CN111549050A - Vitreoscilla hemoglobin expression frame suitable for bacillus and application - Google Patents
Vitreoscilla hemoglobin expression frame suitable for bacillus and application Download PDFInfo
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Abstract
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.
Description
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.
Example 1:
construction of vitreoscilla hemoglobin expression cassettes containing different bacillus cell-penetrating peptides:
1. the P43 promoter, the SPywbN signal peptide, the vhb gene and the TamyL terminator are connected to construct a P43-SPywbN-vhb-TamyL expression cassette, and the sequence of the expression cassette is shown in SEQ ID NO. 1.
2. pHY300PLK vector is obtained by PCR amplification by taking pHY300PLK plasmid as a template, and primers are 300-T5-F: gaattcctgttataaaaaaaggatc and 300-T5-R: tctagaagcttgggcaaagcgtttt are provided.
3. Measuring the concentration of pHY300PLK vector and target gene fragment, calculating the usage amount of the vector and the fragment, preparing a reaction system (using Clon express II recombinant cloning kit, purchased from Nanjing Nodezac Biotech Co., Ltd.) on ice, performing recombination reaction at 37 ℃ for 30min, and immediately cooling on ice; ca2+Transforming the recombinant product into Escherichia coli DH5 α competence, coating thalli on a Tet resistant plate for screening, culturing in an incubator at 37 ℃, carrying out colony PCR verification on a transformant, wherein primers used are pHY-F and pHY-R, if a target band is 1657bp, the next step of sequencing can be carried out, the nucleotide sequence determination of the vector is completed by Wuhan engine biotechnology Limited company, analyzing the sequencing result, if the sequence is consistent with the design, the construction of the expression vector is successful, and the transformant is named as a free expression vector pHY-P43-SPywbN-vhb-Tamy L, and the sequence of the primer used by the colony PCR is as follows:
pHY-F:gtttattatccatacccttac
pHY-R:cagatttcgtgatgcttgtc；
4. by adopting the same method, the inventor selects bacillus Sec type and Tat type cell-penetrating peptides which comprise SacC signal peptide, Vpr signal peptide, aprE signal peptide, SacB signal peptide, BprA signal peptide, YwtF signal peptide, YwoF signal peptide, YbdN signal peptide, YvpA signal peptide, Ywad signal peptide (Sec type cell-penetrating peptide), PhoD signal peptide, TagA signal peptide and TorA signal peptide (Tat type cell-penetrating peptide) to replace SPywbN in pHY-P43-SPywbN-vhb-TamyL vector, and other expression frame elements are completely the same, and the expression frames are inserted into pHY300PLK to construct and obtain different cell-penetrating peptide mediated hyaline hemoglobin expression vectors, which sequentially and respectively: pHY-P43-SPSacC-vhb-TamyL, pHY-P43-SPVpr-vhb-TamyL, pHY-P43-SPAprE-vhb-TamyL, pHY-P43-SPSacB-vhb-TamyL, pHY-P43-SPBprA-vhb-TamyL, pHY-P43-SPYwtF-vhb-TamyL, pHY-P43-SPYwoF-vhb-TamyL, pHY-P43-SPYb865-vhb-TamyL, pHY-P43-SPYvpA-vhb-TamyL, pHY-P43-SPYwad-pHmyL, pHY-P4-SPyvbD-vhb-TamyL, SPYspyP-TamyL-pHY-43-TamyL-TavP-pHY-36yA-TamyL). In addition, as a control group, a hemoglobin expression vector pHY-P43-vhb-TamyL containing no signal peptide was constructed;
5. the hemoglobin expression vector is electrically transformed into bacillus subtilis 168 to construct corresponding signal peptide mediated bacillus subtilis hemoglobin expression strain BS168/pHY-P43-SPywbN-vhb-TamyL, BS168/pHY-P43-SPSacC-vhb-TamyL, BS168/pHY-P43-SPVpr-vhb-TamyL, BS 168/pHY-P43-SPAprae-vhb-TamyL, BS168/pHY-P43-SPSacB-vhb-TamyL, BS168/pHY-P43-SPBprA-vhb-TamyL, BS168/pHY-P43-SPYwtF-vhb-TamyL, BS 168/pHY-P43-SPYywf-vhb-TamyL, SPBS 168/pHY-P43-SPYwoF-vhb-TamyL, SPYb-TamyL-43-SPYb-TamyL, SPYB-pHY-P3527-SPYb-TamyL, SPYb-TamyL and SPYb-TamyL, BS168/pHY-P43-SPYwad-vhb-TamyL, BS168/pHY-P43-SPPhoD-vhb-TamyL, BS168/pHY-P43-SPTagA-vhb-TamyL and BS 168/pHY-P43-SPToRA-vhb-TamyL. In addition, the hemoglobin expression vector pHY-P43-vhb-TamyL without signal peptide was also transferred into Bacillus subtilis 168 to obtain BS 168/pHY-P43-vhb-TamyL.
