High-concentration and high-purity production strain of deacetoxycephalosporin C for 7-ADCA production and production method using the same

Abstract

To develop a bacterial strain that can produce the target substance DAOC at high purity with productivity increased for deacetoxy cephalosporin C (DAOC) and decreased for byproducts.SOLUTION: Disclosed herein are a bacterial strain with excellent deacetoxy cephalosporin C (DAOC) productivity and a use thereof. A mutant bacterial strain with improved DAOC productivity and a use thereof for producing 7-amino deacetoxy cephalosporanic acid (7-ADCA) are provided.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] This application relates to a strain excellent in the ability to produce deacetoxy cephalosporin C (DAOC) and its uses, and more particularly to a mutant strain having an improved ability to produce DAOC and its use for the production of 7-amino deacetoxy cephalosporanic acid (7-ADCA).

Background Art

[0002] Cephalosporin C (hereinafter referred to as "CPC") is a beta-lactam antibiotic produced by some microorganisms such as Acremonium chrysogenum, a filamentous fungus. CPC exhibits antibacterial activity against Gram-negative bacteria through inhibition of cell wall synthesis, but its degree is very weak. Therefore, it is mainly used for producing raw materials for semi-synthetic cephalosporin antibiotics (hereinafter referred to as "cephalosporin antibiotics").

[0003] The raw materials for cephalosporin antibiotics can be mainly classified into 7-amino cephalosporanic acid (hereinafter abbreviated as "7-ACA"), deacetyl 7-amino cephalosporanic acid (hereinafter abbreviated as "D-7-ACA"), 7-amino deacetoxy cephalosporanic acid (hereinafter abbreviated as "7-ADCA"), and the like.

[0004] Currently, as an industrial production method for producing 7-ADCA, a chemical conversion method is used that expands the penam ring, which is the beta-lactam ring of penicillin, to the cephem ring of the cephalosporin series. In particular, in the production of 7-ADCA, there was a major drawback in that organic solvents such as toluene were used in the chemical conversion method involved in the expansion to the cephem ring, generating a large amount of toxic waste, increasing environmental pollution and wastewater treatment costs.

[0005] In the case of recombinant strains that produce existing DAOC at high concentrations, there was a drawback in that the purity decreased due to deacetyl cephalosporin C (DAC), a by-product generated during DAOC production.

[0006] Therefore, there is a need to develop a production strain with high purity of the target substance DAOC by increasing DAOC productivity and decreasing the productivity of by-products.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] In the present application, a strain excellent in the ability to produce deacetoxy cephalosporin C (DAOC) and its use are provided.

[0009] One example provides a novel DAOC-producing strain. In one example, the DAOC-producing strain may be a mutant strain of Acremonium chrysogenum into which an aminotransferase derived from Streptomyces clavuligerus has been introduced. The Acremonium chrysogenum mutant strain may have the ability to produce DAOC.

[0010] Another example provides a method for producing an Acremonium chrysogenum strain with improved DAOC-producing ability and / or a method for improving the DAOC-producing ability of an Acremonium chrysogenum strain, which includes the step of introducing an aminotransferase gene derived from Streptomyces clavuligerus into an Acremonium chrysogenum strain.

[0011] Another example provides a method for producing DAOC, which includes the step of culturing the Acremonium chrysogenum mutant strain.

Means for Solving the Problems

[0012] In the present application, a mutant strain having improved Deacetoxy cephalosporin C (DAOC)-producing ability and its use are provided. The mutant strain may be characterized in that not only does it have high DAOC-producing ability, but also the production amount of by-products (such as Deacetyl cephalosporin C (DAC), etc.) is small and the purity of DAOC is high.

[0013] Definition of Terms As used herein, a polynucleotide (used interchangeably with "gene or nucleic acid molecule") or a polypeptide (used interchangeably with "protein or enzyme") that "comprises a specific nucleic acid sequence or amino acid sequence" or "consists of (essentially) or is represented by a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide necessarily has the specific nucleic acid sequence or amino acid sequence, and includes (or does not exclude) a "substantially equivalent sequence" in which mutations (deletions, substitutions, modifications, and / or additions) are made to the specific nucleic acid sequence or amino acid sequence within the range that maintains the original function and / or intended function of the polynucleotide or polypeptide.

[0014] In one example, a polynucleotide or polypeptide that "comprises a specific nucleic acid sequence or amino acid sequence" or "consists of (essentially) or is represented by a specific nucleic acid sequence or amino acid sequence" means that the polynucleotide or polypeptide (i) necessarily includes the specific nucleic acid sequence or amino acid sequence, or (ii) consists of or necessarily includes an amino acid sequence having an identity of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more (where 100% may optionally be excluded) with the specific nucleic acid sequence or amino acid sequence, and maintains the original function and / or intended function of the specific sequence. In one example, the intended function can be to confer DAOC production ability on a host cell or increase the DAOC production ability of a host cell, and / or to reduce by-products during DAOC production.

[0015] As used herein, the term "sequence identity" refers to the degree of identity with a given nucleic acid sequence or amino acid sequence and can be expressed as a percentage (%). In the case of identity to a nucleic acid sequence, for example, it can be determined using algorithms such as BLAST (see Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90, 5873, 1993) or FASTA by Pearson (see Methods Enzymol., 183, 63, 1990). Based on such an algorithm BLAST, programs called BLASTN and BLASTX have been developed (see http: / / www.ncbi.nlm.nih.gov).

[0016] As used herein, the term "about" is an expression for including all numerical values in a range equivalent to or similar to the numerical value that follows, and can be interpreted as including a range of ±10, ±9, ±8, ±7, ±6, ±5, ±4, ±3, ±2, ±1, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, ±0.05, or ±0.01 based on the numerical value that follows, but is not limited thereto.

[0017] Acremonium chrysogenum has a cephalosporin synthesis pathway, and the final product is CPC (Cephalosporin C). CPC is converted from the precursors L-2-aminoadipate, L-cysteine, and L-valine through N-[(5S)-5-amino-5-carboxylpentanoyl]-L-cysteinyl-D-valine to isopenicillin N and penicillin N. Then, under the catalysis of the expandase (or DAOC synthase) encoded by the CefEF gene, the penam ring is converted to the cephem ring to form DAOC, which is a cephalosporin precursor.

[0018] Deacetoxy cephalosporin C (DAOC) can be represented by the following Chemical Formula 1 and is a precursor of 7-Amino deacetoxy cephalosporanic acid (7-ADCA; Chemical Formula 2), which is an intermediate of cephalosporin antibiotics.

Chemical formula

Chemical formula

[0019] In this specification, the term "DAOC" can be interpreted to mean DAOC (Deacetoxy cephalosporin C) and / or its pharmaceutically acceptable salts. Also, the term "ADCA" is interpreted to mean ADCA (7-Amino deacetoxy cephalosporanic acid) and / or its pharmaceutically acceptable salts.

[0020] As used herein, improved DAOC production ability may mean one or more selected from the group consisting of increased DAOC production, decreased production of deacetyl cephalosporin C (DAC), which is a by-product generated during DAOC production, and decreased DAC / DAOC ratio (%).

