Strain for high-level production of adipic acid with enhanced activity of plant-derived acyl-coa oxidase and method for producing adipic acid using same

Abstract

The present invention relates to a recombinant yeast for high-level production of adipic acid, wherein the activity of endogenous acyl-CoA oxidase is weakened compared to the inherent activity, and the activity of plant-derived acyl-CoA oxidase is enhanced compared to the inherent activity. The recombinant yeast according to the present invention can produce adipic acid at a high yield compared to existing unmodified microorganisms.

Description

Adipic acid high-producing strain with enhanced plant-derived acyl-coa oxidase activity and adipic acid production method using the same

[0001] The present invention relates to a high-producing strain of adipic acid with enhanced plant-derived acyl-CoA oxidase activity and a method for producing adipic acid using the same, and more specifically, to a high-producing recombinant yeast with a beta-oxidation pathway redesigned by introducing a gene encoding Arabidopsis-derived acyl-CoA oxidase and a method for producing adipic acid using the same.

[0002]

[0003] Adipic acid is an aliphatic dicarboxylic acid (DCA) with carboxyl functional groups at both ends of a linear six-carbon chain. It is used as an important platform chemical in various industrial fields, including nylon, lubricants, and food additives. In particular, adipic acid is a key monomer required for nylon-6,6 synthesis. As the scope of nylon utilization and demand continues to expand (expected compound annual growth rate (CAGR) of 5.09% until 2035), adipic acid production is also increasing.

[0004] To date, the majority of adipic acid has been produced through a petrochemical process. This involves reacting phenol with hydrogen at high temperature and pressure to produce cyclohexanol, which is then oxidized through a mixture of cyclohexanol and cyclohexanone. However, this production method 1) consumes a lot of energy and 2) produces nitrous oxide, a more potent greenhouse gas than carbon dioxide, as a byproduct, which can accelerate environmental problems, including global warming. To overcome these issues, eco-friendly production methods that take sustainability into account are being developed, and among these, production methods based on microbial fermentation are being considered as a potential alternative.

[0005]

[0006] Currently, there are no microorganisms that naturally produce adipic acid, so the development of strains through genetic engineering is a research field that must precede the production of bio-adipic acid based on microorganisms. The methods of producing bio-adipic acid that have been developed to date are largely divided into three methods: 1) utilizing metabolic pathway engineering strains that reverse engineer the degradative metabolic pathway for utilizing adipic acid as a substrate into a biosynthetic pathway, 2) utilizing biotransformation technology based on microorganisms specialized in fatty acid degradation from renewable resources containing a large amount of fatty acids, such as vegetable oils, and 3) producing cyclohexanol from phenol extracted from lignin and producing adipic acid using biotransformation technology based on microorganisms such as Pseudomonas.

[0007] Among these, research on bioconversion and production of adipic acid from renewable resources derived from fatty acids has primarily focused on utilizing oleaginous yeasts, which possess exceptional lipolytic capabilities. For example, Verdezyne developed a technology to produce adipic acid from vegetable oils such as coconut oil by developing an engineered strain of Candida tropicalis that optimized its lipolytic and oxidative metabolic pathways. However, this approach is limited by the fact that Candida tropicalis is a pathogenic microorganism with the potential to cause human disease and requires a partial saponification process to break down fatty acid esters.

[0008] Yarrowia lipolytica is an oleaginous yeast with similar lipolytic and body fat accumulation capabilities to Candida tropicalis, and is a safe microorganism classified as Generally Recognized as Safe (GRAS). In particular, because it exists in nature in a haploid form, it is easier to produce a recombinant strain than Candida tropicalis, and its efficient lipolytic enzyme (lipase) secretion system allows for the conversion and utilization of fatty acids in the form of fatty acid esters, making it an optimal alternative microorganism that can complement the limitations of Candida tropicalis.

[0009] Through prior research, our research team developed an engineered strain of Yarrowia lipolytica that selectively expresses an acyl-CoA oxidase within the beta-oxidation pathway and enhances the lipid oxidation pathway (omega-oxidation), producing approximately 1.17 g / L of adipic acid from fatty acid-derived substrates. However, further research is needed to precisely control beta-oxidation activity to minimize further degradation of adipic acid following beta-oxidation.

[0010]

[0011] Meanwhile, some plants, such as Arabidopsis and soybeans, are known to have acyl-CoA oxidases with substrate specificity up to 8 carbon atoms. For example, Arabidopsis has four types of acyl-CoA oxidase genes, and the Acx1 enzyme has maximum activity on a 14-carbon substrate, and the Acx2 enzyme has specificity for a relatively long substrate, with maximum activity particularly on a 18-carbon substrate. The Sacx enzyme has specificity for a relatively short substrate, and ACX3 is known to have specificity for a substrate between 8 and 14 carbon atoms, with maximum activity on a 12-carbon substrate. Acyl-CoA oxidase 1;1 from soybean (Glycine max) also has high substrate specificity for lipids with a length of 8 or more carbon atoms. However, to date, there has been no report of regulating the beta-oxidation activity required for adipic acid production by introducing a plant-derived acyl-CoA oxidase gene based on Yarrowia lipolytica.

[0012] Against this backdrop, the inventors of the present invention developed a mutant strain of the genus Yarrowia capable of producing adipic acid from fatty acid-derived products by precisely controlling the beta-oxidation metabolic pathway, which is a fat decomposition pathway, by introducing a gene encoding a plant-derived foreign acyl-CoA oxidase, thereby completing the present invention.

