Recombinant protein production in Geobacillus sp.

Abstract

To provide a plasmid vector that is suitable for high production of recombinant protein in the genus Geobacillus, and in which toxicity in a coliform bacillus is suppressed.SOLUTION: A plasmid vector for recombinant protein production in the genus Geobacillus includes a promoter having mutation in a base corresponding to the 200th base and the 280th base of sequence number 1 or the 265th base of sequence number 1 in a promoter derived from a gene of cold shock protein CspD of Geobacillus kaustophilus.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This disclosure relates to the production of proteins using genetic engineering techniques. [Background technology]

[0002] A variety of host species, including prokaryotes and eukaryotes, are available for protein production using genetic engineering techniques. However, each host system has its own advantages and disadvantages. For example, Escherichia coli is the most classic host system for protein expression, with many years of research and development underway and a wide range of genetic engineering tools (e.g., expression vectors) available for various experimental conditions. However, it also has disadvantages, such as a relatively limited protein expression level and being unsuitable for the production of secreted proteins.

[0003] Non-Patent Document 1 describes a promoter that can be used to express a recombinant protein in bacteria of the genus Geobacillus and a plasmid vector containing the same. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Extremophiles (2020) 24:147-156 Summary of the Invention [Problem to be solved by the invention]

[0005] The Geobacillus bacteria expression system has several advantageous features. First, it can grow in a wide temperature range from about 40°C (37°C for some strains) to a maximum of about 74°C (80°C for some strains) (the typical optimum temperature is 50-65°C), and is particularly applicable to recombinant protein expression at high temperatures that are impossible with other bacterial hosts (for example, expression of heat-resistant proteins derived from heat-resistant bacteria). Second, a gene introduction method using conjugation transfer or electroporation has been established. Furthermore, the complete genome sequence of Geobacillus kaustophilus species (HTA426 strain) has been determined (Nucleic Acids Res. 32, 6262 (2004)). Third, as described later in this specification, Geobacillus bacteria can produce recombinant proteins at a higher productivity than E. coli, and can also produce secreted proteins. Fourth, as will also be described later in this specification, it has been confirmed that at least certain Geobacillus bacteria can grow robustly in inexpensive media that use corn steep liquor as a nutrient source, making it possible to significantly reduce the costs of culture and protein production compared to other host biological systems.

[0006] The present inventors analyzed the transcriptome of Geobacillus kaustophilus HTA426 strain using RNA-seq and identified highly expressed genes, thereby identifying several candidate promoters suitable for high recombinant protein production in Geobacillus. The promoter of the gene encoding the cold shock protein CspD, which was numbered GK1356 in the annotation of the whole genome sequence described above, showed particularly strong expression driving power.

[0007] However, the promoter of GK1356 (referred to herein as P gk1356It has been found that constructs in which the coding sequence of a recombinant protein is linked to a nucleotide sequence (also called a nucleotide sequence), when introduced into E. coli, can significantly inhibit the growth of the bacterium. This phenomenon is a general trend that has been observed in attempts to use several different recombinant proteins, and it has hindered genetic engineering and cloning procedures in E. coli. gk1356 was seen to cause recombinant protein-based toxicity in E. coli (presumably through overexpression).

[0008] Even if the final protein production is carried out in Geobacillus bacteria, the preliminary genetic engineering steps, including inserting the protein coding sequence of interest downstream of a promoter and cloning the construct, are overwhelmingly carried out in E. coli, and therefore the toxicity of a high-expression promoter in E. coli can significantly reduce the usefulness of the promoter. [Means for solving the problem]

[0009] The present inventors have succeeded in creating and identifying a novel promoter sequence that has the ability to drive high production of recombinant proteins in Geobacillus bacteria while also suppressing toxicity in Escherichia coli, thereby completing the present invention.