[0021] As used herein, the expression "culture of a microorganism" may mean what is obtained by culturing a microorganism in a medium. There is no limitation on the state of the culture. For example, the culture may be, but is not limited to, a liquid culture and / or a solid culture.

[0022] As used herein, a "cultured medium" can be obtained from a culture of a microorganism or after culturing a microorganism in a medium. The cultured medium can contain substances produced and / or secreted by the cultured microorganism. In one specific example, the cultured medium can contain, but is not limited to, a cell-free culture from which the microorganism has been removed from the culture.

[0023] Hereinafter, the present invention will be described in more detail.

[0024] One example provides a novel deacetoxy cephalosporin C (DAOC) producing strain.

[0025] In one example, the DAOC-producing strain may be a mutant strain of Acremonium chrysogenum containing an aminotransferase derived from Streptomyces clavuligerus or a gene encoding the same. The DAOC-producing strain may have an improved DAOC-producing ability as compared to the non-modified strain. As used herein, "improved or excellent DAOC-producing ability" may mean a high DAOC production amount and / or a high DAOC purity (low by-product production amount). Therefore, the DAOC-producing strain may be characterized by a high DAOC purity because it has a higher DAOC production amount and / or a lower by-product production amount such as DAC as compared to the non-modified strain.

[0026] In one specific example, the aminotransferase derived from Streptomyces clavuligerus may be aminotransferase class V-fold PLP-dependent enzyme (CefD; GenBank Accession No. WP_003952494.1; 398aa; SEQ ID NO: 1), but is not limited thereto.

[0027] In another example, the aminotransferase derived from Streptomyces clavuligerus is an enzyme that converts isopenicillin N to penicillin N in the host cell Acremonium chrysogenum. For example, it can additionally contain the C-terminal fragment of isopenicillin N-CoA-synthetase (CefD1; GenBank Accession No. CAD45625.1; 609aa). The C-terminal fragment can contain 3 to 30, 3 to 20, 3 to 15, 3 to 12, 3 to 10, 5 to 30, 5 to 20, 5 to 15, 5 to 12, 5 to 10, 8 to 30, 8 to 20, 8 to 15, 8 to 12, or 8 to 10 consecutive amino acids from the C-terminus of isopenicillin N-CoA-synthetase of Acremonium chrysogenum. The consecutive amino acids can include the 3 amino acids (the 3 C-terminal amino acids; PRL) that are peroxisome targeting signal 1 (PTS1). In one specific example, the C-terminal fragment containing PTS1 can include SEQ ID NO: 2 (SGQSAARPRL), but is not limited thereto. The C-terminal fragment containing PTS1 can be linked to the C-terminus of the aminotransferase derived from Streptomyces clavuligerus.

[0028] The polypeptide containing the aminotransferase derived from Streptomyces clavuligerus and the C-terminal fragment containing PTS1 can be represented by SEQ ID NO: 3, but is not limited thereto.

[0029] In one specific example, the Acremonium chrysogenum mutant strain can be a transformant into which a polynucleotide containing a nucleic acid sequence encoding a polypeptide (for example, the amino acid sequence of SEQ ID NO: 3) containing an aminotransferase derived from Streptomyces clavuligerus and a C-terminal fragment containing PTS1 is introduced, or a recombinant vector containing the polynucleotide, into Acremonium chrysogenum as a host cell. In the transformant, there are no special restrictions on the form of the introduced polynucleotide. For example, the polynucleotide can be inserted into the genome or chromosome of the host cell, or can be independently replicated and / or expressed outside the genome and / or chromosome, but is not limited thereto. The nucleic acid sequence can contain codons optimized for Acremonium chrysogenum, and in one specific example, can be represented by SEQ ID NO: 4, but is not limited thereto. The recombinant vector can be an expression vector capable of being expressed in Acremonium chrysogenum.

[0030] Acremonium chrysogenum as the host cell is a microorganism having the ability to produce deacetoxycephalosporin C (DAOC), which is a precursor of 7-ADCA, one of the raw materials for cephalosporin antibiotics, and can be a wild-type strain or a mutant strain. The mutant strain as the host cell can be an Acremonium chrysogenum strain into which mutations for increasing the DAOC production ability have been introduced.

[0031] In one example, the mutation for increasing the DAOC production ability can be one or more selected from the following, but is not limited thereto: Deletion of the CefEF and / or CefG gene; and Introduction of a foreign CefE gene.

[0032] The foreign CefE gene means a CefE gene derived from various microorganisms other than Acremonium chrysogenum, for example, Amycolatopsis lactamdurans, Gordonia rubripertincta, Mycobacterium abscessus, Microbacterium hydrocarbonoxydans, Nannocystis exedens, Pseudomonas synringae, Streptomyces clavuligerus, Sphingomonas dokdonensis, etc., and can be a CefE gene of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9) microorganisms selected from the group consisting of, but not limited to, these.

[0033] In one example, the CefE gene derived from Amycolatopsis lactamdurans may include, but is not limited to, the nucleic acid sequence of GenBank Accession No. NID Z13974.1; the CefE gene derived from Gordonia rubripertincta may include, but is not limited to, the nucleic acid sequence of GenBank Accession No. NID CP022580.1; the CefE gene derived from Mycobacterium abscessus may include, but is not limited to, the nucleic acid sequence of GenBank Accession No. NID FVPM01000026.1; the CefE gene derived from Microbacterium hydrocarbonoxydans may include, but is not limited to, the nucleic acid sequence of GenBank Accession No. NID JYJB01000010.1; the CefE gene derived from Nannocystis exedens may include, but is not limited to, the nucleic acid sequence of GenBank Accession No. NID FOMX01000018.1; the gene derived from Pseudomonas synringae may include, but is not limited to, the nucleic acid sequence of GenBank Accession No. NID AOJT01001469.1; the gene derived from Streptomyces clavuligerus may include, but is not limited to, the nucleic acid sequence of GenBank Accession No. NID DS570624.1; the CefE gene derived from Sphingomonas dokdonensis may include, but is not limited to, the nucleic acid sequence of GenBank Accession No. NID NBBI01000005.1.

[0034] In one specific example, the foreign CefE gene may be, but is not limited to, the CefE gene of Mycobacterium abscessus (SEQ ID NO: 5), the CefE gene of Sphingomonas dokdonensis (SEQ ID NO: 6), or a combination thereof.

[0035] In other examples, the Acremonium chrysogenum mutant strains provided herein may additionally contain one or more mutations selected from the following, in addition to the mutation by introduction of a polynucleotide containing a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 3 or a recombinant vector containing said polynucleotide as described above: Deletion of the CefEF and / or CefG genes; and Introduction of a foreign CefE gene.

[0036] The foreign CefE gene is as described above.

[0037] As used herein, "gene deletion" may mean removing part or all (i.e., from the start codon to the stop codon) of the respective gene sequences of CefEF and / or CefG as confirmed in the chromosomal sequence of the strain, but is not limited thereto.