Example 2:
the screening method is suitable for screening vitreoscilla hemoglobin expression frames of bacillus:
the bacillus subtilis hemoglobin expression strain obtained in example 1 was subjected to a poly-gamma-glutamic acid fermentation experiment. The specific steps of seed culture are as follows: respectively inoculating the bacillus subtilis strains obtained in the embodiment 1 into LB liquid culture media added with 1 per mill (v/v) Tet antibiotics, and carrying out shake cultivation at 37 ℃ for 12h at 180-300 r/min; then inoculating the activated bacterial liquid into 50mL LB liquid culture medium with the inoculation amount of 1% (v/v), adding 1 ‰ (v/v) Tet antibiotic, and culturing at 230r/min and 37 ℃ for 12h to obtain the seed culture liquid required by fermentation.
The formula of the poly-gamma-glutamic acid fermentation medium is as follows: 80g/L glucose, 30g/L sodium glutamate, 10g/L sodium citrate, 10g/L sodium nitrate, 8g/L ammonium chloride, 1g/L dipotassium hydrogen phosphate, 1g/L zinc sulfate heptahydrate, 1g/L anhydrous calcium chloride, 1g/L magnesium sulfate, 0.15g/L manganese sulfate monohydrate, pH7.2
The fermentation method comprises the following specific steps: the poly-gamma-glutamic acid fermentation medium is filled into a 500mL conical flask (the liquid filling amount of each flask is 50mL), then the seed culture solution is inoculated into the corresponding fermentation medium by the inoculation amount of 3% (v/v), and after the inoculation is finished, the seed culture solution is subjected to shake cultivation at the temperature of 230r/min and 37 ℃ for 36 hours.
The method for detecting the yield of the poly-gamma-glutamic acid comprises the following steps: the method is characterized by comprising the steps of measuring a fermentation liquid sample by adopting an ethanol sedimentation and dry weight method, firstly weighing a certain weight of the fermentation liquid sample, adjusting the pH value to 2.0-3.0 by using an HCl solution, centrifuging at 12000r/min for 10min to remove thalli, adjusting the pH value of a supernatant to be neutral by using NaOH, adding 3 times of volume of absolute ethyl alcohol to precipitate poly gamma-glutamic acid, centrifuging again, collecting precipitate, placing in an oven at 80 ℃ for drying, and measuring the mass of the poly gamma-glutamic acid. The yield of poly-gamma-glutamic acid of each strain after fermentation was calculated according to the dry weight method (specific data are shown in the following table).