[0010] The present disclosure includes the following embodiments. [1] A plasmid vector for recombinant protein production in Geobacillus bacteria, comprising: A promoter derived from the gene encoding the cold shock protein CspD of Geobacillus kaustophilus, which has a mutation in the bases corresponding to the 200th and 280th bases of SEQ ID NO: 1 or the 265th base of SEQ ID NO: 1. Plasmid vector. [2] The plasmid vector according to [1], wherein the bases corresponding to the 200th and 280th bases of SEQ ID NO: 1 are mutated from T to A and A to G, respectively, or the base corresponding to the 265th base of SEQ ID NO: 1 is mutated from T to C. [3] A plasmid vector according to [1] or [2], comprising a nucleic acid of SEQ ID NO: 2 or SEQ ID NO: 3 as a nucleic acid providing the promoter. [4] A plasmid vector according to [3], having an SphI restriction enzyme site at the 3' end of SEQ ID NO:2 or SEQ ID NO:3. [5] The plasmid vector according to any one of [1] to [4], wherein a nucleic acid of a coding sequence encoding a recombinant protein is linked downstream of the promoter. [6] A recombinant protein production system comprising the plasmid vector according to any one of [1] to [5] and a Geobacillus bacterium. [7] The recombinant protein production system described in [6], wherein the plasmid vector is within the Geobacillus bacterium. [8] A promoter derived from the gene encoding the cold shock protein CspD of Geobacillus kaustophilus, which has a mutation in the bases corresponding to the 200th and 280th bases of SEQ ID NO: 1 or the 265th base of SEQ ID NO: 1; A nucleic acid of a coding sequence encoding a recombinant protein linked downstream of the promoter; and culturing a Geobacillus bacterium transformed with a nucleic acid construct comprising the above-mentioned formula (I) to express the recombinant protein. [9] The method according to [8], further comprising, prior to the culturing, transforming the Geobacillus bacterium by introducing a plasmid vector containing the nucleic acid construct into the Geobacillus bacterium. [Brief description of the drawings]

[0011] [Figure 1] Figure 1a shows the results of expressing the venus fluorescent reporter gene in Escherichia coli with the wild-type (pGKE122) or mutant (pGKE123, pGKE124) promoter. E. coli carrying each plasmid was cultured in LB medium at 30°C (black) or 37°C (white) for 24 hours. Figure 1b shows the results of expressing the venus fluorescent reporter gene in Geobacillus kaustophilus with the wild-type (Pgk1356) or mutant (Pm1, Pm2) promoter. Culture was performed in LB medium at 60°C for 24 hours (black), 48 hours (white), and 72 hours (gray). Note that the vertical axes of Figure 1a and b show different relative scales. [Diagram 2] Figure 2 shows an experiment in which secreted xylanase was produced in Geobacillus thermodenitrificans under the Pm2 promoter of pGKE124. Figure 2a shows SDS-PAGE of a sample cultured in LB medium for 24 hours, which indicates that the recombinant xylanase was released outside the cells (arrow). Figure 2b shows a comparison of xylanase production after one week of culture in LB medium (black) and CSL medium (white). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In one aspect, the present disclosure provides a plasmid vector for recombinant protein production in Geobacillus bacteria. Geobacillus bacteria themselves are known and can be found in nature or obtained from various stock centers or sales companies. Geobacillus bacteria are gram-positive bacteria. Examples of Geobacillus bacteria include, but are not limited to, G. kaustophilus, G. thermodenitrificans, G. subterraneus, G. thermoglucosidasius, and G. thermoleovorans. In the present disclosure, the term recombinant gene refers to a gene whose genetic structure has been artificially modified (e.g., linked to a promoter different from the original natural promoter of the gene) and which is typically (but not limited to) expressed in a host different from the original host. The term recombinant protein refers to a protein encoded by such a recombinant gene. The recombinant protein coding sequence and the recombinant protein linked to the mutant promoter of the present disclosure and expressed may also be referred to as a heterologous coding sequence and a heterologous protein. Industrially produced recombinant proteins are typically expressed in higher amounts and / or have altered sequences compared to the corresponding native proteins. A plasmid vector in the present disclosure is a vector that incorporates and carries a recombinant gene and can exist in the form of extrachromosomal DNA, i.e., a plasmid, in a bacterial cell.

[0013] The genome of bacteria such as Geobacillus is densely populated with protein coding regions (also called ORFs), with the spacing between adjacent coding regions, i.e. non-coding regions, typically only a few hundred base pairs. Natural promoters can be defined as sequences present in these non-coding regions that are capable of driving the transcription of downstream coding sequences. Each promoter can be isolated from the genome and independent (e.g., part of a plasmid) and still drive the transcription of downstream coding sequences. The promoters of the present disclosure can include sequences involved in translation (e.g., ribosome binding sites).

