Aldehyde deformylating oxygenase (ADO) mutants and their uses

Abstract

To provide an ADO mutant modified to have high specificity for aldehyde substrates with a specific carbon chain length.SOLUTION: Provided is an aldehyde deformylating oxygenase (ADO) mutant, characterized in that the amino acid sequence at the specificity modification site is modified, and thus the ADO mutant has high specificity for aldehyde substrates having a specific carbon chain length compared to aldehyde substrates having other carbon chain lengths.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to aldehyde deformylating oxygenase (ADO) mutants. More particularly, the present disclosure relates to ADO mutants engineered to have high specificity for aldehyde substrates having specific carbon chain lengths, and to microorganisms into which such ADO mutants have been introduced. [Background technology]

[0002] Aldehyde deformylating oxygenase (ADO) is the only enzyme that can convert aldehydes into alkanes, and is expected to be used in the production of various useful substances including fuels, and is therefore expected to be applied in various fields. Summary of the Invention [Means for solving the problem]

[0003] Thus, the present disclosure provides ADO mutants that have been engineered to have high specificity for aldehyde substrates having specific carbon chain lengths.

[0004] Thus, the present disclosure provides: (Item 1) An aldehyde deformylating oxygenase (ADO) mutant, characterized in that the amino acid sequence of a specificity modification site is modified, thereby having high specificity for an aldehyde substrate having a specific carbon chain length compared to aldehyde substrates having other carbon chain lengths. (Item 2) The ADO variant described in the above item, wherein the ADO comprises NpADO. (Item 3) The ADO mutant according to any one of the preceding claims, wherein the specificity modification site comprises a substrate binding site. (Item 4) The ADO mutant described in any one of the above items, wherein the specificity modification site includes an amino acid site that is located within approximately 5 Å of the substrate when the ADO mutant binds to the substrate to form a three-dimensional structure. (Item 5) The ADO variant of any one of the preceding claims, wherein the amino acid sequence identified based on the affinity of ADO for a substrate is modified. (Item 6) The ADO variant of any one of the preceding claims, wherein the modification comprises multiple mutations. (Item 7) The ADO variant of any one of the preceding items, wherein the modification comprises an amino acid modification corresponding to one or more residues selected from the group consisting of I25, V29, Q111, A122, Y126, W179, and V185 in the amino acid sequence of ADO when aligned with SEQ ID NO:1. (Item 8) The ADO variant of any one of the preceding items, wherein the modification comprises an amino acid modification corresponding to one or more residues selected from the group consisting of V29Y, Q111Y, A122P, Y126S, Y126T, W179R, and V185F in the amino acid sequence of ADO when aligned with SEQ ID NO:1. (Item 9) The ADO mutant according to any one of the preceding items, wherein the modification comprises an amino acid modification corresponding to V29Y / Q111Y / W179R in the amino acid sequence of ADO when aligned with SEQ ID NO:1. (Item A1) A microorganism into which an aldehyde deformylating oxygenase (ADO) or a mutant thereof that specifically reacts with an aldehyde substrate possessed by the microorganism has been introduced. (Item A2) The microorganism described in the above item, wherein the ADO includes NpADO. (Item A3) A microorganism described in any one of the above items, wherein the mutant has an alteration in the amino acid sequence of the specificity alteration site of the ADO. (Item A4) A microorganism described in any one of the above items, wherein the mutant has an amino acid sequence modification at the specificity modification site of the ADO, identified based on the affinity of ADO for a substrate. (Item A5) A microorganism described in any one of the above items, wherein the specificity modification site includes a substrate binding site. (Item A6) A microorganism described in any one of the above items, wherein the specificity modification site includes an amino acid site that is located within approximately 5 Å of the substrate when the ADO mutant binds to the substrate to form a three-dimensional structure. (Item A7) The microorganism according to any one of the preceding items, wherein the modification comprises multiple mutations. (Item A8) The microorganism described in any one of the above items, wherein the mutant comprises an amino acid modification corresponding to one or more residues selected from the group consisting of I25, V29, Q111, A122, Y126, W179, and V185 in the amino acid sequence of ADO when aligned with SEQ ID NO:1. (Item A9) The microorganism according to any one of the preceding paragraphs, wherein the mutant comprises an amino acid modification in the amino acid sequence of ADO that corresponds to one or more residues selected from the group consisting of V29Y, Q111Y, A122P, Y126S, Y126T, W179R, and V185F when aligned with SEQ ID NO:1. (Item A10) The microorganism according to any one of the preceding items, wherein the mutant comprises an amino acid modification in the amino acid sequence of ADO that corresponds to V29Y / Q111Y / W179R when aligned with SEQ ID NO:1.

