Mutant acyl-ACP reductase

Abstract

To provide means capable of improving the production efficiency of alkanes and / or alkenes in hydrocarbon production using acyl-ACP reductase (AAR).SOLUTION: The present invention provides mutant AAR [1] to [3]. Mutant AAR [1] has an amino acid sequence specified in (1) or (2): (1) An amino acid sequence comprising the amino acid sequence represented by Sequence ID No. 1 (amino acid sequence 1), and satisfying one or more requirements selected from (A) to (E): (A) the 11th amino acid of amino acid sequence 1 is an amino acid other than leucine; (B) the 26th amino acid of amino acid sequence 1 is an amino acid other than tyrosine; (C) the 33rd amino acid of amino acid sequence 1 is an amino acid other than phenylalanine; (D) the 40th amino acid of amino acid sequence 1 is an amino acid other than glutamine; and (E) the 61st amino acid of amino acid sequence 1 is an amino acid other than glutamic acid. (2) An amino acid sequence in which one or more amino acids other than the 11th, 26th, 33rd, 40th, and 61st in the amino acid sequence of (1) are mutated.SELECTED DRAWING: None

Description

Technical Field

[0001] Relates to a mutant acyl-ACP reductase.

Background Art

[0002] Much of the energy depends on fossil fuels. However, from the viewpoints of suppressing the depletion of fossil fuels and reducing carbon dioxide emissions, the development of new fuels to replace fossil fuels is important in various industrial fields.

[0003] For example, in the aviation field, crude oil is mainly used as a fossil fuel, and a sustainable aviation fuel (SAF) to replace the fuel is being sought (Non-Patent Document 1). SAF satisfies a plurality of sustainability criteria, and it is required that the physical and chemical properties of the alternative fuel are substantially the same as those of conventional aviation fuel. Currently, there are four main processes for SAF production (Figure 1), and each process of hydrotreatment of esters and fatty acids (HEFA), Fischer-Tropsch synthesis (FT), conversion from alcohol to jet (ATJ), and direct conversion from sugar to hydrocarbon (DSHC) can be mentioned. For example, the HEFA process is the most widely adopted as a SAF production process, and is a process of subjecting triglycerides obtained from vegetable oil or animal fat to hydrotreatment, hydrocracking, and isomerization treatment, removing impurities, and converting long-chain fatty acids into shorter hydrocarbons. Also, among these processes, DSHC-SIP (synthesized iso-paraffins) uses recombinant yeast or Escherichia coli to convert sugar into farnesene (C 15 H 24 )), and then converting farnesene into farnesane (C 15 H 32It is a process of converting into ), and it is known that the obtained product can be used by mixing with conventional aviation fuel. DSHC-SIP has advantages in that it can be carried out in a single fermentation tank and can be carried out in a simpler process compared to the other three processes. However, DSHC-SIP is still cumbersome in that it is necessary to further process farnesene to convert it into farnesane.

[0004] So far, technologies for biosynthesizing desired substances using microorganisms have been known. For example, it is known that cyanobacteria can biosynthesize fatty acids during photosynthesis. Acyl-ACP reductase (AAR; acyl-acyl carrier protein (acyl-ACP) reductase), an important enzyme in fatty acid biosynthesis, is an enzyme that uses a coenzyme (mainly NADPH) to convert acyl-ACP into the corresponding aldehyde. The generated aldehyde is converted into the corresponding alkane or alkene by deformylation by aldehyde deformylating oxygenase (ADO). The conversion by AAR and ADO occurs at the final stage of fatty acid biosynthesis. Since the aldehyde generated by AAR is converted by ADO into the corresponding alkane or alkene, AAR is an important enzyme that determines the composition of fatty acid derivatives in the finally obtained product. Also, it is known that by co-expressing AAR and ADO in Escherichia coli using known recombinant techniques, the above-mentioned conversions by AAR and ADO occur, and hydrocarbons can be produced. Thus, AAR and ADO are useful as enzymes that can easily produce hydrocarbons. On the other hand, in hydrocarbon production using AAR and ADO, it is desirable to produce alkanes more efficiently. For example, the hydrocarbons contained in aviation fuel are mainly alkanes (linear alkanes, branched alkanes, cycloalkanes), and the proportions of olefins and aromatic hydrocarbons are low. Therefore, improving the alkane production efficiency is useful in that the obtained hydrocarbons can be used as they are as biofuels (drop-in biofuels). Also, it is desirable to produce alkanes and alkenes more efficiently. Since it is known that alkenes can be relatively easily converted into alkanes by hydrogenation, it can be said that improving the production efficiency of alkanes and / or alkenes ultimately easily leads to an improvement in alkane production efficiency.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a means for improving the production efficiency of alkanes and / or alkenes in hydrocarbon production using acyl-ACP reductase (AAR).

Means for Solving the Problems

[0007] As a result of intensive studies, the present inventors have found that when hydrocarbons are produced using a mutant acyl-ACP reductase (mutant AAR) having an amino acid sequence represented by SEQ ID NO: 1 and having at least one requirement selected from the following group consisting of (A) to (E), the production efficiency of alkanes and / or alkenes can be improved. (A) The 11th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than leucine (for example, phenylalanine, etc.), (B) The 26th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than tyrosine (for example, glycine, etc.), (C) The 33rd amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than phenylalanine (for example, glycine, etc.), (D) The 40th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than glutamine (for example, lysine, etc.), (E) The 61st amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than glutamic acid (for example, alanine, etc.).

[0008] In addition, as a result of intensive studies, the present inventors have found that when hydrocarbons are produced using a mutant acyl-ACP reductase (mutant AAR) having an amino acid sequence in which 1 to 4 amino acids are deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35, the production efficiency of alkanes and / or alkenes can be improved.

[0009] In addition, as a result of intensive studies, the present inventors have found that when hydrocarbons are produced using a mutant acyl-ACP reductase (mutant AAR) having an amino acid sequence in which an amino acid sequence consisting of 1 to 12 amino acids composed of glutamic acid and / or aspartic acid is linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35, the production efficiency of alkanes and / or alkenes can be improved.

[0010] The present invention has been completed through further studies based on these findings, and the present disclosure includes inventions represented, for example, by the following. Item 1. A mutant acyl-ACP reductase shown in the following [1] to [3] [1] A mutant acyl-ACP reductase having an amino acid sequence shown in the following (1) or (2): (1) An amino acid sequence consisting of the amino acid sequence represented by SEQ ID NO: 1 and having at least one requirement selected from the group consisting of (A) to (E) (A) The 11th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than leucine, (B) The 26th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than tyrosine, (C) The 33rd amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than phenylalanine, (D) The 40th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than glutamine, (E) The 61st amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than glutamic acid, (2) An amino acid sequence in which one or more amino acids other than the 11th, 26th, 33rd, 40th, and 61st amino acids in the amino acid sequence of (1) are deleted, substituted, inserted, or added. [2] A mutant acyl-ACP reductase having an amino acid sequence shown in the following (i) or (ii): (i) An amino acid sequence in which 1 to 4 amino acids are deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 (ii) An amino acid sequence in which one or more amino acids are deleted (excluding the deletion of the first amino acid from the C-terminus of the amino acid sequence of (i)), substituted, inserted, or added in the amino acid sequence of (i). [3] A mutant acyl-ACP reductase having an amino acid sequence shown in the following (a) or (b): (a) An amino acid sequence consisting of 1 to 12 amino acids of glutamic acid and / or aspartic acid is linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 (b) An amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 35 in the amino acid sequence of (a). Item 2. Having at least one requirement selected from the group consisting of the above (A) to (E), In the above (A), the 11th amino acid is phenylalanine, In the above (B), the 26th amino acid is glycine, isoleucine, alanine, methionine, or leucine, In the above (C), the 33rd amino acid is glycine, leucine, valine, or methionine, In the above (D), the 40th amino acid is lysine, tyrosine, valine, histidine, methionine, arginine, or glutamic acid, In the above (E), the 61st amino acid is alanine, The mutant acyl-ACP reductase according to Item 1. Item 3. Comprising at least one requirement selected from the group consisting of the following (B), and (C) and (D): In the said (B), the 26th amino acid is glycine; In the said (C), the 33rd amino acid is glycine; In the said (D), the 40th amino acid is lysine, tyrosine, valine, histidine or methionine; The mutant acyl-ACP reductase according to Item 1 or 2. Item 4. In the said (i), the amino acid sequence is one in which 3 or 4 amino acids are deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. The mutant acyl-ACP reductase according to Item 1. Item 5. In the said (a), the amino acid sequence linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 is an amino acid sequence 2 to 10 amino acids in length consisting of glutamic acid. The mutant acyl-ACP reductase according to Item 1. Item 6. In the said (a), the amino acid sequence linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 is an amino acid sequence 3 to 9 amino acids in length consisting of aspartic acid. The mutant acyl-ACP reductase according to Item 1. Item 7. In the said (a), the amino acid sequence linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 is an amino acid sequence 3 to 6 amino acids in length consisting of glutamic acid and aspartic acid. The mutant acyl-ACP reductase according to Item 1. Item 8. The amino acid sequence of the said (a) is an amino acid sequence 1 to 12 amino acids in length consisting of glutamic acid and / or aspartic acid, which is directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. The mutant acyl-ACP reductase according to Item 1 and any one of Items 5 to 7. Item 9. A polynucleotide encoding the mutant acyl-ACP reductase according to any one of Items 1 to 8. Item 10. A vector containing the polynucleotide according to Item 9. Item 11. Further, the vector according to Item 10, comprising a polynucleotide encoding aldehyde deformylating oxygenase. Item 12. A cell transformed with the vector according to Item 10 or 11. Item 13. A method for producing at least one selected from the group consisting of alkanes and alkenes, using the mutant acyl-ACP reductase according to any one of Items 1 to 8, in the presence of aldehyde deformylating oxygenase.

Advantages of the Invention

[0011] There is provided a mutant AAR capable of improving the production efficiency of alkanes and / or alkenes.

Brief Description of the Drawings

[0012]

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MODE FOR CARRYING OUT THE INVENTION

[0013] Hereinafter, the embodiments included in the present disclosure will be described in more detail. In the present disclosure, "containing" and "including" also include the meanings of "substantially consisting of" and "consisting of".

[0014] 1. Mutant AAR The present disclosure provides a novel acyl-ACP reductase (AAR) that can improve the production efficiency of alkanes and / or alkenes. The novel AAR of the present disclosure has an amino acid sequence different from that of a conventionally known wild-type AAR. Therefore, the novel AAR in the present disclosure may sometimes be described as a mutant AAR. Hereinafter, the mutant AAR may sometimes be described as "the mutant AAR of the present disclosure".

[0015] In one embodiment of the present disclosure, the mutant AAR includes a mutant acyl-ACP reductase having an amino acid sequence shown in the following (1) or (2). Hereinafter, the mutant AAR may sometimes be described as mutant AAR[1].

[0016] Also, in one embodiment of the present disclosure, the mutant AAR includes a mutant acyl-ACP reductase having an amino acid sequence shown in the following (i) or (ii). Hereinafter, the mutant AAR may sometimes be described as mutant AAR[2].

[0017] Also, in one embodiment of the present disclosure, the mutant AAR includes a mutant acyl-ACP reductase having an amino acid sequence shown in the following (a) or (i). Hereinafter, the mutant AAR may sometimes be described as mutant AAR[3].

[0018] Mutant AAR[1] has an amino acid sequence shown in the following (1) or (2): (1) It consists of the amino acid sequence represented by SEQ ID NO: 1 and has at least one requirement selected from the group consisting of (A) to (E). (A) The 11th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than leucine. (B) The 26th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than tyrosine. (C) The 33rd amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than phenylalanine. (D) The 40th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than glutamine. (E) The 61st amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than glutamic acid. (2) An amino acid sequence in which one or more amino acids other than the 11th, 26th, 33rd, 40th, and 61st amino acids in the amino acid sequence of (1) are deleted, substituted, inserted, or added.

[0019] Variant AAR[2] has an amino acid sequence shown in the following (i) or (ii): (i) An amino acid sequence in which 1 to 4 amino acids from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 are deleted (ii) An amino acid sequence in which one or more amino acids are deleted (excluding the deletion of the first amino acid from the C-terminus of the amino acid sequence of (i)), substituted, inserted, or added in the amino acid sequence of (i).

[0020] Variant AAR[3] has an amino acid sequence shown in the following (a) or (b): (a) An amino acid sequence consisting of 1 to 12 amino acids of glutamic acid and / or aspartic acid is linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 (b) An amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 35 in the amino acid sequence of (a).

[0021] · Mutant AAR[1] Variant AAR[1] is a variant acyl-ACP reductase having the amino acid sequence shown in (1) or (2) above.

[0022] In the amino acid sequence represented by SEQ ID NO: 1 (Figure 2), the 11th amino acid is leucine, the 26th amino acid is tyrosine, the 33rd amino acid is phenylalanine, the 40th amino acid is glutamine, and the 61st amino acid is glutamic acid. This amino acid sequence corresponds to the amino acid sequence of AAR derived from conventionally known Synechococcus elongatus PCC 7942 = FACHB-805 (hereinafter sometimes referred to as "wild-type SeAAR"). The nucleotide sequence encoding wild-type SeAAR is also known. For example, this nucleotide sequence is represented by SEQ ID NO: 2 (Figure 3). In the amino acid sequence represented by SEQ ID NO: 1, a sequence in which at least any one of the 11th leucine, 26th tyrosine, 33rd phenylalanine, 40th glutamine, and 61st glutamic acid is substituted with another amino acid is the amino acid sequence shown in (1) above.