[0038] In one specific example, the Acremonium chrysogenum mutant strain may be the strain assigned the accession number KCTC 14989BP, but is not limited thereto.

[0039] The Acremonium chrysogenum mutant strain provided in this specification may have the ability to produce DAOC. The Acremonium chrysogenum mutant strain may have improved DAOC production ability (increased DAOC production and / or increased DAOC purity (e.g., decreased DAC production (e.g., decreased DAC / DAOC ratio (%)))) and / or increased 7-ADCA production performance compared to the unmodified strain. The unmodified strain may mean a wild-type Acremonium chrysogenum strain or a strain of Acremonium chrysogenum into which no mutation has been introduced by the aforementioned mutation, for example, a polynucleotide containing a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 3 or a recombinant vector containing the polynucleotide.

[0040] Another example provides a composition for the production of DAOC and / or 7-ADCA, comprising the Acremonium chrysogenum mutant strain and / or a culture or culture medium of the strain.

[0041] Another example provides a method for producing an Acremonium chrysogenum strain with improved DAOC production ability and / or a method for improving the DAOC production ability of an Acremonium chrysogenum strain, which includes introducing a polynucleotide comprising a nucleic acid sequence encoding a polypeptide (e.g., the amino acid sequence of SEQ ID NO: 3) containing an aminotransferase derived from Streptomyces clavuligerus and a C-terminal fragment containing PTS1 into an Acremonium chrysogenum strain. The produced Acremonium chrysogenum strain has improved DAOC production ability as compared to an Acremonium chrysogenum strain into which a nucleic acid sequence encoding a polypeptide (e.g., the amino acid sequence of SEQ ID NO: 3) containing an aminotransferase derived from Streptomyces clavuligerus and a C-terminal fragment containing PTS1 has not been introduced.

[0042] The method can additionally include one or more steps selected from the following, before, after, or simultaneously with the step of introducing the polynucleotide: Deletion of the CefEF and / or CefG gene; and Introduction of a foreign CefE gene.

[0043] The foreign CefE gene is as described above.

[0044] Another example provides a method for producing deacetoxy cephalosporin C (DAOC), which includes the step of culturing the mutant strain of Acremonium chrysogenum. The culturing step can be carried out in a medium and / or under conditions capable of producing DAOC. The method can additionally include the step of recovering (or separating) DAOC from the culture or the culture medium after the culturing step.

[0045] The mutant strain of Acremonium chrysogenum provided herein has an improved ability to produce DAOC. In one example, the mutant strain of Acremonium chrysogenum may have a 3% or more, 5% or more, 7% or more, 10% or more, 12% or more, or 15% or more increase in the ability to produce DAOC compared to the unmodified strain.

[0046] In another example, the mutant strain of Acremonium chrysogenum has a DAC / DAOC(%) of 10% or less, 9.5% or less, 9% or less, 8.5% or less, 8% or less, 7.5% or less, 7% or less, 6.5% or less, 6% or less, 5.5% or less, 5% or less, 4.5% or less, 4% or less, 3.9% or less, 3.8% or less, 3.7% or less, 3.6% or less, or 3.5% or less, and / or has a DAC / DAOC(%) that is 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 39% or less, 38% or less, 37% or less, 36% or less, or 35% or less of the DAC / DAOC(%) (100%) of the unmodified strain, but is not limited thereto.

[0047] Another example provides a composition for producing 7-ADCA (7-Amino deacetoxy cephalosporanic acid) comprising one or more selected from the group consisting of a mutant strain of Acremonium chrysogenum, a culture of the mutant strain, a culture medium of the culture, and DAOC recovered (separated, purified) from the culture and / or the culture medium.

[0048] Another example provides the use of a composition comprising one or more selected from the group consisting of a mutant strain of Acremonium chrysogenum, a culture of the mutant strain, a culture medium of the culture, and DAOC recovered (separated, purified) from the culture and / or the culture medium for the production of 7-ADCA (7-Amino deacetoxy cephalosporanic acid).

[0049] Another example is a step of treating the culture, culture medium or DAOC recovered (separated, purified) therefrom of the mutant strain of Acremonium chrysogenum with CPC (Cephalosporin C) acylase, which provides a method for producing 7-ADCA (7-Amino deacetoxy cephalosporanic acid).

[0050] The method may additionally include a step of culturing the mutant strain of Acremonium chrysogenum before the step of treating with CPC acylase. The culturing step is as described above and may additionally include a step of recovering DAOC from the culture or the culture medium.

[0051] The step of treating the DAOC culture solution with CPC acylase is a step of producing 7-ADCA through a conversion reaction using CPC acylase. The conversion reaction using the CPC acylase can usually be carried out under exhalation conditions such as shaking culture or rotation by a rotator. In one specific example, the conversion reaction can be carried out under temperature conditions of 5 to 30 °C and / or time conditions of 1 minute to 300 minutes, for example, 1 minute to 120 minutes, but is not limited thereto. The reaction pH can be maintained in the range of 3.0 to 9.0 and can be appropriately adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0052] The 7-ADCA production method can additionally include a step of recovering (separating, harvesting, purifying or collecting) 7-ADCA from the reaction solution after the step of treating with the CPC acylase. Specifically, the recovery of 7-ADCA can be carried out by methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity and size exclusion), etc., but is not limited thereto.

[0053] Another example provides a polypeptide (e.g., the amino acid sequence of SEQ ID NO: 3) comprising an aminotransferase (e.g., SEQ ID NO: 1) derived from Streptomyces clavuligerus and a C-terminal fragment containing PTS1.

[0054] Another example provides a polynucleotide encoding the polypeptide. The polynucleotide can be one containing the nucleic acid sequence of SEQ ID NO: 4.

[0055] Another example provides an expression vector containing the polynucleotide.

[0056] Another example is A polypeptide (e.g., the amino acid sequence of SEQ ID NO: 3) comprising an aminotransferase (e.g., SEQ ID NO: 1) derived from Streptomyces clavuligerus and a C-terminal fragment containing PTS1, A polynucleotide encoding the polypeptide (e.g., SEQ ID NO: 4), and A recombinant vector (expression vector) containing the polynucleotide Provided is the use for the production of one or more DAOC-producing strains selected from the group consisting of and / or for improving the DAOC-producing ability.

[0057] More specifically, A polypeptide (e.g., the amino acid sequence of SEQ ID NO: 3) comprising an aminotransferase (e.g., SEQ ID NO: 1) derived from Streptomyces clavuligerus and a C-terminal fragment containing PTS1, A polynucleotide encoding the polypeptide (e.g., SEQ ID NO: 4), and A recombinant vector (expression vector) containing the polynucleotide Provided is a composition for the production of a DAOC-producing strain containing one or more selected from the group consisting of and / or for improving the DAOC-producing ability.

[0058] The DAOC-producing strain may be Acremonium chrysogenum, and the improvement of the DAOC-producing ability may be an improvement in the DAOC-producing ability of Acremonium chrysogenum.