[0005] It is contemplated that one or more of the above features may be provided in combinations other than those specifically stated.Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0006] Furthermore, features and notable actions and effects of the present disclosure other than those described above will become apparent to those skilled in the art by referring to the following description of the preferred embodiments of the present invention and the drawings. Effect of the Invention

[0007] The present disclosure can provide an ADO mutant modified to have high specificity for an aldehyde substrate having a specific carbon chain length. The present disclosure can also provide a microorganism into which an ADO mutant modified to have high specificity for an aldehyde substrate having a specific carbon chain length has been introduced, and by using such a microorganism, a system for producing alkanes suitable for applications such as fuel and cosmetics can be constructed. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a graph showing the results of activity measurements of ADO mutants against C11 and C15 alkanes in one embodiment of the present disclosure. [Diagram 2] FIG. 2 is a graph showing the results of activity measurements of ADO multiple mutants against C11 and C15 alkanes in one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present disclosure will be described below while showing the best mode. Throughout this specification, the expression of the singular form should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will take precedence.

[0010] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0011] As used herein, "about" means ±10% of the preceding numerical value.

[0012] As used herein, "aldehyde deformylating oxygenase (ADO)" refers to an enzyme that produces hydrocarbons such as alkanes using aldehydes as a raw material, and the origin of the enzyme is not limited. Representative examples include ADO from cyanobacteria. Examples of ADO include OsADO, NpADO, SeADO, 6803ADO, TeADO, 51142ADO, 7425-1ADO, 7425-2ADO, 8801ADO, CwADO, PaADO, LaADO, 9443ADO, LiADO, PmADO, NATL2aADO, 9917-2ADO, SyADO, 7421ADO, and 7336ADO. The types and origins of ADOs are exemplified in, for example, Schirmer, A., et al., (2010), Science 329:559-562, Arai, M., et al., (2018), AEMB., 1080, 119-154, and Kudo et al., (2019), Biotechnology for Biofuels and Bioproducts, 12, 89.

[0013] As used herein, the term "specificity modification site" refers to the substrate binding site in ADO to which the substrate aldehyde binds or its surrounding site, and includes all residues surrounding the substrate and involved in the formation of the substrate pocket. For example, the specificity modification site can include an amino acid site that is within 5 Å from the substrate in the substrate-enzyme three-dimensional structure.

[0014] As used herein, the term "substrate binding site" refers to the site at which an enzyme binds to a substrate.

[0015] In this specification, the term "microorganism" refers to a minute living organism, and includes, for example, prokaryotes such as bacteria and actinomycetes, eukaryotes such as yeast and mold, lower algae, fungi, viruses, etc., as well as cells that exist separately even in multicellular organisms such as animals and plants. In addition, the term "microorganism" includes not only naturally occurring microorganisms, but also those that have been cultured and artificially grown, those that have mutated, or those that have been artificially modified by transformation or other techniques.

[0016] As used herein, "modification" of a gene means that a certain nucleotide (e.g., dC) on a DNA strand is converted to another nucleotide (e.g., dT, dA, or dG) or deleted, or that a nucleotide or nucleotide sequence is inserted or added between certain nucleotides on a DNA strand. As used herein, "modification" includes the substitution or deletion of one or more nucleotides at a targeted site of double-stranded DNA, or the insertion or addition of one or more nucleotides at a targeted site of double-stranded DNA. Here, the double-stranded DNA to be modified is not particularly limited, but is preferably genomic DNA.

[0017] As used herein, "modification of an amino acid corresponding to X" refers to a modification of an amino acid present at a position corresponding to amino acid X in SEQ ID NO: 1 in a particular ADO when the amino acid sequences of various ADOs are aligned.

[0018] When amino acids are represented by single letters, such as Xn (X is the single letter of the amino acid, and n is the residue position of the amino acid), this means that the amino acid X of a particular letter at residue position n in SEQ ID NO:1 is mutated. For example, I25 means that the 25th amino acid in SEQ ID NO:1 is isoleucine, and can indicate the position of the mutation. Also, XnY (X is the single letter of the amino acid, n is the residue position of the amino acid, and Y is the single letter of the amino acid) means that the amino acid X is mutated to the amino acid Y at that residue position in SEQ ID NO:1.