[0013]

[0014] The present invention was completed by confirming the production of adipic acid by a highly adipic acid-producing recombinant yeast in which the activity of endogenous acyl-CoA oxidase is weakened compared to the endogenous activity and the activity of plant-derived acyl-CoA oxidase is enhanced.

[0015]

[0016] The purpose of the present invention is to provide a highly adipic acid-producing recombinant yeast in which the activity of endogenous acyl-CoA oxidase is weakened compared to the endogenous activity and the activity of plant-derived acyl-CoA oxidase is enhanced.

[0017] Another object of the present invention is to provide a composition for producing adipic acid comprising the recombinant yeast and a culture thereof.

[0018] Another object of the present invention is to provide a method for producing adipic acid, comprising a step of culturing the recombinant yeast in a medium.

[0019]

[0020] The recombinant yeast of the present invention is capable of producing adipic acid at a high yield compared to existing unmodified microorganisms.

[0021]

[0022] Figure 1 is a schematic diagram showing an adipic acid production route based on Yarrowia lipolytica according to one embodiment of the present invention.

[0023] Figure 2 is a schematic diagram of a constitutive expression cassette for At.ACX3 insertion according to one embodiment of the present invention.

[0024] FIG. 3 is a schematic diagram of a constitutive expression cassette for insertion of At.ACX3-PTS1 according to one embodiment of the present invention.

[0025] Figure 4 is a schematic diagram of a cassette for POX1 gene deletion according to one embodiment of the present invention.

[0026] Figure 5 is a schematic diagram of a cassette for POX2 gene deletion according to one embodiment of the present invention.

[0027] Figure 6 is a schematic diagram of a cassette for POX3 gene deletion according to one embodiment of the present invention.

[0028] Figure 7 is a schematic diagram of a cassette for POX4 gene deletion according to one embodiment of the present invention.

[0029] Figure 8 is a schematic diagram of a cassette for POX5 gene deletion according to one embodiment of the present invention.

[0030] Figure 9 is a schematic diagram of a cassette for simultaneous insertion of CPR1, ALK5, and FAO1 genes and deletion of the POX6 gene according to one embodiment of the present invention.

[0031] Figure 10 is a diagram comparing adipic acid production for recombinant strains according to one embodiment of the present invention.

[0032]

[0033] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0034] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Furthermore, such equivalents are intended to be encompassed by the present invention.

[0035]

[0036] Yarrowia lipolytica, one of the yeasts of the genus Yarrowia, possesses six types of acyl-CoA oxidase in the beta-oxidation mechanism to produce acetyl-CoA, an energy source, through fat breakdown, and each enzyme is known to have specificity according to the carbon length of the substrate. For example, the POX2 gene can act on a relatively long substrate with a carbon number of 10 or more, and the POX3 gene has specific activity on a relatively short substrate with a carbon number of 8 or less. However, some of the substrate specificities of the six types of POX genes overlap, making precise degradation control according to the carbon number of the substrate difficult.

[0037] The present invention, taking this into account, aims to produce a mutant strain in which the beta-oxidation pathway is redesigned to prevent further decomposition of adipic acid, the target product, and increase production by selectively introducing an acyl-CoA oxidase gene derived from Arabidopsis thaliana, which has high specific activity only for substrates with a carbon length of 8 or more, and the invention is based on the novel confirmation that when a base strain in which six types of acyl-CoA oxidase genes (POX1, 2, 3, 4, 5, 6) originally possessed by Yarrowia lipolytica were deleted and the ACX3 gene derived from Arabidopsis thaliana (At.ACX3) was introduced, the beta-oxidation pathway was precisely redesigned, and adipic acid production was significantly increased compared to the base strain.

[0038]

[0039] One aspect of the present invention provides a highly adipic acid-producing recombinant yeast in which the activity of endogenous acyl-CoA oxidase is weakened compared to the endogenous activity and the activity of plant-derived acyl-CoA oxidase is enhanced.

[0040] The term "endogenous acyl-CoA oxidase" of the present invention refers to an enzyme that catalyzes the reaction of acyl-CoA to trans-2,3-dehydroacyl-CoA, and refers to an enzyme that is endogenously contained in a microorganism containing acyl-CoA oxidase, and for example, refers to an enzyme that catalyzes the beta-oxidation pathway as an oxidase that is endogenously contained in a microorganism of the genus Yarrowia.

[0041] The genes encoding the endogenous acyl-CoA oxidase include POX1, POX2, POX3, POX4, POX5, and POX6, and it is known that a strain lacking all of POX1 to POX6 has completely inhibited activity of beta oxidation (β). In the present invention, the endogenous acyl-CoA oxidase may have, include, or be essentially consisting of an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12, and specifically, may mean a protein encoded by a POX1 to POX6 gene alone or in combination, and more specifically, the acyl-CoA oxidase is encoded by a POX1 to POX6 gene, and the POX1 gene has SEQ ID NO: 1, the POX2 gene has SEQ ID NO: 3, the POX3 gene has SEQ ID NO: 5, and the POX4 gene has SEQ ID NO: 7, POX5 gene may include a polynucleotide sequence of sequence number 9, POX6 gene may include a polynucleotide sequence of sequence number 11, but is not limited thereto.

[0042] In one embodiment of the present invention, it was confirmed that the adipic acid production concentration of the BSHF038 strain, in which POX1 to POX6 were simultaneously deleted to completely inhibit the activity of oxidase, was significantly inhibited.

[0043]

[0044] The term "plant-derived acyl-CoA oxidase" of the present invention refers to an enzyme derived from a plant among acyl-CoA oxidases that catalyze the beta-oxidation pathway.

