Secretion signal peptide gene derived from bacterium of genus hydrogenophilus

Abstract

The DNA (a), (b) or (c) mentioned below is a novel secretion signal peptide gene which can be used for the secretion / production of proteins by microorganisms. (a) DNA which encodes a peptide comprising an amino acid sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, or 151; (b) DNA which encodes a peptide comprising an amino acid sequence having 90% or higher identity with SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, or 151 and capable of acting as a secretion signal peptide; and (c) DNA which encodes a peptide comprising an amino acid sequence having a structure such that one to five amino acid resides are deleted, substituted, inserted, or added in an amino acid sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, or 151 and capable of acting as a secretion signal peptide.

Description

Secretory signal peptide gene from Hydrogenophilus bacteria

[0001] The present invention relates to a novel secretory signal peptide gene, a transformant or recombinant cell into which this secretory signal peptide gene has been introduced, and a method for producing a target protein using this transformant or recombinant cell.

[0002] Traditionally, various hosts such as Escherichia coli, yeast, and Bacillus subtilis have been used for microbial protein production using genetic engineering technology. Protein production using these hosts relies on sugars derived from biomass as the carbon source. The use of biomass has drawbacks, including high costs due to the complex process required to convert biomass into sugars, and the environmental impact of its industrial use.

[0003] The Paris Agreement, adopted in 2015, calls for rapid reductions in global greenhouse gas emissions. In accordance with this agreement, Japan aims to reduce its greenhouse gas emissions, including carbon dioxide and methane, by 46% by 2030 compared to 2013 levels.

[0004] As part of research into decarbonization, gases such as carbon dioxide, methane, and carbon monoxide are attracting attention as carbon feedstocks with a higher degree of sustainability, and there is growing interest in technologies for producing valuable materials using microorganisms that utilize these gases. Among these, there are high hopes for fixing and effectively utilizing carbon dioxide, which contributes significantly to global warming. Fixing carbon dioxide to produce materials does not have the disadvantages of using sugars derived from biomass as a carbon feedstock.

[0005] Hydrogen-oxidizing bacteria (also known as "hydrogen bacteria") can grow using carbon dioxide as the sole carbon source, utilizing the chemical energy generated by the reaction of hydrogen and oxygen. This allows them to produce chemical products using a mixture of oxygen, hydrogen, and carbon dioxide as raw materials, allowing for efficient carbon dioxide organic conversion. Furthermore, they can be cultured in simple media. While hydrogen bacteria generally grow slowly, the growth rate of Hydrogenophilus bacteria is remarkably fast. The Mitsubishi Research Institute Bulletin No. 34, 1999, evaluated Hydrogenophilus bacteria, stating, "Their growth rate is so high that it cannot be compared to the carbon dioxide fixation capacity of plants, clearly demonstrating the high carbon dioxide fixation capacity of microorganisms." Thus, Hydrogenophilus bacteria possess particularly excellent carbon dioxide fixation capabilities among organisms capable of carbon dioxide fixation, and there is a demand for using Hydrogenophilus bacteria as hosts for substance production.

[0006] In protein production by microorganisms, secretory production of proteins has advantages such as making the purification of the produced proteins very easy, and therefore many efforts have been made to have microorganisms secrete and produce proteins. To date, techniques for secreting and producing proteins in bacteria of the genus Bacillus (Non-Patent Document 1), the methanol-utilizing yeast Pichia pastoris (Non-Patent Document 2), the coryneform bacterium Corynebacterium glutamicum (Patent Document 1), and filamentous fungi of the genus Aspergillus (Non-Patent Document 3) have been reported, but no techniques for secreting and producing proteins in hydrogen-producing bacteria, including bacteria of the genus Hydrogenophilus, have been reported.

[0007] For a protein to be secreted outside the cell, it must first pass through the cell membrane via a membrane permeabilization mechanism (translocon) on the cell membrane. The secretory signal peptide at the N-terminus of the protein plays a crucial role in the translocon's recognition of the secretory protein. Microbial secretory signal peptides are generally approximately 20 amino acids long, with many basic amino acids near the N-terminus followed by a series of hydrophobic amino acids. Finally, this secretory signal peptide is cleaved by a signal peptidase on the cell membrane. The cleavage site, the C-terminus, typically contains small amino acids. Membrane passage can occur in conjunction with protein synthesis on ribosomes bound to the translocon, or after precursor protein synthesis in the cytoplasm, it passes into the periplasm via the translocon with the addition of the Sec complex. Thus, numerous proteins are involved in the secretion of a single protein. The structure of the secretory signal peptide significantly influences the secretion efficiency of a secretory protein, in part by determining the secretory pathway it chooses.

[0008] U.S. Patent No. 4,965,197

[0009] Microbiology Reviews, 57,109-137(1993)Bio / technology, 11, 905-910(1993)Bio / technology, 6, 1419-1422(1988)Journal of Molecular Biology, 362, 393-402(2006)

[0010] An objective of the present invention is to provide a novel secretory signal peptide gene that can be used for the secretory production of proteins by microorganisms, particularly, a novel secretory signal peptide gene that can be used for the secretory production of proteins by bacteria of the genus Hydrogenophilus.

[0011] As described in the Examples section, the present inventors analyzed proteins contained in the culture supernatant of Hydrogenophilus thermorteolus, and based on the data, performed in silico searches and function predictions. From the proteins, proteins having secretory signal sequences at the N-terminus were selected, and as a result, a large number of secretory signal peptides encoded in the genome of Hydrogenophilus thermorteolus were discovered.

[0012] The present invention was completed based on the above findings and provides the following [1] to

[11] : [1] A DNA of the following (a), (b), or (c): (a) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, (b) DNA encoding a peptide containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, (c) DNA encoding a peptide that contains an amino acid sequence having 90% or more identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and functions as a secretory signal peptide. 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,[2] DNA encoding a peptide that contains an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, or 151, and that functions as a secretory signal peptide. [3] DNA according to [1], which is the DNA of (d) or (e) below: (d) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, (e) DNA containing the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,or 152, and encoding a peptide that functions as a secretory signal peptide. [3] A secretory signal peptide gene comprising the DNA of [1] or [2]. [4] A vector comprising the DNA of [1] or [2]. [5] A vector for expressing a target protein, comprising a target protein gene downstream of the DNA of [1] or [2]. [6] A transformant obtained by introducing into a host microorganism a DNA fragment comprising the DNA of [1] or [2] and the structural gene of the target protein located downstream thereof, or the vector of [5]. [7] The transformant of [6], wherein the host microorganism is a bacterium of the genus Hydrogenophilus. [8] A method for producing a target protein, comprising the steps of culturing the transformant of [7] using carbon dioxide as substantially the only carbon source and recovering the protein from the medium. [9] A genetic recombinant in which the DNA of [1] or [2] has been introduced upstream of the structural gene of the target protein on the genome of a microorganism.

[10] The genetic recombinant according to [9], wherein the microorganism is a bacterium of the genus Hydrogenophilus.

[11] A method for producing a target protein, comprising culturing the genetic recombinant according to

[10] using carbon dioxide as substantially the only carbon source, and recovering the protein from the medium.