[0019] As used herein, the term "gene" is interpreted in the broadest sense and refers to a character string of nucleic acid or a sequence of a substance that carries it (e.g., nucleotides such as DNA or RNA), and preferably refers to a sequence or a substance that contains a sequence that exerts some function. For example, in addition to those that code for proteins, adjacent transcriptional regulatory regions such as promoters and enhancers that control the timing and amount of transcription of a transcript as a transcription factor binding site, and adjacent transcriptional regulatory regions such as promoters and enhancers that control the timing and amount of transcription of a transcript as a transcription factor binding site, are also included.

[0020] In this specification, "introduction" of a gene refers to the introduction of an exogenous or endogenous gene, preferably a functional gene, into, for example, a chromosomal genome, etc., by an appropriate introduction technique. A gene can be introduced using a vector such as a phage or a plasmid, and natural transformation, conjugation, protoplast-PEG, electroporation, etc. can also be used for the introduction of a gene. In addition, by utilizing a target gene recombination method known in the art, an exogenous functional gene can be introduced by replacing it with an endogenous functional gene. Note that an exogenous functional gene is a gene that does not originally exist in the chromosomal genome of the organism, and may be a gene derived from another organism or a synthetic gene produced by PCR or the like. The introduction of a gene also includes conversion to a desired gene by genome editing of an existing genome.

[0021] In this specification, the term "carbon chain length" refers to the number of carbon atoms constituting an alkyl group. The carbon chain length refers to the length of the longest continuous carbon chain in a molecule, and the carbon chain may be either linear or branched.

[0022] As used herein, the term "aldehyde substrate" refers to an aldehyde that can be a substrate for ADO, and is a general term for organic compounds having a structure in which one hydrogen atom is substituted for the carbonyl carbon in the molecule.

[0023] As used herein, the term "aldehyde substrate having a (specific) carbon chain length" refers to an aldehyde substrate having any carbon chain length.

[0024] As used herein, the term "aldehyde substrate possessed by a microorganism" refers to an aldehyde substrate that can be possessed by a microorganism, and most microorganisms can synthesize fatty acid aldehydes as intermediate products in fatty acid synthesis.

[0025] In this specification, "specificity" refers to an enzyme showing activity or reactivity to a certain substrate. Therefore, "specificity to a certain substrate" can be defined as not only catalyzing a substrate having a certain carbon chain length, but having higher reactivity than other substrates. Therefore, in this specification, when ADO shows reactivity to a substrate having a different carbon chain length than a substrate having a certain carbon chain length, it can be said that it has specificity to the substrate having the other carbon chain length.

[0026] As used herein, the term "form a three-dimensional structure" refers to the formation of an enzyme-substrate complex by the binding of a substrate to an enzyme. In the present disclosure, the three-dimensional structure is formed, for example, when an ADO mutant binds to a substrate.

[0027] As used herein, "an amino acid site within about (X) Å from a substrate" refers to all amino acids that are located about (X) Å away from the substrate in an enzyme-substrate complex, and all amino acids that are located at positions less than about (X) Å. The amino acid site within about (X) Å from a substrate can be calculated or determined by measurement. The distance from the substrate position to the amino acid site can be experimentally determined by analyzing the crystal structure of the enzyme and substrate. For example, the distance from the substrate to the amino acid residue can be determined by drawing the three-dimensional structure from PDB data using protein structure drawing software such as MOE, Pymol, or Chimera.

[0028] As used herein, "identified based on affinity" refers to calculating the binding energy between the substrate and the enzyme, and identifying based on the results of such affinity calculation. A state of high affinity refers to a state in which the enzyme and the substrate are more strongly bound by binding energies such as intermolecular forces and electrostatic interactions. In the present disclosure, the effect of the introduction of a mutation on the affinity with the substrate is evaluated by calculating the difference in binding energy between the mutant and wild-type enzymes.

[0029] In this specification, "multiple mutations" usually refers to multiple mutations, but may also refer to a single mutation. By combining multiple mutation sites, the effects of each mutation can be synergistically achieved, and it is possible to design mutants with high activity and specificity. In the present disclosure, the affinity between a mutant containing multiple mutations and a substrate can be calculated.

[0030] (Preferred embodiment) Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. In addition, the following embodiments of the present disclosure can be used alone or in combination.