[0023] The 11th amino acid of (A), the 26th amino acid of (B), the 33rd amino acid of (C), the 40th amino acid of (D), and the 61st amino acid of (E) may be any of the known amino acids that can form a protein as long as they are, as described above, an amino acid other than leucine, an amino acid other than tyrosine, an amino acid other than phenylalanine, an amino acid other than glutamine, and / or an amino acid other than glutamic acid, respectively. That is, within this limit, the 11th, 26th, 33rd, 40th, and 61st amino acids are each independently alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), selenocysteine (U), pyrrolysine (O), or valine (V).

[0024] The 11th amino acid of the above (A) may be any amino acid within this limit, but preferably phenylalanine or the like is exemplified.

[0025] The 26th amino acid of the above (B) may be any amino acid within this limit, but preferably glycine, isoleucine, alanine, methionine, leucine or the like is exemplified. As the 26th amino acid, more preferably glycine, isoleucine, alanine or the like is exemplified.

[0026] The 33rd amino acid of the above (C) may be any amino acid within this limit, but preferably glycine, leucine, valine, methionine or the like is exemplified.

[0027] The 40th amino acid of the above (D) may be any amino acid within this limit, but preferably lysine, tyrosine, valine, histidine, methionine, arginine, glutamic acid or the like is exemplified.

[0028] The 61st amino acid of the above (E) may be any amino acid within this limit, but preferably alanine or the like is exemplified.

[0029] The above (1) amino acid sequence may have at least one requirement selected from the group consisting of (A) to (E). The amino acid sequence of the above (1) preferably has 1 to 4 requirements selected from the group consisting of (A) to (E), and more preferably has 1, 2 or 3 requirements.

[0030] Although not limiting the present disclosure, the following combinations are more preferably exemplified as the 11th, 26th, 33rd, 40th and 61st amino acids in the amino acid sequence of the above (1).

[0031] [A] L11 Mutant AAR [A1]The 11th is phenylalanine (F), the 26th is tyrosine (Y), the 33rd is phenylalanine (F), the 40th is glutamine (Q), and the 61st is glutamic acid (E) (this may be referred to as "L11F mutant AAR").

[0032] [B] Y26 Mutant AAR [B1]The 11th is leucine (L), the 26th is glycine (G), the 33rd is phenylalanine (F), the 40th is glutamine (Q), and the 61st is glutamic acid (E) (this may be referred to as "Y26G mutant AAR"). [B2]The 11th is leucine (L), the 26th is alanine (A), the 33rd is phenylalanine (F), the 40th is glutamine (Q), and the 61st is glutamic acid (E) (this may be referred to as "Y26A mutant AAR"). [B3]The 11th is leucine (L), the 26th is methionine (M), the 33rd is phenylalanine (F), the 40th is glutamine (Q), and the 61st is glutamic acid (E) (this may be referred to as "Y26M mutant AAR"). [B4]The 11th is leucine (L), the 26th is leucine (L), the 33rd is phenylalanine (F), the 40th is glutamine (Q), and the 61st is glutamic acid (E) (this may be referred to as "Y26L mutant AAR"). [B5]The 11th is leucine (L), the 26th is isoleucine (I), the 33rd is phenylalanine (F), the 40th is glutamine (Q), and the 61st is glutamic acid (E) (this may be referred to as "Y26I mutant AAR").

[0033] [C] F33 Mutant AAR [C1]The 11th is leucine (L), the 26th is tyrosine (Y), the 33rd is glycine (G), the 40th is glutamine (Q), and the 61st is glutamic acid (E) (this may be referred to as "F33G mutant AAR"). [C2]The 11th is leucine (L), the 26th is tyrosine (Y), the 33rd is leucine (L), the 40th is glutamine (Q), and the 61st is glutamic acid (E) (this may be referred to as "F33L mutant AAR"). [C3]The 11th is leucine (L), the 26th is tyrosine (Y), the 33rd is valine (V), the 40th is glutamine (Q), and the 61st is glutamic acid (E) (this may be referred to as "F33V mutant AAR"). [C4]The 11th is leucine (L), the 26th is tyrosine (Y), the 33rd is methionine (M), the 40th is glutamine (Q), and the 61st is glutamic acid (E) (this may be referred to as "F33M mutant AAR").

[0034] [D] Q40 Mutant AAR [D1]The 11th is leucine (L), the 26th is tyrosine (Y), the 33rd is phenylalanine (F), the 40th is lysine (K), and the 61st is glutamic acid (E) (this may be referred to as "Q40K mutant AAR"). [D2]The 11th is leucine (L), the 26th is tyrosine (Y), the 33rd is phenylalanine (F), the 40th is tyrosine (Y), and the 61st is glutamic acid (E) (this may be referred to as "Q40Y mutant AAR"). [D3]The 11th is leucine (L), the 26th is tyrosine (Y), the 33rd is phenylalanine (F), the 40th is valine (V), and the 61st is glutamic acid (E) (this may be referred to as "Q40V mutant AAR"). [D4]The 11th is leucine (L), the 26th is tyrosine (Y), the 33rd is phenylalanine (F), the 40th is arginine (R), and the 61st is glutamic acid (E) (this may be referred to as "Q40R mutant AAR"). [D5]The 11th is leucine (L), the 26th is tyrosine (Y), the 33rd is phenylalanine (F), the 40th is glutamic acid (E), and the 61st is glutamic acid (E) (this may be referred to as "Q40E mutant AAR").

[0035] [E] E61 Mutant AAR [E1]Position 11 is leucine (L), position 26 is tyrosine (Y), position 33 is phenylalanine (F), position 40 is glutamine (Q), and position 61 is alanine (A) (this may be referred to as "E61A mutant AAR").

[0036] [F] Y26 / Q40 Mutant AAR [F1]Position 11 is leucine (L), position 26 is glycine (G), position 33 is phenylalanine (F), position 40 is methionine (M), and position 61 is glutamic acid (E) (this may be referred to as "Y26G / Q40M mutant AAR"). [F2]Position 11 is leucine (L), position 26 is glycine (G), position 33 is phenylalanine (F), position 40 is histidine (H), and position 61 is glutamic acid (E) (this may be referred to as "Y26G / Q40H mutant AAR"). [F3]Position 11 is leucine (L), position 26 is glycine (G), position 33 is phenylalanine (F), position 40 is valine (V), and position 61 is glutamic acid (E) (this may be referred to as "Y26G / Q40V mutant AAR"). [F4]Position 11 is leucine (L), position 26 is glycine (G), position 33 is phenylalanine (F), position 40 is tyrosine (Y), and position 61 is glutamic acid (E) (this may be referred to as "Y26G / Q40Y mutant AAR").

[0037] [G] Y26 / F33 / Q40 Mutant AAR [G1]Position 11 is leucine (L), position 26 is glycine (G), position 33 is glycine (G), position 40 is methionine (M), and position 61 is glutamic acid (E) (this may be referred to as "Y26G / F33G / Q40M mutant AAR").

[0038] In the present disclosure, for amino acid sequences, nucleotide sequences, and their individual components, simplified symbols using alphabetical notation may be used, all in accordance with common practice in the fields of molecular biology and genetic engineering. Further, in the present disclosure, in order to concisely indicate mutations in amino acid sequences, notations such as "Y26", "Y26G", etc. may be used. As described above, in the AAR (wild-type SeAAR) derived from Synechococcus elongatus PCC 7942 = FACHB-805, the 26th amino acid in SEQ ID NO: 1 is tyrosine. For example, the "Y26 mutant AAR" indicates that the 26th tyrosine (Y) has been substituted with an amino acid other than tyrosine, and "Y26G" indicates the substitution of the 26th tyrosine (Y) with glutamine (G). That is, for example, "Y26G" indicates the type of amino acid residue before substitution, its location, and the type of amino acid residue after substitution. The 11th, 33rd, 40th, 61st amino acids in SEQ ID NO: 1, and the amino acids at other positions are also explained in the same way. Also, unless otherwise specified, the SEQ ID NOs. correspond to the SEQ ID NOs. described in the Sequence Listing. Also, in the case of multiple mutants, the above notations may be connected with " / " (for example, it can be denoted as "Y26G / Q40M").

[0039] Also, in the present disclosure, the amino acid sequence constituting the mutant AAR[1] may be an amino acid sequence in which one or more amino acids other than the 11th, 26th, 33rd, 40th, and 61st amino acids in the amino acid sequence described in (1) above are deleted, substituted, inserted, or added, as described in (2) above.

[0040] Here, "a plurality" is exemplified by, for example, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 18, 2 to 15, 2 to 13, 2 to 10, 2 to 8, 2 to 5, 2 to 4, 2, or 3.

[0041] The mutated amino acid may be either a natural amino acid or an artificial amino acid, for example. Examples of amino acids include hydrophobic amino acids, hydrophilic amino acids, basic amino acids, acidic amino acids, branched-chain amino acids, aromatic amino acids, sulfur-containing amino acids, etc. More specifically, examples of amino acids include, for example, A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, U, O, or V, etc. Techniques for deleting, substituting, inserting, and / or adding one or more amino acids in a specific amino acid sequence are known.

[0042] As the mutation, substitution is preferably exemplified, and substitution between amino acids that are structurally and / or chemically similar (conservative substitution) is more preferably exemplified. Examples of conservative substitutions include substitution between basic amino acids (H, K, R), substitution between acidic amino acids (D, E), substitution between neutral non-polar amino acids (A, V, L, I, P, F, M, W), substitution between neutral polar amino acids (G, N, Q, S, T, Y, C), substitution between aromatic amino acids (W, F, H, Y), substitution between sulfur-containing amino acids (C, M), substitution between β-branched chain amino acids (V, L, I), etc., but are not limited thereto.

[0043] Also, as the mutation, it is preferably to have a sequence identity of 80% or more with the amino acid sequence shown in the above (1), more preferably 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more of amino acid sequences are exemplified. The sequence identity is an example of a preferable value when comparing the amino acid sequence shown in the above (1) including the 11th, 26th, 33rd, 40th, and 61st amino acids with the mutated amino acid sequence.

[0044] In the present disclosure, the identity of amino acid sequences refers to the degree of coincidence of amino acid sequences of two or more comparable amino acid sequences with respect to each other. The level of amino acid sequence identity can be evaluated by any means known in the art. For example, it can be calculated using analytical tools that are commercially available or accessible through telecommunications lines (the Internet). As an example, the identity of amino acid sequences can be calculated using the default (initial setting) parameters in the homology algorithm BLAST (Basic Local Alignment Search Tool) on the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ).

[0045] The amino acid sequence in (2) preferably has amino acid mutations at positions other than the 11th, 26th, 33rd, 40th, and 61st positions within a range where production improvement of alkanes and / or alkenes equivalent to or higher than that of the amino acid sequence shown in (1) can be achieved. The range where production improvement of alkanes and / or alkenes equivalent to or higher than that can be achieved means that when, in the amino acid sequence shown in (1), the 11th, 26th, 33rd, 40th, and 61st amino acids are not mutated, and at least one amino acid at positions other than the 11th, 26th, 33rd, 40th, and 61st positions (that is, the 1st to 10th, 12th to 25th, 27th to 32nd, 34th to 39th, 41st to 60th, and 62nd to 341st positions of the amino acid sequence shown in (1)) is mutated to form a mutated amino acid sequence, when hydrocarbons are produced under the same conditions except for the difference in whether the amino acid sequence shown in (1) or the mutated amino acid sequence is used, it means the range where alkanes and / or alkenes can be produced with the same or higher efficiency as when the amino acid sequence shown in (1) is used when the mutated amino acid sequence is used. In mutant AAR[1], hydrocarbon production (production procedure, measurement, calculation, etc.) follows the procedure of Test Example 1 described below. The production efficiency will be described in the same manner as below.

[0046] In the variant AAR[1] of the present disclosure, the amino acid may be further modified as long as the effects of the present disclosure are not hindered. Examples of the modification include acylation, acetylation, alkylation, amidation, formylation, and the like.

[0047] · Mutant AAR[2] Variant AAR[2] is a variant acyl-ACP reductase having the amino acid sequence shown in the above (i) or (ii).

[0048] The amino acid sequence of the above (i) is an amino acid sequence in which 1 to 4 amino acids are deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35.

[0049] The amino acid sequence represented by SEQ ID NO: 35 is a sequence encoding the amino acid sequence of wild-type SeAAR, and in the amino acid sequence represented by SEQ ID NO: 1, the 11th amino acid is leucine, the 26th amino acid is tyrosine, the 33rd amino acid is phenylalanine, the 40th amino acid is glutamine, and the 61st amino acid is glutamic acid.

[0050] As the amino acid sequence of the above (i), preferably, an amino acid sequence in which 2 to 4 amino acids are deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 is exemplified, and more preferably, an amino acid sequence in which 3 or 4 amino acids are deleted is exemplified.

[0051] In the present disclosure, the amino acid sequence constituting variant AAR[2] may be an amino acid sequence in which 1 or more amino acids are deleted (provided that deletion of the first amino acid from the C-terminus of the amino acid sequence of the above (i) is excluded), substituted, inserted, or added in the amino acid sequence of the above (i) as described in the above (ii).

[0052] Here, "a plurality" is exemplified by, for example, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 18, 2 to 15, 2 to 13, 2 to 10, 2 to 8, 2 to 5, 2 to 4, 2 or 3. The mutated amino acid may be, for example, either a natural amino acid or an artificial amino acid, and the amino acid is described in the same manner as in the description of the "mutant AAR [1]". Further, substitution is preferably exemplified as the mutation, and conservative substitution is more preferably exemplified. Conservative substitution is described in the same manner as above.