[0059] The nucleic acid sequences described herein can be modified in various ways within the coding region without changing the amino acid sequence and / or function of the protein expressed from the coding region, taking into account the codons preferred by the microorganism in which the target polypeptide is to be expressed, e.g., Acremonium chrysogenum, due to the degeneracy of the codons.

[0060] The introduction of the polynucleotide or vector can be appropriately selected and carried out by those skilled in the art using known transformation methods. In this specification, the term "transformation" means introducing a vector containing a nucleic acid molecule encoding a target protein (foreign protein) into a host cell so that the protein encoded by the nucleic acid molecule can be expressed in the host cell. As long as the transformed nucleic acid molecule can be expressed in the host cell, it can be inserted into the chromosome of the host cell and / or located extrachromosomally. Further, the nucleic acid molecule includes DNA and / or RNA encoding the target protein. As long as the nucleic acid molecule can be introduced into the host cell and expressed, there is no limitation on its introduced form. For example, the nucleic acid molecule can be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its own expression. The expression cassette can usually contain expression regulatory elements such as a promoter operably linked to the nucleic acid molecule, a transcription termination signal, a ribosome binding site, and / or a translation termination signal. The expression cassette can be in the form of an expression vector capable of self-replication. Also, the nucleic acid molecule can be introduced into the genome of the host cell in its own form and operably linked to the sequences necessary for expression in the host cell. The term "operably linked" as used above can mean that an expression regulatory element (e.g., a promoter) and a nucleic acid molecule are functionally linked so that the expression regulatory element can regulate the transcription (e.g., initiate transcription) of the nucleic acid molecule encoding the target protein (foreign protein). Operable linkage can be carried out using known genetic recombination techniques in the art, for example, by ordinary site-specific DNA cleavage and ligation, but is not limited thereto.

[0061] The method of transforming (introducing) the polynucleotide into a host cell can be accomplished by any method of introducing a nucleic acid molecule into a cell (microorganism), and an appropriate transformation technique known in the art can be appropriately selected and carried out depending on the host cell. Examples of the known transformation methods include, but are not limited to, electroporation, calcium phosphate (CaPO 4 ) precipitation method, calcium chloride (CaCl 2 ) precipitation method, microinjection, polyethylene glycol (PEG) precipitation method (polyethylene glycol-mediated uptake), DEAE-dextran method, cationic liposome method, lipofection, lithium acetate-DMSO method, heat shock method, particle gun bombardment, silicon carbide whiskers, sonication, etc.

[0062] As used herein, the term "vector" means a DNA construct containing the nucleotide sequence of a nucleic acid molecule encoding the target protein operably linked to appropriate regulatory sequences so that the target protein can be expressed in a suitable host. The regulatory sequences can include a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding an appropriate mRNA ribosome binding site, and / or a sequence for regulating the termination of transcription and / or translation. After being transformed into an appropriate host microorganism, the vector can be expressed regardless of the host microorganism's genome or integrated into the host microorganism's genome.

[0063] The vectors that can be used in this specification are not particularly limited as long as they can replicate within a host cell, and can be selected from all commonly used vectors. Examples of commonly used vectors include plasmids, cosmids, viruses, bacteriophages, etc. in their natural or recombinant states. For example, as the vector, as a phage vector or cosmid vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. can be used, and as a plasmid vector, pBC series (e.g., pBC-KS(+)), pBR series (e.g., pBR322, pBR325), pUC series (e.g., pUC118 and pUC119), pBluescriptII series, pGEM series, pTZ series, pCL series, pET series (e.g., pET-22b(+)), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5), etc. can be used, but are not limited thereto.

[0064] The vector can additionally contain a selection marker for confirming the presence or absence of the chromosomal insertion. The selection marker is for selecting cells transformed with the vector, that is, for confirming the presence or absence of the insertion of the polynucleotide, and can be selected and used from genes that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other phenotypes, so that transformed cells can be selected.

Advantages of the Invention

[0065] In this specification, a hybrid with the CefD nucleic acid sequence and the PTS1 nucleic acid sequence is first performed, and by introducing the CefD-PTS1 fusion gene into Acremonium chrysogenum, the steps in which CefD1, CefD2, and thioesterase act are reduced in one step to optimize the metabolic pathway, and the content of DAC, which is an impurity in the DAOC production process, can be epoch-makingly reduced, and the production ability of DAOC, which is the target product, can be increased to a significant level.

Brief Description of the Drawings

[0066]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0067] Hereinafter, the present invention will be described more specifically with reference to examples, which are merely illustrative and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art that the examples described below can be modified without departing from the essential gist of the invention.

[0068] Example 1: Preparation of D7px-introduced Strain

[0069] 1.1. Preparation of Parent Strain As a parent strain for the introduction of D7px, an Acremonium chrysogenum strain producing deacetoxycephalosporin C (DAOC) was prepared with reference to the examples described in Korean Registered Patent No. 10-2194740 (incorporated herein by reference).

[0070] Briefly, first, a strain (strain 3-7) in which the CefE3 (genbank PID SKX81615.1) coding DNA (SEQ ID NO: 5) of Mycobacterium abscessus was introduced into a high CPC-producing Acremonium chrysogenum strain (deposit number: KCTC 13922BP) lacking the CefEF and CefG genes and a strain (strain 8-60) in which the CefE8 (genbank PID OWK28829.1) coding DNA (SEQ ID NO: 6) of Sphingomonas dokdonensis was introduced were prepared. By the "transformation test method" described in Korean Registered Patent No. 10-2194740, protoplasts of the prepared strains 3-7 and 8-60 were prepared, and after counting the prepared protoplasts, 250 μL of a 60% (w / v) PEG solution was added to 1 × 10 7 protoplasts of each strain and mixed, and then left on ice for 40 minutes. Then, 2.5 mL of a 60% PEG solution was further added and mixed, and then left at room temperature for 40 minutes, and then spread on an LB-sucrose plate medium containing an antibiotic. The plate medium was cultured at 28°C until colonies were formed for 14 days.

[0071] For the strain obtained as described above, the presence or absence of CefE3 was confirmed using the primers of SEQ ID NO: 7 (5’-cgcttgagca gacatcacca tgacggacat cggtgaac-3’) and SEQ ID NO: 8 (5’-gctaagcttt tatcagccga cggttatggc-3’), and the presence or absence of CefE8 was confirmed using the primers of SEQ ID NO: 9 (5’-cgcttgagca gacatcacca tgcatcgcgc gggcggc-3’) and SEQ ID NO: 10 (5’-gctaagcttt tatcacttct tgatgagac-3’). It was confirmed that a fused strain (named E3-E8) into which both CefE3 of Mycobacterium abscessus and CefE8 of Sphingomonas dokdonensis were introduced was obtained.