TABLE 1 production of 168 poly-gamma-glutamic acid by Bacillus subtilis containing different vitreoscilla hemoglobin expression cassettes
| Bacterial strains | Yield of poly-gamma-glutamic acid (g/L) |
| BS168/pHY-P43-SPywbN-vhb-TamyL | 36.75 |
| BS168/pHY-P43-SPSacC-vhb-TamyL | 27.32 |
| BS168/pHY-P43-SPVpr-vhb-TamyL | 29.75 |
| BS168/pHY-P43-SPAprE-vhb-TamyL | 30.43 |
| BS168/pHY-P43-SPSacB-vhb-TamyL | 31.24 |
| BS168/pHY-P43-SPBprA-vhb-TamyL | 29.54 |
| BS168/pHY-P43-SPYwtF-vhb-TamyL | 31.25 |
| BS168/pHY-P43-SPYwoF-vhb-TamyL | 24.32 |
| BS168/pHY-P43-SPYbdN-vhb-TamyL | 21.46 |
| BS168/pHY-P43-SPYvpA-vhb-TamyL | 28.43 |
| BS168/pHY-P43-SPYwaD-vhb-TamyL | 30.43 |
| BS168/pHY-P43-SPPhoD-vhb-TamyL | 28.43 |
| BS168/pHY-P43-SPTagA-vhb-TamyL | 26.54 |
| BS168/pHY-P43-SPTorA-vhb-TamyL | 31.25 |
| BS168/pHY-P43-vhb-TamyL | 28.43 |
The results show that when the YwbN signal peptide is adopted, the vitreoscilla hemoglobin expression effect is the best (namely the vitreoscilla hemoglobin expression frame shown in SEQ ID NO. 1), the poly-gamma-glutamic acid promotion effect is the most obvious, and meanwhile, not all Tat-type signal peptides are suitable for the expression of the vitreoscilla hemoglobin in bacillus, and no principle can be deduced as to which signal peptide is suitable for the hemoglobin expression.
Example 3:
the application of the P43-SPywbN-vhb-TamyL expression cassette in improving the fermentation yield of the bacillus subtilis poly gamma-glutamic acid is as follows:
in this example, the ability of Bacillus subtilis BS168/pH Y-P43-SPywbN-vhb-TamyL to produce poly-gamma-glutamic acid was examined for different poly-gamma-glutamic acid fermentation medium formulations (while inoculating Bacillus subtilis BS168/pH Y-P43-vhb-TamyL as a control to these 21 media), and the formulation of 21 groups of media was specifically shown in Table 2:
TABLE 2 different poly-gamma-glutamic acid fermentation medium formulas
The specific steps of seed culture are as follows: inoculating the bacillus subtilis strain BS168/pHY-P43-SPywbN-vhb-TamyL obtained in the example 1 and a control strain bacillus subtilis strain BS168/pHY-P43-vhb-TamyL into an LB liquid culture medium added with 1 per thousand (v/v) Tet antibiotic, and carrying out shake culture at 37 ℃ at 180-300 r/min for 12 hours; then inoculating the activated bacterial liquid into 50mL LB liquid culture medium with the inoculation amount of 1% (v/v), simultaneously adding 1 ‰ (v/v) Tet antibiotic, and shake-culturing at 230r/min and 37 ℃ for 12h to obtain a seed culture solution required by fermentation;
the fermentation method comprises the following specific steps: the different poly-gamma-glutamic acid fermentation media in table 2 were filled into 500mL Erlenmeyer flasks (50 mL liquid content per flask), then the seed culture solution was inoculated into the corresponding fermentation media at an inoculum size of 3% (v/v), and after inoculation, shaking cultured at 37 ℃ at 230r/min for 36 h.
The method for detecting the yield of the poly-gamma-glutamic acid comprises the following steps: the method is characterized by comprising the steps of measuring a fermentation liquid sample by adopting an ethanol sedimentation and dry weight method, firstly weighing a certain weight of the fermentation liquid sample, adjusting the pH value to 2.0-3.0 by using an HCl solution, centrifuging at 12000r/min for 10min to remove thalli, adjusting the pH value of a supernatant to about 7.0 by using an NaOH solution, adding 3 times of anhydrous ethanol to enable poly gamma-glutamic acid to be sedimentated, centrifuging again to collect sediment, placing in an oven at 80 ℃ for drying, and measuring the dry weight of the poly gamma-glutamic acid. And (3) calculating the yield of the poly-gamma-glutamic acid in the fermented bacterial liquid (see the specific data in the table 3).
TABLE 3 Poly-gamma-glutamic acid yields after fermentation with different medium recipes
As can be seen from Table 3, under the same seed fermentation and different production fermentations, compared with the Bacillus subtilis BS168/pHY-P43-vhb-TamyL of the prior art, the production fermentation of poly-gamma-glutamic acid by using the Bacillus subtilis strain BS168/pHY-P43-SPywbN-vhb-TamyL of the invention has greatly improved (at least 21.85 percent improved) yield, which shows that: the P43-SPywbN-vhb-TamyL expression cassette has great application value in the aspect of improving the yield of poly gamma-glutamic acid of bacillus subtilis.