[0031] In one aspect of the present disclosure, there is provided an aldehyde deformylating oxygenase (ADO) mutant, characterized in that the amino acid sequence of a specificity modification site is modified, thereby having high specificity for an aldehyde substrate having a specific carbon chain length compared to aldehyde substrates having other carbon chain lengths.

[0032] Aldehyde deformylating oxygenase (ADO) is the only enzyme that can synthesize alkanes from aldehydes. The physical properties of alkanes vary depending on the carbon number, and they can be used for various purposes such as fuels and cosmetics. Therefore, by modifying the substrate specificity of ADO, it is possible to create an ADO series that can mass-synthesize only alkanes with specific carbon chain lengths. To date, there have been few studies on the substrate specificity of ADO, and although studies using mutants with modified ADO substrate specificity have already been reported, most of these studies target substrates with short carbon chains (aldehydes with carbon numbers (C) 4-8) and are not capable of synthesizing alkanes of any carbon chain length.

[0033] In one embodiment of the present disclosure, an ADO mutant can be provided in which an amino acid modification, preferably a single amino acid substitution mutation, has been introduced into the substrate binding site or the periphery of the substrate pocket of ADO based on affinity calculations. By performing affinity calculations between such an ADO mutant and a substrate, it is possible to provide a mutant capable of synthesizing an alkane of a desired carbon chain length, such as a mutant having a higher affinity for C12 aldehyde (medium substrate) than for C16 aldehyde (long substrate).

[0034] In one embodiment of the present disclosure, the ADO is not particularly limited as long as it is an enzyme that can synthesize alkanes using aldehydes as substrates. For example, ADO can include, but is not limited to, OsADO, NpADO, SeADO, 6803ADO, TeADO, 51142ADO, 7425-1ADO, 7425-2ADO, 8801ADO, CwADO, PaADO, LaADO, 9443ADO, LiADO, PmADO, NATL2aADO, 9917-2ADO, SyADO, 7421ADO, and 7336ADO. In one embodiment, the ADO can preferably be NpADO from Nostoc punctiforme PCC73102. The types and origins of ADOs are exemplified in, for example, Schirmer, A., et al., (2010), Science 329:559-562, Arai, M., et al., (2018), AEMB., 1080, 119-154, and Kudo et al., (2019), Biotechnology for Biofuels and Bioproducts, 12, 89.

[0035] In one embodiment, ADO having high activity against linear aldehydes such as C12, C14, C16, or C18 can be used as the ADO. By using ADO having high enzymatic activity against any substrate, it is possible to obtain an ADO mutant that reacts specifically with an aldehyde of a desired carbon chain length and has high enzymatic activity against the substrate even when modified. In one embodiment of the present disclosure, a mutant having improved Km (substrate affinity) can be provided while substantially maintaining the kcat (catalytic efficiency) of ADO.

[0036] In one embodiment, the ADO mutant of the present disclosure may have a modified amino acid sequence at the specificity modification site, for example, the amino acid sequence at the substrate binding site to which the aldehyde, which is the substrate of ADO, binds or at a site nearby the site may be modified. In one embodiment, the specificity modification site may typically be the substrate binding site, but may also be a site nearby the substrate binding site, and may include all residues surrounding the substrate and involved in the formation of the substrate pocket. For example, in one embodiment, the specificity modification site may include an amino acid site that is within about 10 Å, about 9 Å, about 8 Å, about 7 Å, about 6 Å, about 5 Å, about 4 Å, or about 3 Å from the substrate when the ADO mutant and the substrate bind to form a three-dimensional structure. Whether or not the specificity modification site is within XÅ can be determined by analyzing the crystal structure of the enzyme and the substrate, and selecting an amino acid molecule that is within a specified distance from the coordinate point of the molecule that constitutes the substrate. For example, the distance from the substrate to the amino acid residue can be examined by depicting the three-dimensional structure from PDB data using protein structure depiction software such as MOE, Pymol, and Chimera. For example, you can calculate the distance between two points in three-dimensional coordinates: A ,Y A ,Z A ) and point B(X B , Y B , Z B ) distance (D A-B ) can be calculated based on the following formula: D A-B 2 =(X A -X B ) 2 +(Y A -Y B ) 2 +(Y A -Y B ) 2

[0037] In one embodiment, the amino acid sequence to be modified in the ADO mutant of the present disclosure can be specified based on the affinity between ADO and the substrate, and such affinity calculation can be performed, for example, by drawing the three-dimensional structure from PDB data using protein structure drawing software such as Molecular operating environments (MOE), Pymol, Chimera, etc. The affinity calculation is based on the MM / GBVI binding energy calculation method (see, for example, Labute, P,. (2008), J Comput Chem 29: 1693-1698), and a high affinity state refers to a state in which the enzyme and the substrate are more strongly bound by binding energy such as intermolecular forces and electrostatic interactions.

