcctag attgg 35 <210> 41 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_16 <400> 41 gag attaagatat gtcgccctta aagcg 35 <210> 42 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer_VB7_17 <400> 42 tctgcactac gcagggttac tgcaggcgaa ggtgc 35 <210> 43 <211> 861 < 212> DNA < 213> Artificial Sequence <220> <223> birA gene of Corynebacterium glutamicum (861bp) <400> 43 atgtcgccct taaagcgcgc ttttcgacgc gaccccacta cattggcttc catgaacgtt 60 gacatttcac gatccagaga gccgctaaac gttgagctcc tgaaggaaaa attgctccaa 120 aacggtgact ttggccaggt catttacgaa aaagtgacag gctccactaa tgctgacttg 180 ctggcacttg caggttctgg cgctccaaac tggacggtga aaactgtcga gtttcaagat 240 catgcgcgtg ggcgactcggtgg tctgcccctg agggttccca aacaatcgtg 300 tctgtgctcg ttcaactatc tattgatcaa gtggaccgga ttggcactat tccactcgcg 360 gcgggactcg ctgtcatgga tgcgttgaat gacctcggtg tggaaggtgc cggactgaaa 420 tggcccaacg atgttcaaat ccacggcaag aaactctgcg gcatcctggt ggaagccacc 480 ggctttgatt ccaccccaac agttgtcatc ggttggggca ctaatatcag cctgactaaa 540 gaggagcttc ctgttcctca tgcaacttcc ctcgcattgg aaggtgttga ag tcgacaga 600 accacattcc ttattaatat gctcacacat ctgcatactc gactggacca gtggcagggt 660 ccaagtgtgg attggctcga tgattaccgt gcggtatgtt ccagtattgg ccaagatgtt 720 cgagtgcttc tacctgggga taaagaactc ttaggtgaag cgatcggtgt cg cgactggc 780 ggagaaattc gtgttcgcga tgcttcgggc accgttcaca ccctcaacgc cggtgaaatt 840acgcaccttc gcctgcagta a 861

Claims (12)

delete delete delete delete Containing at least one selected from the group consisting of a polypeptide variant having biotin synthase activity, in which the amino acid at the third position of sequence number 1, alanine, is substituted with threonine, and a polynucleotide encoding the polypeptide variant;
A microorganism having increased biotin production activity compared to a microorganism comprising at least one member selected from the group consisting of a polypeptide of sequence number 1 and a polynucleotide encoding the polypeptide. In claim 5, a microorganism wherein the polypeptide variant comprises an amino acid sequence of SEQ ID NO: 21. In claim 5, the microorganism is a microorganism having at least one activity further enhanced from the group consisting of 7,8-diamino-pelargonic acid aminotransferase, 8-amino-7-oxononanoate synthase, malonyl-ACP O-methyltransferase, and dethiobiotin synthetase. In claim 5, the microorganism is a microorganism of the genus Escherichia ( Escherichia sp.). In claim 8, the microorganism of the genus Escherichia is Escherichia coli . A composition for producing biotin, comprising a microorganism according to any one of claims 5 to 9. A method for producing biotin, comprising a step of culturing a microorganism according to any one of claims 5 to 9 in a medium. A method for producing biotin, wherein the method further comprises a step of recovering biotin from a cultured medium or microorganism.