ISOLATED POLYNUCLEOTIDE INCLUDING PROMOTER REGION, HOST CELL INCLUDING THE SAME AND METHOD OF EXPRESSING A TARGET GENE USING THE HOST CELL

Abstract

An isolated polynucleotide including a promoter region derived from a bacterium of the genus Paracoccus, a recombinant host cell including the isolated polynucleotide, and a method of expressing a target gene by culturing a recombinant host cell comprising a polynucleotide comprising the promoter region and a target gene operably linked to the promoter region, under conditions in which the target gene is expressed.

Claims

1. An isolated polynucleotide comprising a promoter region comprising the nucleotide sequence of SEQ ID NO: 1.

2. The isolated polynucleotide of claim 1, wherein the isolated polynucleotide consists of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

3. The isolated polynucleotide of claim 1, wherein the isolated polynucleotide is a vector.

4. The isolated polynucleotide of claim 1, wherein a target gene is operably linked to the promoter region.

5. The isolated polynucleotide of claim 4, wherein the target gene encodes a target protein.

6. The isolated polynucleotide of claim 5, wherein the target protein has the amino acid sequence of SEQ ID NO: 3.

7. A recombinant host cell comprising an isolated polynucleotide comprising a promoter region comprising the nucleotide sequence of SEQ ID NO: 1.

8. The recombinant host cell of claim 7, wherein the isolated polynucleotide has been introduced to the recombinant host cell.

9. The recombinant host cell of claim 7, wherein the recombinant host cell is a bacterial cell.

10. The recombinant host cell of claim 7, wherein the recombinant host cell is different from the cell from which the promoter is derived.

11. The recombinant host cell of claim 7, wherein the recombinant host cell is a bacterial cell of the genus Paracoccus or the genus Escherichia.

12. The recombinant host cell of claim 7, wherein the recombinant host cell is Paracoccus versutus or Escherichia coli.

13. The recombinant host cell of claim 7, wherein the isolated polynucleotide is a vector.

14. The recombinant host cell of claim 7, wherein a target gene is operably linked to the promoter region.

15. The recombinant host cell of claim 14, wherein the target gene encodes a target protein.

16. The recombinant host cell of claim 14, wherein the target protein has the amino acid sequence of SEQ ID NO: 3.

17. A method for expressing a target gene, the method comprising: culturing a recombinant host cell comprising an isolated polynucleotide comprising a promoter region and a target gene operably linked to the promoter region, under conditions in which the target gene is expressed, wherein the promoter region comprises the nucleotide sequence of SEQ ID NO: 1.

18. The method of claim 17, wherein the recombinant host cell is a bacterial cell.

19. The method of claim 17, wherein the target gene encodes a target protein.

20. The method of claim 17, wherein the culturing is performed under aerobic conditions.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0041] FIG. 1 illustrates a map of a pBBR-122 vector;

[0042] FIG. 2 illustrates a map of a pB_PEPCK vector; and

[0043] FIG. 3 is a graph of relative fluorescence versus test sample, which shows the results of measuring fluorescence emitted from mRFP protein expressed by each promoter in Paracoccus versutus.

DETAILED DESCRIPTION

[0044] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modifies the entire list of elements and does not modify the individual elements of the list.

[0045] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, including “at least one,” unless the content clearly indicates otherwise. “At least one” is not to be construed as limiting “a” or “an.” “or” means “and/or.” As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and/or “comprising,” or “includes” and/or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

[0046] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0047] Hereinafter, the present disclosure will be described in more detail with reference to the Examples. However, these Examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these Examples.

Example 1: Exploration and Identification of Paracoccus versutus Promoter

[0048] In this Example, genes were selected from a denitrifying microorganism Paracoccus versutus (strain DSM 582) and putative promoters of each of the genes were isolated, respectively. Vectors including each promoter operably linked to a reporter protein-encoding gene was constructed, and each vector was introduced into the host strain to examine promoter strength.

(1) Exploration of Promoter

[0049] Polynucleotides which were considered to include a promoter were obtained from gene regions encoding glyceraldehyde-3-phosphate dehydrogenase (gapD), phosphoglycerate kinase (pgk), or phosphoenolpyruvate carboxykinase (pepck) of Paracoccus versutus. The promoter region was amplified by PCR using primers designed for in-fusion cloning, and then used for vector construction. The primer sets used for PCR amplification of the promoter regions of the gapD, pgk, and pepck genes were polynucleotides having the sequences of SEQ ID NOS: 5 and 6; SEQ ID NOS: 7 and 8; and SEQ ID NOS: 9 and 10, respectively.

(2) Construction of Vector

[0050] A vector including the obtained promoter polynucleotide was constructed. The vector was constructed as follows.

[0051] A pB-GAP vector was constructed as follows. A pBBR-122 vector (Mo Bi Tec) as a template and a primer set having the sequences of SEQ ID NOS: 11 and 12 were used to amplify the pBBR-122 vector. The genomic DNA of Paracoccus versutus strain was used as a template with the primer set of SEQ ID NOS: 5 and 6 to amplify the promoter region of the gapD gene. The primer set having the nucleotide sequences of SEQ ID NOS: 13 and 14 was used to amplify mRFP, which was then cloned into the amplified vector backbone using an In-fusion GD cloning kit (Takara), thereby constructing a pB-GAP vector.

[0052] The vector region was amplified using the pB-GAP vector as a template and a primer set of SEQ ID NOS: 11 and 12, and each of the promoter regions amplified using a primer set of SEQ ID NOS: 9 and 10 or a primer set of SEQ ID NOS: 7 and 8 were cloned using an In-Fusion GD cloning kit (Takara) to construct pB-PEPCK and pB-PGK vectors, respectively.

[0053] FIG. 1 illustrates a map of the pBBR-122 vector. mRFP represents monomeric red fluorescent protein (RFP).

[0054] FIG. 2 illustrates a map of the pB_PEPCK vector.

(3) Comparative Evaluation of Promoter Performance

[0055] Vectors including the mRFP gene downstream of the promoter polynucleotides of three genes derived from Paracoccus versutus were constructed as described in (2), and each of the vectors was introduced into Paracoccus versutus strain. The introduction was performed by electroporation. As control groups, buffer and pBBR-122 vector were introduced into Paracoccus versutus strain in the same manner, respectively.

[0056] Each strain was cultured in an LB medium under conditions of 30° C. and with shaking at 230 rpm, for 18 hr. After culturing, the strain was isolated and quantified at A600 of 1. Fluorescence was measured at excitation of 485 nm and emission of 590 nm using Spectramax M5 to determine mRFP expression. The results are shown in Table 1 and FIG. 3. FIG. 3 shows results of measuring fluorescence emitted from mRFP protein expressed by each promoter in Paracoccus versutus.

TABLE-US-00001 TABLE 1 pepck gapD pgk PBS promoter promoter promoter Sample buffer pBBR122 (pB_PEPCK) (pB-GAP) (pB-PGK) Relative 3.13 1.78 242.08 4.35 13.75 fluores- ence

[0057] As shown in Table 1 and in FIG. 3, PBS buffer, pBBR122, gapD promoter, and pgk promoter showed very low mRFP fluorescence, whereas the pepck promoter showed significantly high fluorescence. These results indicate that the pepck promoter is a very strong promoter capable of efficiently expressing a foreign (heterologous) target protein.

[0058] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope as defined by the following claims.