阅读说明：本技术 适用于芽胞杆菌的透明颤菌血红蛋白表达框及应用 (Vitreoscilla hemoglobin expression frame suitable for bacillus and application ) 是由 陈守文 张清 蔡冬波 陈耀中 杨帆 马昕 于 2020-05-18 设计创作，主要内容包括：本发明属于生物技术与发酵工程领域,提供了适用于芽胞杆菌的透明颤菌血红蛋白表达框及应用,本发明将透明颤菌血红蛋白VHb与枯草芽胞杆菌中YwbN的信号肽SPywbN以及枯草芽胞杆菌启动子P43、地衣芽胞杆菌中淀粉酶终止子TamyL相连,组成VHb蛋白表达框P43-SPywbN-<I>vhb-</I>TamyL。将该表达框应用于三种芽胞杆菌(枯草芽胞杆菌168、解淀粉芽胞杆菌LX-12、地衣芽胞杆菌DW2)中,与常规的单独强化表达VHb菌株相比,分别使得聚γ-谷氨酸、伊枯草菌素A、杆菌肽的产量提高21.85%、18.77%和23.40%,本发明为透明颤菌血红蛋白VHb在芽胞杆菌中高效表达和广泛应用提供了理论指导。(The invention belongs to the field of biotechnology and fermentation engineering, and provides a vitreoscilla hemoglobin expression frame suitable for bacillus and application thereofBlock P43-SPywbN- vhb‑ TamyL. The expression frame is applied to three kinds of bacillus (bacillus subtilis 168, bacillus amyloliquefaciens LX-12 and bacillus licheniformis DW2), compared with a conventional single intensified expression VHb strain, the yield of poly-gamma-glutamic acid, iturin A and bacitracin is respectively improved by 21.85%, 18.77% and 23.40%, and the invention provides theoretical guidance for efficient expression and wide application of vitreoscilla hemoglobin VHb in bacillus.)

1. The vitreoscilla hemoglobin expression frame suitable for bacillus is shown in SEQ ID No. 1.

2. The use of the vitreoscilla hemoglobin expression cassette of claim 1 for increasing the fermentation efficiency of bacillus, wherein the expression vector is an expression vector suitable for bacillus.

3. The use of claim 2, wherein said bacillus comprises bacillus subtilis, bacillus amyloliquefaciens, or bacillus licheniformis.

4. The use of claim 3, wherein the Bacillus subtilis is Bacillus subtilis 168, the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens LX-12, and the Bacillus licheniformis is Bacillus licheniformis DW 2.

5. The use of the vitreoscilla hemoglobin expression cassette of claim 1 to enhance fermentation of bacillus subtilis 168 to produce poly-gamma-glutamic acid.

6. The use of the vitreoscilla hemoglobin expression cassette of claim 1 to increase the fermentation production of iturin a by bacillus amyloliquefaciens LX-12.

7. The use of the vitreoscilla hemoglobin expression cassette of claim 1 to enhance the fermentative production of bacitracin by bacillus licheniformis DW 2.

8. The use according to any one of claims 5 to 6, wherein the expression vector used in the application process is pHY300PLK vector.

Technical Field

The invention belongs to the field of biotechnology and fermentation engineering, and particularly relates to a vitreoscilla hemoglobin expression frame suitable for bacillus and application thereof.

Background

Vitreoscilla hemoglobin (VHb) is the first hemoglobin found in bacteria and is derived from Vitreoscilla, a gram-negative bacterium. In its natural state, VHb exists as a homodimer, with two subunits forming 6 alpha-helices (A, B, E, F, G, H) of 146 amino acid residues, a relative molecular weight of 15775, and each containing one molecule of B-type heme. Under the condition of hypoxia, VHb can be combined with oxygen to generate conformational change and can be dissociated with the oxygen to transmit the oxygen to a respiratory chain so as to regulate the activity of the respiratory chain terminal oxidase. VHb has been successfully expressed in various host cells (E.coli, Pseudomonas, tobacco, rice, zebrafish, etc.) for improving animal and plant cell characteristics, improving bacterial growth in high oxygen consumption or oxygen limited high density fermentations.

Although vitreoscilla hemoglobin has been successfully and heterogeneously expressed in Bacillus (Albizzia et al, microbiological bulletin, 2008,11: 1703-.