Example 4:
the application of the P43-SPywbN-vhb-TamyL expression cassette in improving the fermentation yield of the bacillus amyloliquefaciens iturin A is as follows:
the hemoglobin expression vectors pHY-P43-SPywbN-vhb-TamyL and pHY-P43-vhb-TamyL are respectively transformed into the bacillus amyloliquefaciens LX-12 to obtain the bacillus amyloliquefaciens LX-12/pHY-P43-SPywbN-vhb-TamyL and LX-12/pHY-P43-vhb-TamyL. In this example, the ability of Bacillus amyloliquefaciens strain LX-12/pHY-P43-SPywbN-vhb-TamyL to produce iturin A was examined for different iturin A fermentation medium formulations (while Bacillus amyloliquefaciens LX-12/pHY-P43-vhb-TamyL was inoculated into these 13 media as a control), and the 13 group media formulations were specifically shown in Table 4:
TABLE 4 different iturin A fermentation medium formulations
The specific steps of seed culture are as follows: inoculating a bacillus amyloliquefaciens strain LX-12/pHY-P43-SPywbN-vhb-TamyL and a contrast strain bacillus amyloliquefaciens LX-12/pHY-P43-vhb-TamyL into an LB liquid culture medium added with 1 per mill (v/v) Tet antibiotic, and performing shake culture at the temperature of 230r/min and 37 ℃ for 12 hours; then inoculating the activated bacterial liquid into 50mL LB liquid culture medium with the inoculation amount of 1% (v/v), simultaneously adding 1 ‰ (v/v) Tet antibiotic, and culturing at 30 ℃ at 230r/min for 12h to obtain a seed culture solution required by fermentation;
the method comprises the following specific steps of iturin A fermentation: the different fermentation media of the iturin A in the table 4 are filled into 500mL conical flasks (the liquid filling amount of each flask is 150mL), then the seed culture solution is inoculated into the corresponding fermentation medium with the inoculation amount of 3% (v/v), after the inoculation, the seed culture solution is subjected to shake cultivation at the temperature of 28 ℃ of 250r/min for 72h, and the fermentation liquid is collected for the yield detection of the iturin A.
The method for detecting the yield of the iturin A comprises the following steps: and detecting the sample by using a High Performance Liquid Chromatography (HPLC) method. The HPLC system was an Agilent 1260 liquid chromatograph, a chromatographic column Lichrospher C18 (specification: 5 μm, 25 cm. times.4.6 mm), a mobile phase of 10mmol/L ammonium acetate/acetonitrile 65:35(v/v), a sample size of 10 μ L, a detection wavelength of 210nm, and a flow rate of 1.0 mL/min. The yield of iturin A in the fermented broth was calculated according to this method (see Table 5 for data).
TABLE 5 yield of iturin A after fermentation of different medium formulations
As can be seen from Table 5, under the same seed fermentation and different production fermentations, the yield of iturin A produced by fermentation using the Bacillus amyloliquefaciens strain LX-12/pHY-P43-SPywbN-vhb-TamyL of the present invention is greatly increased (by at least 18.77%) compared to the control strain of the prior art, i.e., Bacillus amyloliquefaciens LX-12/pHY-P43-vhb-TamyL, indicating that: the P43-SPywbN-vhb-TamyL expression cassette has great application value in the aspect of improving the yield of iturin A of bacillus amyloliquefaciens.