[0053] Although not limiting the present disclosure, as the amino acid sequence of (ii) above, it may be an amino acid sequence in which 1 to 4 amino acids are deleted from the C-terminus of the amino acid sequence described in (1) or (2) of the mutant AAR [1].

[0054] Further, as the mutation, that is, as the amino acid sequence of (ii) above, it preferably has a sequence identity of 80% or more with an amino acid sequence in which 1 to 4 amino acids are deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 (hereinafter sometimes referred to as the "pre-mutated amino acid sequence"), more preferably 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more. The sequence identity is an example of a preferable value when comparing the pre-mutated amino acid sequence and the mutated amino acid sequence.

[0055] The amino acid sequence of (ii) preferably has an amino acid mutation in the amino acid sequence lacking 1 to 4 amino acids from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35, within the range where production improvement of alkanes and / or alkenes equivalent to or higher than that of the amino acid sequence shown in (i) can be achieved. The range where production improvement of alkanes and / or alkenes equivalent to or higher than that can be achieved means that when the amino acid sequence in (i) is mutated (mutated amino acid sequence), and hydrocarbons are produced under the same conditions except for the difference in whether the amino acid sequence in (i) or the mutated amino acid sequence is used, the mutated amino acid sequence can produce alkanes and / or alkenes at the same or higher efficiency as when the amino acid sequence in (i) is used. Hydrocarbon production (production procedure, measurement, calculation, etc.) follows the procedure of Test Example 4 described below. Production efficiency is explained in the same manner as described below. In mutant AAR[2] as well, as long as the effects of the present disclosure are not hindered, the amino acid may be further modified, and examples of the modification are explained in the same manner as described above.

[0056] · Mutant AAR[3] Mutant AAR[3] is a mutant acyl-ACP reductase having the amino acid sequence shown in (a) or (i).

[0057] The amino acid sequence of (a) is an amino acid sequence 1 to 12 amino acids in length consisting of glutamic acid and / or aspartic acid, linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35.

[0058] The amino acid sequence represented by SEQ ID NO: 35 is explained in the same manner as described above.

[0059] An amino acid sequence 1 to 12 amino acids in length consisting of glutamic acid and / or aspartic acid (hereinafter sometimes referred to as "amino acid sequence E / D") is, in other words, an amino acid sequence consisting of glutamic acid and / or aspartic acid, in which glutamic acid and / or aspartic acid are consecutively arranged in a total of 1 to 12. As the amino acid sequence E / D, preferably, an amino acid sequence 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids in length consisting of glutamic acid and / or aspartic acid is exemplified. The amino acid sequence E / D may be an amino acid sequence consisting of glutamic acid, an amino acid sequence consisting of aspartic acid, or an amino acid sequence in which glutamic acid and aspartic acid are mixed.

[0060] When the amino acid sequence E / D is an amino acid sequence consisting of glutamic acid, the amino acid length (number of amino acid residues) of the sequence is more preferably 2 to 11, still more preferably 3 to 10, particularly preferably 3 to 9, particularly more preferably 3 to 5, and particularly still more preferably 3 or 4.

[0061] When the amino acid sequence E / D is an amino acid sequence consisting of aspartic acid, the amino acid length of the sequence is more preferably 3 to 10, still more preferably 3 to 9, particularly preferably 3 to 6, particularly more preferably 3 to 5, and particularly still more preferably 3 or 4.

[0062] When the amino acid sequence E / D is an amino acid sequence in which glutamic acid and aspartic acid are mixed, the amino acid length of the sequence is more preferably 2 to 11, still more preferably 3 to 10, particularly preferably 3 to 9, particularly more preferably 3 to 6, and particularly still more preferably 3, 4 or 5.

[0063] When the amino acid sequence E / D is an amino acid sequence in which glutamic acid and aspartic acid are mixed, the number of glutamic acid residues and the number of aspartic acid residues constituting the amino acid sequence E / D may be the same or different, and the order in which glutamic acid and aspartic acid are consecutive is also not limited. As the amino acid sequence E / D, an amino acid sequence in which 1 aspartic acid and 3 glutamic acids are consecutive in order from the N-terminus, an amino acid sequence in which 2 aspartic acids and 2 glutamic acids are consecutive in order from the N-terminus, etc. are preferably exemplified.

[0064] It is preferably exemplified that one or two amino acid sequences E / D are linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. When two amino acid sequences E / D are linked, the amino acid sequence represented by SEQ ID NO: 35, the amino acid sequence E / D (first amino acid sequence E / D), and the amino acid sequence E / D (second amino acid sequence E / D) are arranged in this order from the N-terminal side to the C-terminal side, and the amino acid sequences E / D (first amino acid sequence E / D and second amino acid sequence E / D) are linked via a linker. The linker will be described in the same manner as described later. The amino acid sequence of the first amino acid sequence E / D and the amino acid sequence of the second amino acid sequence E / D may be the same or different. It is preferably exemplified that one amino acid sequence E / D is linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35.

[0065] The amino acid sequence represented by SEQ ID NO: 35 and the amino acid sequence E / D are linked directly or via a linker, preferably directly linked by a peptide bond. The linker is not limited as long as it does not interfere with the effects of the present disclosure, and examples include a linker consisting only of glycine (G), a linker consisting only of serine (S), a GS linker, an EAAAK linker, etc. The length of the linker is not particularly limited, and preferably the number of amino acid residues of the linker is 2 to 10, more preferably 2 to 5, still more preferably 2 or 3, etc. As the GS linker, preferably (G4S)n is exemplified, and n is exemplified as an integer of 1 or 2. As the EAAAK linker, (EAAAK)n is exemplified, and n is exemplified as an integer of 1 or 2 (EAAAK is shown in SEQ ID NO: 36). The linker may be used alone or in combination of two or more.

[0066] In addition, the amino acid sequence constituting the mutant AAR[3] in the present disclosure may be an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 35 in the amino acid sequence described in (a) as described in (i) above.

[0067] Here, "a plurality" is exemplified by, for example, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 18, 2 to 15, 2 to 13, 2 to 10, 2 to 8, 2 to 5, 2 to 4, 2 or 3. The mutated amino acid may be, for example, either a natural amino acid or an artificial amino acid, and the amino acid is described in the same manner as in the description of the "mutant AAR[1]". In addition, substitution is preferably exemplified as the mutation, and conservative substitution is more preferably exemplified. Conservative substitution is described in the same manner as above.

[0068] Although not limiting the present disclosure, as the amino acid sequence of (i) above, an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 35 may be the amino acid sequence described in (1) or (2) of the mutant AAR [1]. Further, an amino acid sequence in which one or more amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 35 may be an amino acid sequence in which 1, 2, 3, 4 or 5 or more consecutive amino acids are deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Preferably, an amino acid sequence in which 4 or 5 or more consecutive amino acids are not deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 is exemplified, and more preferably, an amino acid sequence in which 1, 2 or 3 or more consecutive amino acids are not deleted from the C-terminus is exemplified.

[0069] Further, as the mutation, it is preferably an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 35, more preferably 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more sequence identity. The sequence identity is an example of a preferable value when comparing the amino acid sequence represented by SEQ ID NO: 35 with the amino acid sequence after mutation.

[0070] The amino acid sequence of (i) preferably has an amino acid mutation in the amino acid sequence represented by SEQ ID NO: 35 within a range where improvement in the production of alkanes and / or alkenes equivalent to or higher than that of the amino acid sequence shown in (a) can be achieved. The range where improvement in the production of alkanes and / or alkenes equivalent to or higher than that can be achieved means that, in the amino acid sequence of (a), when the sequence is mutated only in the amino acid sequence represented by SEQ ID NO: 35 (mutated amino acid sequence), and hydrocarbons are produced under the same conditions except for the difference in whether the amino acid sequence of (a) or the mutated amino acid sequence is used, it means the range where alkanes and / or alkenes can be produced with the same or higher efficiency as when the amino acid sequence of (a) is used. Hydrocarbon production (production procedures, measurement, calculation, etc.) follows the procedure of Test Example 4 described below. Production efficiency is explained in the same manner as described below. In the mutant AAR[3] as well, as long as the effects of the present disclosure are not hindered, the amino acid may be further modified, and examples of the modification are explained in the same manner as described above.

[0071] The mutant AARs (mutant AARs [1] to [3]) of the present disclosure can be produced by any genetic engineering method known in the art. For example, a mutant AAR having the target amino acid sequence (the mutant AAR of the present disclosure) can be produced by appropriately designing the base sequence encoding the target amino acid sequence, incorporating the base sequence into an arbitrary vector, etc., and transforming and expressing it in a host cell. Also, the base sequence, amino acid sequence, etc. can also be produced according to known genetic engineering methods, chemical synthesis methods of peptides (for example, azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DDC method, active ester method, carbodiimidazole method, oxidation-reduction method, etc.) based on the information of the target sequence, etc. as needed.

[0072] As described above, it is known that AAR uses a coenzyme (mainly NADPH) to convert acyl-ACP into the corresponding aldehyde, and the generated aldehyde is converted into the corresponding alkane and alkene by deformylation with ADO (Figure 4). Therefore, the mutant AAR of the present disclosure is preferably used in combination with ADO. It is also known that hydrocarbons can be produced from glucose through this conversion pathway by co-expressing AAR and ADO using host cells such as Escherichia coli. In addition, since cells such as Escherichia coli are rich in acyl-ACP, hydrocarbons can be easily produced by co-expressing AAR and ADO using such cells. Therefore, in the present disclosure, hydrocarbons may be produced in the same manner as known procedures except that the mutant AAR of the present disclosure is used instead of the known AAR, and co-expressing the mutant AAR and ADO of the present disclosure using host cells such as Escherichia coli is preferably exemplified as a simple production means for producing hydrocarbons. The mutant AAR of the present disclosure may be used alone or in combination of two or more.

[0073] When the mutant AAR of the present disclosure produces hydrocarbons in combination with wild-type ADO, the production efficiency of at least one selected from the group consisting of alkanes and alkenes (i.e., alkanes and / or alkenes) can be improved as compared with the case where hydrocarbons are produced in the same manner by combining wild-type SeAAR and wild-type ADO.

[0074] Whether the production efficiency of alkanes and / or alkenes can be improved can be confirmed by incorporating the nucleotide sequence encoding the mutant AAR and the nucleotide sequence encoding the wild-type ADO (preferably ADO derived from Nostoc punctiforme PCC 73102) into a vector, introducing the resulting vector into Escherichia coli BL21(DE3), culturing the obtained transformant in a medium to express the mutant AAR and the wild-type ADO, and producing hydrocarbons. In this case, the total production amount of alkanes (especially tridecane and pentadecane) relative to the total production amount of hydrocarbons (especially tridecane, pentadecane, and heptadecene) produced using the nucleotide sequence encoding the mutant AAR in this way (that is, the production ratio of alkanes to the total production amount of alkanes and alkenes) is higher than the production ratio of alkanes determined by producing hydrocarbons in the same manner except that the nucleotide sequence encoding the wild-type SeAAR is used instead of the nucleotide sequence encoding the mutant AAR. In such a case, the mutant AAR is determined to be able to improve the production efficiency of alkanes and / or alkenes (especially, it can improve the production efficiency of alkanes).

[0075] Alternatively, when the total production amount of alkanes (especially tridecane and pentadecane) produced using the nucleotide sequence encoding the mutant AAR in this way (that is, the alkane production amount) is higher than the alkane production amount determined by producing hydrocarbons in the same manner except that the nucleotide sequence encoding the wild-type SeAAR is used instead of the nucleotide sequence encoding the mutant AAR, the mutant AAR is determined to be able to improve the production efficiency of alkanes and / or alkenes (especially, it can improve the production efficiency of alkanes). Or, when the total production amount of alkenes (especially heptadecene) produced using the nucleotide sequence encoding the mutant AAR in this way (that is, the alkene production amount) is higher than the production amount of alkenes determined by producing hydrocarbons in the same manner except that the nucleotide sequence encoding the wild-type SeAAR is used instead of the nucleotide sequence encoding the mutant AAR, the mutant AAR is determined to be able to improve the production efficiency of alkanes and / or alkenes (especially, it can improve the production efficiency of alkenes).

[0076] Alternatively, when the total production amount of alkanes and alkenes (particularly tridecane, pentadecane, and heptadecene) produced using the nucleotide sequence encoding the mutant AAR in this manner is greater than the total production amount of alkanes and alkenes determined by producing hydrocarbons in the same manner except that the nucleotide sequence encoding the wild-type SeAAR is used instead of the nucleotide sequence encoding the mutant AAR, the mutant AAR is determined to be able to improve the production efficiency of alkanes and / or alkenes (particularly, able to improve the production efficiency of alkanes). Regarding these production efficiencies, more detailed procedures and the like may follow the examples described later, such as Example 1.

[0077] Thus, the mutant AAR of the present disclosure has an effect of improving the production efficiency of alkanes and / or alkenes. More specifically, as will be described later, the mutant AAR of the present disclosure has at least one of the effects of improving the alkane production ratio, improving the alkane production amount, and improving the total production amount of alkanes and alkenes. More preferably, the mutant AAR of the present disclosure has at least one of the effects of improving the alkane production ratio and improving the production amount of alkanes and / or alkenes, and still more preferably has both effects of improving the alkane production ratio and improving the alkane production amount.