[0072] 1.2. D7px gene synthesis and cloning Based on the CefD protein sequence of Streptomyces clavuligerus, an actinomycete that produces cephalosporin antibiotics, the gene sequence was resynthesized by changing it to the codons optimized in Acremonium chrysogenum (hereinafter referred to as "D7") and the coding sequence of 10 consecutive amino acid sites (including 3 amino acids of peroxisome targeting signal 1 (PTS1)) present at the C-terminus of the CefD1 protein of Acremonium chrysogenum were prepared. The coding sequence of 10 amino acids (SEQ ID NO: 2) of CefD1 containing the PTS1 sequence was ligated to the 3' end (corresponding to the C-terminal codon) of the D7 sequence, and this was named D7px (amino acid sequence: SEQ ID NO: 3; DNA sequence: SEQ ID NO: 4).

[0073] To produce D7px, a codon sequence that can optimize the expression of CefD of Streptomyces clavuligerus in Acremonium chrysogenum was found through sequence analysis, and a hybrid with the CefD1 sequence and PTS1 sequence of CefD was first performed. By introducing D7px into Acremonium chrysogenum, the step where CefD1, CefD2, and thioesterase act was reduced in one step to optimize the metabolic pathway. It was confirmed that the content of DAC, an impurity in the DAOC production process, was epoch-makingly reduced, and the target product, DAOC, also increased significantly.

[0074] The gene sequence of D7px and the protein sequence encoded thereby are as follows.

[0075] D7 amino acid sequence (398 aa) (SEQ ID NO: 1) MAVADWEEAR GRMLLDPTVV NLNTGSGGPL PRSAFERVTG FRAHLAAEPM DFLLREVPAL LWQARESLAR LIGGDPLRLA LATNVTAAVN LVASSLRLEA PGEILLSDDE YTPMRWCWER VARRHGLELR TFRLPELPSD PAEITAAAVA AMGPRTRLFF FSHVVSTTGL ILPAAELCEE ARARGITTVV DGAHAPGFLD LDLSRIPCDF YAGSGHKWLL APTGVGFLHL APGRLEELEP TQVSWAYEPP EGSGPPAARD RFGSTPGLRR LECEGTRDIC PWLATPESID FQAELGPGAI RARRRELTDH ARRLLADRPG RTLLTPDSPE LSGGMVAYRL PPGTDAAELR RGLWERFRIE AAVAEQPPGP VLRISANFYT TEEEIDRLAD ALDALTGE

[0076] Amino acid sequence of 10 amino acid sites of CefD1 containing the PTS1 sequence (10 aa) (SEQ ID NO: 2) SGQSAARPRL

[0077] D7px amino acid sequence (408 aa) (SEQ ID NO: 3) MAVADWEEAR GRMLLDPTVV NLNTGSGGPL PRSAFERVTG FRAHLAAEPM DFLLREVPAL LWQARESLAR LIGGDPLRLA LATNVTAAVN LVASSLRLEA PGEILLSDDE YTPMRWCWER VARRHGLELR TFRLPELPSD PAEITAAAVA AMGPRTRLFF FSHVVSTTGL ILPAAELCEE ARARGITTVV DGAHAPGFLD LDLSRIPCDF YAGSGHKWLL APTGVGFLHL APGRLEELEP TQVSWAYEPP EGSGPPAARD RFGSTPGLRR LECEGTRDIC PWLATPESID FQAELGPGAI RARRRELTDH ARRLLADRPG RTLLTPDSPE LSGGMVAYRL PPGTDAAELR RGLWERFRIE AAVAEQPPGP VLRISANFYT TEEEIDRLAD ALDALTGESG QSAARPRL

[0078] D7px coding DNA sequence (SEQ ID NO: 4) atggccgttg cggactggga ggaggcgcgc ggccgcatgc tcctggatcc caccgttgtc 60 aatctaaaca cggggtcggg aggcccgctc cccaggtccg ccttcgagag ggtcacaggt 120 ttccgtgccc acctggccgc ggagccgatg gacttcctgc ttcgcgaggt gcctgctctt 180 ctctggcagg ccagggagag cctcgcccgc ctgatcggtg gcgatccctt gcggctcgcc 240 ctctggcagg ccagggagag cctcgcccgc ctgatcggtg gcgatccctt gcggctcgcc 240 ctggcgacca atgtcacggc agccgtgaat ctggtcgcca gttcgctgcg tctcgaggca 300 ctggcgacca atgtcacggc agccgtgaat ctggtcgcca gttcgctgcg tctcgaggca 300 ccgggtgaga ttcttctgtc ggacgacgaa tacacaccca tgagatggtg ctgggagcgc 360 ccgggtgaga ttcttctgtc ggacgacgaa tacacaccca tgagatggtg ctgggagcgc 360 gtggcccgta ggcatggctt ggagctccgc acattccgcc tgccagagct gccaagcgat 420 gtggcccgta ggcatggctt ggagctccgc acattccgcc tgccagagct gccaagcgat 420 ccggctgaaa tcactgcagc cgcagttgct gcaatgggac cgcggacgcg actctttttc 480 ccggctgaaa tcactgcagc cgcagttgct gcaatgggac cgcggacgcg actctttttc 480 ttctcccacg tcgtttcgac cacgggtctc atactccccg ctgcggagct ctgcgaagag 540 ttctcccacg tcgtttcgac cacgggtctc atactccccg ctgcggagct ctgcgaagag 540 gcccgagccc gtggcatcac gacggtcgtc gatggcgccc atgcccctgg ttttctggat 600 gcccgagccc gtggcatcac gacggtcgtc gatggcgccc atgcccctgg ttttctggat 600 ctcgacctca gccgtatccc gtgcgacttt tacgccggtt ccggtcataa gtggctcctg 660 ctcgacctca gccgtatccc gtgcgacttt tacgccggtt ccggtcataa gtggctcctg 660 gcacccactg gcgttggctt cctccacttg gcgccgggcc gcctagagga actcgagcct 720 gcacccactg gcgttggctt cctccacttg gcgccgggcc gcctagagga actcgagcct 720 acacaggtct catgggccta cgagcctcca gagggctcgg gccctccggc ggcgcgagac 780 acacaggtct catgggccta cgagcctcca gagggctcgg gccctccggc ggcgcgagac 780 cggtttggga gtaccccggg cctgcggagg ctcgagtgcg agggcacgcg agatatctgc 840 cggtttggga gtaccccggg cctgcggagg ctcgagtgcg agggcacgcg agatatctgc 840 ccctggctcg ccacccccga aagtatcgac ttccaagcag agctaggtcc cggggctatc 900 ccctggctcg ccacccccga aagtatcgac ttccaagcag agctaggtcc cggggctatc 900 cgtgcgcgtc gtcgcgagct gaccgaccat gcgcggcgcc tgctcgccga ccgtcctggg 960 aggaccctcc ttactcctga ctcgccggag ctctcaggcg gaatggttgc ttacaggttg 1020 ccacccggca ccgacgcagc cgagctgcgc cgtggcctgt gggagcgatt tcgcatcgag 1080 gccgccgtcg ccgaacagcc gccgggcccc gtgctacgca tctccgcgaa tttctatacc 1140 accgaggagg agattgatcg actcgccgac gccctcgacg cccttacggg agagtccggc 1200 cagtcggcgg ctcggccaag actctaatga 1230

[0079] When cloning the D7px gene, gene amplification using the PCR method was carried out according to the manual enclosed at the time of purchasing DNA polymerase. Generally, Pfu-X polymerase (Solgent, Republic of Korea) was used as the DNA polymerase. Restriction enzymes, T4 DNA ligase, and Klenow fragment were purchased from NEB (USA) and used according to the enclosed manual. QIAprep Spin Miniprep Kit, QIAquick PCR Purification Kit, and QIAquick Gel Extraction Kit (Qiagen, Netherlands) were used for plasmid DNA purification, PCR product purification, DNA extraction from agarose gel, etc.