[0013] Measures to curb the increase in carbon dioxide include reducing carbon dioxide emissions and fixing emitted carbon dioxide. To reduce carbon dioxide emissions, solar, wind, geothermal, and other energy sources are being used in place of fossil fuels. However, in reality, even these energy sources have not been able to sufficiently curb the increase in carbon dioxide. Therefore, it is necessary to promote the fixation or resource recovery of emitted carbon dioxide. Carbon dioxide can be fixed physically or chemically, but if it is fixed using living organisms, it can be used to produce organic matter that can be used as food, feed, fuel, and other raw materials for chemical products. In other words, carbon dioxide itself can be directly converted into a valuable resource. This can solve both the two problems of global warming caused by increased carbon dioxide and the difficulty in securing food, feed, fuel, and raw materials for chemical products.

[0014] Hydrogen bacteria, which can grow using carbon dioxide as their sole carbon source, can use the chemical energy generated by the reaction between hydrogen and oxygen to grow on a mixture of oxygen, hydrogen, and carbon dioxide as their raw material, so they can efficiently organicize carbon dioxide and can be cultivated in a simple medium. While hydrogen bacteria generally grow slowly, Hydrogenophilus bacteria grow at a significantly faster rate. The Mitsubishi Research Institute Bulletin No. 34 1999 evaluated Hydrogenophilus bacteria, saying, "Their growth rate is so high that it cannot be compared to the carbon dioxide fixation ability of plants, and clearly demonstrates the high carbon dioxide fixation ability of microorganisms."

[0015] Although the secretory signal peptide of the present invention is derived from the genome of Hydrogenophilus thermoluteolus, it is believed to function in a wide range of microorganisms, including bacteria. It functions efficiently in bacteria belonging to the genus Hydrogenophilus, particularly Hydrogenophilus thermoluteolus. By introducing a DNA fragment containing the secretory signal peptide gene of the present invention ligated upstream of a gene encoding a target protein into a Hydrogenophilus bacterium, the target protein is produced as a fusion product with the secretory signal peptide by culturing the resulting transformant. Subsequent processing results in the secretion of the mature protein without the secretory signal peptide from the cells. Therefore, the present invention allows the target protein to accumulate in the medium, facilitating protein recovery. Furthermore, since fewer impurities are produced than when the target protein is recovered by disrupting the bacterial cells, the target protein can be easily purified. Therefore, proteins can be produced efficiently. As described above, Hydrogenophilus bacteria grow very rapidly using carbon dioxide as the sole carbon source. Therefore, the target protein can be produced extracellularly using the secretory signal peptide gene of the present invention to produce the target protein extremely efficiently.

[0016] Furthermore, research on protein secretion using Bacillus bacteria has revealed that the secretory signal sequence required for efficient secretion varies greatly depending on the protein being expressed, and that it is desirable to optimize the secretory signal sequence for each protein whose secretion is desired (Non-Patent Document 4). In this regard, the present invention provides 76 secretory signal peptides that function efficiently in Hydrogenophilus bacteria, allowing one to easily select from these secretory signal peptides a secretory signal peptide suitable for the secretory production of a target protein.

[0017] FIG. 1 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry. FIG. 2 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry. FIG. 3 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry. FIG. 4 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry. FIG. 5 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry. FIG. 6 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry. FIG. 7 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry. FIG. 1 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry. FIG. 2 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry. FIG. 3 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry. FIG. 4 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry. FIG. 5 shows the results of matching the amino acid sequences of predicted secreted proteins derived from Hydrogenophilus thermorteolus with the amino acid sequences of peptides detected by mass spectrometry.

[0018] The present invention will be described in detail below. (1) Secretory signal peptide gene The present invention relates to the secretory signal peptide genes of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, DNA encoding a peptide comprising the amino acid sequence of 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, or 151, in particular, DNA encoding a peptide comprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, DNA encoding a peptide consisting of the amino acid sequence of 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, or 151 is provided.Representative examples of these DNAs include SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, DNA comprising the nucleotide sequence of 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, or 152, in particular, DNAs comprising the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, Examples of such a DNA include DNA consisting of the nucleotide sequence of 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, or 152.

[0019] The present invention also relates to the following sequences: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, or 151, and in particular, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, Also provided are DNAs encoding peptides that function as secretory signal peptides, the peptides having an amino acid sequence that has 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, or 151.

[0020] Furthermore, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, The amino acid sequence of SEQ ID NO: 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, or 151 includes amino acid sequences in which 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is deleted, substituted, inserted, or added, and particularly includes SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, The present invention also provides DNA encoding a peptide that functions as a secretory signal peptide, which comprises an amino acid sequence in which 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of the first or second ..."Conservative substitutions" include substitutions between Val, Ile, Leu, Ala, and Met; substitutions between Asp and Glu; substitutions between Asn and Gln; substitutions between Ser, Thr, Gly, and Ala; substitutions between Lys, Arg, and His; and substitutions between Phe, Tyr, and Trp, of which substitutions between Gly and Ala; substitutions between Val, Ile, and Leu; substitutions between Asp and Glu; substitutions between Asn and Gln; substitutions between Ser and Thr; substitutions between Lys and Arg; and substitutions between Phe and Tyr are preferred.

[0021] Representative examples of these homologous DNAs include SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, DNA comprising a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, or 152, in particular SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, or 152, and the DNA has a nucleotide sequence that is 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identical to the sequence of the gene encoding the secretory signal peptide.

[0022] The DNA of the present invention described above can be used as a secretory signal peptide gene.

[0023] In the present invention, a DNA fragment in which a test DNA is ligated adjacent to the 5' end of any non-secreted protein gene encoded on the genome of Hydrogenophilus thermorteolus is introduced into the host Hydrogenophilus thermorteolus, and the test DNA is judged to function as a secretory signal peptide gene if the transformant secretes a greater amount of the protein into the culture supernatant than the host strain upon cultivation. In the present invention, a non-secreted protein refers to a protein that is not predicted as a secretory protein by either the secretory signal peptide prediction programs PrediSi or SignalP.

[0024] SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, A DNA sequence encoding a protein containing the amino acid sequence of 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, or 151 may have various base substitutions in the coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region, taking into account codon degeneracy or codon preference in the host microorganism (particularly bacteria, especially hydrogen bacteria, especially bacteria of the genus Hydrogenophilus).

[0025] In the present invention, the identities of base sequences and amino acid sequences are values ​​calculated using GENETYX ver. 17 (GENETYX).

[0026] In the present invention, a DNA that contains a nucleotide sequence that is 90% to less than 100% identical to the nucleotide sequence of a certain DNA and encodes a peptide that functions as a secretory signal peptide is referred to as a homolog of that DNA. Furthermore, a DNA that contains an amino acid sequence that is 90% to less than 100% identical to the amino acid sequence of a peptide encoded by a certain DNA and encodes a peptide that functions as a secretory signal peptide is referred to as a homolog of that DNA. Furthermore, a DNA that contains an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of a peptide encoded by a certain DNA and encodes a peptide that functions as a secretory signal peptide is referred to as a homolog of that DNA. Furthermore, in the present invention, a peptide that contains an amino acid sequence that is 90% to less than 100% identical to the amino acid sequence of a certain peptide and functions as a secretory signal peptide is referred to as a homolog of that peptide. Furthermore, a peptide that contains an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of a certain peptide and functions as a secretory signal peptide is referred to as a homolog of that peptide.