[0045] The plant-derived acyl-CoA oxidase may be, for example, an Arabidopsis-derived acyl-CoA oxidase or a soybean-derived acyl-CoA oxidase, and may include another plant-derived acyl-CoA oxidase having similar activity.

[0046] The above Arabidopsis-derived acyl-CoA oxidase may be acyl-CoA oxidase 3, and the soybean-derived acyl-CoA oxidase may be soybean-derived acyl-CoA oxidase 1, but is not limited thereto.

[0047] The above Acyl-CoA oxidase may include a codon-optimized sequence, and specifically may have, include, or consist essentially of the amino acid sequence of SEQ ID NO: 20 or 22, and may include, without limitation, an amino acid sequence having the activity of the plant-derived Acyl-CoA oxidase, a partial peptide thereof, or a fragment thereof. Specifically, the plant-derived Acyl-CoA oxidase may be encoded by the At.ACX3 gene.

[0048] The above At.ACX3 gene may have, include, or consist essentially of the polynucleotide sequence of SEQ ID NO: 19 or 21, and may include, without limitation, any polynucleotide sequence encoding a protein having the activity of the plant-derived acyl-CoA oxidase, and may also include a part thereof.

[0049] The above plant-derived acyl-CoA oxidase may be expressed in a peroxisome in a recombinant yeast with enhanced activity of the above oxidase.

[0050] The term "peroxisome" in the present invention refers to an intracellular organelle found in protozoa, yeast, fungi, plant seeds and leaves, and vertebrate livers and kidneys, which catalyzes the breakdown of very long-chain fatty acids through beta-oxidation and plays an important role in reducing reactive oxygen species such as hydrogen peroxide. It is a soluble body abundant in liver and kidney cells, and has lattice-shaped granules inside that contain oxidative enzymes, and is used interchangeably with peroxisomes.

[0051] The plant-derived acyl-CoA oxidase may be characterized in that a peroxisome targeting sequence is operably linked to a gene encoding the plant-derived acyl-CoA oxidase so that it is expressed in a peroxisome in a recombinant yeast.

[0052] The above peroxisome targeting sequence may include the polynucleotide sequence of SEQ ID NO: 23, consist of the polynucleotide sequence, or consist essentially of the polynucleotide sequence, and may include any sequence capable of targeting peroxisomes without limitation.

[0053] The above peroxisome targeting sequence may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 24, and may include any amino acid sequence capable of targeting peroxisomes without limitation.

[0054] The above peroxisome targeting sequence may be linked to the C-terminus of a gene sequence encoding a plant-derived acyl-CoA oxidase.

[0055]

[0056] In the present invention, it may mean that the activity of acyl-CoA oxidase derived from the genus Yarrowia is weakened or inactivated; and the activity of acyl-CoA oxidase derived from a plant is enhanced or activated, and specifically, it may mean that the activity of a gene encoding acyl-CoA oxidase is weakened, deleted or inactivated; and the activity of a gene encoding acyl-CoA oxidase derived from a plant is enhanced, increased or activated.

[0057] For the purposes of the present invention, the recombinant yeast in which the endogenous acyl-CoA oxidase activity is weakened or inactivated; and the activity of a gene encoding plant-derived acyl-CoA oxidase is enhanced, increased, or activated is characterized by a significant increase in adipic acid production compared to the BSHF038 strain in which the endogenous acyl-CoA oxidase activity is weakened or inactivated or the BSHF038 strain modified to increase adipic acid production.

[0058]

[0059] In the present invention, even if a DNA, RNA, polynucleotide, gene or protein is described as having a base sequence or amino acid sequence of a specific sequence number, it is obvious that a DNA, RNA, polynucleotide, gene or protein having a sequence in which some of the sequences are deleted, modified, substituted, conservatively substituted or added may also be used in the present invention, if it has the same or corresponding activity as the DNA, RNA, polynucleotide, gene or protein consisting of the base sequence or amino acid sequence of the sequence number.

[0060] In addition, as long as it has the same or corresponding activity as the DNA, RNA, polynucleotide, gene or protein of the base sequence or amino acid sequence represented by a specific sequence number, a sequence having 80% or more homology or identity with the base sequence or amino acid sequence represented by a specific sequence number may also be included in the scope of the present invention, but is not limited thereto. Specifically, it may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with the base sequence or amino acid sequence of a specific sequence number, and it is obvious that a sequence in which some sequences are deleted, modified, substituted or added is also included in the scope of the present invention as long as it exhibits corresponding efficacy or activity.

[0061] As used herein, the terms "homology" or "identity" refer to the degree to which two given amino acid sequences or base sequences are related to each other, and may be expressed as a percentage. The terms "homology" and "identity" are often used interchangeably.

[0062] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences are generally capable of hybridizing under moderate or high stringency conditions, typically at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or its full length. Hybridization is also contemplated for polynucleotides that contain degenerate codons in place of codons in the polynucleotide.

[0063] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example as in 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 used. (including 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, BLAST from the National Center for Biotechnology Information database, or ClustalW can be used to determine homology, similarity, or identity.

[0064] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using the GAP computer program, as 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 defines the GAP 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 unary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a univariate comparison matrix, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48: 443. 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 a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Therefore, as used herein, the term "homology" or "identity" refers to the relevance between sequences.

[0065] The term "complementary" is used herein to describe the relationship between nucleotide bases capable of hybridizing with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the present invention may also encompass isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0066]

[0067] In the present invention, the term "protein activity inhibition, protein activity inactivation, or protein activity weakening compared to intrinsic activity" means that the activity is inhibited, inactivated, or reduced compared to the activity of a protein that a microorganism originally has in its natural state, and may also include cases where the activity is completely inhibited or eliminated, and the inhibition or weakening may be used interchangeably with terms such as down-regulation, decrease, reduce, and attenuation.