[0038] In one embodiment, the ADO mutant of the present disclosure can be modified to have high specificity for an aldehyde substrate having a specific carbon chain length, and can synthesize an alkane having any desired carbon chain length substantially more than an alkane having another carbon chain length, such as an ADO mutant that synthesizes more C11 alkanes than C15 alkanes (high specificity for C12 aldehydes), or more C17 alkanes than C13 alkanes (high specificity for C18 aldehydes). Thus, by changing the amino acid modification site in ADO, a mutant having high specificity for an aldehyde substrate having any desired carbon chain length can be obtained.

[0039] In one embodiment, when creating a mutant with higher affinity for C12 aldehyde than for C16 aldehyde, amino acid modifications can be made to the amino acid sequence of ADO corresponding to one or more residues selected from the group consisting of I25, V29, Q111, A122, Y126, W179, and V185 when aligned with SEQ ID NO:1.

[0040] In one embodiment, when creating a mutant with higher affinity for C12 aldehyde than for C16 aldehyde, amino acid modifications can be made to the amino acid sequence of ADO corresponding to one or more residues selected from the group consisting of V29Y, Q111Y, A122P, Y126S, Y126T, W179R, and V185F when aligned with SEQ ID NO:1.

[0041] In one embodiment, the modification in the ADO mutant of the present disclosure may be one amino acid or multiple mutations of multiple amino acids. For example, in one embodiment, the ADO mutant of the present disclosure may have two, three, four, or five amino acids modified, and the modified amino acids may be adjacent or distant. By performing multiple mutations, it may be possible to obtain mutants with higher activity and specificity. As shown in the examples of the present application, in the present disclosure, a mutant with higher affinity for C12 aldehyde than for C16 aldehyde was produced, and it was found that the activity of the 29 / 111 / 185 mutant against C12 aldehyde was improved by about 1.4 times compared to the V29Y and Q111Y mutants.

[0042] In one embodiment, when creating a mutant with higher affinity for C12 aldehyde than for C16 aldehyde, amino acid modifications can be made in the amino acid sequence of ADO that correspond to V29Y / Q111Y / W179R when aligned with SEQ ID NO:1.

[0043] In one embodiment of the present disclosure, the ADO mutants of the present disclosure can catalyze only aldehyde substrates having a specific carbon chain length. In another embodiment, the ADO mutants of the present disclosure can be made to have high catalytic activity for aldehyde substrates having a first carbon chain length, while also having higher catalytic activity for aldehyde substrates having a second carbon chain length than for aldehyde substrates having a third carbon chain length. In one embodiment, when an ADO mutant having high catalytic activity for aldehyde substrates having multiple specific carbon chain lengths is used, alkanes having a desired carbon chain length can be selectively recovered, for example, by fractional distillation utilizing the difference in boiling point.

[0044] In another aspect of the present disclosure, a microorganism is provided into which an aldehyde deformylating oxygenase (ADO) or a mutant thereof that specifically reacts with an aldehyde substrate possessed by the microorganism is introduced. By using such a microorganism, it is possible to selectively obtain a target alkane because of high specificity for an aldehyde substrate having a target carbon chain length. In one embodiment of the present disclosure, the microorganism of the present disclosure can have any of the characteristics described elsewhere in this specification.

[0045] Representative examples of the microorganisms disclosed herein include cyanobacteria, but other examples include microorganisms that use carbon as a source of energy, such as necator, cyanobacteria, microalgae, and yeast.

[0046] In one embodiment, when Escherichia coli is used as the microorganism of the present disclosure, alkane synthesis is possible by co-expression of ADO and AAR located upstream thereof. Alkane production using microorganisms can be performed, for example, by referring to Arai, M., et al., (2018), AEMB., 1080, 119-154, and Wang, J. and Zhu, K. (2018), Curr Opin Biotechnol., 50: 11-18.