[0027] (2) Vector The vector of the present invention is a vector comprising the DNA of the present invention capable of functioning as the secretory signal peptide gene described above. Examples of the vector include a plasmid vector, a viral vector, a cosmid, a fosmid, and a BAC. The vector may comprise a DNA capable of autonomously replicating in a host microorganism, particularly a bacterium, particularly a bacterium of the genus Hydrogenophilus. Examples of DNA capable of autonomous replication in Hydrogenophilus bacteria include broad-host-range vectors such as pRK415 (GenBank: EF437940.1), pBHR1 (GenBank: Y14439.1), pMMB67EH (ATCC 37622), pCAR1 (NCBI Reference Sequence: NC_004444.1), pC194 (NCBI Reference Sequence: NC_002013.1), pK18mobsacB (GenBank: FJ437239.1), and pUB110 (NCBI Reference Sequence: NC_001384.1), as well as genetically modified versions of these vectors (e.g., pCAMO-6). Among these, pCAMO-6 is preferred. pCAMO-6 can be prepared by those skilled in the art according to the Examples. Examples of promoters contained in the vector include the tac promoter, lac promoter, trc promoter, and each of the OXB1, OXB11 to OXB20 promoters from Oxford Genetics. Examples of terminators contained in the vector include the rrnB T1T2 terminator of the Escherichia coli rRNA operon, the bacteriophage λt0 transcription terminator, and the T7 terminator.

[0028] The vectors of the present invention contain an insertion site for a target protein gene downstream of the secretory signal peptide gene. The structural gene for a target protein (hereinafter also referred to as the "target protein gene") can be inserted into this gene insertion site, either directly or via multiple nucleotides (multiple nucleotides being a multiple of three) without altering the reading frame during translation, to create a target protein expression vector that can be used to secrete and produce the protein in a host. A protein in which the secretory signal peptide and the target protein are fused is expressed from this expression vector. Any number of nucleotides may be interposed between the secretory signal peptide gene and the target protein gene, as long as the secretory signal peptide is processed normally and the target protein is secreted. The number of intervening nucleotides is preferably 300 or less, 90 or less, 30 or less, or 9 or less. For example, a restriction enzyme recognition sequence may be intervened.

[0029] The target protein gene may be a foreign gene (heterologous gene) that is not present in the genome of the host microorganism, or an endogenous gene that is present in the genome of the host microorganism, with a foreign gene (heterologous gene) being preferred. Furthermore, the target protein may be either a non-secreted protein or a secreted protein. When the target protein to be inserted into the vector of the present invention is a naturally secreted protein, DNA encoding a portion other than the secretory signal peptide may be used as the target protein gene. The secretion efficiency of the target protein can be improved by expressing the portion other than the secretory signal peptide as a protein fused with a secretory signal peptide that has a higher secretion efficiency than the naturally occurring secretory signal peptide.

[0030] A secretory signal peptide capable of efficiently secreting a target protein can be selected as follows: the amount of target protein secreted into the culture supernatant is compared between a transformant obtained by introducing a test vector containing a secretory signal peptide gene and a target protein gene downstream thereof into a host microorganism and a transformant obtained by introducing a control vector that is identical to the test vector except that it does not contain the secretory signal peptide gene, and a secretory signal peptide gene that increases the amount of target protein secreted in the culture supernatant is selected.

[0031] (2) Transformant The transformant of the present invention is a transformant obtained by introducing into a host microorganism a DNA fragment containing the DNA of the present invention that functions as the secretory signal peptide gene described above and a structural gene for a target protein located downstream thereof. In other words, the transformant of the present invention is a microorganism containing an exogenous DNA fragment containing the DNA of the present invention that functions as the secretory signal peptide gene described above and a structural gene for a target protein located downstream thereof. The DNA fragment may be directly introduced into the host microorganism, or a vector incorporating the DNA fragment (the vector of the present invention described above) may be introduced into the host microorganism. The DNA fragment can be introduced using a common method for introducing exogenous genes into microorganisms, such as the calcium chloride method, the rubidium chloride method, or the electric pulse method (electroporation).

[0032] Examples of host microorganisms include prokaryotes such as bacteria and archaea, and eukaryotes such as fungi, plants, prokaryotes, and algae. Among these, prokaryotes, bacteria, hydrogen bacteria, and bacteria of the genus Hydrogenophilus are preferred. Examples of Hydrogenophilus bacteria include Hydrogenophilus thermoluteolus, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus thiooxidans, Hydrogenophilus sp. Mar3, and Hydrogenophilus sp. Z1038. Among these, Hydrogenophilus thermoluteolus is preferred because it has the highest growth rate and carbon dioxide fixation ability among carbon dioxide fixation microorganisms. Hydrogenophilus bacteria can be easily isolated from all over the world. A preferred strain of Hydrogenophilus thermorteolus is the TH-1 (NBRC 14978) strain. Hydrogenophilus thermorteolus TH-1 (NBRC 14978) exhibits the highest growth rate of any carbon-fixing microorganism (Agricultural and Biological Chemistry, 41, 685-690 (1977)) (doubling in one hour). The Hydrogenophilus thermorteolus NBRC 14978 strain has been internationally deposited under the Budapest Treaty and is publicly available.

[0033] The host microorganism may be a microorganism isolated from nature, or may be a microorganism that has been genetically modified from a microorganism isolated from nature. Modifications can be performed for purposes such as enabling high expression of introduced genes. For example, in bacteria, such modifications can be performed by removing (curing) endogenous plasmids in the bacteria. Methods for removing endogenous plasmids are well known, including methods that utilize plasmid incompatibility, such as the use of chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide; destabilization by introducing a plasmid with the same replication origin as the endogenous plasmid; and destabilization by disrupting factors involved in the plasmid partition system.

[0034] (3) Genetic Recombinant The genetic recombinant of the present invention is a genetic recombinant in which a DNA of the present invention functioning as a secretory signal peptide gene is inserted into the 5'-terminus of a structural gene of a non-secreted protein on the genome of a microorganism, either directly or via multiple nucleotides (multiple nucleotides being a multiple of three), or in which the secretory signal peptide gene of a secreted protein has been replaced with a DNA of the present invention functioning as a secretory signal peptide gene. The microorganism is as described for the host microorganism of the transformant of the present invention. The number of nucleotides that can be inserted between the structural gene of a non-secreted protein or the gene of a portion of a secreted protein other than the secretory signal peptide and the DNA of the present invention is the same as described for the vector of the present invention. In either case, when the DNA of the present invention is introduced upstream of the structural gene of a non-secreted protein or when the DNA of the present invention is introduced by replacing the secretory signal peptide gene of a secreted protein, the DNA of the present invention can be introduced by homologous recombination using the nucleotide sequences on both sides of the desired insertion site on the genome. When the DNA of the present invention is inserted upstream of the structural gene of a non-secreted protein present on the genome, the protein will be secreted. Furthermore, when substituting the gene for the original secretory signal peptide of a secretory protein with the secretory signal peptide gene of the present invention, the secretion efficiency of the protein can be improved by selecting a secretory signal peptide gene suitable for secretion of the protein from the DNA of the present invention.