[0068] The above inhibition or weakening may also include cases where the activity of the protein itself is reduced or partially or completely eliminated compared to the activity of the protein originally possessed by the microorganism due to mutation of the gene encoding the protein, cases where the overall level of protein activity within the cell is lower than that of the natural strain due to inhibition of expression or translation of the gene encoding the protein, cases where the gene is not expressed at all, and cases where the gene is expressed but has no activity.

[0069] Inhibition or weakening of such protein activity can be achieved by various methods well known in the art. Examples of such methods include a method of replacing a gene encoding the protein on a chromosome with a mutated gene such that the activity of the enzyme is reduced, including cases where the activity of the protein is eliminated; a method of modifying an expression control sequence of a gene encoding the protein; a method of deleting all or part of a gene on a chromosome encoding the protein; a method of introducing an antisense oligonucleotide (e.g., antisense RNA) that complementarily binds to a transcript of the gene on the chromosome and inhibits translation from the mRNA into a protein; a method of artificially adding a sequence complementary to the SD sequence in front of the SD sequence of the gene encoding the protein to form a secondary structure, thereby making ribosome attachment impossible; and a RTE (Reverse transcription engineering) method of adding a promoter to the 3' end of the ORF (open reading frame) of the corresponding sequence so that reverse transcription occurs; and the like, and combinations thereof can also be achieved, but are not particularly limited by the above examples.

[0070] Specifically, the method for deleting part or all of the gene encoding the protein can be performed by replacing the polynucleotide encoding the endogenous target protein in the chromosome with a polynucleotide or marker gene having a partial nucleic acid sequence deleted through a vector for chromosomal insertion into bacteria. As an example, a method of deleting the gene by homologous recombination can be used. In addition, the term "part" as described above varies depending on the type of polynucleotide, but may specifically be 1 to 300, preferably 1 to 100, and more preferably 1 to 50, but is not particularly limited thereto.

[0071] In addition, the method of modifying the expression control sequence can be performed by inducing a mutation in the expression control sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the nucleic acid sequence to further weaken the activity of the expression control sequence, or by replacing it with a nucleic acid sequence having weaker activity. The expression control sequence includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and translation.

[0072] In addition, the method of modifying the gene sequence on the chromosome can be performed by inducing a mutation in the gene sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof to further weaken the activity of the protein, or by replacing the gene sequence with a gene sequence modified to have weaker activity or a gene sequence modified to have no activity.

[0073]

[0074] The term "enhancement of protein activity compared to intrinsic activity" or "introduction of protein" in the present invention can also be expressed as "increased activity" and means that a prior to genetic modification microorganism or a non-modified or unmodified microorganism is enhanced compared to before modification due to introduction of a foreign gene, etc., and the enhancement can be used interchangeably with terms such as activation, up-regulation, overexpression, and increase.

[0075] The term "endogenous" in the present invention refers to the original state of the parent strain prior to the change in phenotype, when the phenotype of a microorganism changes due to genetic mutation caused by natural or artificial factors. The increased activity may include both the introduction of an exogenous protein and the enhancement of the activity of an endogenous protein. The increased / enhanced activity of the protein may be achieved by the increased / enhanced expression of a gene.

[0076] Specifically, in the present invention, active enhancement is

[0077] 1) Increase in the copy number of the polynucleotide encoding the above protein,

[0078] 2) Modification of the expression control sequence to increase the expression of the above polynucleotide,

[0079] 3) Modification of the polynucleotide sequence on the chromosome to enhance the activity of the above protein;

[0080] 4) Introduction of a foreign polynucleotide exhibiting the activity of the above protein or a codon-optimized variant polynucleotide of the above polynucleotide, or

[0081] 5) It can be performed by a method of transforming it to be strengthened by a combination of these, but is not limited thereto.

[0082]

[0083] The above 1) increase in the copy number of the polynucleotide may be performed in a form operably linked to a vector, or by insertion into a chromosome in a host cell, but is not particularly limited thereto. Specifically, the increase may be performed by introducing a polynucleotide encoding the protein of the present invention into a host cell by being operably linked to a vector capable of replicating and functioning independently of the host, or by introducing the polynucleotide into a host cell by being operably linked to a vector capable of inserting the polynucleotide into a chromosome in the host cell, thereby increasing the copy number of the polynucleotide in the chromosome of the host cell.

[0084] Next, 2) Modification of the expression control sequence to increase the expression of the polynucleotide may be performed by, but is not particularly limited to, inducing a sequence mutation in the nucleic acid sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the expression control sequence, or by replacing the nucleic acid sequence with a nucleic acid sequence having stronger activity. The expression control sequence may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, etc.

[0085] In the present invention, promoter strengthening may be performed by substituting the endogenous promoter of each of the FAA1, LCB1, TSC10, and LCB3 genes with a TEF (termination elongation factor) promoter and a TEF intron, and specifically, the TEF promoter and the TEF intron may include the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. In addition, 3) modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but may be performed by inducing a mutation in the expression regulatory sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further strengthen the activity of the polynucleotide sequence, or by replacing it with a polynucleotide sequence that has been improved to have stronger activity.

[0086] In addition, 4) introduction of a foreign polynucleotide sequence can be performed by introducing a foreign polynucleotide encoding a protein exhibiting the same / similar activity as the protein, or a codon-optimized mutant polynucleotide thereof, into a host cell. The foreign polynucleotide can be used without limitation in its origin or sequence as long as it exhibits the same / similar activity as the protein. In addition, the introduced foreign polynucleotide can be introduced into a host cell by optimizing its codons so that optimized transcription and translation can occur within the host cell. The introduction can be performed by a person skilled in the art appropriately selecting a known transformation method, and the introduced polynucleotide can be expressed within the host cell, thereby producing a protein and increasing its activity.