Example 5:
the application of the P43-SPywbN-vhb-TamyL expression cassette in improving the fermentation yield of bacitracin of Bacillus licheniformis:
the hemoglobin expression vectors pHY-P43-SPywbN-vhb-TamyL and pHY-P43-vhb-TamyL are respectively transformed into Bacillus licheniformis DW2 to obtain Bacillus licheniformis DW2/pHY-P43-SPywbN-vhb-TamyL and DW 2/pHY-P43-vhb-TamyL. In this example, the ability of Bacillus licheniformis strain DW2/pHY-P43-SPywbN-vhb-TamyL to produce bacitracin was examined for different bacitracin fermentation media formulations (while Bacillus licheniformis strain DW2/pHY-P43-vhb-TamyL was inoculated into these 11 media as a control), and the 11 media formulations were specifically shown in Table 6:
TABLE 6 different bacitracin fermentation media formulations
| Recipe number | Bean pulp (g/L) | Corn starch (g/L) | CaCO3(g/L) | (NH4)2SO4(g/L) |
| 1 | 80 | 30 | 4 | 0.5 |
| 2 | 60 | 30 | 4 | 0.5 |
| 3 | 100 | 30 | 4 | 0.5 |
| 4 | 80 | 15 | 4 | 0.5 |
| 5 | 80 | 45 | 4 | 0.5 |
| 6 | 80 | 30 | 2 | 0.5 |
| 7 | 80 | 30 | 6 | 0.5 |
| 8 | 80 | 30 | 8 | 0.5 |
| 9 | 80 | 30 | 4 | 0.25 |
| 10 | 80 | 30 | 4 | 0.75 |
| 11 | 80 | 30 | 4 | 1 |
The specific steps of seed culture are as follows: inoculating Bacillus licheniformis strain DW2/pHY-P43-SPywbN-vhb-TamyL and control strain Bacillus licheniformis strain DW2/pHY-P43-vhb-TamyL into LB liquid culture medium added with 1 per mill (v/v) Tet antibiotic, and performing shake culture at 230r/min and 37 ℃ for 12 h; then inoculating the activated bacterial liquid into 50mL LB liquid culture medium with the inoculation amount of 1% (v/v), simultaneously adding 1 ‰ (v/v) Tet antibiotic, and shake-culturing at 230r/min and 37 ℃ for 12h to obtain a seed culture solution required by fermentation;
the fermentation method comprises the following specific steps: the different bacitracin fermentation media in table 6 were filled into 500mL Erlenmeyer flasks (liquid content per flask was 70mL), then the seed culture broth was inoculated into the corresponding fermentation media at an inoculum size of 2% (v/v), after inoculation, shaking culture was carried out at 28 ℃ at 230r/min for 48h, and the fermentation broth was collected for bacitracin yield detection.
The method for detecting the yield of bacitracin comprises the following steps: and detecting the sample by using a High Performance Liquid Chromatography (HPLC) method. The HPLC system is Agilent 1260 liquid chromatograph, chromatographic column Hypersil BDS C18(5 μm,4.6 mm. times.250 mm), mobile phase A: B35: 65 (phase A: 100mL pH6.0 phosphate buffer solution to 300mL water mixed well; phase B: 520mL methanol to 40mL acetonitrile mixed well); the sample volume is 20 mu L; the detection wavelength is 254 nm; the flow rate was 1.0 mL/min. The yield of bacitracin in the fermented broth was calculated according to this method (see Table 7 for data).
TABLE 7 bacitracin yields after fermentation with different medium formulations
As can be seen from Table 7, under the same seed fermentation and different production fermentations, the yield of bacitracin was greatly increased (at least by 23.40%) using the Bacillus licheniformis strain DW2/pHY-P43-SPywbN-vhb-TamyL of the present invention, compared to the control strain Bacillus licheniformis strain DW2/pHY-P43-vhb-TamyL of the prior art. Description of the drawings: the P43-SPywbN-vhb-TamyL expression cassette has important application value in the aspect of improving the yield of bacillus licheniformis bacitracin.