[0035] (4) Protein Production Method The method for producing a target protein of the present invention comprises the steps of culturing the transformant or genetic recombinant of the present invention described above and recovering the target protein from the culture medium. Taking hydrogen bacteria (particularly bacteria of the genus Hydrogenophilus) as an example, the culture step involves culturing the transformant or genetic recombinant in an inorganic or organic medium while supplying a gas containing carbon dioxide, preferably a mixed gas containing hydrogen, oxygen, and carbon dioxide. The gas supplied is preferably a mixed gas consisting of hydrogen, oxygen, and carbon dioxide, but other gases may be present within the range that allows efficient production of the target protein.

[0036] Hydrogen bacteria (particularly bacteria of the genus Hydrogenophilus) can grow using hydrogen as an energy source and carbon dioxide as the sole carbon source, and therefore can efficiently fix carbon dioxide by producing a target protein using substantially only carbon dioxide as a carbon source (particularly, using only carbon dioxide). Therefore, in the method of the present invention, it is preferable to use an inorganic medium that does not contain carbon sources such as organic matter or carbonates, i.e., to culture using substantially only carbon dioxide as a carbon source (particularly, using carbon dioxide as the only carbon source). In the present invention, "using carbon dioxide as substantially the sole carbon source" and "using carbon dioxide as the sole carbon source" encompass cases where unavoidable amounts of other carbon sources are mixed in.

[0037] The pH of the culture medium used for culture is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. Within this range, bacterial growth and mixed gas solubility in the medium are high, allowing for highly efficient production of the target protein. When performing batch culture, the mixed gas can be sealed in a sealed culture vessel and cultured by static or shaking, with shaking culture being preferred because it improves the solubility of the mixed gas in the medium. When performing continuous culture, the mixed gas can be continuously supplied to the culture vessel while being cultured by shaking, or the transformed or genetically modified organism can be cultured while introducing the mixed gas into the medium by bubbling. The volume ratio of hydrogen, oxygen, and carbon dioxide in the supply gas (hydrogen:oxygen:carbon dioxide) is preferably 1.75 to 7.5:1:0.25 to 3, more preferably 5 to 7.5:1:1 to 2, and even more preferably 6.25 to 7.5:1:1.5. Within this range, bacterial growth is favorable and the target protein can be efficiently produced. The supply rate of the mixed gas or raw material gas should be 10 to 60 L / hour, preferably 10 to 40 L / hour, and more preferably 10 to 20 L / hour per liter of medium. Within this range, the growth of the transformant or genetically modified organism is favorable, the target protein can be produced efficiently, and waste of the mixed gas is reduced. The culture temperature is preferably 35 to 55°C, more preferably 37 to 52°C, and even more preferably 50 to 52°C. Within this range, the growth of the transformant is favorable and the target protein can be produced efficiently.

[0038] By culturing as described above, a fusion protein of the secretory signal peptide and the target protein is produced, processed intracellularly, and the target protein is secreted extracellularly. The target protein can be recovered by recovering the culture medium, but it can also be separated and purified from the reaction solution by known methods. Examples of such known methods include methods that utilize differences in solubility, such as salting out or solvent precipitation; methods that utilize differences in molecular weight, such as dialysis, ultrafiltration, or gel electrophoresis; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reversed-phase high-performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing. While the culture using a liquid medium has been described above, the transformant or genetic recombinant of the present invention may also be cultured on a solid medium and the target protein recovered from the solid medium.

[0039] Methods for confirming the isolated and purified protein include, for example, Western blotting, activity measurement, etc. The structure of the purified protein can also be clarified by amino acid analysis, amino-terminal analysis, primary structure analysis, etc.

[0040] The present invention will now be described with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0041] (1) Identification of secreted proteins contained in the culture supernatant of Hydrogenophilus bacteria (1-1) Preparation of protein samples from the culture supernatant Hydrogenophilus thermorteolus TH-1 strain was cultured in liquid medium A [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4 7H2O 0.014 g, MgSO4 7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4 7H2O 28 mg, CuSO4 5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4 5H2O 4.0 mg, CoCl2 6H2O 4.0 mg dissolved in 1 L of distilled water (pH 7.0). The bacteria were inoculated into a test tube containing 5 mL of ethanol (7.0) using a platinum loop, and the tube was filled with a 7.5:1:1.5 H2:O2:CO2 gas mixture and cultured at 52°C. This was used to inoculate and culture three 1 L medium bottles containing 50 mL of liquid medium A, yielding a total of 150 mL of seed culture medium. This seed culture was added to liquid medium A in a 3 L jar fermenter to make a 1.5 L culture volume, and approximately 25 mL of bacterial liquid was harvested after 8 hours of culture. The harvested culture medium was centrifuged, and the supernatant was passed through a 0.22 μm pore size filter to completely remove the bacterial cells. It was then centrifuged using an ultrafiltration unit (Amicon Ultra 15 mL, 10,000 MWCO) to reduce the volume to approximately 1 / 80 of its original volume. A 4 μL sample of this concentrated culture supernatant was analyzed by 5-20% SDS-PAGE, and many proteins were detected by staining with Coomassie Brilliant Blue G-250. Using molecular weight markers as indicators, the gel portion corresponding to a molecular weight of 40 kDa or less and 15 kDa or more, and the gel portion corresponding to a molecular weight of 40 kDa or more and 75 kDa or less were excised and cut into pieces approximately 1 mm square. These were designated Sample 1 and Sample 2, respectively.

[0042] (1-2) Protein Identification by Liquid Chromatography / Mass Spectrometry Identification of the proteins contained in Samples 1 and 2 was performed by MS Bioworks (Ann Arbor, Michigan, USA) using the following procedure. Polyacrylamide gel fragments, which were protein samples, were washed with 25 mM ammonium carbonate solution and then dehydrated by immersion in acetonitrile. Subsequently, reduction treatment with 10 mM dithiothreitol and alkylation (carbamidomethylation) with 50 mM iodoacetamide were performed to cleave disulfide bonds in the protein and prevent their reformation. The gel fragments were digested with porcine trypsin (Promega) at 37°C for 4 hours, after which the reaction was stopped by adding formic acid. The trypsin-digested peptide mixture leached from the resulting gel was collected and used for analysis.

[0043] The resulting tryptic peptide mixture was analyzed using a nanoLC-MS / MS system combining an ACQUITY UPLC M-Class liquid chromatography system (Waters) and an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). A 75 μm internal diameter analytical column packed with 2.4 μm diameter XSelect CSH C18 resin was used for liquid chromatography, and the flow rate was set to 350 nL / min. The mass spectrometer was operated at a resolution of 60,000 FWHM for MS analysis and 15,000 FWHM for MS / MS analysis, with data acquisition switching between MS and MS / MS every 3 seconds.