[0014] The plasmid vector of this embodiment contains a promoter for driving transcription of the recombinant gene leading to production of the recombinant protein, which promoter is based on the promoter of the gene for the cold shock protein CspD of Geobacillus kaustophilus. The protein is referred to as CspD, while the gene can also be referred to as cspD. The cspD gene of Geobacillus kaustophilus was found in the annotation of the whole genome sequence of the above-mentioned HTA426 strain, and is also called GK_RS07225 or GK1356 (Note that in https: / / www.ncbi.nlm.nih.gov / gene / 3186394, which states that it was "discontinued on 7-Apr-2017," the product of the same gene was written as CspB instead of CspD. In https: / / www.ncbi.nlm.nih.gov / protein / WP_003251474.1, which has a date of 18-JAN-2021, it has been changed to CspD.). The present inventors have discovered that this cspD gene is one of the most abundantly expressed genes in the HTA426 strain.

[0015] In this embodiment, a promoter derived from Geobacillus kaustophilus cspD is used that has mutations in the bases corresponding to the 200th and 280th bases of SEQ ID NO: 1 or the 265th base of SEQ ID NO: 1 (i.e., they are replaced with non-wild-type bases). A wild-type promoter has the sequence of SEQ ID NO: 1, but it has been found that this wild-type Geobacillus promoter often causes toxicity to E. coli in E. coli. The present invention is based on the unexpected new finding that by mutating the specific bases, toxicity in E. coli can be suppressed while maintaining high expression ability in the genus Geobacillus. Although the mechanism is unclear, since cspD was originally identified as one of the most powerful promoters, it is highly likely that the toxicity is due to excessive expression levels in E. coli, and it is believed that the mutations at the specific sites alleviate the binding of expression activating factors in E. coli. The characteristic of suppressing toxicity in E. coli eliminates the difficulties in constructing, manipulating, maintaining, cloning, screening, etc., plasmid vector constructs using E. coli, and in the conjugal transfer of plasmid vectors from E. coli to Geobacillus bacteria.

[0016] SEQ ID NO: 1 corresponds to the reverse complementary strand of bases 1376819 to 1377098 in the entire genome sequence BA000043.1 of the HTA426 strain registered in GenBank. In one embodiment, the bases corresponding to bases 200 and 280 of SEQ ID NO: 1 are mutated from T to A and A to G, respectively. In one embodiment, the base corresponding to base 265 of SEQ ID NO: 1 is mutated from T to C.

[0017] More specifically, the plasmid vector may contain the nucleic acid of SEQ ID NO:2 or SEQ ID NO:3 as a nucleic acid providing a promoter for driving transcription of a recombinant gene. In SEQ ID NO:2, the bases corresponding to bases 200 and 280 of SEQ ID NO:1 are mutated from T to A and A to G, respectively. In SEQ ID NO:3, the base corresponding to base 265 of SEQ ID NO:1 is mutated from T to C. More specifically, the plasmid vector may have, for example, a SphI restriction enzyme site at the 3' end of SEQ ID NO:2 or SEQ ID NO:3. As is commonly understood by those skilled in the art, a restriction enzyme site is a short nucleic acid sequence that is specifically recognized and cleaved by an individual restriction enzyme. For example, the promoter itself can be incorporated into the plasmid vector or the desired recombinant gene coding sequence can be inserted downstream of the promoter through cleavage / ligation of this SphI restriction enzyme site, but this is just one example of a specific configuration of a plasmid vector. Similarly, a HindIII restriction enzyme site may be placed at the 5' end of SEQ ID NO:2 or SEQ ID NO:3, but this is just one example.

[0018] The nucleic acid of the coding sequence encoding the recombinant protein can be linked downstream of the above-mentioned promoter. This means that the coding sequence is arranged so that its transcription is driven by this promoter. Such operably linking of any coding sequence to a given promoter is within the ordinary skill of a person skilled in the art. A plasmid vector lacking the nucleic acid of the coding sequence of this recombinant protein is a so-called "empty vector", into which any recombinant protein coding sequence can be inserted.

[0019] The plasmid vector of this embodiment can contain various additional elements in addition to the promoter described above. Examples of such additional elements include, but are not limited to, a transcription terminator, a replication origin, a drug resistance gene for clone selection, and a multi-cloning site for inserting a recombinant gene. Those skilled in the art can select an appropriate combination of additional elements based on their general knowledge and place them in the plasmid vector.