[0087] Finally, 5) the method of modifying to be strengthened by a combination of the above 1) to 4) can be performed by applying at least one of the following methods together: increasing the copy number of a polynucleotide encoding the protein, modifying an expression regulatory sequence to increase its expression, modifying the polynucleotide sequence on a chromosome, and modifying a foreign polynucleotide exhibiting the activity of the protein or a codon-optimized mutant polynucleotide thereof.

[0088]

[0089] In the present invention, the term "unmodified microorganism" does not exclude a strain that contains a mutation that may occur naturally in a microorganism, and means a natural strain itself, a microorganism that does not contain a deletion or overexpression of the protein, a microorganism that has not been transformed with a vector designed to delete or overexpress the protein, or a microorganism before modification such as transformation.

[0090] The term "vector" of the present invention refers to a DNA construct containing a polynucleotide sequence encoding a target protein operably linked to a suitable regulatory sequence so as to enable expression of the target protein in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. The vector, after being transformed into a suitable host cell, can replicate or function independently of the host genome, and can be integrated into the genome itself. For example, a vector for intracellular chromosomal integration can be used to replace a polynucleotide encoding a target protein in a chromosome with a mutated polynucleotide. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination.

[0091] The vector of the present invention 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 pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0092] The term "transformation" of the present invention refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed within the host cell. The transformed polynucleotide may be located within the chromosome of the host cell or outside the chromosome, as long as it can be expressed within the host cell. Furthermore, the polynucleotide includes DNA and RNA encoding the target protein. 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 the elements necessary for autonomous expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. Additionally, 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.

[0093] Additionally, the term "operably linked" in the present invention means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target protein of the present invention.

[0094] The method for transforming the vector of the present invention includes any method for introducing nucleic acids into cells, and can be performed by selecting an appropriate standard technique known in the art depending on the host cell. Examples thereof include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0095]

[0096] In the present invention, the term "adipic acid-high-producing recombinant yeast" includes all yeast that has undergone genetic modification, either naturally or artificially, and is a yeast in which a specific mechanism has been weakened or strengthened due to causes such as the insertion of an external gene or the strengthening or inactivation of the activity of an endogenous gene, and may be a microorganism in which genetic mutation has occurred for the production of the desired adipic acid or in which the activity has been weakened, inhibited, inactivated, strengthened, or the activity has been strengthened by the introduction of a foreign gene. For the purpose of the present invention, the yeast that produces adipic acid may refer to a microorganism in which the adipic acid biosynthesis pathway has been strengthened or the degradation pathway has been weakened, and which can produce adipic acid in excess compared to a wild type or unmodified microorganism.

[0097]

[0098] Specifically, it may refer to a recombinant yeast in which the activity of acyl-CoA oxidase endogenous to yeast of the genus Yarrowia is inhibited, suppressed, or inactivated through selective weakening or deletion of a gene, or in which the beta-oxidation pathway by a foreign enzyme, plant-derived acyl-CoA oxidase, is strengthened through strengthening or introduction of a gene, and more specifically, it may refer to a recombinant yeast in which POX, a gene encoding an endogenous acyl-CoA oxidase of the genus Yarrowia, is weakened or deleted, and the At.ACX3 gene, a gene encoding a plant-derived acyl-CoA oxidase, is introduced, or the metabolic pathway for biosynthesizing adipic acid can be redesigned to be expressed in a peroxisome by additionally including a peroxisome targeting sequence, thereby increasing the production of adipic acid and increasing the production of accumulated adipic acid, but is not limited thereto. In the present invention, the “recombinant microorganism with increased adipic acid production” may be used interchangeably with “microorganism having adipic acid production ability” or “microorganism producing adipic acid.”

[0099] For the purposes of this application, the recombinant microorganism with enhanced adipic acid production is characterized by increased adipic acid production. This is significant because, unlike wild-type or unmodified microorganisms that either cannot produce adipic acid or produce trace amounts of adipic acid, the gene expression regulation of the present invention can increase adipic acid production.

[0100] The recombinant microorganism with increased adipic acid production is not particularly limited in type as long as it can produce adipic acid, but may be yeast, and more specifically, may be Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Neurospora crassa, Candida tropicalis, Yarrowia lipolytica, etc., and more specifically, may be Yarrowia lipolytica, but any microorganism belonging to yeast capable of producing adipic acid may be included without limitation.

[0101] In addition, in the present invention, the parent strain of the recombinant microorganism with increased adipic acid production is not particularly limited as long as it is a microorganism that produces adipic acid, and the microorganism that produces adipic acid may be a microorganism in which a gene that weakens the adipic acid biosynthesis pathway is inactivated, and / or the adipic acid biosynthesis pathway or the production of adipic acid precursor is enhanced, in order to increase the production of adipic acid.

[0102] The parent strain of the above microorganism may be modified to enhance the adipic acid biosynthetic pathway or the production of adipic acid precursors by introducing a strong promoter or genetic mutation into the upstream region of the gene to enhance the expression of the gene, but is not limited thereto. Furthermore, methods for removing feedback inhibition of adipic acid, increasing the expression of adipic acid-related operons, or conferring resistance to adipic acid analogs may be used. However, the present invention is not limited thereto, and adipic acid production capacity may be enhanced using any gene expression control method known in the art.