[0044] The MS and MS / MS data obtained by the mass spectrometer were processed using Mascot (Matrix Science software). Specifically, using Mascot, the proteins to which the amino acid sequences of the tryptic peptides belonged were identified by comparing and matching (MS / MS ion search) the MS / MS spectra, consisting of the measured masses of multiple fragment ions generated by cleavage and fragmentation of individual tryptic peptides, with hypothetical MS / MS spectra calculated from the amino acid sequences of proteins encoded in the genome of Hydrogenophilus thermorteolus obtained from the protein database (GenBank). The settings used for comparison and matching using Mascot were as follows: Cleavage enzyme: Trypsin Database species: Hydrogenophilus thermolteolus Fixed amino acid modification: Carbamidomethylation (cysteine) Possible amino acid modifications: Oxidation (methionine), acetylation (N-terminus), pyroglutamylation (N-terminus glutamine), deamination (aspartic acid, glutamine) Mass tolerance for tryptic peptides: 10 ppm or less Mass tolerance for fragment ions generated by MS / MS: 0.02 Da or less

[0045] Proteins were identified as present in the culture supernatant if their amino acid sequences were assigned by at least two independent MS / MS spectra and the FDR (False Discovery Rate) calculated from the mass measurement error was 1% or less. As a result of the analysis, 337 proteins were identified from sample 1, which contained proteins of approximately 40 kDa or less, and 206 proteins were identified from sample 2, which contained proteins of approximately 40 kDa or more. The total number of identified proteins, excluding duplicates, was 502.

[0046] (1-3) Identification of Secreted Proteins In addition to proteins secreted from the bacterial cells, the culture supernatant is expected to contain many proteins derived from bacterial cells disrupted by lysis during cultivation. Therefore, not all proteins of approximately 15 kDa or larger in the culture supernatant identified by Mascot are secreted proteins. Therefore, we searched for secretory signal peptides using the amino acid sequences encoded in the genome of Hydrogenophilus thermorteolus for the 502 proteins identified in the culture supernatant using the secretory signal peptide prediction programs PrediSi (http: / / www.predisi.de) and SignalP (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ). Of these, 79 proteins were predicted to contain secretory signal peptides. PrediSi is described in "Hiller et al. Nucleic Acids Research, 32, W375-W379 (2004)," and SignalP is described in "Teufel et al. Nature Biotechnology, 40, 1023-1025 (2022)." These publications have been cited 193 times and 201 times, respectively, indicating that these programs are highly reliable.

[0047] To further improve accuracy, the present inventors compared and examined the amino acid sequences of secretory proteins predicted by the secretory signal peptide prediction program with the amino acid sequences of peptides present in the culture supernatant identified by mass spectrometry. Secretory signal peptides are usually removed during secretion, and therefore are not usually included in proteins present in the culture supernatant identified by mass spectrometry. Therefore, even if a protein is predicted to be a secretory protein by the secretory signal peptide prediction program, if its N-terminal portion is assigned to a peptide present in the culture supernatant, i.e., if the N-terminal portion of the amino acid sequence of the protein predicted to be a secretory protein contains a sequence matching the amino acid sequence of a peptide present in the culture supernatant, the protein is considered not to be a secretory protein.

[0048] The matching results are shown in Figures 1 to 12. The amino acid sequences predicted as secretory signal sequences are shown in bold, and the amino acid sequences identified by mass spectrometry from culture supernatant samples are underlined. The N-terminal amino acid sequences of proteins No. 1 to 76 do not contain sequences that match the amino acid sequences of peptides present in the culture supernatant. Therefore, these proteins were determined to be secretory proteins. The amino acid sequences shown in bold in the N-terminal portions of proteins No. 1 to 76 are the amino acid sequences of secretory signal peptides No. 1 to 76 predicted by a secretory signal peptide prediction program. The amino acid sequences of secretory signal peptides No. 1 to 76 contained in secretory proteins No. 1 to 76 (sequences with odd-numbered SEQ ID NOs. 1 to 152) and examples of the nucleotide sequences encoding them (sequences with even-numbered SEQ ID NOs. 1 to 152) are shown in Tables 1 to 4.

[0049]

[0050] On the other hand, three proteins, protein Nos. 77–79, contained sequences in their N-terminus that matched the amino acid sequences of peptides present in the culture supernatant. Therefore, they were unlikely to be secreted proteins. Furthermore, this finding suggests that it is not particularly difficult to assign the amino acid sequences of peptides present in the culture supernatant to sequences near the N-terminus of proteins. Generally, if two or more amino acid sequences of peptides present in the sample are assigned to the protein's amino acid sequence with high mass accuracy, the protein can be determined to be present in the sample. For example, proteins Nos. 66 and 69–76 contain two amino acid sequences that match the amino acid sequences of peptides present in the culture supernatant (protein No. 71 has two consecutive underlines). However, considering that the FDR (False Discovery Rate) calculated from mass measurement error was less than 1%, it is safe to assume that these proteins are present in the culture supernatant.

[0051] Secretory proteins No. 1 to 76 shown in Figures 1 to 12 were identified by two or more MS / MS spectra. Among them, secretory proteins No. 1 to 46 having signal peptides consisting of the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91 have ten or more spectra, among which the spectra of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91 have ten or more spectra. More than 40 spectra were obtained for secretory proteins No. 1 to No. 16, which have signal peptides consisting of amino acid sequences of 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31. The number of MS / MS spectra (spectral count) assigned to an identified protein is known to have a strong positive correlation with the protein abundance, and this correlation is widely used in relative quantification methods. Therefore, among the 76 signal peptides listed in Tables 1 to 4, signal peptides Nos. 1 to 46, which consist of the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91, are derived from proteins secreted in greater amounts than the other signal peptides and are therefore more preferred signal peptides. Signal peptides Nos. 1 to 16, each consisting of the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, are derived from proteins secreted in even greater amounts and are therefore considered to be even more preferred signal peptides.

[0052] (2) Construction of Plasmid Vector (pCAMO-6) The construction method of the plasmid vector pCAMO-6 used to introduce a gene encoding a fusion protein consisting of a secretory signal sequence and a protein to be expressed is described below. (2-1) Preparation of the tac promoter DNA fragment Using the plasmid pMAL-c5X (New England BioLabs) as a template, PCR was performed using the following pair of primers to amplify the tac promoter DNA fragment. Unless otherwise noted below, PCR was performed according to standard procedures using a Thermo Fisher Scientific 2720 Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as the reaction reagent. Primers for amplifying the tac promoter: (a-1) 5'-TTTTATAACCCGGGCCATCGACTGCACGGTGCACC-3' (SEQ ID NO: 153) (b-1) 5'-TGCTAGCACTGTTTCCTGTGTGAAATTGTTATCCG-3' (SEQ ID NO: 154). Primer (a-1) contained a SmaI restriction enzyme site, as underlined. The reaction mixture prepared above was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 0.3 kbp corresponding to the tac promoter was detected. This fragment was excised from the agarose gel, and the gel was frozen and thawed to recover the DNA.