[0020] In one aspect, the present disclosure provides a method for producing a recombinant protein. The method includes culturing a Geobacillus bacterium transformed with a nucleic acid construct comprising the above-mentioned mutant cspD promoter and a nucleic acid of a coding sequence encoding a recombinant protein linked downstream of the promoter, to express the recombinant protein. The transformed Geobacillus bacterium may be a bacterium carrying a plasmid vector (e.g., any of the plasmid vectors described herein) comprising the nucleic acid construct, or may be a stable transformant established by inserting the nucleic acid construct (promoter-coding sequence cassette) from the plasmid vector into a host genome chromosome.

[0021] Those skilled in the art can appropriately culture Geobacillus bacteria based on their general knowledge and the disclosure of this specification. Cultivation in a liquid medium is preferred. The culture time is typically 12 hours or more, for example, 18 hours or more, or 24 hours or more. The upper limit of the culture time is not particularly limited, since the culture can be continued indefinitely by adding fresh medium. For example, the culture can be continued for 48 hours, one week, one month, or even several months. The culture temperature is typically 50 to 65°C.

[0022] As the liquid medium, LB medium, which is often used for Escherichia coli, is suitable. Furthermore, it has been found that the Geobacillus bacteria of the present disclosure grow robustly in corn steep liquor (CSL) medium using CSL as a nutrient source, and are capable of high recombinant protein production. Corn steep liquor is a by-product obtained in the process of producing starch (corn starch) from corn. Since CSL medium is inexpensive, about one-third the price per liter compared to LB medium, it is a great advantage that it can be used for recombinant protein production.

[0023] The mutant cspD promoter of the present disclosure does not require any particular manipulation for expression induction, and recombinant protein expression and production can be achieved simply by culturing and growing the transformant Geobacillus bacterium.

[0024] The expressed recombinant protein can be recovered from inside and / or outside the bacterial cells. In the case of a protein expressed inside the bacterial cells, the bacterial cells (cells) are usually lysed before the protein is recovered. In the case of a secretory protein, the extracellular fraction can be separated from the intracellular fraction after cultivation, thereby obtaining the recombinant protein in high purity in the former fraction. Regardless of whether it is in the extracellular fraction or the intracellular fraction, the recombinant protein of interest is usually found to be the most abundant protein in the sample. Those skilled in the art can separate and further purify the produced recombinant protein based on their general knowledge.

[0025] The method may further comprise the step of transforming the Geobacillus bacterium by introducing a plasmid vector containing the nucleic acid construct into the host Geobacillus bacterium prior to the culture. Methods for transforming Geobacillus bacteria by introducing a given plasmid into the bacterium are known to those skilled in the art, and for example, conjugative transfer or electroporation may be used.

[0026] The present disclosure further provides a recombinant protein production system comprising the above-mentioned plasmid vector and Geobacillus bacteria. In one embodiment, the system may be provided in the form of a kit comprising the plasmid vector and Geobacillus bacteria. A user can incorporate a target recombinant protein coding sequence into the plasmid vector as necessary, and / or introduce the plasmid vector containing the target recombinant protein coding sequence into Geobacillus bacteria. The kit may include instructions for performing one or more of these operations. In another embodiment, the system is provided in a state in which the plasmid vector is in the Geobacillus bacteria. In that case, it is preferable that the coding sequence nucleic acid of the recombinant protein is already linked downstream of the promoter in the plasmid vector, and the system can be regarded as a recombinant protein production device. EXAMPLES

[0027] [Construction and identification of mutant promoters] A comprehensive analysis of all mRNAs transcribed by Geobacillus kaustophilus HTA426 strain growing at 60°C was performed using RNA-seq, and GK1356 was identified as one of the most abundantly transcribed genes. gk1356 We have constructed an E. coli-Geobacillus bacterial shuttle vector, pGKE122, designed to have a promoter downstream of which a coding sequence of a recombinant protein of interest can be ligated. However, it was found that there are many heterologous coding sequences that cannot be inserted downstream of this promoter in the context of E. coli (or that, if inserted, will not allow E. coli to grow). Examples of such heterologous coding sequences include at least venus, bgaB, xynA, PH1171, and amyE. gk1356 It was suspected that overexpression of heterologous proteins by might cause toxicity in E. coli.