[0080] For cloning, transformation of Escherichia coli DH5alpha (Thermo Fisher) was performed using the heat shock method with competent cells. Escherichia coli was inoculated into LB broth (BD Difco) and cultured until the OD 600 reached 0.4 - 0.6, and then the cells were harvested by centrifugation (4°C, 4000 rpm). The harvested cells were washed 4 times with ice-cooled 0.1 M calcium chloride solution to prepare competent cells. For heat shock transformation, 100 - 500 ng of plasmid DNA (collectively referred to as the plasmid used for subcloning in the transformation process) was prepared. The plasmid DNA and 100 μL of competent cells were mixed and reacted on ice for 30 minutes, then given a heat shock at 42°C for 30 seconds, immediately placed on ice and cooled for 2 minutes, and then 1 mL of LB broth was added. After culturing at 37°C for 1 hour, it was spread on an LB plate containing an antibiotic and statically cultured overnight at 37°C to obtain transformants.

[0081] The gene of D7px was amplified from the template secured by commissioning synthesis. Primers 5’- CGCTTGAGCAGACATCACC ATGGCCGTTGCGGACTGGG-3’ (SEQ ID NO: 11) and 5’- TATGAATTC TCATTAGAGTCTTGGCCGAG-3’ (SEQ ID NO: 12) were used to amplify D7px. The promoter PEP3 (SEQ ID NO: 13) was amplified from pB-HCXEP3 (SEQ ID NO: 16) using primers 5’-GCAACTAGTGCGGCCGCCCTTGTATCTCTACACACAGGC-3’ (SEQ ID NO: 14) and 5’-GGTGATGTCTGCTCAAGCG-3’ (SEQ ID NO: 15).

[0082] Since the amplified D7px gene and the promoter PEP3 have overlapping sequences, overlapping PCR (sewing PCR) was performed to ligate them. Using the amplified D7px fragment and PEP3 fragment as templates, PCR was carried out using primer SEQ ID NO: 12 and SEQ ID NO: 14 to obtain a PEP3-D7px fragment in which PEP3 and D7px were ligated. To have a terminator sequence, the terminator vector pB-TtrpC (SEQ ID NO: 17) was used. The PEP3-D7px fragment was cleaved with SpeI and HindIII and inserted into the same site of pB-TtrpC to complete pB-D7pxcast (SEQ ID NO: 18).

[0083] After amplifying the D7px gene cassette from pB-D7pxcast using primers T3 (SEQ ID NO: 19) and T7 (SEQ ID NO: 20), it was inserted into the PmeI restriction site of pB-HF (SEQ ID NO: 21) by blunt-end ligation. The completed plasmid was named pB-HFD7px (SEQ ID NO: 22). The structure of the completed expression vector is shown in Figure 1. The plasmid has a hygromycin antibiotic marker and is configured to be able to remove the marker with the flp-FRT system.

[0084] 1.3. Transformation for D7px gene introduction To remove the antibiotic marker from the parental strain E3-E8 prepared in Example 1.1, it was subcultured on an antibiotic LB plate medium containing 2% xylose, and total DNA was extracted from the colonies that appeared after 10 days, and the presence or absence of the deletion of the marker gene cassette was confirmed by PCR. After repeating the above steps to select a strain in which all the antibiotic marker gene cassettes were lost, the pB-HFD7px vector prepared in Example 1.2 was transformed and introduced, and then the obtained transformants were compared for DAOC productivity through test tube culture to select a high-productivity strain for fermentation tests.

[0085] Preparation of the pB-HFD7px vector for transformation into Acremonium chrysogenum was carried out according to the manual of the QIAprep Spin Miniprep Kit (Qiagen, Netherlands).

[0086] Transformation into Acremonium chrysogenum was performed using the PEG (polyethylene glycol) method. After spreading the fungal strains (E3 - E8) on an LB plate to obtain the cells, they were cultured at 28°C for 7 days. The obtained mycelia were cut into squares with a side length of 5 - 7 mm using a flame-sterilized scalpel and inoculated into TB broth (12 g / L tryptone, 24 g / L yeast extract, 9.4 g / L K 2 HPO 4 、2.2 g / L KH 2 PO 4 、4 g / L glycerol) at 4 - 6 pieces per culture medium, and the inoculated medium was cultured at 28°C for 3 days. For cell collection, the culture broth was centrifuged (4°C, 4000 rpm), and then the supernatant was discarded and washed once with 0.6 M MgSO 4 .

[0087] The cell mass was measured for the preparation of protoplasts, and the total volume containing 2% lysing enzyme (Sigma-Aldrich, L1412, USA) was made 4 times the cell mass. After reacting at 30 °C and 100 rpm for 3 hours, the same volume of separation buffer A (0.6 M sorbitol, 100 mM Tris-Cl, pH 7.0) was overlaid, and the protoplasts were separated by centrifugation (4 °C, 1,800 g). After transferring the protoplast layer to a new centrifuge tube, the same volume of separation buffer B (1.2 M sorbitol, 100 mM Tris-Cl, pH 7.5) was mixed, and the protoplasts were recovered by centrifugation (4 °C, 1,800 g). The supernatant was discarded and the protoplasts were washed once with MSC solution (1 M sorbitol, 10 mM MOPS, pH 6.5, 10 mM CaCl 2 ) and then counted through a microscope to secure 1×10 7 protoplasts.

[0088] For transformation, 1×10 7Individual protoplasts were mixed with 1 - 5 μg of DNA (pB-HFD7px) and 50 μL of a 60% (w / v) PEG solution (prepared by dissolving polyethylene glycol 6000 in the MSC solution at a concentration of 60% (w / v)), and reacted on ice for 20 minutes. After additionally mixing 500 μL of the 60% PEG solution, the reaction was carried out at room temperature for 20 minutes, and then spread on an LB-sucrose plate (0.8M sucrose, 2% (w / v) agar) containing an appropriate antibiotic for the selection of transformants. The medium was cultured at 28°C for 14 days to ensure transformants into which pB-HFD7px was successfully introduced. Since the vector pB-HFD7px used as an example in this example is designed so that it cannot exist in plasmid form when introduced into cells, most of the obtained transformants have the D7px gene integrated into the chromosome. However, this is only an example for carrying out the present invention, and the possibility that the introduced gene exists in plasmid form is not excluded, and it does not matter in whatever form it exists.