[0078] In the present disclosure, the ADO used for the conversion of aldehydes is not limited, and any ADO may be used as long as the effects of the present disclosure are not hindered. ADO derived from cyanobacteria is preferably exemplified as the ADO. Although not limiting the present disclosure, examples of cyanobacteria include Nostoc such as Nostoc punctiforme PCC 73102; Synechococcus such as Synechococcus elongatus PCC 7942; Synechocystis such as Synechocystis sp. PCC 6803; Prochlorococcus such as Prochlorococcus marinus str. MIT 9313; Gloeobacter such as Gloeobacter violaceus PCC 7421; Synechococcus such as Synechococcus sp. PCC 7336, Synechococcus sp. PCC 6312; Microcystis such as Microcystis aeruginosa PCC 9443; Thermosynechococcus such as Thermosynechococcus elongatus BP-1; Cyanothece such as Cyanothece sp. PCC 7425; Synechocystis such as Synechocystis sp. PCC 7509; Cyanobium such as Cyanobium sp. PCC 7001; Microcoleus such as Microcoleus sp. PCC 7113; Gloeocapsa such as Gloeocapsa sp. PCC 73106; Halothece such as Halothece sp., etc. The amino acid sequences and nucleotide sequences of ADOs derived from these are known and can be easily obtained from conventionally known sequence databases such as NCBI, UniPort (https: / / www.uniprot.org), etc.The ADO may be a wild-type ADO as long as it can convert aldehyde into hydrocarbon, or it may be a mutant ADO with some mutations. As an example, the amino acid sequence encoding ADO derived from Nostoc punctiforme PCC 73102 (hereinafter sometimes referred to as "wild-type NpADO") and the nucleotide sequence encoding the amino acid sequence can be represented by SEQ ID NO: 3 and SEQ ID NO: 4 (Figure 5). The ADO may be used alone or in combination of two or more.

[0079] In the present disclosure, the improvement in the production of alkane and / or alkene, the improvement in the production efficiency of alkane and / or alkene, and the ability to efficiently produce alkane and / or alkene are used in the same meaning. First, when producing hydrocarbon by combining the mutant AAR of the present disclosure and the wild-type ADO, compared with the case of producing hydrocarbon in the same way by combining the wild-type AAR and the wild-type ADO, it means that the alkane production ratio and / or alkene production ratio in the produced hydrocarbon are high. That is, this means that the ratio of the production amount of alkane and / or alkene in the produced hydrocarbon amount has improved. Hereinafter, this may be described as "improvement in alkane production ratio" and / or "improvement in alkene production ratio".

[0080] Second, the improvement in the production of the alkane and / or alkene, etc. means that when producing hydrocarbon by combining the mutant AAR and the wild-type ADO, compared with the case of producing hydrocarbon in the same way by combining the wild-type AAR and the wild-type ADO, the amount of the produced alkane and / or alkene is large. That is, this means that the production amount of alkane and / or alkene has improved. Hereinafter, this may be described as "improvement in the production amount of alkane and / or alkene".

[0081] Further, the improvement in the production of the alkane and / or alkene, etc. means, thirdly, that when hydrocarbons are produced by combining the mutant AAR and the wild-type ADO, the total amount of the produced alkane and alkene is larger than that when hydrocarbons are produced in the same manner by combining the wild-type AAR and the wild-type ADO. That is, this means that the total production amount of the alkane and alkene has been improved. Hereinafter, this may be described as "improvement in the total production amount of the alkane and alkene".

[0082] Therefore, the mutant AAR of the present disclosure has at least one effect of improving the alkane production ratio, improving the alkene production ratio, improving the alkane production amount, and improving the total production amount of the alkane and alkene. More preferably, the mutant AAR of the present disclosure has at least one of the effects of improving the alkane production ratio and improving the alkane production amount. More preferably, the mutant AAR of the present disclosure has both effects of improving the alkane production ratio and improving the alkane production amount.

[0083] More specifically explaining the improvement in the alkane production ratio, the improvement in the production ratio means that when Escherichia coli BL21(DE3) is transformed with a plasmid into which a base sequence encoding AAR and a base sequence encoding wild-type NpADO are introduced in the same procedure as in Test Example 1 or 4 described later, and then cultured to produce hydrocarbons, and the production ratio of the alkane in the produced hydrocarbons is calculated, the ratio of the alkane production amount in the produced hydrocarbon amount is higher when the mutant AAR of the present disclosure is used than when the wild-type SeAAR is used as the AAR. Here, the improvement in the production ratio preferably means that the ratio of the total production amount of the alkane (tridecane and pentadecane) to the total production amount of the alkane and alkene (tridecane, pentadecane, and heptadecene) produced in the same procedure as in Test Example 1 or 4 described later is high. The hydrocarbon content ratio is preferably determined in the same procedure as in Test Example 1 or 4.

[0084] Without being limited thereto, as the conditions for the production, Escherichia coli BL21(DE3) is transformed using a vector in the same procedure as in Test Example 1 or 4 described below to form colonies, the colonies are inoculated into an LB liquid medium containing an antibiotic (100 μg / mL of sodium ampicillin), and after shaking culture overnight at 37°C, the obtained culture is added to a modified M9 medium, and further a trace metal solution is added, followed by shaking culture at pH 7.2 and 37°C, and then IPTG is added, and shaking is carried out at 24°C for 24 hours to produce hydrocarbons. The improvement in the above-mentioned alkene production ratio and the second and third improvements are preferably exemplified in the same manner.

[0085] The degree of improvement in the alkane production ratio is not limited, but if determined based on Test Example 1 or 4 described below, when the mutant AAR and the wild-type NpADO are used in combination, the alkane production ratio is preferably improved by 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 10 times or more, etc. compared to the case where the wild-type AAR and the wild-type NpADO are used in combination. The upper limit is not limited, but preferably 100 times or less, 50 times or less, 30 times or less, 20 times or less, etc. are exemplified. From this, as the range of the improvement, 1.5 times or more and 100 times or less, 2 times or more and 100 times or less, 3 times or more and 50 times or less, 4 times or more and 30 times or less, 10 times or more and 20 times or less, etc. are preferably exemplified. As an example, the alkane production ratio was improved by about 4.36 times when the Y26A mutant AAR was used in Test Example 1 described below, and was improved by about 3.45 times when the Q40V mutant AAR was used.

[0086] More specifically explaining the improvement in alkane production amount, the improvement in the production amount means that when Escherichia coli BL21(DE3) is transformed with a plasmid into which a nucleotide sequence encoding AAR and a nucleotide sequence encoding wild-type NpADO are introduced in the same procedure as in Test Example 1 or 4 described below, and then cultured to produce hydrocarbons and the alkane production amount is calculated, as AAR, when using the mutant AAR of the present disclosure, the produced alkane production amount is larger than when using the wild-type AAR. Here, the improvement in the production amount preferably means that the amount of alkanes (tridecane and pentadecane) produced in the same procedure as in Test Example 1 or 4 described below is larger. The alkane production amount is preferably determined in the same procedure as in Test Example 1 or 4.

[0087] The degree of improvement in alkane production amount is not limited, but based on Test Example 1 or 4 described below, when determined, the alkane production amount when using the mutant AAR and the wild-type NpADO in combination is preferably exemplified as being 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, etc. higher than the production amount when using the wild-type AAR and the wild-type NpADO in combination. The upper limit value is not limited, but preferably exemplified as 300 times or less, 250 times or less, 200 times or less, 150 times or less, 100 times or less, etc. From this, as the improvement range, preferably exemplified are 1.5 times or more and 300 times or less, 2 times or more and 250 times or less, 3 times or more and 200 times or less, 4 times or more and 150 times or less, 5 times or more and 100 times or less, etc. As an example, the alkane production amount was improved by about 42 times when using the Y26G mutant AAR in Test Example 1 described below, and was improved by about 64 times when using the Y26G / Q40M mutant AAR.

[0088] Similarly, although the degree of improvement in alkene production is not limited, if determined based on Test Example 1 or 4 described below, it is preferably exemplified that the alkene production when using a combination of the mutant AAR and the wild-type NpADO is 1.2 times or more, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, etc. of the production when using a combination of the wild-type AAR and the wild-type NpADO. The upper limit is not limited, but preferably exemplified as 20 times or less, 15 times or less, 10 times or less, etc. From this, as the range of improvement, 1.2 times or more and 20 times or less, 1.5 times or more and 15 times or less, 2 times or more and 10 times or less, etc. are preferably exemplified.

[0089] More specifically regarding the improvement in the total production of alkanes and alkenes, the improvement in the total production means that, in the same procedure as Test Example 1 or 4 described below, Escherichia coli BL21(DE3) is transformed with a plasmid into which the nucleotide sequence encoding AAR and the nucleotide sequence encoding wild-type NpADO are introduced, and then cultured to produce hydrocarbons, and when calculating the production amounts of alkanes and alkenes, the total production amount of the produced alkanes and alkenes is larger when using the mutant AAR of the present disclosure than when using the wild-type AAR. Here, the improvement in the total production amount preferably means that the amounts of alkanes and alkenes (tridecane, pentadecane, and heptadecene) produced in the same procedure as Test Example 1 or 4 described below are larger. The total production amount of alkanes and alkenes is preferably determined in the same procedure as Test Example 1 or 4.

[0090] When using the obtained hydrocarbons as biofuel (drop-in biofuel) as they are, although it is preferable that the production ratio and / or the production amount of alkanes is high, it is known that alkenes can be relatively easily converted into alkanes by hydrogenation. Therefore, it can be said that the improvement in the total production amount of alkanes and alkenes and / or the improvement in the production amount of alkanes also lead to an improvement in alkane production efficiency.

[0091] The degree of improvement in the total production amount of alkanes and alkenes is not limited, but if determined based on Test Example 1 or 4 described later, the total production amount when using the mutant AAR and the wild-type NpADO in combination is 1.2 times or more, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, etc. of the total production amount when using the wild-type AAR and the wild-type NpADO in combination, which is preferably exemplified. The upper limit is not limited, but 20 times or less, 15 times or less, 10 times or less, etc. are preferably exemplified. From this, as the improvement range, 1.2 times or more and 20 times or less, 1.5 times or more and 15 times or less, 2 times or more and 10 times or less, etc. are preferably exemplified. As an example, the total production amount of alkanes and alkenes was improved by about 3.2 times when the Y26G mutant AAR was used in Test Example 1 described later, and was improved by about 6.2 times when the Y26G / Q40M mutant AAR was used.

[0092] From this, it can also be said that the present disclosure provides a method for efficiently producing alkanes and / or alkenes using the mutant AAR. From this, it can be said that the present disclosure provides a method for producing alkanes and / or alkenes using the mutant AAR in the presence of ADO. As the method, a method of culturing a transformant such as Escherichia coli exemplified in Test Example 1 or 4 is preferably exemplified. In this case, a plasmid such as pT7Cm15A_aACP that highly expresses acyl-ACP may be incorporated.

[0093] In particular, the present disclosure uses the mutant AAR to efficiently produce pentadecane (C 15 H 32 ), tridecane (C 13 H 28 ) and / or heptadecene (C 17 H 34 ) compared with the case of using the wild-type AAR. Furthermore, the present disclosure uses the mutant AAR to efficiently produce pentadecane (C 15 H 32 ) and tridecane (C 13 H 28It can also be said that a method for producing ) is provided. This process can be referred to as the pentadecane (especially linear pentadecane) and tridecane (especially linear tridecane) synthesis process (DSHC-SLPT). Furthermore, it can also be said that the present disclosure provides a method for efficiently producing heptadecene using the mutant AAR as compared with the case of using the wild-type AAR.

[0094] In yet another embodiment, the present disclosure provides a polynucleotide encoding the mutant AAR of the present disclosure. The polynucleotide encoding the mutant AAR of the present disclosure refers to, for example, a polynucleotide from which the protein of the mutant AAR of the present invention can be obtained when expressed by a conventional method using the polynucleotide. The polynucleotide can be any nucleic acid polymer such as DNA or RNA.

[0095] The polynucleotide is not limited as long as it encodes the mutant AAR of the present disclosure. Preferably, in the base sequence represented by SEQ ID NO: 2 or 5, a polynucleotide composed of a base sequence into which a mutation according to the amino acid mutation shown in the above (1) or (2) is introduced is exemplified. Further, as the polynucleotide, preferably, a polynucleotide composed of a base sequence in which a base sequence encoding 1 to 4 amino acids is deleted from the 3'-end of the base sequence represented by SEQ ID NO: 2 or 5, or a polynucleotide composed of a base sequence into which a mutation according to the amino acid mutation shown in the above (ii) is introduced in the polynucleotide is exemplified. Further, as the polynucleotide, preferably, a polynucleotide composed of a base sequence in which a base sequence encoding the amino acid sequence E / D is linked to the 3'-end of the base sequence represented by SEQ ID NO: 2 or 5, or a polynucleotide composed of a base sequence in which a base sequence encoding the amino acid sequence E / D is linked to the 3'-end of a base sequence into which a mutation according to the amino acid mutation shown in the above (a) is introduced in the base sequence represented by SEQ ID NO: 2 or 5 is exemplified. According to a codon table known in the art, etc., a base sequence corresponding to a predetermined amino acid sequence can be easily determined. Further, the polynucleotide encoding the mutant AAR of the present disclosure also includes polynucleotides that differ due to codon degeneracy.