[0103]

[0104] In one embodiment, the recombinant yeast may be a highly adipic acid-producing recombinant yeast in which the POX1 to POX6 genes inherent in yeast of the genus Yarrowia are deleted and the At.ACX3 gene derived from Arabidopsis thaliana is introduced.

[0105] In another embodiment, the recombinant yeast may be a highly adipic acid-producing recombinant yeast in which the POX1 to POX6 genes endogenously possessed by yeast of the genus Yarrowia are deleted, the Arabidopsis-derived At.ACX3 gene is introduced, and a peroxisome targeting sequence is linked to the C-terminus of the Arabidopsis-derived At.ACX3 gene.

[0106] In another embodiment, the recombinant yeast may be a high-adipic acid-producing recombinant yeast in which the POX1 to POX6 genes endogenously possessed by yeast of the genus Yarrowia are deleted, the Arabidopsis-derived At.ACX3 gene is introduced, a peroxisome targeting sequence is linked to the C-terminus of the Arabidopsis-derived At.ACX3 gene, and the KU70 gene and MHY1 gene are additionally deleted.

[0107] In the present invention, the term "KU70" gene is an ATP-dependent DNA helicase II subunit involved in the non-homologous end joining (NHEJ) activity of a wild-type Yarrowia lipolytica strain, and is one of the genes that cleaves and repairs DNA double strands. Generally, by forcibly deleting the KU70 gene in a wild-type Yarrowia lipolytica, the efficiency of homologous recombination is increased, thereby improving the efficiency of introducing or deleting foreign genes.

[0108] In the present invention, the term "MHY1" gene is a gene that acts as a key regulator of the heteroplastic transition to hyphae form, and controls hyphal growth, thereby enabling the strain to grow in a colony form.

[0109] In another embodiment, the recombinant yeast may be a high-adipic acid-producing recombinant yeast in which the POX1 to POX6 genes endogenously possessed by yeast of the genus Yarrowia are deleted, the Arabidopsis-derived At.ACX3 gene is introduced, a peroxisome targeting sequence is linked to the C-terminus of the Arabidopsis-derived At.ACX3 gene, the KU70 gene and the MHY1 gene are additionally deleted, and an omega-oxidation pathway-related gene is additionally introduced.

[0110] In the present invention, the term omega oxidation pathway refers to a pathway that substitutes a fatty acid terminal with a carboxyl group, and the omega oxidation pathway related gene may be CPR1, ALK5, FAO1, or a combination thereof.

[0111] In the present invention, the term "CPR1" gene encodes cytochrome P450 reductase (NADPH-dependent cytochrome P450 reductase), and the hydroxyl functional group insertion mechanism, which is the first step of the omega oxidation pathway, acts complementarily with cytochrome P450 monooxygenase.

[0112] In the present invention, the term "ALK5" gene is one of 12 types of cytochrome P450 monooxygenase possessed by Yarrowia lipolytica, and the effect of increasing adipic acid production through activity enhancement was confirmed in a previously published patent.

[0113] In the present invention, the term "FAO1" gene is a gene encoding fatty alcohol oxidase, an enzyme that acts in the second step of the omega oxidation pathway.

[0114] In one embodiment of the present invention, it was confirmed that a strain in which the POX1 to POX6 genes inherent to a Yarrowia strain were deleted and the Arabidopsis-derived At.ACX3 gene was introduced increased the adipic acid production by about 1.7 times, or about 75%, compared to a control group in which the At.ACX3 gene was not strengthened. In particular, in the case of a recombinant yeast in which the peroxisome targeting sequence was not linked, the adipic acid production was very low at the same level as the control strain despite the introduction of a gene encoding an Arabidopsis-derived acyl-CoA oxidase, whereas in the case of a recombinant yeast in which the peroxisome targeting sequence was linked to the C-terminus of the Arabidopsis-derived At.ACX3 gene, a significantly superior adipic acid production was confirmed to increase.

[0115]

[0116] Another aspect of the present invention provides a composition for producing adipic acid comprising the recombinant yeast or a culture thereof.

[0117] The above “recombinant yeast” and “adipic acid” are as described above.

[0118] In the present invention, the term "culture thereof" refers to a yeast culture, and refers to a substance excreted by the yeast into the medium during the culturing process, and may include a product made by drying the yeast and a mixture within the culture medium. For example, it may include the yeast itself and digestive enzymes, vitamin B complex, chelate minerals, nucleic acids, etc. excreted by the yeast into the medium.

[0119]

[0120] Another aspect of the present invention provides a method for producing adipic acid, comprising the step of culturing the yeast in a medium.

[0121] The above “adipic acid” is as described above.

[0122]

[0123] The term "cultivation" in the present invention refers to growing the microorganism under appropriately controlled environmental conditions. The culturing process of the present invention can be performed using any suitable medium and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, or fed-batch, but is not limited thereto.

[0124] In the present invention, the term "medium" refers to a material containing nutrients necessary for culturing the microorganism as a main component, 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 invention may be any medium used for culturing general microorganisms without particular limitation, but the microorganism of the present invention may be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid, and / or vitamin.

[0125] In the present invention, the carbon source may include carbohydrates such as glucose, 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 bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to 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.

[0126] 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.

[0127] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. 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 suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0128] In the present invention, during the cultivation of microorganisms, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, an antifoaming agent such as fatty acid polyglycol ester can be used to suppress bubble formation. In addition, to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or to maintain anaerobic and microaerobic states, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, but is not limited thereto.

[0129] The temperature of the medium may be, but is not limited to, 20°C to 50°C, specifically 30°C to 37°C. The incubation period may continue until the desired amount of useful material is produced, and specifically may be, but is not limited to, 10 to 100 hours.