[0089] To obtain spores, the obtained transformants were picked with a flame-sterilized scalpel, then clamped and placed in a 1.5 mL e-tube. After adding 200 μL of 0.85% NaCl, the cells were disrupted using a pestle. The cells were spread on a spore medium (starch 24 g / L, glycine 1.2 g / L, polypeptone 4 g / L, yeast extract 0.3 g / L, casein 8 g / L, ammonium sulfate 6 g / L, dipotassium phosphate 1.2 g / L, magnesium sulfate 0.6 g / L, agar 20 g / L, pH 7.0) and cultured at 28°C for 14 days. The spores were scraped with an inoculation loop, suspended in 20% (w / v) glycerol, and stored in a -80°C freezer.

[0090] The obtained mutant strain of Acremonium chrysogenum into which the D7px gene was introduced was deposited with the Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center, Iksan, Jeollabuk-do, Republic of Korea on June 2, 2022, and was assigned the accession number KCTC 14989BP.

[0091] Example 2. Selection of High DAOC-Producing Strains The production of DAOC (Deacetoxy cephalosporin C) and DAC (Deacetyl cephalosporin C) and the conversion amount of 7-ADCA of the transformants prepared in Example 1.3 were confirmed.

[0092] For the selection of strains with high DAOC production ability and low DAC production amount among the obtained transformants, test tube culture was carried out in a medium containing 15 g / L of sugar, 15 g / L of soytone, 5 g / L of ammonium sulfate, 10 g / L of methionine, 10 g / L of calcium carbonate, 10 g / L of yeast extract, 5 g / L of glucose, 2 g / L of magnesium sulfate, and 50 g / L of methyl oleate. The spores (1x10 7 individuals) collected in Example 1.3 were inoculated into test tubes and cultured at 28 °C and 200 rpm for 10 days, and 0.5 mL of the culture solution was collected. For the analysis of the production amounts of DAOC and DAC, 25 μL of the supernatant and 975 μL of tertiary distilled water were mixed, filtered through a 0.2-μm size filter, and analyzed by HPLC.

[0093] The HPLC analysis conditions were as follows. The HPLC instrument used was Shimadzu LC10Avp, and analysis was performed using a ZORBAX Eclipse Plus C18 (Analytical 4.6 mm × 250 mm, 5-Micron) column. The mobile phase was 20 mM ammonium acetate: acetonitrile (95:5), pH 7.0, the flow rate was 0.8 mL / min, the column temperature was 40 °C, and analysis was performed using a UV detector at 220 nm. The above HPLC analysis conditions were commonly used for the analysis of DAOC, DAC, and 7-ADCA in Example 2 and Example 3.

[0094] In addition, the ratio of DAC to DAOC (DAC / DAOC, %) was calculated by dividing the DAC area value by the DAOC area value. A smaller DAC / DAOC value indicates a higher production amount of DAOC, which is the target product, and a lower production amount of DAC, which is an impurity in the DAOC production process.

[0095] In addition, CPC acylase was added to the culture solution to perform the conversion of DAOC to 7-ADCA by an enzymatic reaction. The CPC acylase used was the mutant CPC acylase (PM2 mutant; encoded by SEQ ID NO: 23) disclosed in Korean Patent Publication No. 10-2014-0094150 (incorporated herein by reference). More specifically, in a culture test tube, 14% (v / v) aqueous ammonia was added to the DAOC culture solution prepared for the HPLC analysis and adjusted to pH 8.0, and then liquid CPC acylase was added at a final concentration of 30 U / mL and reacted at 15°C and 200 rpm for 1 hour. Thereafter, the conversion amount of 7-ADCA (g / L) was analyzed by HPLC. More specifically, after centrifuging the reaction solution and taking 25 μL of the supernatant, it was mixed with 975 μL of triple-distilled water and diluted 40-fold. The diluted fermentation broth was filtered through a 0.2 μm filter and then analyzed by HPLC. The HPLC analysis conditions followed the content described above.

[0096] The production amount of DAOC (g / L) obtained above and the ratio of DAC, which is an impurity in the DAOC production process (DAC / DAOC, %) are shown in FIG. 2 and Table 1. Also, the obtained conversion amount of 7-ADCA (g / L) is shown in Table 1.

[0097]

Table 1

[0098] In FIG. 2 and Table 1, E3-E8 are the parent strains (control group) into which D7px has not been introduced, and ED-1, ED-2, ED-3, ED-4, and ED-5 are all transformed strains into which D7px has been introduced into E3-E8.

[0099] As shown in Figure 2 and Table 1, for all five transformed strains tested, the productivity of the target product DAOC increased by 9 - 17% compared to the control group E3 - E8, and the DAC / DAOC(%) was also at the level of 2.55 - 3.54%, which was significantly lower than that of the control group E3 - E8 (about 10.5%). Also, as shown in Table 1, for all five transformed strains tested, the conversion amount of DAOC to 7 - ADCA increased by about 6 - 17% compared to the control group E3 - E8.

[0100] Example 3. Confirmation of DAOC production ability, DAC / DAOC ratio, and 7 - ADCA conversion amount During the culture period of the ED - 4 strain, which was confirmed to have the highest DAOC production amount and the lowest by - product DAC production amount in the above example, the DAOC production ability and DAC / DAOC ratio were confirmed. Also, the 7 - ADCA conversion amount at the maximum culture period was measured. The E3 - E8 strain was used as the control group.

[0101] DAOC production proceeded in stages of primary seed culture, secondary seed culture, and main culture. For the primary seed culture, 4 to 6 colonies were inoculated into the primary seed culture medium (28.5 g / L soybean powder, 25 mL / L corn steep liquor, 35 g / L sucrose, 5 g / L glucose, 5 g / L calcium carbonate, 0.8 mL / L antifoaming agent) according to colony size, and then cultured at 30 °C and 200 rpm for 63 hours. For the secondary seed culture, the entire primary seed culture broth was inoculated into a fermenter filled with the secondary seed culture medium (the same as the primary seed culture medium but with 5 mL / L soybean oil added). Starting with the conditions of 30 °C, 35% DO, 400 rpm, and 1.0 vvm air, 6% glucose was supplied by the growth of the bacterial cells, and the culture was carried out for 68 hours while gradually increasing the stirring speed. For the main culture, 200 mL of the secondary seed culture broth was inoculated into a fermenter filled with the main culture medium (23 g / L peanut powder, 50 mL / L corn steep liquor, 1.5 g / L methionine, 70 g / L dextrin, 35 g / L corn meal, 13 g / L calcium sulfate, 60 mL / L soybean oil, 13 g / L ammonium sulfate, 9 g / L fructose syrup, 10 g / L calcium carbonate, 0.5 mL / L antifoaming agent). The culture was started with the conditions of 28 °C, 35% DO, 400 rpm, and 1.0 vvm air, the pH was adjusted to 5.4 to 5.7 using aqueous ammonia, and the culture was carried out while gradually increasing the stirring speed by the growth of the bacterial cells. After 2 days of culture, the culture temperature was lowered from 28 °C to 25 °C, and then fermentation was completed on the 7th to 8th day while gradually supplying 5% to 10% soybean oil by the growth of the bacterial cells.