[0028] Therefore, we used the error-prone PCR technique to gk1356 We attempted to obtain a mutant promoter that suppresses toxicity in E. coli and maintains the same function as the wild-type promoter in Geobacillus by randomly introducing mutations into the promoter sequence. Error-prone PCR was performed in the presence of 7 mM MgCl2 and 0.3 mM MnCl2 using 2 mM dATP, 2 mM dGTP, 10 mM dCTP, 10 mM dTTP, and regular Taq DNA polymerase (New England Biolabs Japan).

[0029] Plasmid pGKE74-P, which has a structure in which the promoter after error-prone PCR is linked to the coding sequence of the fluorescent reporter gene venus gk1356When the -venus library was introduced into Escherichia coli DH5α, approximately 1,700 colonies were obtained. Some of these colonies were fluorescent and some were not. When the mixed plasmids extracted from the colonies were reintroduced into Geobacillus thermodenitrificans K1041 by electroporation, approximately 7,000 colonies were obtained. Of these, 24 clones that emitted strong fluorescence at 60°C and 65°C were selected and cultured in liquid at 55°C, but fluorescence was not observed in 14 clones. When the plasmids were extracted from the 24 clones and reintroduced into Escherichia coli, the 14 colonies produced a mixture of fluorescent and non-fluorescent colonies. On the other hand, all colonies of the remaining 10 clones emitted fluorescence.

[0030] In the subsequent experiments, we used the plasmids from 10 clones that showed stable fluorescent expression. We extracted these plasmids and determined their sequences. Five of the 10 clones had no mutations in the promoter region, while the remaining five had mutations in the promoter region.

[0031] When a plasmid carrying any of these five mutant promoter regions instead of the promoter region of pGKE122-venus was introduced into E. coli, P m1 and P m2 Only the plasmids carrying either of the two mutant promoters, called pGKE123-venus and pGKE124-venus, produced a large number of colonies (over 1,000) that were fluorescent and fluorescent in all colonies. The remaining three plasmids produced less than 10 colonies, suggesting that the mutations did not suppress toxicity in E. coli. m1 and P m2 It is possible that the other mutations may have introduced some other cause of instability other than toxicity.

[0032] The sequences of the mutant promoters carried by pGKE123 and pGKE124 are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively. In pGKE123 and pGKE124, these mutant promoter sequences are inserted between the HindIII-SphI site. In pGKE123-venus and pGKE124-venus, the venus coding sequence is inserted downstream of the SphI site.

[0033] [Characterization of mutant promoters] E. coli DH5α transformed with pGKE123-venus or pGKE124-venus was cultured in LB medium for 24 hours to examine the functionality of the mutant promoters in E. coli (Fig. 1a). m1 and P m2 It was confirmed that the toxicity in E. coli was suppressed by using mutant promoters, and that the venus recombinant protein coding sequence inserted under those promoters could be moderately expressed and stably maintained in E. coli. Furthermore, it was shown that the expression of the recombinant gene could be suppressed by culturing E. coli at a relatively low temperature (30°C) (black graph).

[0034] Figure 1b shows the P m1 or P m2 Expression of the venus fluorescent reporter linked to a mutant promoter in Geobacillus kaustophilus was demonstrated using a strain established by inserting the reporter cassette from a plasmid vector into a specific location in the chromosomal genome. m1 or P m2 The mutant promoter is the wild-type P gk1356 It was confirmed that the promoter had high expression potential in Geobacillus, similar to that of the promoter.

[0035] Various Geobacillus thermophiles were transformed with pGKE123-venus or pGKE124-venus and cultured in LB medium at 50-60°C for 24 hours. m1 and P m2The mutant promoter was shown to be able to support high production of recombinant protein (Table 1). The values ​​in Table 1 indicate the amount of venus protein produced in mg / L, i.e., recombinant protein production per L of medium.

[0036] [Table 1]

[0037] We attempted to produce various recombinant proteins using the G. thermodenitrificans K1041 strain and the pGKE124 host-vector system. The coding sequences of the heterologous proteins shown in Table 2 were inserted into the pGKE124 plasmid vector. m2 The gene was inserted downstream of the promoter and introduced into strain K1041 by electroporation. The transformants were cultured in CSL medium at 55°C for 24 hours. In addition, the transformants were cultured in CSL medium at 55°C until they reached the growth phase, and then cultured at 40°C for 24 hours. The proteins in the crude extract (intracellular) and in the culture medium (extracellular) were subjected to SDS-PAGE and image processing to evaluate the amount of recombinant protein produced.