[0047] (General technology) The molecular biological, biochemical and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999).These methods are described in Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Special Edition of Experimental Medicine: "Gene Introduction & Expression Analysis Experimental Methods" Yodosha, 1997, etc., and the relevant parts (possibly in their entirety) of these are incorporated herein by reference.

[0048] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as those of GeneArt, GenScript, Integrated DNA Technologies (IDT), etc. can be used. In addition, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Bioconjugate Techniques, Academic Press, etc., the relevant portions of which are incorporated herein by reference.

[0049] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within the range of" "two values", the range includes the two values ​​themselves. All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth.

[0050] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims. EXAMPLES

[0051] Example 1: Activity measurement in ADO mutants Plasmids A DNA fragment encoding an ADO homolog was constructed by GenScript. Codons were optimized for high expression in E. coli. For expression of ADO in E. coli, the pET28a(+) vector (Novagen) was used, into which the codon-optimized DNA fragment of ADO was cloned via the NdeI and XhoI restriction sites. A T7 promoter, lac operator, and ribosome binding site were placed upstream of the ADO gene. NpADO mutants were constructed according to the protocol of the QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA).

[0052] For the production of medium-chain alkanes, two plasmids were used, expressed in E. coli BL21(DE3) or BL21(DE3)pLysS as host cells. The first plasmid, pCDFDuet-1 (Novagen), expressing the acyl-ACP thioesterase BTE from Umbellularia californica and E. coli fadD, was used to produce C12:0 fatty acids and acyl-CoA in the host cells. The second plasmid, pETDuet-1 (Novagen), expressing the fatty acyl-CoA reductase FAR and an ADO mutant with high specificity for medium-chain alkanes, was used to overproduce undecane (C11:0).

[0053] Protein overexpression and purification For the expression of ADO, the pET28a plasmid encoding ADO was transformed into E. coli BL21(DE3) competent cells. The cells were incubated in Luria-Bertani (LB) medium containing 25 μg / mL kanamycin. When the OD600nm reached about 0.7 to 0.8, the culture was induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG). The culture was further cultured at 18°C ​​and 180 rpm for 18 hours. The culture was collected by centrifugation at 8000×g and 4°C for 15 minutes. The collected cells were suspended in binding buffer (20 mM Tris-HCl, 100 mM NaCl, 5 mM imidazole, pH 8.0) and lysed with FastBreak (registered trademark) Cell Lysis Reagent (Promega Japan, Tokyo, Japan). The lysate was centrifuged at 20380×g and 4°C for 30 minutes. The supernatant was added to TALON Resin (Clontech Laboratories, Inc.) and gently stirred on a shaker for 20 min at 15°C to bind the his-tagged ADO protein to the resin. The resin was washed with washing solution, and the ADO protein was eluted after adding elution solution (20 mM Tris-HCl, 100 mM NaCl, 500 mM imidazole, pH 8.0) and incubating at 15°C for 20 min. The elution solution was exchanged with HEPES buffer containing 100 mM HEPES and 100 mM KCl, pH 7.2, and the ADO reaction was carried out.

[0054] To confirm the expression of ADO, SDS-PAGE was performed on 12% polyacrylamide gels using Coomassie Blue R-250 staining. Proteins were concentrated and the concentration was measured by the Bradford method using bovine serum albumin as a standard.

[0055] Enzyme assays ADO activity measurements were performed according to a previous study [Bao et al., 2016]. The reaction buffer was pH 7.2 and contained 100 mM HEPES, 100 mM KCl, 1 mM NADH, 75 μM PMS, 1 mg / ml catalase, 100 μM ferrous sulfate, 200 μM C18, 16, 14, 12 aldehydes, 1 mg / ml BSA, and 0.8% Triton. An enzyme solution of 5 μM ADO was added to the reaction buffer and gently stirred at 37 °C for 1 h. C16 alkane was used as an internal standard for C11, 13, 15, and 17 alkanes. The reaction solution and ethyl acetate were mixed by vortexing for 30 min. The organic phase was centrifuged at 13,000 × g for 10 min at 4 °C to separate the aqueous phase, which was then collected in a glass vial for GC-MS measurement. All enzyme assays were repeated in triplicate.