[0102] For the analysis of the DAOC and DAC production amounts in the fermentation broth, 1 mL of the culture broth was taken, diluted 100 times, centrifuged at 14,000 rpm for 10 minutes, and the supernatant was filtered through a 0.2 μm syringe filter for use in HPLC analysis. The HPLC analysis conditions followed the content described in Example 2 above. The ratio of DAC to DAOC (DAC / DAOC, %) was calculated by dividing the DAC area value by the DAOC area value.

[0103] The obtained DAOC production amount (g / L) was shown in Figure 3 and Table 2, and the DAC / DAOC ratio (%) was shown in Figure 4 and Table 3.

[0104]

Table 2

[0105]

Table 3

[0106] As shown in FIGS. 3 and 4 and Tables 2 and 3, it was revealed that the ED-4 strain into which the D7px gene was introduced had a higher DAOC production amount and a significantly lower DAC / DAOC ratio compared to the E8-E3 strain as the control group.

[0107] In addition, the culture solution was treated with CPC acylase to perform the conversion from DAOC to 7-ADCA by an enzymatic reaction. The CPC acylase used was the mutant CPC acylase (PM2 mutant; encoded by SEQ ID NO: 23) disclosed in Korean Patent Publication No. 10-2014-0094150 (incorporated herein by reference). More specifically, after adding 14% (v / v) aqueous ammonia to the fermenter filled with the fermentation culture solution at the maximum culture time to adjust the pH to 8.0, liquid CPC acylase was added at a final concentration of 30 U / mL, and the reaction was carried out for 1 hour with stirring at 800 rpm under the condition of a temperature of 15°C. Then, the 7-ADCA conversion amount (g / L) was analyzed by HPLC. More specifically, for the analysis of the 7-ADCA conversion amount of the fermentation culture solution, 1 mL of the reaction solution was taken and diluted 100-fold, and after centrifugation at 14,000 rpm for 10 minutes, the supernatant was filtered through a 0.2-um syringe filter and used for HPLC analysis. The HPLC analysis conditions followed the content described in Example 2 above.

[0108] Based on the obtained results, the final DAOC production amount, DAC / DAOC ratio, and 7-ADCA conversion amount (g / L) were described in Table 4 below.

[0109]

Table 4

[0110] As shown in Table 4, the ED-4 strain into which the D7px gene was introduced had a higher DAOC production amount compared to the E8-E3 strain, which was the control group. It was also revealed that not only was the DAC / DAOC ratio significantly lower, but the 7-ADCA conversion amount was also higher.

[0111] The mutant strain ED-4 of Acremonium chrysogenum into which the D7px gene was introduced was deposited with the Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center, Iksan, Jeollabuk-do, Republic of Korea on June 2, 2022, and was assigned the accession number KCTC 14989BP.

[0112] From the above description, those skilled in the art to which this application pertains will be able to understand that this application can be implemented in other specific forms without changing its technical idea and essential features. In this regard, it must be understood that all the examples described above are illustrative in all aspects and not restrictive. The scope of this application should be interpreted to include the meaning and scope of the claims described below, as well as all changes or modified forms derived from the equivalent concepts, rather than the detailed description above.

[0113] [Accession Number] Name of the depository institution: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center Accession number: KCTC14989BP Date of deposit: 20220602 TIFF0007691779000007.tif237168

Claims

**Claim 1** (1) The CefD protein of Streptomyces clavuligerus and (2) A C-terminal fragment containing 10 consecutive amino acids at the C-terminus of the CefD1 protein of Acremonium chrysogenum, a polypeptide containing the same, or a mutant strain of Acremonium chrysogenum containing a gene encoding the same, wherein there is a deletion of the CefEF gene and the CefG gene; and additionally, there is an introduction of a foreign CefE gene, the C-terminal fragment of the CefD1 protein contains a peroxisome targeting signal 1 (PTS1) and is linked to the C-terminus of the CefD protein, a mutant strain of Acremonium chrysogenum. **Claim 2** The mutant strain of Acremonium chrysogenum according to claim 1, wherein the CefD protein is represented by SEQ ID NO:

1. **Claim 3** The mutant strain of Acremonium chrysogenum according to claim 1, wherein the C-terminal fragment of the CefD1 protein is represented by SEQ ID NO:

2. **Claim 4** The mutant strain of Acremonium chrysogenum according to claim 1, wherein the polypeptide is represented by SEQ ID NO:

3. **Claim 5** The mutant strain of Acremonium chrysogenum according to claim 1, wherein the gene encoding the polypeptide is represented by SEQ ID NO:

4. **Claim 6** The exogenous CefE gene is one or more CefE genes selected from the group consisting of Mycobacterium abscessus, Sphingomonas dokdonensis, Amycolatopsis lactamdurans, Gordonia rubripertincta, Microbacterium hydrocarbonoxydans, Nannocystis exedens, Pseudomonas syringae, and Streptomyces clavuligerus, and the Acremonium chrysogenum mutant strain according to claim 1.

7. The Acremonium chrysogenum mutant strain according to claim 1, which has the accession number KCTC 14989BP.

8. The Acremonium chrysogenum mutant strain according to any one of claims 1 to 7, having one or more characteristics selected from the following: Increased production of deacetoxycephalosporin C (DAOC) compared to the non-mutant strain, Decreased production of deacetylcephalosporin C (DAC) compared to the non-mutant strain, and Decreased DAC / DAOC ratio (%) compared to the non-mutant strain.

9. A composition for producing deacetoxycephalosporin C (DAOC), comprising the Acremonium chrysogenum mutant strain according to any one of claims 1 to 7.

10. A method for producing deacetoxycephalosporin C (DAOC) comprising the step of culturing a mutant strain of Acremonium chrysogenum according to any one of claims 1 to 7.

11. A composition for producing 7-amino deacetoxycephalosporanic acid (7-ADCA) comprising a mutant strain of Acremonium chrysogenum according to any one of claims 1 to 7.

12. The step of culturing a mutant strain of Acremonium chrysogenum according to any one of claims 1 to 7, and A method for producing 7-ADCA comprising the step of treating a culture of the mutant strain of Acremonium chrysogenum obtained in the culturing step or the culture medium of the culture with CPC (Cephalosporin C) acylase.

13. A polypeptide represented by SEQ ID NO:

3.

14. A polynucleotide encoding the polypeptide according to claim 13.

15. The polynucleotide according to claim 14, represented by SEQ ID NO:

4.

16. An expression vector comprising a polynucleotide encoding the polypeptide represented by SEQ ID NO:

3.

17. A polypeptide represented by SEQ ID NO: 3, a polynucleotide encoding the polypeptide, and A composition for producing a strain having an improved DAOC production ability, comprising one or more selected from the group consisting of the polynucleotide and the expression vector containing the polynucleotide.

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