[0096] In a further embodiment, the present disclosure provides a vector comprising the polynucleotide. Examples of the vector include vectors commonly used in the fields of genetic engineering and protein engineering. The vector may be appropriately selected according to the purpose of use (cloning, protein expression) and the type of host cell. The origin, such as bacteria like Escherichia coli, yeast, or virus, is not limited, and any vector such as a plasmid, phage, or cosmid may be used. Examples of the vector include pET3a, pET8c, pET21b, pET23b, pUC118, pUC18, pUC8, pBR322, pB325, pAT153, pBluescript, pLED-M1, p73, pGW7, etc. A polynucleotide encoding ADO may be connected to the vector. Therefore, the present disclosure also provides a vector comprising a polynucleotide encoding a mutant AAR and a polynucleotide encoding ADO. A promoter may be further connected to the vector as needed, for example, a promoter suitable for the host cell may be selected. Base sequences such as enhancers, splicing signals, polyA addition signals, drug resistance genes, and marker genes such as Green Fluorescent Protein (GFP) may be connected to the vector. These are connected etc. at any position of the vector according to the purpose.

[0097] Examples of the host cell include Escherichia coli and yeast, with Escherichia coli being preferably exemplified. Examples of Escherichia coli include Escherichia coli BL21(DE3), DH5α, JM109, HB101, XL1Blue, PR1, HS641(DE3), etc. It is preferably exemplified that a polynucleotide encoding the mutant AAR of the present disclosure is inserted into the vector to obtain an expression vector, and further, the host cell is transformed with the expression vector. Therefore, in a further embodiment, the present disclosure provides a cell transformed with the vector. According to a conventionally known protein expression procedure, the cell can be cultured in a medium to express the mutant AAR of the present disclosure. When a polynucleotide encoding ADO is incorporated into the cell, not only the mutant AAR of the present disclosure but also ADO can be expressed by the culture.

[0098] The medium is not limited as long as the cell can be cultured, and examples thereof include conventionally known media such as LB medium, M9 medium, modified M9 medium, 2×YT medium, and TB medium. The medium can be either a liquid medium or a solid medium. The medium may be formulated with components such as antibiotics (e.g., ampicillin, kanamycin, chloramphenicol, etc.), trace metal solutions (e.g., a solution containing ZnCl2, FeCl3·6H2O, CuSO4·5H2O, Na2MoO4·2H2O, and H3BO3), and expression induction components (e.g., Isopropyl β-D-1-thiogalactopyranoside (IPTG)) as necessary. From the viewpoint of more efficiently performing protein (enzyme) expression and the conversion of acyl-ACP to the corresponding aldehyde in the medium, it is preferably exemplified that a liquid medium is used during the expression and the conversion.

[0099] While not limiting the present disclosure, as an example of the culturing, expression, and conversion procedures, the following procedures are exemplified. After culturing and transforming host cells using the vector, the resulting culture is spread on an agar medium containing an antibiotic to form colonies. The colonies are inoculated into a liquid medium containing an antibiotic (such as LB liquid medium), cultured at 4 to 50 °C (preferably 30 to 40 °C) for 1 hour to 1 week (preferably 8 to 20 hours), then the culture is added to a liquid medium (such as modified M9 medium), and a trace metal solution is further added, followed by culturing at 4 to 50 °C (preferably 10 to 40 °C) and pH 4 to 9 (preferably pH 5 to 8), and then IPTG is added and cultured at 4 to 50 °C (preferably 10 to 37 °C) to perform the expression and conversion. The time from the start of culturing in the liquid medium (such as modified M9 medium) to the completion of culturing in the presence of IPTG is not limited either, and for example, 1 hour to 2 weeks (preferably 2 to 72 hours) is exemplified.

[0100] Also, a vector incorporating a nucleotide sequence encoding mutant AAR is prepared, the vector is transformed into a host cell, and the transformant thus obtained is cultured in the same manner as described above, whereby mutant AAR can be expressed in the medium, and acyl-ACP is converted to the corresponding aldehyde by mutant AAR. By contacting ADO with the culture containing the mutant AAR and aldehyde thus obtained, the aldehyde can also be converted to the corresponding hydrocarbon by ADO. Also in this case, a hydrocarbon product with an improved alkane production ratio, alkane production amount, and / or total production amount of alkane and alkene can be easily obtained.

[0101] In these cases, from the viewpoint of more easily obtaining a hydrocarbon product in which at least one of the alkane production ratio, alkane production amount, and total production amount of alkane and alkene is improved, it is preferably exemplified to obtain a transformant using an expression vector incorporating a nucleotide sequence encoding mutant AAR and a nucleotide sequence encoding ADO, and co-express mutant AAR and ADO in the medium to perform the above conversion.

[0102] The expressed mutant AAR may be fractionated and / or purified as needed. Also, the expressed ADO may be fractionated and / or purified as needed. Using the obtained mutant AAR and further ADO, conversion of acyl-ACP to aldehyde and further conversion of aldehyde to hydrocarbon can also be carried out. The fractionation and / or purification may be performed according to conventional known protein fractionation and / or purification procedures. For example, as the procedure, cells are disrupted to extract a crude enzyme solution. Any known method may be used for disrupting the cells, for example, physical disruption methods such as ultrasonic treatment, French press, and glass bead disruption, and lytic enzymes such as lysozyme can be used. Any method may be used to obtain a purified enzyme from the obtained crude enzyme solution. For example, it can be isolated by subjecting it to centrifugation, ultracentrifugation, ultrafiltration, salting out, dialysis, ion exchange column chromatography, adsorption column chromatography, affinity chromatography, gel filtration column chromatography, etc.

[0103] The cells may be provided in the form of a reagent in combination with any other components (any additives such as stabilizers and preservatives). By using the cells or the reagent, the mutant AAR can be expressed more simply, and thus the production efficiency of alkanes and / or alkenes can be improved more simply.

[0104] The mutant AAR may be provided in the form of an enzyme preparation or a kit. The enzyme preparation and the kit may contain other components as needed in addition to the mutant AAR. Examples of other components include ADO (enzyme), acyl-ACP, excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, oils and fats, etc. Also, the kit may include instructions for use when converting acyl-ACP to aldehyde using the mutant AAR, and further instructions for use when converting aldehyde to hydrocarbon using ADO. The instructions for use may be those in which the URL of a web page, a read code, etc. are described, and the usage procedures, etc. may be obtained via the URL, the read code, etc.

[0105] Thus, the variant AAR of the present disclosure is useful for improving the production efficiency of alkanes and / or alkenes.

[0106] From these, according to the present disclosure, a variant AAR capable of improving the production efficiency of alkanes and / or alkenes can be provided. Further, according to the present disclosure, by using the variant AAR, alkanes and / or alkenes can be produced more efficiently. Although cyanobacteria are known to produce hydrocarbons during photosynthesis, according to the present disclosure, alkanes and / or alkenes can be produced even in the dark.

[0107] Also, as described in Test Example 3 below, when the variant AAR (AAR mutant) of the present disclosure was used, compared with the case of using the wild-type SeAAR, the supply of linear aldehydes (e.g., hexadecanal) to ADO was promoted, and thus it was reasonably understood that the production ratio and / or production amount of alkanes were improved. From this, according to the present disclosure, it can be said that aldehydes (which can also be said to be precursors of SAF) converted to alkanes by ADO can be efficiently provided, and this leads to an improvement in alkane production efficiency (especially linear alkanes of C13, C15, and / or C17). Therefore, the present disclosure can be said to provide a novel DSHC process that can more simply convert sugars to alkanes. Thus, the variant AAR of the present disclosure, which can improve the production efficiency of alkanes and / or alkenes, can contribute to the development of SAF.

[0108] Also, as described in Test Example 4 below, when the variant AAR (AAR mutant) of the present disclosure was used, compared with the case of using the wild-type SeAAR, the protein solubility in the expression of the variant AAR was improved, and the stability was also improved. From this, it can be said that the improvement in the solubility (suppression of inclusion body formation) and / or stability of the variant AAR of the present disclosure contributes to the improvement of the production efficiency of alkanes and / or alkenes, and according to the present disclosure, alkanes and / or alkenes can be efficiently provided even when using host cells such as Escherichia coli. Also in this regard, the variant AAR of the present disclosure can contribute to the development of SAF.

Example

[0109] Hereinafter, embodiments of the present disclosure will be described more specifically with examples, but the embodiments of the present disclosure are not limited to the following examples.

[0110] Unless otherwise specified in the following test examples, restriction enzymes and DNA ligase were obtained from New England Biolabs Japan Co., Ltd. The primers shown in Table 1 were obtained from Integrated DNA Technologies (Singapore). PCR products were obtained from Toyobo Co., Ltd. Gel extraction kits and plasmid-miniprep kits were obtained from Nippon Genetics Co., Ltd. Alkanes (C8H 18 ~C 20 H 42 ) and alkene (C 17 H 34 ) calibration standards were obtained from Merck (Germany) and Tokyo Chemical Industry Co., Ltd., respectively. Plasmid pET-21b(+) was obtained from Novagen (Germany).

[0111]

Table 1

[0112] Test Example 1 <Test procedure> 1. Construction of Plasmids Introducing Polynucleotides of Mutant AAR PCR amplification and the like were performed, and the nucleotide sequence encoding AAR derived from Synechococcus elongatus PCC 7942 = FACHB-805 (hereinafter, wild-type SeAAR) and the nucleotide sequence encoding NpADO were cloned into plasmid pET-21b(+), respectively, to prepare plasmid pET7SeAAR and plasmid pET7NpADO. In this test example, the nucleotide sequence represented by SEQ ID NO: 5 was used as the nucleotide sequence encoding wild-type SeAAR. SEQ ID NO: 5 encodes an amino acid sequence in which the 11th amino acid is leucine, the 26th amino acid is tyrosine, the 33rd amino acid is phenylalanine, the 40th amino acid is glutamine, and the 61st amino acid is glutamic acid in the amino acid sequence represented by SEQ ID NO: 1. The nucleotide sequence represented by SEQ ID NO: 6 was used as the nucleotide sequence encoding NpADO. SEQ ID NO: 6 encodes wild-type NpADO represented by SEQ ID NO: 3. The SphI site was PCR-amplified using plasmid pET7SeAAR as a template, and plasmid pET7NpADO was amplified from the T7 promoter to the T7 terminator, and the T7NpADO fragment was incorporated into the pET7SeAAR fragment to prepare plasmid pET7SeAAR-T7NpADO (Figure 7). This was named the basic plasmid. Further, using this plasmid as a template plasmid, a plasmid (test plasmid) into which a nucleotide sequence encoding a mutant AAR was introduced instead of the nucleotide sequence 5 encoding wild-type SeAAR was prepared by cloning a PCR fragment by homologous recombination. More specifically, regarding the test plasmid, a plasmid encoding mutant SeAAR was prepared by circularizing a DNA fragment obtained by PCR using plasmid pET7SeAAR as a template and mutant primers with the part where mutation was to be introduced changed. Furthermore, a plasmid (test plasmid) into which a nucleotide sequence encoding mutant SeAAR and a nucleotide sequence encoding wild-type NpADO were introduced was prepared by incorporating the T7NpADO fragment into the plasmid encoding mutant SeAAR. In the basic plasmid and the test plasmid, the nucleotide sequence encoding NpADO is the same.

[0113] The nucleotide sequence encoding the mutant AAR introduced into the test plasmid is the nucleotide sequence encoding the amino acid sequence of the following mutant AAR.

[0114] Base Sequence Encoding Mutant AAR [1-A] Y26 Mutant AAR [1-A1] Y26G Mutant AAR (Example 1) A nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is glycine (G), the 33rd amino acid is phenylalanine (F), the 40th amino acid is glutamine (Q), and the 61st amino acid is glutamic acid (E). [1-A2] Y26A Mutant AAR (Example 2) A nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is alanine (A), the 33rd amino acid is phenylalanine (F), the 40th amino acid is glutamine (Q), and the 61st amino acid is glutamic acid (E). [1-A3] Y26M Mutant AAR (Example 3) A nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is methionine (M), the 33rd amino acid is phenylalanine (F), the 40th amino acid is glutamine (Q), and the 61st amino acid is glutamic acid (E). [1-A4] Y26L Mutant AAR (Example 4) A nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is leucine (L), the 33rd amino acid is phenylalanine (F), the 40th amino acid is glutamine (Q), and the 61st amino acid is glutamic acid (E).

[0115] [1-B] Q40 Mutant AAR [1-B1] Q40Y Mutant AAR (Example 5) A base sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is tyrosine (Y), the 33rd amino acid is phenylalanine (F), the 40th amino acid is tyrosine (Y), and the 61st amino acid is glutamic acid (E). [1-B2] Q40V Mutant AAR (Example 6) A base sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is tyrosine (Y), the 33rd amino acid is phenylalanine (F), the 40th amino acid is valine (V), and the 61st amino acid is glutamic acid (E).

[0116] [1-C] E61 Mutant AAR [1-C1] E61A Mutant AAR (Example 7) A base sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is tyrosine (Y), the 33rd amino acid is phenylalanine (F), the 40th amino acid is glutamine (Q), and the 61st amino acid is alanine (A).

[0117] [1-D] Y26 / Q4 Mutant AAR [1-D1] Y26G / Q40M Mutant AAR (Example 8) A base sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is glycine (G), the 33rd amino acid is phenylalanine (F), the 40th amino acid is methionine (M), and the 61st amino acid is glutamic acid (E). [1-D2] Y26G / Q40H Mutant AAR (Example 9) A base sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is glycine (G), the 33rd amino acid is phenylalanine (F), the 40th amino acid is histidine (H), and the 61st amino acid is glutamic acid (E). [1-D3] Y26G / Q40V Mutant AAR (Example 10) A base sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is glycine (G), the 33rd amino acid is phenylalanine (F), the 40th amino acid is valine (V), and the 61st amino acid is glutamic acid (E). [1-D4] Y26G / Q40Y Mutant AAR (Example 11) A base sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is glycine (G), the 33rd amino acid is phenylalanine (F), the 40th amino acid is tyrosine (Y), and the 61st amino acid is glutamic acid (E).