[0038] As shown in Table 2, it was confirmed that various heterologous proteins derived from mesophilic, thermophilic, and hyperthermophilic bacteria from a wide range of bacterial genera can be produced with this system. Of these, it was particularly notable that xylanase, known as a secreted protein from Geobacillus bacteria, was produced in the culture medium at a high concentration of over 0.5 g / L. Furthermore, it was shown that recombinant protein production is possible even at a relatively low temperature of 40°C. The values ​​in Table 2 indicate the amount of recombinant protein produced in mg / L.

[0039] [Table 2]

[0040] Figure 2 shows the expression of xylanase, a secretory protein derived from the thermophilic bacterium G. thermodenitrificans, in the G. thermodenitrificans host by the P-transfection of pGKE124. m2 The following shows an experiment in which xylanase was produced under the control of a promoter. In this experiment, LB medium or CSL medium was used and cultured at 55°C for one week. Samples were taken every 24 hours, and the recombinant xylanase released into the culture medium was analyzed and quantified by SDS-PAGE (Figure 2a). High production of over 1.0 g / L was achieved on the fourth day of culture in LB medium, and a similar production level was obtained in CSL medium, which is significantly cheaper than LB medium (Figure 2b). CSL medium is a 1% (v / v) Corn Steep Liquor (CSL) (Sigma-Aldrich Japan) aqueous solution with minimal medium elements (0.3 g / L K2SO4, 2.5 g / L Na2HPO4·12H2O, 1 g / L NH4Cl, 0.1% v / v trace element solution, 0.4 g / L MgSO4, 3 mg / L MnCl2·4H2O, 5 mg / L CaCl2·2H2O, 7 mg / L FeCl3·6H2O, 10 mM Tris-HCl, pH 7.5). The composition of the trace element solution is 400 mg / L ZnSO4·7H2O, 200 mg / L CuSO4·5H2O, 250 mg / L EDTA·2Na, 50 mg / L CoCl2·6H2O, 10 mg / L H3BO3, and 10 mg / L NiCl2·6H2O.

[0041] It has been reported that amylase can be produced at a high concentration of 3.0 g / L or more in high-secretory strains such as Brevibacillus and Bacillus, but it is rare to find other secretory proteins that are produced at a concentration of 1.0 g / L or more. The high production of xylanase in this example is very remarkable.

[0042] The above examples demonstrate various utilities of the embodiments of the present invention, but the invention is not limited to these specific embodiments.

Claims

1. A plasmid vector for recombinant protein production in Geobacillus bacteria, comprising: A promoter derived from the gene encoding the cold shock protein CspD of Geobacillus kaustophilus, which has a mutation in the base corresponding to the 200th and 280th bases of SEQ ID NO: 1 or the 265th base of SEQ ID NO:

1. Plasmid vector.

2. The plasmid vector of claim 1, wherein the bases corresponding to bases 200 and 280 of SEQ ID NO:1 are mutated from T to A and A to G, respectively, or the base corresponding to base 265 of SEQ ID NO:1 is mutated from T to C.

3. 3. A plasmid vector according to claim 1, comprising the nucleic acid of SEQ ID NO: 2 or SEQ ID NO: 3 as the nucleic acid providing the promoter.

4. The plasmid vector according to claim 3 , which has an SphI restriction enzyme site at the 3′ end of SEQ ID NO:2 or SEQ ID NO:

3.

5. The plasmid vector according to any one of claims 1 to 4, wherein a nucleic acid of a coding sequence encoding a recombinant protein is linked downstream of the promoter.

6. A recombinant protein production system comprising the plasmid vector according to any one of claims 1 to 5 and a Geobacillus bacterium.

7. The recombinant protein production system according to claim 6 , wherein the plasmid vector is present in the Geobacillus bacterium.

8. A promoter derived from the gene encoding the cold shock protein CspD of Geobacillus kaustophilus, which has a mutation in the bases corresponding to the 200th and 280th bases of SEQ ID NO: 1 or the 265th base of SEQ ID NO: 1; A nucleic acid of a coding sequence encoding a recombinant protein linked downstream of the promoter; and culturing a Geobacillus bacterium transformed with a nucleic acid construct comprising the above-mentioned formula (I) to express the recombinant protein.

9. The method according to claim 8, further comprising transforming the Geobacillus bacterium by introducing a plasmid vector containing the nucleic acid construct into the Geobacillus bacterium prior to the culturing.

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