[0053] (2-2) Preparation of DNA Fragment of Multicloning Region To prepare the DNA fragment of the multicloning region, PCR was performed using the following pairs of primers to generate the first and second halves. This method does not use template DNA; the 3' ends of each pair of primers anneal and extend to form double-stranded DNA. Primers for preparing the first half of the multicloning region: (a-2) 5'-GGAAACAGTGCTAGCAGATCTGGAGGAGAAACGCATAT-3' (SEQ ID NO: 155) (b-2) 5'-CAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCGATATCAGCATATGCGTTTCTCCTCCAGA-3' (SEQ ID NO: 156) The sequences of the 20 bases on the 3' side of primers (a-2) and (b-2) are complementary to each other. Primers for preparing the second half of the multicloning region: (a-3) 5'-ACAAGCTTGCGGCCGCACTGCAGCACCATCACCACCATCATTGATAAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCT-3' (SEQ ID NO: 157) (b-3) 5'-TATTTGAATCGAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGT-3' (SEQ ID NO: 158). The 3'-20 base pairs of primers (a-3) and (b-3) are complementary to each other. The reaction mixture prepared above was subjected to agarose gel electrophoresis, and approximately 0.1 kbp DNA fragments corresponding to the first and second halves of the multicloning region were detected. Each DNA fragment was excised from the agarose gel, and the DNA was recovered from the gel by freezing and thawing.

[0054] Overlap extension PCR was performed using the recovered DNA fragments corresponding to the first and second halves of the multicloning region as templates. The 5' 20 base sequences of primers (b-2) and (a-3) used to amplify this template DNA fragment are complementary to each other. For overlap extension PCR, a combination of primers (a-2) and (b-3) was used to generate DNA for the multicloning region. The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 0.2 kbp corresponding to the multicloning region was detected and recovered from the gel.

[0055] (2-3) Preparation of a DNA fragment containing the rrnB terminator and the replication origin of pUC19 Using the plasmid pMAL-c5X (New England BioLabs) as a template, PCR was performed with the following pair of primers to amplify a DNA fragment of the rrnB terminator. Primers for rrnB terminator amplification: (a-4) 5'-TAACTCGATTCAAATAAAACGAAAGGCTCAGTCGA-3' (SEQ ID NO: 159) (b-4) 5'-CCTAGATCCGCGGAGTTTGTAGAAACGCAAAAAGG-3' (SEQ ID NO: 160) Furthermore, PCR was performed with the following pair of primers to amplify a DNA fragment of the DNA replication origin using the plasmid pUC19 as a template. Primers for amplifying the DNA replication origin of pUC19: (a-5) 5'-ACAAACTCCGCGGATCTAGGTGAAGATCCTTTTTG-3' (SEQ ID NO: 161) (b-5) 5'-CGTCCGCGGCCAGCAAAAGGCCAGGAACCGTAAAA-3' (SEQ ID NO: 162). The resulting reaction solution was subjected to agarose gel electrophoresis, detecting a DNA fragment of approximately 0.3 kbp for the rrnB terminator and an approximately 0.8 kbp for the DNA replication origin of pUC19. Each DNA fragment was excised from the agarose gel, and the gel was frozen and thawed to recover the DNA.

[0056] Overlap extension PCR was performed using the recovered rrnB terminator and a DNA fragment corresponding to the pUC19 DNA replication origin as templates. The 5' 20 base pairs of primers (b-4) and (a-5) used to amplify these template DNA fragments are complementary to each other. For overlap extension PCR, a combination of primers (a-4) and (b-5) was used to generate a DNA fragment linking the rrnB terminator and the pUC19 replication origin. The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 1.0 kbp corresponding to the DNA linking the rrnB terminator and the pUC19 replication origin was detected and recovered from the gel.

[0057] (2-4) Preparation of a DNA fragment of the neomycin / kanamycin resistance gene Using the plasmid pK18mobsacB (GenBank: FJ437239.1) [Gene, 145, 69-73 (1994)] containing the neomycin / kanamycin resistance gene (hereinafter sometimes referred to as "nptII") sequence as a template, PCR was performed with the following pair of primers to amplify a DNA fragment containing the nptII gene sequence: Primers for amplifying the nptII gene: (a-6) 5'-TTGCTGGCCGCGGACGTAGAAAGCCTGTCCGCAGA-3' (SEQ ID NO: 163) (b-6) 5'-GGCCCGGGTTATAAAAGCCAGTCATTAGGCCTATC-3' (SEQ ID NO: 164) Primer (b-6) contained an SmaI restriction enzyme site as underlined. The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 1.0 kbp corresponding to the nptII gene was detected and recovered from the gel.

[0058] (2-5) Construction of Circular Plasmids: The DNA fragments prepared in steps (1-1) to (1-4) above share 16-bp homologous sequences with the ends of DNA fragments (1-4) and (1-2), with the ends of DNA fragment (1-2) with the ends of DNA fragments (1-1) and (1-3), with the ends of DNA fragment (1-3) with the ends of DNA fragments (1-2) and (1-4), and with the ends of DNA fragment (1-4) with the ends of DNA fragments (1-3) and (1-1). Therefore, the DNA fragments (1-1), (1-2), (1-3), and (1-4) can be ligated into a circular DNA by Gibson Assembly (Gibson et al., Nature Methods 6(5):343-345), a DNA ligation method that does not use restriction enzymes or ligases. To ligate the DNA fragments prepared in (1-1) to (1-4) into circular DNA, Gibson assembly was performed using New England Biolabs' Gibson Assembly Master Mix. The resulting reaction mixture was used to transform Escherichia coli JM109 using the calcium chloride method and plated on LB solid medium containing 50 μg / mL kanamycin. The strain grown on the medium was cultured in liquid culture using standard methods, and plasmid DNA was extracted from the culture medium and reacted with the restriction enzyme SmaI. Agarose gel electrophoresis of this reaction mixture revealed a DNA fragment of approximately 2.3 kbp, corresponding to the size of the single DNA fragment prepared in (1-1) to (1-4), confirming their ligation. This circular plasmid DNA, which replicates in E. coli, was designated pCAMO-1.

[0059] (2-6) Construction of Plasmid Vector (pCAMO-4) Hydrogenophilus thermorteolus TH-1 strain was inoculated into a test tube containing 5 mL of liquid medium A using a platinum loop. The test tube was then cultured at 50°C under a gas mixture of H2:O2:CO2 = 7.5:1:1.5. The endogenous plasmid pTH1 [Microbiol Resour Announcement. 2018 Aug 16;7(6)] was isolated from the culture medium using the standard alkaline SDS method. The approximately 66 kbp pTH-1 fragment was digested with the restriction enzymes ScaI and PvuII, and the plasmid pCAMO-1 was digested with the restriction enzyme SmaI and ligated to each other using T4 DNA ligase (Takara Bio Inc.).

[0060] The resulting ligation solution was used to transform the Hydrogenophilus thermorteolus TH-1 strain (NBRC 14978) by electroporation. The transformants were grown on solid medium A containing 50 μg / mL kanamycin [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4 7H2O 0.014 g, MgSO4 7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4 7H2O 28 mg, CuSO4 5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4 5H2O 4.0 mg, CoCl2 6H2O 4.0 mg, and 15 g of agar dissolved in 1 L of distilled water (pH 7.0). 7.0) and cultured for 60 hours at 50°C in a chamber filled with a gas mixture of H2:O2:CO2 = 7.5:1:1.5. Seven strains grown on solid medium A were inoculated using a platinum loop into test tubes containing 5 mL of liquid medium A containing 50 μg / mL kanamycin. The test tubes were filled with a gas mixture of H2:O2:CO2 = 7.5:1:1.5 and cultured with shaking at 52°C. Plasmid DNA was extracted from the culture medium and subjected to agarose gel electrophoresis. The results confirmed that all seven strains contained the same plasmid DNA of approximately 5.5 kbp.