[0118] [1-E] Wild-Type SeAAR (Comparative Example 1) The base sequence represented by SEQ ID NO: 5. SEQ ID NO: 5 is a base sequence encoding an amino acid sequence in which the 11th amino acid in the amino acid sequence represented by SEQ ID NO: 1 is leucine (L), the 26th amino acid is tyrosine (Y), the 33rd amino acid is phenylalanine (F), the 40th amino acid is glutamine (Q), and the 61st amino acid is glutamic acid (E).

[0119] 2. Preparation of Transformants and Hydrocarbon (Alkane and / or Alkene) Biosynthesis Escherichia coli BL21(DE3) was used as the host cell. The cells were cultured in Luria-Bertani (LB) liquid medium, and the plasmid prepared as described above (basic plasmid (Comparative Example 1) or each test plasmid (Examples 1 to 11)) was transformed into Escherichia coli BL21(DE3). The obtained transformant was cultured at 37 °C for 16 hours on an LB agar medium containing 100 μg / mL sodium ampicillin. The obtained colonies were inoculated into an LB liquid medium (5 mL) containing 100 μg / mL sodium ampicillin and shaken overnight at 160 rpm and 37 °C. Next, the obtained medium (culture) was placed in a 250 mL flask containing 100 mL of modified M9 liquid medium (3 g / L KH2PO4, 6 g / L Na2HPO4, 3.4 g / L KCl, 0.5 g / L NaCl, 0.45 g / L CaCl2, 2 g / L NH4Cl, 0.25 g / L MgSO4, 20 g / L yeast extract, 10 mg / L thiamine, 20 g / L glucose and 100 μg / mL ampicillin sodium), and a trace-metal solution (containing 2 mg / L ZnCl2, 27 mg / L FeCl3·6H2O, 1.9 mg / L CuSO4·5H2O, 2 mg / L Na2MoO4·2H2O and 0.5 mg / L H3BO3) was added. The pH of the obtained medium (mixture) was adjusted to 7.2 with Tris buffer. The initial OD 600 was 0.02, and the OD 600 after shaking at 200 rpm and 37 °C was 0.5 - 0.8. Furthermore, Isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.12 mM) was added to the medium, and the mixture was shaken at 24 °C and 200 rpm for 24 hours to induce protein expression.

[0120] 3. Gas Chromatography-Mass Spectrometry (GC-MS) of Synthesized Hydrocarbons Ethyl acetate (500 μL) was added to the culture solution (1 mL) in which protein expression was induced in the above Procedure 2, and alkanes and alkenes were extracted. The extract was subjected to GC / MS analysis using a GCMS-QP5050A system (manufactured by Shimadzu Corporation). The oven temperature was maintained at 100°C for 5 minutes, then heated to 120°C at a rate of 20°C / min, then heated to 190°C at a rate of 7°C / min, and finally heated to 320°C at a rate of 30°C / min. The temperatures of the injector and detector were maintained at 260°C and 250°C, respectively, and the injection volume was 1 μL. Calibration curves were prepared using tridecane and pentadecane as alkane standards and heptadecene as an alkene standard. The regression equation was created by the external standard method.

[0121] <Results> 1. Results of Y26, Q40 or E61 Mutant AAR (Examples 1-7, Single-Site Mutations) Figure 8 shows the results of introducing single-site mutations into amino acid residues Y26, Q40, or E61. The Y26, Q40, or E61 mutant AARs are mutant AARs in which only one amino acid is substituted in the amino acid sequence encoding wild-type SeAAR.

[0122] When wild-type SeAAR (Comparative Example 1) was used, the total production amount of alkanes (tridecane and pentadecane) and alkenes (heptadecene) was 27.17 mg / L, and the production amount of alkanes (tridecane and pentadecane) among them was 1.68 mg / L.

[0123] In contrast, as shown in the left graph of Fig. 8, when the Y26G mutant AAR was used, the total production amount of alkanes and alkenes was 89.34 mg / L, which was approximately 3.29 times higher than that when the wild-type SeAAR was used. Also, as shown in the right graph of Fig. 9, when the Y26G mutant AAR was used, the production amount of alkanes (tridecane and pentadecane) among them was 70.93 mg / L, and the production amount of alkanes increased by approximately 42.2 times compared to the case when the wild-type SeAAR was used. Further, as shown in the right graph of Fig. 9, the production amount of alkanes accounted for approximately 6.2% of the total production amount of alkanes and alkenes when the wild-type SeAAR was used, whereas it was approximately 79.4% when the Y26G mutant AAR was used. In the latter case, the production ratio of alkanes in the total production amount of alkanes and alkenes increased significantly (by approximately 12.8 times). Thus, when the Y26G mutant AAR was used, the production efficiency of alkanes and / or alkenes could be improved.

[0124] In the case of the Y26A mutant AAR, as shown in the left graph of Fig. 8, the total production amount of alkanes and alkenes was 29.49 mg / L, and the total production amount of alkanes and alkenes increased by approximately 1.09 times compared to the case when the wild-type SeAAR was used. Also, in the Y26A mutant AAR, both the production ratio of alkanes in the total production amount of alkanes and alkenes and the production amount of alkanes increased. Thus, when the Y26A mutant AAR was used, the production efficiency of alkanes and / or alkenes could also be improved.

[0125] In the Y26M mutant AAR, as shown in the left graph of Figure 8, the total production amount of alkanes and alkenes was 14.02 mg / L. Compared with the case of using the wild-type SeAAR, the total production amount of alkanes and alkenes decreased, but the production ratio of alkanes in the total production amount and the production amount of alkanes increased compared with the case of using the wild-type SeAAR. Also in the case of using the Y26L mutant AAR, the total production amount of alkanes and alkenes was 10.12 mg / L. Compared with the case of using the wild-type SeAAR, it decreased, but the production ratio of alkanes in the total production amount and the production amount of alkanes increased compared with the case of using the wild-type SeAAR. From this, it was found that when using the Y26M mutant AAR or the Y26L mutant AAR, although an increase in the production amount of hydrocarbons was not observed compared with the case of using the wild-type SeAAR, hydrocarbon production with a high proportion of alkanes became possible, and also the production amount of alkanes was increased. Thus, when using the Y26M mutant AAR or the Y26L mutant AAR, the production efficiency of alkanes and / or alkenes could be improved.

[0126] Also, as shown in the middle graph of Figure 8, in the Q40Y mutant AAR, the total production amount of alkanes and alkenes was 77.76 mg / L. Compared with the case of using the wild-type SeAAR, the total production amount of alkanes and alkenes increased significantly. Also, the production amount of alkanes in the total production amount increased significantly. In the Q40V mutant AAR, the total production amount of alkanes and alkenes was 6.75 mg / L. Compared with the case of using the wild-type SeAAR, it decreased, but the production amount of alkanes in the total production amount of alkanes and alkenes increased compared with the case of using the wild-type SeAAR. From this, it was found that when using the Q40Y mutant AAR or the Q40V mutant AAR, the production efficiency of alkanes and / or alkenes could be improved compared with the case of using the wild-type SeAAR.

[0127] Also, as shown in the graph on the right side of FIG. 8, in the E61A mutant AAR, the total production amount of alkane and alkene is 13.31 mg / L. Compared with the case of using the wild-type SeAAR, the total production amount of alkane and alkene decreased. However, compared with the case of using the wild-type SeAAR, the production amount of alkane and the production amount of alkane in the total production amount of alkane and alkene increased. From this, it was found that even when the E61A mutant AAR is used, the production efficiency of alkane and / or alkene can be improved compared with the case of using the wild-type SeAAR.

[0128] 2. Results of Y26 and Q40 Mutant AAR (Examples 8-7, Double-Site Mutations) The graph on the left side of FIG. 9 shows the results of introducing double-site mutations at amino acid residues Y26 and Q40. The Y26 and Q40 mutant AAR is a mutant AAR in which two amino acids are substituted in the amino acid sequence encoding the wild-type SeAAR.

[0129] Since the production efficiency of alkane was most improved when the Y26G mutant AAR was used in the single-site mutation, a double-site mutation having the Y26G mutation and the mutation at Q40 was prepared, and the influence on the production of alkane was examined.

[0130] In the Y26G / Q40M mutant AAR, as shown in the left graph of Fig. 9, the total production of alkanes and alkenes increased to 167.3 mg / L. Among them, the production of alkanes was 107.77 mg / L (right graph of Fig. 9). Compared with the case of using the wild-type SeAAR, the total production of alkanes and alkenes increased by about 6.2 times, and the production of alkanes increased by about 64 times. In addition, the production of alkanes accounted for about 6.2% of the total production of alkanes and alkenes when using the wild-type SeAAR, while when using the Y26G / Q40M mutant AAR, it increased significantly to about 64.4%, which was about 10.4 times that when using SeAAR. When using each of the mutant AARs of Y26G / Q40H mutant AAR, Y26G / Q40V mutant AAR, and Y26G / Q40Y mutant AAR, compared with the case of using the wild-type SeAAR, the total production of alkanes and alkenes and the production of alkanes increased, and the production of alkanes accounted for in the total production of alkanes and alkenes also increased significantly.

[0131] From this, it was found that even when the double-site mutation at Y26 and Q40 was made, the production efficiency of alkanes and / or alkenes could be improved.

[0132] Test Example 2 <Test procedure> In the same way as in Test Example 1, mutant AAR was prepared, and the production of alkanes and / or alkenes was measured and calculated. The nucleotide sequence encoding the mutant AAR introduced into the test plasmid in Test Example 2 is the nucleotide sequence encoding the amino acid sequence of the following mutant AAR. Although the primers for mutants such as L11F are not described in this document, appropriate amplification was performed using commercially available primers in the same way as in Test Example 1.

[0133] [2-A] L11 Mutant AAR [2-A1] L11F Mutant AAR (Example 12) A base sequence encoding the amino acid sequence represented by SEQ ID NO:1, wherein the 11th amino acid of the amino acid sequence is phenylalanine (F), the 26th amino acid is tyrosine (Y), the 33rd amino acid is phenylalanine (F), the 40th amino acid is glutamine (Q), and the 61st amino acid is glutamic acid (E).

[0134] [2-B] Y26 Mutant AAR [2-B1] Y26I Mutant AAR (Example 13) A base sequence encoding the amino acid sequence represented by SEQ ID NO:1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is isoleucine (I), the 33rd amino acid is phenylalanine (F), the 40th amino acid is glutamine (Q), and the 61st amino acid is glutamic acid (E).

[0135] [2-C] F33 Mutant AAR [2-C1] F33M Mutant AAR (Example 14) A base sequence encoding the amino acid sequence represented by SEQ ID NO:1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is tyrosine (Y), the 33rd amino acid is methionine (M), the 40th amino acid is glutamine (Q), and the 61st amino acid is glutamic acid (E). [2-C2] F33V Mutant AAR (Example 15) A base sequence encoding the amino acid sequence represented by SEQ ID NO:1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is tyrosine (Y), the 33rd amino acid is valine (V), the 40th amino acid is glutamine (Q), and the 61st amino acid is glutamic acid (E). [2-C3] F33L Mutant AAR (Example 16) A base sequence encoding the amino acid sequence represented by SEQ ID NO:1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is tyrosine (Y), the 33rd amino acid is leucine (L), the 40th amino acid is glutamine (Q), and the 61st amino acid is glutamic acid (E).

[0136] [2-D] Q40 Mutant AAR [2-D1] Q40R Mutant AAR (Example 17) A base sequence encoding the amino acid sequence represented by SEQ ID NO:1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is tyrosine (Y), the 33rd amino acid is phenylalanine (F), the 40th amino acid is arginine (R), and the 61st amino acid is glutamic acid (E). [2-D2] Q40E Mutant AAR (Example 18) A base sequence encoding the amino acid sequence represented by SEQ ID NO:1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is tyrosine (Y), the 33rd amino acid is phenylalanine (F), the 40th amino acid is glutamic acid (E), and the 61st amino acid is glutamic acid (E). [2-D3] Q40K Mutant AAR (Example 19) A base sequence encoding the amino acid sequence represented by SEQ ID NO:1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is tyrosine (Y), the 33rd amino acid is phenylalanine (F), the 40th amino acid is lysine (K), and the 61st amino acid is glutamic acid (E).

[0137] [2-E]Y26 / F33 / Q40 Variant AAR [2-E1]Y26G / F33G / Q40M Variant AAR (Example 20) A base sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the 11th amino acid of the amino acid sequence is leucine (L), the 26th amino acid is glycine (G), the 33rd amino acid is glycine (G), the 40th amino acid is methionine (M), and the 61st amino acid is glutamic acid (E).

[0138] <Result> The results are shown in Fig. 10. As can be understood from Fig. 10, in any of the mutant AARs of Examples 12 to 20, compared with the case of using the wild-type SeAAR, the total production amount of alkane (tridecane and pentadecane) and alkene (heptadecene) increased, the production ratio of alkane in the total production amount increased, and the production amount of alkane also increased. From this, it was found that the production efficiency of alkane and / or alkene can also be improved by these mutant AARs.