[0056] Quantitative analysis of alkanes using gas chromatography-mass spectrometry (GC-MS) C18, 16, 14, and 12 alkanes were quantified using a Shimadzu gas chromatograph mass spectrometer GCMS-QP2020NX (Shimadzu, Kyoto, Japan). Samples were injected splitless into a DB-5MS UI column (30 m × 0.25 mm × 0.25 um). The GC-MS program was as follows: After holding at 80 °C for 4 min, the temperature was increased to 290 °C at 40 °C / min and held at 290 °C for 1 min. The GC-MS interface temperature was 290 °C, and the ion source temperature was 230 °C. The flow rate of the He carrier gas was 1.50 ml / min. An alkane standard solution containing C8 to C20 (Sigma-Aldrich) was used to determine the retention time and fragmentation pattern of the alkane product peaks.

[0057] Alkane production in engineered Escherichia coli Alkane production was performed in E. coli according to previous studies. BL21(DE3)pLysS competent cells were transformed with pCDFDuet-1 co-expression vector containing both UcBTE and FadD genes, and pETDuet-1 co-expression vector containing both AAR and ADO genes, and inoculated onto LB agarose plates with 50ug / ml spectromycin and 50ug / ml carbenicillin. The colonies were precultured in M9 minimal medium containing 3% glucose, 50ug / ml spectinomycin, and 50ug / ml carbenicillin. The precultures were inoculated into M9 minimal medium containing 100μM ferric sulfate and cultured in 96 deep-well plate maximizer (TAITEC, Tokyo, Japan). Protein expression was then induced with 1mM IPTG, followed by incubation at 37℃ for 20-30 hours.

[0058] The culture was lysed, and alkanes in the lysate were extracted with ethyl acetate. For detection of alkane peaks by GC-MS, the oven temperature was held at 75°C for 5 min, increased to 290°C at a rate of 10°C / min, and then held at 290°C for an additional 9 min. C16 alkane was used as an internal standard. Pure alkane standards were used for identification and quantification of each alkane.

[0059] System Analysis and Computer Design A total of 150 orthologues of ADO were found on the BLAST server using the 9313ADO amino acid sequence as a query. Multiple sequence alignment of the 150 ADO amino acid sequences was performed using MAFFT. A phylogenetic tree of cyanobacteria based on the ADO amino acid sequence was created using NJplot. The structure of ADO from Nostoc punctiforme (PDB ID: 5uxi) was used as a template to design ADO mutants. Complex structures of ADO and C16 or C12 aldehydes were created, and mutants were designed using Molecular operating environments (MOE). The affinity of ADO mutants to aldehydes was also calculated using the Residue Scan program in MOE. Residues close to the substrate pocket of ADO were targeted, and saturation mutations were introduced into these residues, changing them to 19 amino acids excluding the original amino acid.

[0060] The results are shown in Figures 1 and 2. The upper graph in Figure 1 shows the results of ADO activity toward C16 aldehyde, and the lower graph shows the results of ADO activity toward C12 aldehyde. Activity values ​​were calculated by dividing the amount of C15 alkane and C11 alkane, which are the products, by the concentration of ADO protein. For the samples, I25F to M194F on the left side are single amino acid substitutions of NpADO, V184F(Se) is the benchmark strain, and WT on the far right is the wild type of NpADO. Of these, V29Y, Q111Y, and A122P showed no C15 alkane activity at all, while only a certain amount of C11 alkane activity was detected. Y126S(T), W179R, and V185F showed high activities for both C15 alkane and C11 alkane, indicating that their activities were improved compared to the benchmark strain.

[0061] In Figure 2, multiple mutants were prepared by combining the mutations that showed high activity in Figure 1, and activity measurements were performed in the same manner. As a result, it was found that the 29 / 111 / 185 mutant, which combines V29Y, Q111Y, and V185F, was able to synthesize only C11 alkanes in the greatest amount.

[0062] (Example 2: Synthesis of alkanes with desired carbon chain length using microorganisms) The genes required for C11 alkane synthesis are introduced into E. coli. Below, we will create E. coli capable of mass-synthesizing C11 alkanes using two strategies. 1.Introduce two types of genes, ADO and its upstream AAR gene. 2.UcFatB, which can synthesize C12 fatty acids, and FadD, which can synthesize acyl-CoA → aldehyde from that, are introduced in addition to ADO.

[0063] For cultivation, experimental conditions will be optimized on a small scale (96-well deep well plate (2 ml size)). Finally, the cultivation will be scaled up and large-scale cultivation will be carried out in jars. The target alkanes will be confirmed by analyzing the components in the culture medium using GC-MS. As for the recovery method, since it is known that E. coli secretes alkanes into the culture medium, C11 alkanes in the medium will be recovered by fractional distillation. The microorganisms used are not limited to E. coli, and other microorganisms (such as necator and cyanobacteria that use CO2 as a raw material) can also be used.