[0061] Using the extracted plasmid as a template, PCR was performed using the following pair of primers, one on each side of the SmaI restriction site of pCAMO-1, to amplify the DNA fragment of the endogenous plasmid pTH1 inserted into the SmaI restriction site of pCAMO-1. Primers for amplifying the inserted pTH1 DNA fragment: (a-7) 5'-AATTGTCAGATAGGCCTAATGACTGGCTTTTATAA-3' (SEQ ID NO: 165) (b-7) 5'-TGACGCCAGAAGCATTGGTGCACCGTGCAGTCGAT-3' (SEQ ID NO: 166). The resulting reaction solution was subjected to agarose gel electrophoresis, detecting a DNA fragment of approximately 3.2 kbp. In other words, the resulting plasmid contained a DNA fragment of approximately 3.2 kbp, a portion of pTH1, inserted into the SmaI restriction site of the pCAMO-1 plasmid. This approximately 3.2 kbp DNA fragment contains a replication origin that functions in Hydrogenophilus thermorteolus cells, so the resulting plasmid replicates in Hydrogenophilus thermorteolus cells. The constructed plasmid was named pCAMO-4.

[0062] (2-7) Construction of Plasmid Vector (pCAMO-5) The following pair of oligonucleotides were mixed in equimolar amounts and slowly cooled from 98°C to 20°C to prepare a double-stranded DNA fragment to which the oligonucleotides were annealed. The ends of this DNA fragment were equivalent to the protruding ends obtained by cleavage with the restriction enzymes BglII and NdeI. This DNA fragment and a fragment obtained by cleaving pCAMO-4 with the restriction enzymes BglII and NdeI were ligated together using T4 DNA ligase. (a-8) 5'-GATCTGGAGGAGAAACGCA-3' (SEQ ID NO: 167) (b-8) 5'-TATGCGTTTCTCCTCCA-3' (SEQ ID NO: 168) The constructed plasmid was named pCAMO-5.

[0063] (2-8) Construction of Plasmid Vector (pCAMO-6) Using the endogenous plasmid pTH-1 as a template, PCR was performed with the following pair of primers to amplify a DNA fragment of the mazF gene encoding a plasmid stabilization factor. Primers for amplifying the mazF gene: (a-9) 5'-GAGGCCATCTAGGCCATGAGTAAGTCTGACGGAAC-3' (SEQ ID NO: 169) (b-9) 5'-ATCCGGCACCCATATCTGAACCGGACGCAAACCCG-3' (SEQ ID NO: 170)

[0064] PCR was performed using the endogenous plasmid pTH-1 as a template and the following pair of primers to amplify a DNA fragment of the mazE gene encoding a plasmid stabilization factor: Primers for amplifying the mazE gene: (a-10) 5'-TTCAGATATGGGTGCCGGATACCCGCCGCCCGGGC-3' (SEQ ID NO: 171) and (b-10) 5'-AAGGCCTTCATGGCCTTATTTCGCGATTCCCAAGA-3' (SEQ ID NO: 172).

[0065] The 3' end of the mazF DNA fragment shares a 20-bp homologous sequence with the 5' end of the mazE DNA fragment. Therefore, the mazF and mazE DNA fragments can be ligated by PCR. The DNA fragments prepared above were mixed, and PCR was performed using primers (a-9) and (b-10) to amplify the ligated DNA fragment as a template. The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 0.7 kbp corresponding to the ligated DNA of the mazF and mazE DNA fragments was detected and recovered from the gel.

[0066] The prepared DNA fragment (approximately 0.7 kbp) and the plasmid pCAMO-5 were digested with the restriction enzyme SfiI and ligated together using T4 DNA ligase. The constructed plasmid was designated pCAMO-6.

[0067] (3) Construction of a secretory expression plasmid for a gene (3-1) Preparation of a secretory signal peptide gene fragment To construct a vector for expressing a fusion gene in which a gene consisting of a nucleotide sequence with an odd-numbered SEQ ID NO: 1 to 152 is fused to the gene for the target protein to be secreted, DNA fragments containing each nucleotide sequence with an odd-numbered SEQ ID NO: 1 to 152 are prepared as follows. Oligonucleotides consisting of nucleotide sequences in which GCAT is added to the 5' end of each nucleotide sequence with an odd-numbered SEQ ID NO: 1 to 152 are prepared as the sense strand, and oligonucleotides consisting of nucleotide sequences in which CGAC is added to the 5' end of each nucleotide sequence complementary to the nucleotide sequences with an odd-numbered SEQ ID NO: 1 to 152 are prepared as the antisense strand. Using SEQ ID NO: 1 as an example, the oligonucleotide pairs prepared are as follows: Two oligonucleotides, one for the sense strand and one for the antisense strand, are mixed and the 5' ends are phosphorylated with T4 polynucleotide kinase. The mixture is then incubated at 95°C for 10 minutes and slowly cooled to allow the two strands to anneal and form a double-stranded fragment. This reaction can also be performed with a larger number of oligonucleotides, for example, 152 oligonucleotides to generate 76 DNA fragments containing odd-numbered nucleotide sequences from 1 to 152, allowing the creation of a library of secretory signal peptide genes. The resulting double-stranded gene fragments are inserted into the expression vector pCAMO-6.

[0068] Gene expression plasmids were constructed using the Golden Gate system (NEB). MuGFP (green fluorescent protein) was used as the target protein to be secreted. To perform DNA ligation using the Golden Gate system, the plasmid vector pCAMO-6 was amplified by PCR. The following primers were used for PCR: Primers for amplification of the plasmid vector pCAMO-6: (a-11) 5'-GGCTACGGTCTCAATTCGAGCTCCGTCGACAAGC-3' (SEQ ID NO: 175) (b-11) 5'-GGCTACGGTCTCGCATATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 176). The restriction enzyme DpnI was added to the reaction mixture and incubated at 37°C for 30 minutes to digest the template vector DNA. The linearized vector DNA fragment obtained by amplified DNA was then subjected to agarose gel electrophoresis and excised. The vector DNA fragment was then recovered from the agarose gel using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0069] A DNA encoding the green fluorescent protein muGFP was prepared by adding terminal sequences necessary for cloning (before the start codon: GCGGGTCTCAT (SEQ ID NO: 177), after the stop codon: GAATTCGGAGACCCGC (SEQ ID NO: 178)) to obtain a DNA (SEQ ID NO: 179) by gene synthesis (Twist Biotechnology).

[0070] Using the New England Biolabs NEBridge Golden Gate Assembly Kit (BsaI-HF v2), the synthesized muGFP gene DNA fragment and the linear DNA fragment of pCAMO-6 were ligated together. The restriction enzyme BsaI included in the kit cleaved near each end, generating sticky ends (TATG, GAAT), which were then used for ligation with T4 DNA ligase to join the DNA. The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, plated onto LB medium containing 50 μg / mL kanamycin, and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 37°C with shaking. Plasmid DNA was extracted from the culture medium. The sequence of the gene inserted into the plasmid was analyzed by Sanger analysis at Eurofins Genomics, Inc., to confirm its match with the database sequence. This plasmid was designated pCAMO-6-muGFP.