[0139] Test Example 3 In Test Examples 1 and 2, it was found that the production efficiency of alkane and / or alkene can be improved by amino acid mutations in AAR. The present inventors conducted various studies on AAR, and among them, noticed that the substrate tunnel formed by AAR affects the production efficiency of alkane and / or alkene. Below, it is shown that the cross-sectional area of the substrate tunnel of the AAR is expanded in the mutant AAR in which the production of alkane and / or alkene is improved.

[0140] <Test Procedure> · Determination of the AAR and tunnel cross-sectional area used In this test example, the tunnel cross-sectional area of the wild-type AAR was compared with that of three AAR mutants. In this test example, the same wild-type SeAAR as in Test Example 1 was used as the wild-type AAR. As the three AAR mutants, the Y26G mutant AAR, Q40Y mutant AAR, and Y26G / Q40M mutant AAR prepared in Test Example 1 were used.

[0141] The tunnel structures of wild-type SeAAR and AAR mutants were determined using the known software CAVER Analyst 2.0 (BETA 2) ver.2.0, HCI Laboratory, Faculty of Informatics, Masaryk University). The probe radius was set to 0.9 Å, and the default settings were adopted, starting from the amino acid residues C294, Y59, and V17 of each AAR (the amino acid positions correspond to SEQ ID NO: 1). Using MGLTools 1.5.7, all water molecules were removed, hydrogen atoms were added, and according to CAVER Analyst 2.0, the tunnel profiles (lower part of Figure 11) and tunnel cross-sectional views (upper part of Figure 11) of wild-type SeAAR and AAR mutants were obtained.

[0142] In the tunnel analysis, the structural information of each AAR predicted by Alphafold2 (DeepMind) was used. Alphafold2 is a well-known software in this field that can predict the three-dimensional structure of proteins from amino acid sequences. The said tunnel profile (lower part of Figure 11) was created by performing molecular docking with hexadecanal (C 16 H 32 O) as the substrate and the amino acid residue C294 of AAR as the active site. In this test example, the docking pose with the lowest free energy was selected.

[0143] In selecting the docking pose with the lowest free energy, a molecular docking simulation was performed according to a conventionally known procedure. The crystal structure of wild-type SeAAR was obtained from the Protein Data Bank (PDB ID: 6JZU). In the simulation, the resolution of the crystal structure was 2.18 Å. Hexadecanal was used as the ligand for wild-type SeAAR and the AAR variant. The structure of hexadecanal was obtained from PubChem (Kim, S. et al., PubChem in 2021: new data content and improved web interfaces. Nucleic Acids Research. 2021, 49, 1388-1395). Minimization of the ligand energy was performed using the MM2 calculation function of Chem3D (ver.4.0, CambridgeSoft, https: / / www.hulinks.co.jp / software / chem / chembio / section_01 / chem3d). AutoDock 4.2 (https: / / autodock.scripps.edu / ) was used for the molecular docking simulation. Before docking the protein and the ligand, the protein was processed and protonated using the H++ server, all water molecules were removed, and hydrogen atoms were added. Residues C294, Y59, and V17 were defined as flexible residues, and the Lamarckian Genetic Algorithm (LGA) method was used for the protein-ligand docking simulation. The interaction energy between the ligand and the receptor was calculated using MGLTools 1.5.7. In this way, the docking pose with the lowest free energy was selected.

[0144] The tunnel cross-sectional area was determined as follows. The 26th amino acid Y (meaning that the 26th X in SEQ ID NO: 1 is Y, and hereinafter, the amino acid positions follow SEQ ID NO: 1 in the same manner), the 40th amino acid Q, the 23rd amino acid Y, and the 43rd amino acid V of the wild-type SeAAR were connected, and the narrowest substrate tunnel region among the planes crossing the inside of the substrate tunnel was specified, and a substrate tunnel cross-sectional view was created. The substrate tunnel region was used as the substrate tunnel cross-section. The obtained substrate tunnel cross-sectional view is shown in the upper part a of FIG. 11. The area of the largest circle (a perfect circle) that fits within the substrate tunnel cross-section in the upper part a of FIG. 11 was determined, and this was used as the substrate tunnel cross-sectional area of the wild-type SeAAR.

[0145] The tunnel cross-sectional area of the Y26G mutant AAR was determined as follows. The 26th amino acid G, the 40th amino acid Q, the 23rd amino acid Y, and the 43rd amino acid V of the Y26G mutant AAR were connected, and the narrowest substrate tunnel region among the planes crossing the inside of the substrate tunnel was specified, and a substrate tunnel cross-sectional view was created. The substrate tunnel region was used as the substrate tunnel cross-section. The obtained substrate tunnel cross-sectional view is shown in the upper part b of FIG. 11. The area of the largest circle that fits within the substrate tunnel cross-section in the upper part b of FIG. 11 was determined, and this was used as the substrate tunnel cross-sectional area of the Y26G mutant AAR.

[0146] The tunnel cross-sectional area of the Q40Y mutant AAR was determined as follows. The 26th amino acid Y, the 40th amino acid Y, the 23rd amino acid Y, and the 43rd amino acid V of the Q40Y mutant AAR were connected, and the narrowest substrate tunnel region among the planes crossing the inside of the substrate tunnel was specified, and a substrate tunnel cross-sectional view was created. The substrate tunnel region was used as the substrate tunnel cross-section. The obtained substrate tunnel cross-sectional view is shown in the upper part c of FIG. 11. The area of the largest circle that fits within the substrate tunnel cross-section in the upper part c of FIG. 11 was determined, and this was used as the substrate tunnel cross-sectional area of the Q40Y mutant AAR.

[0147] The determination of the tunnel cross-sectional area of the Y26G / Q40M mutant AAR was performed as follows. The 26th amino acid G, the 40th amino acid M, and the 23rd amino acid Y that constitute the Y26G / Q40M mutant AAR were connected, and the narrowest substrate tunnel region among the planes crossing the inside of the substrate tunnel was specified, and a substrate tunnel cross-sectional view was created. The obtained substrate tunnel cross-sectional view is shown in the upper part d of FIG. 11. The area of the largest circle that fits into the substrate tunnel cross-section in the upper part d of FIG. 11 was determined, and this was taken as the substrate tunnel cross-sectional area of the Y26G / Q40M mutant AAR.

[0148] The substrate tunnel cross-sectional areas thus obtained were compared.

[0149] · Evaluation of improvement in alkane and / or alkene production efficiency In the same manner as in Test Example 1, the alkane and / or alkene production efficiencies of the wild-type strain SeAAR, Y26G mutant AAR, Q40Y mutant AAR, and Y26G / Q40M mutant AAR were determined.

[0150] <Results> The results are shown in FIGS. 11 and 13. In FIG. 11, a shows the results of the wild-type strain SeAAR, b shows the results of the Y26G mutant AAR, c shows the results of the Q40Y mutant AAR, and d shows the results of the Y26G / Q40M mutant AAR. The upper part shows the substrate tunnel cross-sectional view, and the lower part shows the substrate tunnel profile. As illustrated in FIG. 12, the area of the largest circle (a perfect circle) that fits into the substrate tunnel cross-section of the cross-sectional view was taken as the substrate tunnel cross-section. FIG. 13 shows the values of the radius and cross-sectional area of each cross-sectional view calculated according to the CAVER Analyst 2.0.

[0151] As can be understood from the upper part of FIG. 11 and FIG. 13, the substrate tunnel cross-sectional areas of the three mutant AARs were expanded compared to the substrate tunnel cross-sectional area of the wild-type AAR. Further, as a result of performing alkane and alkene production using these AARs, as shown in Test Example 1, the production efficiencies of alkanes and / or alkenes were improved in the three mutant AARs compared to the case of using the wild-type AAR.

[0152] The present inventors have found from these results that it can be said that there is a correlation between the expansion of the substrate tunnel cross-sectional area and the improvement of alkane and / or alkene production efficiency. From this, the expansion of the substrate tunnel cross-sectional area can be a useful index in determining mutant candidates useful for improving alkane and / or alkene production efficiency, and thus, it was confirmed that it can be useful for more easily screening AAR mutant candidates useful for improving alkane and / or alkene production efficiency.

[0153] In order to further investigate the reasons why the production efficiency of alkanes and / or alkenes is improved by the mutant AAR (particularly the reasons for the improvement in the production ratio of alkanes and / or the production amount of alkenes), the inventors conducted various analyses. Electrostatic complementarity is one of the parameters for evaluating the selectivity and affinity of proteins for ligands. The inventors created and compared electrostatic potential (ESP) surfaces according to known procedures (Figure 14). The ESP surface is created by moving a probe with a specific charge on the molecular surface, the ESP energy at each point on the molecular surface is measured, and that point is colored according to the ESP energy. Figure 14 is a diagram in which the molecular structures of hexadecanal and cis-9-octadecenaal were optimized at the B3LYP / 6-311G(d) level using Gaussian 16 software. The ESP surface of wild-type SeAAR (Figure 14) was created using PyMol, and the ESP surfaces of hexadecanal and cis-9-octadecenaal (upper right in Figure 14) were created using Multiwfn 3.6. The action of AAR produces the corresponding aldehydes (such as cis-9-octadecenaal and hexadecanal) from acyl-ACP. From Figure 14, it can be speculated that the shape of the tunnel affects the substrate specificity of AAR. In particular, the substrate tunnel of wild-type SeAAR is curved, which is speculated to be more suitable for cis-9-octadecenaal with a bent shape than for hexadecanal with a linear shape. One reason for this is, for example, that the bulky structure of the side chain of the Y26 residue suppresses the binding of linear hexadecanal to the active site (left and lower right in Figure 14). From this, it was predicted that it is difficult for linear hexadecanal to be supplied to ADO, and as a result, the production efficiency of alkanes is low in wild-type SeAAR.

[0154] In the aforementioned tunnel analysis, it was found that the substrate tunnels of the Y26G mutant AAR, Q40Y mutant AAR, and Y26G / Q40M mutant AAR (b, c, d in Fig. 11) were enlarged, especially near the exits of the respective substrate tunnels (near Y26G and Q40M). Considering in light of Fig. 14, in the AAR mutations capable of expanding the tunnel cross-sectional area, the supply of hexadecanal to ADO, which was hindered in the wild-type SeAAR, was promoted, suggesting that this improved the production efficiency of alkanes and / or alkenes (particularly the production ratio of alkanes and / or the production amount of alkenes).

[0155] Test Example 4 In this test example, the restriction enzyme and NEBuilder were obtained from New England Biolabs Japan, Ltd. The primers shown in Table 2 were obtained from Integrated DNA Technologies (Singapore). The enzyme for PCR was obtained from Toyobo Co., Ltd. The kits for plasmid separation and gel purification were obtained from Nippon Genetics Co., Ltd. The calibration standards for alkanes (C 13 H 28 、C 15 H 32 ) and alkenes (including C 17 H 34 ) were obtained from Nacalai Tesque, Inc. and Tokyo Chemical Industry Co., Ltd., respectively. The plasmid pET-21b(+) was obtained from Novagen (Germany).

[0156]

Table 2

[0157] <Test procedure> 1. Construction of plasmids introduced with polynucleotides of variant AAR In the same manner as in Test Example 1, PCR amplification and the like were performed to clone the nucleotide sequence encoding AAR derived from Synechococcus elongatus PCC 7942 = FACHB-805 (hereinafter, wild-type SeAAR) and the nucleotide sequence encoding NpADO (ADO derived from Nostoc punctiforme PCC 73102) into the plasmid pET-21b(+), respectively, to prepare plasmid pET7SeAAR and plasmid pET7NpADO. In the same manner as in Test Example 1, the nucleotide sequence represented by SEQ ID NO: 5 was used as the nucleotide sequence encoding wild-type SeAAR, and the nucleotide sequence represented by SEQ ID NO: 6 was used as the nucleotide sequence encoding NpADO. Next, in the same manner as in Test Example 1, plasmid pET7SeAAR-T7NpADO (basic plasmid) was prepared. Also, in the same manner as in Test Example 1, a plasmid (test plasmid) into which a nucleotide sequence encoding a mutant AAR was introduced instead of the nucleotide sequence encoding wild-type SeAAR was prepared. A model diagram of plasmid pET7SeAAR-T7NpADO constructed in Test Example 4 is shown in FIG. 15.

[0158] The nucleotide sequence encoding the mutant AAR is the nucleotide sequence encoding the following amino acid sequence.

[0159] Example 21: SeAAR-Del3 (Del in Fig. 16) 3 ) An amino acid sequence in which three amino acids are deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 22: SeAAR-Del4 (Del in Figs. 15 and 16) 4 ) An amino acid sequence in which four amino acids are deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35.

[0160] Example 23: SeAAR-E1 (E in Figs. 15 and 16) 1 ) A nucleotide sequence encoding an amino acid sequence in which one glutamic acid is directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 24: SeAAR-E2 (E in Figs. 15 and 16) 2 ) A base sequence encoding an amino acid sequence of 2 amino acids in length consisting of glutamic acid and directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 25: SeAAR-E3 (E in Figs. 15 and 16) 3 ) A base sequence encoding an amino acid sequence of 3 amino acids in length consisting of glutamic acid and directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 26: SeAAR-E4 (E in Figs. 15 and 16) 4 ) A base sequence encoding an amino acid sequence of 4 amino acids in length consisting of glutamic acid and directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 27: SeAAR-E6 (E in Figs. 15 and 16) 6 ) A base sequence encoding an amino acid sequence of 6 amino acids in length consisting of glutamic acid and directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 28: SeAAR-E9 (E in Figs. 15 and 16) 9 ) A base sequence encoding an amino acid sequence of 9 amino acids in length consisting of glutamic acid and directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35.