[0064] (Note) Although the present disclosure has been illustrated using the preferred embodiments thereof, it is understood that the present disclosure should be interpreted in scope only by the claims. It is understood that the patents, patent applications, and other documents cited in this specification are incorporated by reference into this specification in the same manner as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0065] According to the present disclosure, it is possible to provide an ADO mutant modified to have high specificity for an aldehyde substrate having a specific carbon chain length, and a microorganism into which such an ADO mutant has been introduced, and therefore, by using such an ADO mutant or microorganism, a system for producing alkanes suitable for applications such as fuels and cosmetics can be constructed, and thus applications in fields using hydrocarbons are expected. [Sequence List Free Text]

[0066] SEQ ID NO: 1: Amino acid sequence of NpADO

Claims

1. An aldehyde deformylating oxygenase (ADO) mutant characterized in that the amino acid sequence at the specificity modification site is modified, thereby having high specificity for aldehyde substrates having a specific carbon chain length compared to aldehyde substrates having other carbon chain lengths.

2. The ADO variant of claim 1 , wherein the ADO comprises NpADO.

3. The ADO mutant of claim 1 or 2, wherein the specific modification site comprises a substrate binding site.

4. The ADO mutant according to claim 1 or 2, wherein the specific modification site comprises an amino acid site that is located within approximately 5 Å of the substrate when the ADO mutant and the substrate bind to form a three-dimensional structure.

5. The ADO variant of claim 1 or 2, wherein the amino acid sequence identified based on the affinity of ADO for a substrate is modified.

6. The ADO mutant of claim 1 or 2, wherein the modification comprises multiple mutations.

7. 3. The ADO variant of claim 1 or 2, wherein the modification comprises a modification of an amino acid corresponding to one or more residues selected from the group consisting of I25, V29, Q111, A122, Y126, W179, and V185 in the amino acid sequence of ADO when aligned with SEQ ID NO:

1.

8. 3. The ADO variant of claim 1 or 2, wherein the modification comprises an amino acid modification corresponding to one or more residues selected from the group consisting of V29Y, Q111Y, A122P, Y126S, Y126T, W179R, and V185F in the amino acid sequence of ADO when aligned with SEQ ID NO:

1.

9. 3. The ADO variant of claim 1 or 2, wherein the modification comprises an amino acid modification corresponding to V29Y / Q111Y / W179R in the amino acid sequence of ADO when aligned with SEQ ID NO:

1.

10. A microorganism into which an aldehyde deformylating oxygenase (ADO) or a mutant thereof that specifically reacts with an aldehyde substrate possessed by the microorganism has been introduced.

11. The microorganism of claim 10 , wherein the ADO comprises NpADO.

12. The microorganism of claim 10, wherein the mutant has an alteration in the amino acid sequence of the specific alteration site of the ADO.

13. The microorganism of claim 10, wherein the mutant has an amino acid sequence modification at a specific modification site of the ADO, identified based on the affinity of the ADO for a substrate.

14. The microorganism of claim 12 , wherein the specific modification site comprises a substrate binding site.

15. The microorganism described in claim 12, wherein the specific modification site comprises an amino acid site that is located within approximately 5 Å of the substrate when the ADO mutant and the substrate bind to form a three-dimensional structure.

16. The microorganism according to any one of claims 12 to 15, wherein the modification comprises multiple mutations.

17. The microorganism of any one of claims 10 to 15, wherein the mutant comprises an amino acid modification corresponding to one or more residues selected from the group consisting of I25, V29, Q111, A122, Y126, W179, and V185 in the amino acid sequence of ADO when aligned with SEQ ID NO:

1.

18. 16. The microorganism of any one of claims 10 to 15, wherein the mutant comprises an amino acid modification in the amino acid sequence of ADO corresponding to one or more residues selected from the group consisting of V29Y, Q111Y, A122P, Y126S, Y126T, W179R, and V185F when aligned with SEQ ID NO:

1.

19. The microorganism according to any one of claims 10 to 15, wherein the mutant comprises amino acid modifications corresponding to V29Y / Q111Y / W179R in the amino acid sequence of ADO when aligned with SEQ ID NO: 1.

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