The protein YwbN with unknown function in the bacillus subtilis is a protein secreted according to a Tat secretion pathway, and comprises a double arginine signal peptide, and the N end of the protein contains a double arginine (RR/KR) secondary structure. However, the function of the YwbN protein is currently unknown. In previous studies, one could increase the expression level of the target protein by screening for signal peptides. However, relevant literature studies also suggest that the screening of cell-penetrating peptides for different proteins of interest is not universal, i.e., one needs to select an appropriate cell-penetrating peptide for a particular protein of interest. However, there is no literature report that the transmembrane peptide YwbN affects the synthesis of the protein of interest.

In addition, although there are studies on coupled expression of hemoglobin with Tat-type cell-penetrating peptide, oxygen supply efficiency and PHA yield in Halomonas were improved (Ouyang et al, Metab Eng,2018,45: 20-31). However, the article does not list specific types of cell-penetrating peptides. In addition, the halomonas has no similarity with the genome information of the bacillus, and the physiological forms are greatly different. Therefore, efficient expression of vitreoscilla hemoglobin in bacillus and efficient synthesis of metabolites based on efficient expression of vitreoscilla hemoglobin cannot be performed according to prior results.

The applicant finds that the vitreoscilla hemoglobin VHb and the cell-penetrating peptide are coupled and expressed, so that the oxygen transfer efficiency of cells can be improved, and the synthesis of metabolic products is facilitated. The Vitreoscilla hemoglobin gene VHb is connected with a signal peptide SPywbN of unknown functional protein YwbN in bacillus subtilis, a bacillus subtilis promoter P43 and a bacillus licheniformis amylase terminator TamyL to form a VHb expression frame P43-SPywbN-VHb-TamyL. The expression frame is applied to three kinds of bacillus (bacillus subtilis 168, bacillus amyloliquefaciens LX-12 and bacillus licheniformis DW2) to respectively improve the yield of poly-gamma-glutamic acid, iturin A and bacitracin. The research result of the invention provides guidance for the efficient expression and wide application of Vitreoscilla hemoglobin VHb in bacillus in industrial production.

Disclosure of Invention

The invention aims to provide a vitreoscilla hemoglobin expression frame suitable for bacillus, wherein the nucleotide sequence of the expression frame is shown in SEQ ID No. 1.

It is another object of the present invention to provide the use of vitreoscilla hemoglobin expression cassettes suitable for use with Bacillus. In order to achieve the purpose, the invention adopts the following technical measures:

the vitreoscilla hemoglobin expression frame suitable for bacillus is shown in SEQ ID No. 1.

The application of vitreoscilla hemoglobin expression frame in improving the fermentation efficiency of bacillus comprises introducing an expression vector containing vitreoscilla hemoglobin expression frame into bacillus, and carrying out conventional fermentation production on the transgenic strain.

The sequence of the vitreoscilla hemoglobin expression frame is shown in SEQ ID NO. 1;

the expression vector is suitable for bacillus;

in the above application, preferably, the bacillus includes bacillus subtilis, bacillus amyloliquefaciens and bacillus licheniformis.

In the above application, preferably, the bacillus subtilis is bacillus subtilis 168, the bacillus amyloliquefaciens is bacillus amyloliquefaciens LX-12, and the bacillus licheniformis is bacillus licheniformis DW 2;

the application comprises the application of the vitreoscilla hemoglobin expression frame in improving the fermentation production of poly-gamma-glutamic acid by bacillus subtilis 168;

the application comprises the application of the vitreoscilla hemoglobin expression frame in improving the fermentation production of iturin A by the bacillus amyloliquefaciens LX-12;

the application comprises the application of the vitreoscilla hemoglobin expression frame in improving the fermentation production of bacitracin by bacillus licheniformis DW 2;

in the above application, preferably, the expression vector is pHY300PLK vector.

Compared with the prior art, the invention has the following advantages:

the vitreoscilla hemoglobin gene VHb is connected with a signal peptide S PywbN of unknown functional protein YwbN in bacillus subtilis, a bacillus subtilis promoter P43 and a bacillus licheniformis amylase terminator TamyL to form a VHb expression frame P43-SPywbN-VHb-TamyL, and the yield of poly-gamma-glutamic acid, iturin A and bacitracin of three kinds of bacillus (bacillus subtilis 168, bacillus amyloliquefaciens LX-12 and bacillus licheniformis DW2) containing the expression frame is improved to different degrees. The result of the invention shows that the construction of the VHb expression frame P43-SPywbN-VHb-TamyL is a method for improving the vitreoscilla hemoglobin expression efficiency in bacillus or has reference significance for wider industrial production of bacillus.

Detailed Description

The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.