[0130] Adipic acid produced by the above culture may be released into the medium or may remain within the cells without being released.

[0131]

[0132] The above method for producing adipic acid may include a step of recovering adipic acid from a cultured microorganism or medium.

[0133] The method for recovering adipic acid produced in the above-described culturing step of the present invention may be to collect the desired adipic acid from the culture solution using a suitable method known in the art, depending on the culturing method. For example, centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC may be used, and the desired adipic acid may be recovered from the medium or microorganism using a suitable method known in the art.

[0134] Additionally, the recovery step may include a purification process, which may be performed using any suitable method known in the art. Accordingly, the recovered adipic acid may be in purified form or a microbial fermentation broth containing adipic acid (Introduction to Biotechnology and Genetic Engineering, AJ Nair., 2008).

[0135] Another aspect of the present invention provides a use of the recombinant yeast for producing adipic acid.

[0136]

[0137] Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, these examples and experimental examples are intended to exemplify the present invention, and the scope of the present invention is not limited to these examples and experimental examples.

[0138]

[0139] Example 1. Construction of a constitutive expression cassette for insertion of the Arabidopsis thaliana-derived acyl-CoA oxidase 3 gene.

[0140] In the present invention, to introduce the Arabidopsis-derived acyl-CoA oxidase 3 (At.ACX3, 2,049 bp) gene, a codon-optimized form of the gene based on Yarrowia lipolytica was synthesized. Thereafter, gene cloning was performed so that the gene is constantly expressed by the TEFint promoter (PTEFint) and the TDH3 terminator (TTDH3).

[0141] In addition, in order to compare the activity differences according to the intracellular location where the Arabidopsis-derived acyl-CoA oxidase is expressed, a gene cloning was performed that included the peroxisomal targeting sequence (peroxisomal targeting signal1, PTS1, 5`-GGTGGTGGTTCTTCTAAACTA-3`) of Yarrowia lipolytica at the C-terminus of the At.ACX3 gene.

[0142] Next, to enable the PTEFint-At.ACX3-TTDH3 or PTEFint-At.ACX3-PTS1-TTDH3 gene cassettes to be inserted into the Yarrowia lipolytica IntC3 locus, the URA3 gene was used as an auxotrophic marker and an IntC3 locus-based At.ACX3 or At.ACX3-PTS1 gene insertion cassette having 1 kb-long homologous regions at both ends was constructed as shown in FIGS. 2 and 3, respectively (FIGS. 2 and 3).

[0143]

[0144] Example 2. Production of Yarrowia lipolytica strain lacking the internal acyl-CoA oxidase gene.

[0145] In order to confirm the activity of At.ACX3, a plant-derived foreign gene, introduced into Y. lipolytica, a strain was created in which all six genes (POX1, 2, 3, 4, 5, and 6) encoding acyl-CoA oxidases in the beta-oxidation pathway in Y. lipolytica were deleted. The deletion cassettes for each gene were used as a sample, and the genomic DNA of the Po1f strain (purchased from ATCC, strain number MYA-2613) was used to amplify -1000 bp at the 5' position and 1000 bp at the 3' position based on the open reading frame (ORF) of each POX gene by PCR. Then, a gene deletion cassette was created using the URA3 gene as an auxotrophic marker.

[0146] The completed cassette was amplified by PCR and sequentially transformed into the BSHF002 strain to complete the BSHF053 strain, which lacks all six acyl-CoA oxidase genes.

[0147] Specifically, the BSHF002 strain was constructed using the BSHF003 strain, which has the KU70 gene, MHY1 gene, and POX3 gene all deleted from the Yarrowia lipolytica Po1f strain, as a parent strain. First, a cassette for the deletion of the POX2 gene was amplified by PCR and transformed into the BSHF003 strain to create the BSHF004 strain. Then, transformation of each deletion cassette was sequentially performed to create the BSHF005 strain, which has the POX4 gene deleted in the BSHF004 strain, the BSHF006 strain, which has the POX5 gene deleted in the BSHF005 strain, and the BHSF007 strain, which has the POX1 gene deleted in the BSHF006 strain. Finally, the BSHF007 strain was transformed with a POX6 deletion cassette containing the CPR1, ALK5, and FAO1 genes, which are omega-oxidation factors for increasing adipic acid production, to produce the BSHF053 strain, which has a POX6 gene deletion and an enhanced omega-oxidation pathway (see Figs. 4 to 9).

[0148]

[0149] Example 3. Construction of strains inserting At.ACX3-PTS1 or At.ACX3 constitutive expression cassettes.

[0150] Example 3-1. Construction of strain with At.ACX3-PTS1 constant expression cassette insertion

[0151] The At.ACX3 gene cassette containing the peroxisome targeting sequence produced in Example 1 at the C-terminus was amplified by PCR and transformed into the BSHF053 strain using the same method as in Example 2 to produce the BSHF057 strain.

[0152]

[0153] Example 3-2. Construction of strain with At.ACX3 constitutive expression cassette insertion

[0154] The At.ACX3 gene cassette, which did not include the peroxisome targeting sequence produced in Example 1, was amplified by PCR and transformed into the BSHF053 strain using the same method as in Example 2 to produce the BSHF058 strain.

[0155]

[0156] Example 4. Confirmation of adipic acid production by At.ACX3 insertion strain

[0157] To confirm the adipic acid production capacity of the BSHF057 and BSHF058 strains produced in Example 3, a culture evaluation was conducted. The parent strain BSHF053 strain served as the control group, and the adipic acid-producing strain BSHF038 (see Bioresource Technology 391 (2024) 129920), developed through a previous study, served as the comparison group. For reference, various strains developed in the above-mentioned previous study were referenced in the present study.