[0071] To ligate the secretion signal gene fragment using the Golden Gate system, the plasmid vector pCAMO-6-muGFP was amplified by PCR. The following primers were used for PCR: Primers for amplification of the plasmid vector pCAMO-6-muGFP: (a-12) 5'-GGCTACGGTCTCAGTCGAAAGGCGAAGAACTCTTC-3' (SEQ ID NO: 180) (b-12) 5'-GGCTACGGTCTCGATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 181). The BsaI restriction enzyme recognition sequence (GGTCTC) is underlined, and the sticky end resulting from BsaI cleavage is double underlined. The restriction enzyme DpnI was added to the reaction mixture and incubated at 37°C for 30 minutes to digest the template vector DNA. The linearized vector DNA fragment obtained by amplified vector was subjected to agarose gel electrophoresis and excised from the gel. The vector DNA fragment was then recovered from the agarose gel using the GEL / PCR Purification Mini Kit (FAVORGEN).

[0072] (3-2) Preparation of a Plasmid Containing a Secretory Signal Peptide Gene: Using the New England Biolabs NEBridge Golden Gate Assembly Kit (BsaI-HF v2), the DNA fragment of the secretory signal peptide gene prepared in section (3-1) Preparation of the Secretory Signal Peptide Gene Fragment was ligated to the linear DNA fragment of pCAMO-6-muGFP. The BsaI restriction enzyme included in the kit cleaves the vector DNA near its terminus, creating sticky ends, which are then used for ligation with T4 DNA ligase. This generates a plasmid containing the DNA encoding muGFP, flanked by a three-nucleotide oligonucleotide, at the 3' end of the secretory signal peptide gene. The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, and the resulting mixture was plated on LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours. Each strain growing on the LB medium was individually cultured to extract plasmids, and the gene sequence was confirmed if necessary. When gene fragments derived from multiple genes are mixed, a large number of different clones grown in LB medium are collected together as needed, and plasmids are extracted to prepare an expression library of secretory signal peptides.

[0073] (4) Hydrogenophilus thermorteolus transformants (4-1) Gene introduction and selection. Hydrogenophilus thermorteolus TH-1 strain was transformed by electroporation with pCAMO-6-muGFP and pCAMO-6-muGFP, each containing a DNA fragment of the secretory signal peptide gene. The transformants were then plated on LB solid medium containing 50 μg / mL kanamycin and cultured at 52°C for 24 hours. Each transformant grown on LB solid medium was inoculated into liquid medium A containing 50 μg / mL kanamycin using a platinum loop. A gas mixture of H2:O2:CO2 = 7.5:1:1.5 was supplied during the culture, and the transformants were cultured with shaking at 52°C for 24 hours. The green fluorescent protein in the culture supernatant was quantified at an excitation wavelength of 488 nm and an emission wavelength of 509 nm. Compared to strains transformed with pCAMO-6-muGFP (a vector containing a secretory signal peptide gene), which lacks a secretory signal peptide gene, we confirm that the fluorescence intensity at 509 nm of the culture supernatant is higher in strains transformed with pCAMO-6-muGFP (a control vector) containing an inserted DNA fragment of the secretory signal peptide gene. Here, muGFP is used as the target protein. However, if the target protein cannot be quantified by the fluorescence intensity or absorbance of the culture supernatant, quantification can be performed using methods such as measuring the enzyme activity of the culture supernatant, SDS-polyacrylamide electrophoresis, or Western blotting. This allows us to evaluate whether the secretory signal peptide is suitable for secreting the target protein and select a secretory signal peptide that is suitable for the target protein.

[0074] (4-2) Protein Production. The transformants selected by the above method, which were transformed with a vector containing a secretory signal peptide gene appropriate for the target protein (pCAMO-6, which contains both the secretory signal peptide gene and the target protein gene), and the transformant (NC strain) containing a control vector (pCAMO-6 containing the target protein gene but no secretory signal peptide gene), were inoculated into liquid medium A containing 50 μg / mL kanamycin using a platinum loop. A gas mixture of H2:O2:CO2 = 7.5:1:1.5 was supplied during the culture, and the transformants were cultured with shaking at 52°C for 24 hours. After the culture, the culture supernatant was obtained by centrifugation (4°C, 5,000 g, 10 minutes). The target protein was produced in the culture supernatant of the transformant transformed with the vector containing the secretory signal peptide gene appropriate for the target protein, unlike the culture supernatant of the NC strain transformed with the control vector.

[0075] Hydrogenophilus bacterial transformants and recombinants incorporating the secretory signal peptide gene of the present invention together with a target protein gene can be prepared by referring to the description in the specification, including the Examples. The other strains described herein are either internationally deposited under the Budapest Treaty, held by institutions from which they can be freely distributed, commercially available, or can be prepared by those skilled in the art based on the present specification and are publicly available.

[0076] The DNA of the present invention functions as a secretory signal peptide gene in various microorganisms, enabling efficient extracellular production of a target protein. In particular, a transformant or genetic recombinant obtained by introducing a DNA fragment in which a gene for a target protein is secretibly linked to the DNA of the present invention into a bacterium of the genus Hydrogenophilus can efficiently produce a target protein extracellularly using carbon dioxide as the sole carbon source, thereby enabling efficient production of a target protein while solving global warming caused by increased carbon dioxide.

Claims

1. DNA that is (a), (b), or (c) below: (a) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, (b) DNA encoding a peptide containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, or 151, and which functions as a secretory signal peptide. (c) DNA encoding a peptide having an amino acid sequence having 90% or more identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,A DNA encoding a peptide that contains an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, inserted or added in the amino acid sequence of 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149 or 151, and functions as a secretory signal peptide, 2. The DNA of claim 1, which is the DNA of (d) or (e) below. (d) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, (e) DNA containing the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, DNA comprising a nucleotide sequence having 90% or more identity to 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, or 152, and encoding a peptide that functions as a secretory signal peptide.

3. A secretory signal peptide gene comprising the DNA according to claim 1 or 2.

4. A vector comprising the DNA of claim 1 or 2.

5. A vector for expressing a target protein, comprising a target protein gene downstream of the DNA according to claim 1 or 2.

6. A transformant obtained by introducing into a host microorganism a DNA fragment comprising the DNA according to claim 1 or 2 and a structural gene of a target protein located downstream thereof, or the vector according to claim 5.

7. The transformant according to claim 6, wherein the host microorganism is a Hydrogenophilus bacterium.

8. A method for producing a target protein, comprising the steps of culturing the transformant according to claim 7 using carbon dioxide as substantially the only carbon source, and recovering the protein from the medium.

9. A genetic recombinant in which the DNA according to claim 1 or 2 has been introduced upstream of a structural gene of a target protein on the genome of a microorganism.

10. The genetic recombinant according to claim 9, wherein the microorganism is a Hydrogenophilus bacterium.

11. A method for producing a target protein, comprising the steps of culturing the genetic recombinant according to claim 10 using carbon dioxide as substantially the only carbon source, and recovering the protein from the medium.

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