[0161] Example 29: SeAAR-D1 (D in Figs. 15 and 16) 1 ) A base sequence encoding an amino acid sequence in which 1 aspartic acid is directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 30: SeAAR-D2 (D in Figs. 15 and 16) 2 ) A base sequence encoding an amino acid sequence of 2 amino acids in length consisting of aspartic acid and directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 31: SeAAR-D3 (D in Figs. 15 and 16) 3 ) A base sequence encoding an amino acid sequence of 3 amino acids in length consisting of aspartic acid and directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 32: SeAAR-D4 (D in Figs. 15 and 16) 4 ) A base sequence encoding an amino acid sequence 4 amino acids in length consisting of aspartic acid, directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 33: SeAAR-D56 (D in Figs. 15 and 16) 6 ) A base sequence encoding an amino acid sequence 6 amino acids in length consisting of aspartic acid, directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 34: SeAAR-D9 (D in Figs. 15 and 16) 9 ) A base sequence encoding an amino acid sequence 9 amino acids in length consisting of aspartic acid, directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35.

[0162] Example 35: SeAAR-E1D3 (D in Figs. 15 and 16) 1 D 3 ) A base sequence encoding an amino acid sequence in which 1 aspartic acid and 3 glutamic acids are consecutive in order from the N-terminus (an amino acid sequence 4 amino acids in length consisting of glutamic acid and aspartic acid), directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Example 36: SeAAR-E2D2 (D in Figs. 15 and 16) 2 D 2 ) A base sequence encoding an amino acid sequence in which 2 aspartic acids and 2 glutamic acids are consecutive in order from the N-terminus (an amino acid sequence 4 amino acids in length consisting of glutamic acid and aspartic acid), directly linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35. Comparative Example 2 (WT in Figs. 15 and 16) A base sequence encoding the amino acid sequence represented by SEQ ID NO: 35 (the base sequence represented by SEQ ID NO: 5).

[0163] 2. Preparation of transformants and hydrocarbon (alkane and / or alkene) biosynthesis Escherichia coli BL21(DE3) was used as the host cell. The cells were cultured in Luria-Bertani (LB) liquid medium, and the plasmids prepared as described above (each test plasmid (Examples 21 to 31 and Comparative Examples 2 to 4)) were transformed into Escherichia coli BL21(DE3). The obtained transformants were cultured at 37 °C for 16 hours on an LB agar medium containing 100 μg / mL sodium ampicillin. The obtained colonies were inoculated into an LB liquid medium (5 mL) containing 100 μg / mL sodium ampicillin and shaken overnight at 160 rpm and 37 °C. Then, the obtained medium (culture) was cultured in 100 mL of modified M9 liquid medium (containing 3 g / L KH2PO4, 6 g / L Na2HPO4, 3.4 g / L KCl, 0.5 g / L NaCl, 0.45 g / L CaCl2, 2 g / L NH4Cl, 0.25 g / L MgSO4, 20 g / L yeast extract, 10 mg / L thiamine, 20 g / L glucose and 100 μg / mL ampicillin sodium), and a trace-metal solution (containing 2 mg / L ZnCl2, 27 mg / L FeCl3·6H2O, 1.9 mg / L CuSO4·5H2O, 2 mg / L Na2MoO4·2H2O, and 0.5 mg / L H3BO3) was added. The pH of the obtained medium (mixture) was adjusted to 7.2 with Tris buffer. The initial OD 600 of the medium was 0.02, and the OD 600 after shaking at 200 rpm and 37 °C was 0.6 - 0.7. Furthermore, IPTG (0.12 mM) was added to the medium, and the mixture was shaken at 24 °C and 200 rpm for 24 hours to induce protein expression. After the culture, the culture was collected, and the alkane and alkene production amounts were evaluated for wild-type SeAAR and mutant SeAAR.

[0164] 3. Gas chromatography-mass spectrometry (GC-MS) of the synthesized hydrocarbons The amounts of the produced alkanes and alkenes were quantified by GC-MS analysis. Specifically, ethyl acetate (750 μL) was added to the culture solution (750 μL) in which protein expression was induced in the above-mentioned procedure 2 to extract alkanes and alkenes. The extract was subjected to GC / MS analysis under the same conditions as the GC-MS analysis in Test Example 1 using a GCMS-QP5050A GC-MS system. Similar to Test Example 1, tridecane and pentadecane were used as alkane standards, and heptadecene was used as an alkene standard to identify and quantify alkanes and alkenes.

[0165] <Results> The results are shown in Table 3 and Figure 16. Figure 16 was created based on Table 3.

[0166]

Table 3

[0167] As shown in Table 3 and Figure 16, the production amounts of alkanes and / or alkenes increased in the mutant SeAARs of Examples 21 to 36 as compared with the wild-type SeAAR (WT, Comparative Example 2). For example, the total production amount (112.71 mg / L) of alkanes and alkenes in SeAAR-Del3 of Example 21, which is an example of the mutant AAR, increased by about 4.5 times the total production amount (25.37 mg / L) of alkanes and alkenes in the wild-type SeAAR (WT) of Comparative Example 2. Also, for example, the total production amount (159.25 mg / L) of alkanes and alkenes in SeAAR-E3 of Example 25, which is an example of the mutant AAR, increased by about 6.3 times the total production amount (25.37 mg / L) of alkanes and alkenes in the wild-type SeAAR (WT) of Comparative Example 2. Also, for example, the total production amount (139.38 mg / L) of alkanes and alkenes in SeAAR-D4 of Example 32, which is an example of the mutant AAR, increased by about 5.5 times the total production amount (25.37 mg / L) of alkanes and alkenes in the wild-type SeAAR (WT) of Comparative Example 2.

[0168] From Table 3 and FIG. 16, it was found that in the mutant AARs of Examples 21 to 36, alkanes or alkenes can be produced more efficiently. Such an improvement in production efficiency leads to an improvement in the production efficiency of biofuels.

[0169] Also, although not shown in the results, four amino acids were deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35, and a 4-amino acid-long amino acid sequence consisting of glutamic acid or a 4-amino acid-long amino acid sequence consisting of aspartic acid was directly linked to the C-terminus of the amino acid sequence after deletion to construct mutant AARs (SeAAR-del4-E4, SeAAR-del4-D4) and the same tests were conducted. As a result, in SeAAR-del4-E4 and SeAAR-del4-D4 as well, the production amount of alkanes or alkenes increased as compared with wild-type SeAAR. In addition, the production amounts of alkanes and alkenes were further improved in the mutant AARs (SeAAR-E4 and SeAAR-D4) of Examples 26 and 32 as compared with SeAAR-del4-E4 and SeAAR-del4-D4. Considering this, when linking an amino acid sequence consisting of glutamic acid and / or aspartic acid to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35, it is suggested that it is more preferable in terms of improving the production efficiency of alkanes and / or alkenes when linking the amino acid sequence consisting of glutamic acid and / or aspartic acid without deleting four or more amino acids from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35.

[0170] Furthermore, the solubility and stability of some of the mutant AARs obtained as described above were confirmed. Solubility was evaluated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Specifically, each plasmid was transformed into E. coli in the same manner as described above. After culturing for 24 hours, the sample with the lowest OD600 value (wild-type SeAAR in this test) was selected, and the OD600 values of all mutant AAR cultures were also adjusted. Then, 3 mL of the culture solution of each sample was centrifuged to collect the cells, which were resuspended in 20 mM Tris-HCl buffer. The cells were disrupted by sonication at 200 W for 40 minutes with a 30-second ON / OFF cycle on an ice water bath. Next, the disrupted cells were centrifuged at 15,000 rpm at 4°C for 10 minutes to separate them into a soluble fraction and an insoluble fraction. For both fractions, the protein concentration was measured according to the Bradford method and denatured at 95°C for 5 minutes in 20 mM Tris-HCl buffer containing 2% SDS and 6% 2-mercaptoethanol. After applying 20 μg of the denatured solution and the protein standard to the gel, the gel was stained with Coomassie Brilliant Blue R-250 to visualize the proteins. As a result, an increase in the amount of protein in the soluble fraction was observed in the mutant AARs (especially SeAAR-E3 and SeAAR-E4) compared with wild-type SeAAR, and thus it was confirmed that the solubility was improved in the mutant AARs.

[0171] The stability was evaluated by differential scanning fluorimetry (DSF) using a CFX96 touch real-time PCR detection system (Bio-Rad, USA) after purifying the wild-type SeAAR and the mutant AAR (amino acid sequence-linked mutant AAR consisting of glutamic acid) expressed as described above. An assay solution (25 μL) containing wild-type SeAAR or mutant AAR (10 μL; 20 μM), 50× SYPRO Orange dye (2.5 μL; Invitrogen, USA), and potassium phosphate buffer (12.5 μL; pH 7.2) was prepared. Also, a control solution containing Orange dye and potassium phosphate buffer was prepared and used for background correction. Each solution was placed in a 96-well plate (Bio-Rad, USA) and heated from 20°C to 90°C at a rate of 0.5°C every 5 seconds. Fluorescence intensity was measured at 0.5°C intervals using the FRET scan mode. As a result, the thermal stability was improved in the mutant AAR compared to the wild-type SeAAR, and the thermal stability was most improved in SeAAR-E3.

[0172] In protein expression using host cells such as Escherichia coli, inclusion body formation reduces protein production efficiency. Therefore, improving solubility, i.e., increasing the amount of protein in the solubilized fraction, is important for efficiently producing the desired protein. In addition, the stability of proteins, particularly the thermal stability of enzymes, is important for efficiently causing enzyme reactions. From the results of this test example, it was confirmed that the mutant AAR is useful for increasing the production efficiency of the desired protein (enzyme) and for improving the stability of the desired protein. In addition, such an improvement in production efficiency and / or stability leads to an improvement in the production efficiency of alkanes and / or alkenes by the mutant AAR, and ultimately leads to an improvement in the production efficiency of biofuels.

Claims

1. A mutant acyl-ACP reductase shown in the following [1] to [3] ［１］ A mutant acyl-ACP reductase having an amino acid sequence shown in the following (1) or (2): (1) An amino acid sequence consisting of the amino acid sequence represented by SEQ ID NO: 1 and having at least one requirement selected from the group consisting of (A) to (E) (A) The 11th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than leucine. (B) The 26th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than tyrosine. (C) The 33rd amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than phenylalanine. (D) The 40th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than glutamine. (E) The 61st amino acid of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than glutamic acid. (2) An amino acid sequence in which one or more amino acids other than the 11th, 26th, 33rd, 40th, and 61st amino acids in the amino acid sequence of (1) are deleted, substituted, inserted, or added ［２］ A mutant acyl-ACP reductase having an amino acid sequence shown in the following (i) or (ii): (i) An amino acid sequence in which 1 to 4 amino acids from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 are deleted (ii) An amino acid sequence in which one or more amino acids are deleted (except for the deletion of the first amino acid from the C-terminus of the amino acid sequence of (i)), substituted, inserted, or added in the amino acid sequence of (i) ［３］ A mutant acyl-ACP reductase having an amino acid sequence shown in the following (a) or (b): (a) An amino acid sequence having a length of 1 to 12 amino acids consisting of glutamic acid and / or aspartic acid is linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 (b) An amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 35 in the amino acid sequence of (a).

2. Comprising at least one requirement selected from the group consisting of (A) to (E), wherein the 11th amino acid in (A) is phenylalanine, and the 26th amino acid in (B) is glycine, isoleucine, alanine, methionine, or leucine In (C), the 33rd amino acid is glycine, leucine, valine, or methionine, In (D), the 40th amino acid is lysine, tyrosine, valine, histidine, methionine, arginine, or glutamic acid, In (E), the 61st amino acid is alanine, The mutant acyl-ACP reductase according to claim 1.

3. Comprising at least one requirement selected from the group consisting of (B), and (C) and (D), In (B), the 26th amino acid is glycine, In (C), the 33rd amino acid is glycine, In (D), the 40th amino acid is lysine, tyrosine, valine, histidine, or methionine, The mutant acyl-ACP reductase according to claim 1.

4. In (i), it is an amino acid sequence in which 3 or 4 amino acids are deleted from the C-terminus of the amino acid sequence represented by SEQ ID NO: 35, The mutant acyl-ACP reductase according to claim 1.

5. In (a), the amino acid sequence linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 is an amino acid sequence 2 to 10 amino acids in length consisting of glutamic acid, The mutant acyl-ACP reductase according to claim 1.

6. In (a), the amino acid sequence linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 is an amino acid sequence 3 to 9 amino acids in length consisting of aspartic acid, The mutant acyl-ACP reductase according to claim 1.

7. In (a), the amino acid sequence linked to the C-terminus of the amino acid sequence represented by SEQ ID NO: 35 is an amino acid sequence 3 to 6 amino acids in length consisting of glutamic acid and aspartic acid, The mutant acyl-ACP reductase according to claim 1.

8. A polynucleotide encoding the mutant acyl-ACP reductase according to any one of claims 1 to 7.

9. A vector containing the polynucleotide according to claim 8.

10. Furthermore, the vector according to claim 9, which contains a polynucleotide encoding aldehyde deformylating oxygenase.

11. A cell transformed with the vector according to claim 9.

12. A method for producing at least one selected from the group consisting of alkanes and alkenes using the mutant acyl-ACP reductase according to claim 1 in the presence of aldehyde deformylating oxygenase.

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