[0158] Specifically, each strain was cultured for 24 hours at a culture temperature of 30°C and a stirring speed of 200 rpm in a 250 mL baffled flask containing 20 mL of YP complex medium containing 2% glucose. Then, the cells in the culture medium corresponding to OD600=2 were inoculated into 50 mL of YP complex medium containing 2% glucose and cultured for another 24 hours. The cells inoculated into 20 mL of YPDP50 complex medium containing 5% glucose at OD600=40 and cultured for an additional 24 hours. After adding 1% (v / v) of the culture volume as a substrate and culturing for 96 hours, samples were collected from the culture and the concentration of adipic acid in the culture medium was quantified by HPLC. The results are shown in Fig. 10.

[0159] As can be seen in Fig. 10, in the BSHF053 strain, which has a complete lack of acyl-CoA oxidase in Yarrowia lipolytica, the adipic acid production was very low at the level of 40 mg / L, and it was confirmed that although the omega-oxidation pathway was strengthened, adipic acid was hardly produced due to a deficiency in beta-oxidation.

[0160] The BSHF038 strain developed in the previous study was confirmed to produce adipic acid in an amount (1,027 mg / L) similar to the previous experimental results.

[0161] The BSHF057 strain, an experimental strain with a peroxisome targeting sequence, which is a high-adipic acid producing strain of the present invention, showed an adipic acid production (1,794 mg / L) that was approximately 1.7 times higher than that of the control strain BSHF038, whereas the BSHF058 strain, into which the At.ACX3 gene without a peroxisome targeting sequence was inserted, showed a very low adipic acid production at the same level as that of the control strain, despite the introduction of an acyl-CoA oxidase derived from Arabidopsis thaliana.

[0162]

[0163] These results indicate that the lipolytic metabolic pathway of Y. lipolytica, which is completely inhibited due to a deficiency of acyl-CoA oxidase, can be restored by introduction of an acyl-CoA oxidase from Arabidopsis, and in particular, the exogenous acyl-CoA oxidase exhibits activity when targeted to the peroxisome, the main organelle where beta-oxidation occurs.

[0164] In particular, compared to the BSHF038 strain studied in a previous study, the new strain of the present invention showed a significant increase in adipic acid production. Based on this, it was confirmed that a more effective adipic acid-producing strain can be constructed by utilizing the At.ACX3 gene compared to the native oxidase of Yarrowia lipolytica. These results suggest that the Arabidopsis-derived acyl-CoA oxidase 3 expressed from the At.ACX3 gene has precise substrate specificity.

[0165]

[0166] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. The activity of endogenous acyl-CoA oxidase is weakened compared to the intrinsic activity; A highly adipic acid-producing recombinant yeast with enhanced activity of plant-derived acyl-CoA oxidase compared to the endogenous activity.

2. A recombinant yeast producing a high amount of adipic acid in the first paragraph, wherein the plant is Arabidopsis thaliana, soybean, or a combination thereof.

3. A recombinant yeast according to claim 1, wherein the plant-derived acyl-CoA oxidase is expressed in a peroxisome within the recombinant yeast.

4. In the third paragraph, the recombinant yeast is characterized in that a peroxisome targeting sequence is operably linked to a gene encoding the plant-derived acyl-CoA oxidase so that the plant-derived acyl-CoA oxidase is expressed in a peroxisome in the recombinant yeast.

5. In the first paragraph, the recombinant yeast is a recombinant yeast in which the KU70 gene and the MHY1 gene are additionally deleted.

6. In the first paragraph, the recombinant yeast is a recombinant yeast in which an omega oxidation pathway-related gene is additionally strengthened.

7. A recombinant yeast according to claim 6, wherein the omega oxidation pathway related gene is CPR1, ALK5, FAO1 or a combination thereof.

8. A recombinant yeast according to claim 1, wherein the acyl-CoA oxidase comprises an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12.

9. A recombinant yeast according to claim 8, wherein the acyl-CoA oxidase is encoded by a gene of POX1 to POX6.

10. A recombinant yeast according to claim 8, wherein the acyl-CoA oxidase is encoded by any one gene sequence selected from the group consisting of sequence numbers 1, 3, 5, 7, 9, and 11.

11. A recombinant yeast according to claim 1, wherein the plant-derived acyl-CoA oxidase comprises an amino acid sequence of SEQ ID NO: 20 or 22.

12. A recombinant yeast according to claim 11, wherein the plant-derived acyl-CoA oxidase is encoded by a gene sequence of SEQ ID NO: 19 or 21.

13. A recombinant yeast according to claim 4, wherein the peroxisome target sequence comprises a polynucleotide sequence of SEQ ID NO:

23.

14. A recombinant yeast according to claim 13, wherein the peroxisome targeting sequence is linked to the C-terminus of a gene sequence encoding a plant-derived acyl-CoA oxidase.

15. A recombinant yeast according to claim 1, wherein the yeast is of the genus Yarrowia.

16. A recombinant yeast according to claim 1, wherein the yeast is Yarrowia lipolytica.

17. A composition for producing adipic acid comprising the recombinant yeast of any one of claims 1 to 16 or a culture thereof.

18. A method for producing adipic acid, comprising a step of culturing the recombinant yeast of any one of claims 1 to 16 in a medium.

19. A method for producing adipic acid, wherein the method further comprises a step of recovering adipic acid from the cultured yeast or medium.

20. Use of the recombinant yeast of any one of claims 1 to 16 for producing adipic acid.

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