Sequence listing
<110> university of Hubei
<120> vitreoscilla hemoglobin expression frame suitable for bacillus and application
<160>18
<170>SIPOSequenceListing 1.0
<210>1
<211>1379
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60
taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120
tttgcgtttt taccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240
gcgattatgt aaaatataaa gtgatagcgg taccattata ggtaagagag gaatgtacac 300
atgaaatgag cgatgaacaa aaaaagccag aacaaattca tagacgggac attttaaaat 360
ggggagcgat ggcgggggca gccgttgcga tcggtgccag cggtctcggc ggtctcgctc 420
cgcttgttca gactgcgatg ttagaccagc aaaccattaa catcatcaaa gccactgttc 480
ctgtattgaa ggagcatggc gttaccatta ccacgacttt ttataaaaac ttgtttgcca 540
aacaccctga agtacgtcct ttgtttgata tgggtcgcca agaatctttg gagcagccta 600
aggctttggc gatgacggta ttggcggcag cgcaaaacat tgaaaatttg ccagctattt 660
tgcctgcggt caaaaaaatt gcagtcaaac attgtcaagc aggcgtggca gcagcgcatt 720
atccgattgt cggtcaagaa ttgttgggtg cgattaaaga agtattgggc gatgccgcaa 780
ccgatgacat tttggacgcg tggggcaagg cttatggcgt gattgcagat gtgtttattc 840
aagtggaagc agatttgtac gctcaagcgg ttgaataaaa gagcagagag gacggatttc 900
ctgaaggaaa tccgtttttt tattttgccc gtcttataaa tttctttgat tacattttat 960
aattaatttt aacaaagtgt catcagccct caggaaggac ttgctgacag tttgaatcgc 1020
ataggtaagg cggggatgaa atggcaacgt tatctgatgt agcaaagaaa gcaaatgtgt 1080
cgaaaatgac ggtatcgcgg gtgatcaatc atcctgagac tgtgacggat gaattgaaaa 1140
agcttgttca ttccgcaatg aaggagctca attatatacc gaactatgca gcaagagcgc 1200
tcgttcaaaa cagaacacag gtcgtcaagc tgctcatact ggaagaaatg gatacaacag 1260
aaccttatta tatgaatctg ttaacgggaa tcagccgcga gctggaccgt catcattatg 1320
ctttgcagct tgtcacaagg aaatctctca atatcggcca gtgcgacggc attattgcg 1379
<210>2
<211>66
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
atgaaaaaga gaatgattca gatggggatc ataggggcta tgatgttccc ggaagccttt 60
tccgca 66
<210>3
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
ttgagaaaaa gtatcgtgcg ctattttgtt atggctttta ttctattatt tgcgttatcc 60
acattcctca ccggagtgca ggca 84
<210>4
<211>81
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60
atggcattca gcgattccgc t 81
<210>5
<211>87
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
atgaacatca aaaacattgc taaaaaagcg tcagccttaa ccgttgctgc ggcactgctg 60
gccggaggtg cgccgcaaac ctttgca 87
<210>6
<211>96
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
gtgaagaaaa agccattatt cagcacattc atgtgcgctg cactcatcgg ttcacttctc 60
gctccggctg ctgtgcaggc tgaaacaggc acgaca 96
<210>7
<211>93
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
atgctacgat cgcagcgaac gaagaaaaag agactaagaa aatgggtgaa atactcactg 60
tttttcattg ccttaatcct gacggcgacg gca 93
<210>8
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
atgagaaaat ggtattttat tttatcagcg tgtattttag tttctgttat catcgctttt 60
gct 63
<210>9
<211>75
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
atgaaaaaaa gtttgtttct tttcgtgttc agtgtgtttt tgatggcgat tccagcattt 60
tcggcttcgg caaat 75
<210>10
<211>87
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
ttgaaaaaaa tcgtgtctat cctatttatg ttcggtttgg ttatgggttt cagccagttt 60
cagccatcaa ccgtttttgc agctgac 87
<210>11
<211>90
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
atgaagagaa aaatgatgat gttcggattg gcgctatcga tcattgcagg cggcgtggtc 60
gccgatggaa cggggaatgc agctcaagcg 90
<210>12
<211>138
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
atgaaaaaac tgagcgagga aagcctcaag gacaatacgt ttgaccgccg ccgctttatt 60
caaggggccg gcaaaatagc cgggctttcg ctcggacttg cgatcgcgca atcgatgggg 120
gcaatggaag tcaatgca 138
<210>13
<211>108
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
atgctgcaaa cggaaacggt tcatcatctt gcatatgtta acggggattt gcccggattt 60
ttgaatcatc ttgaaaaatc ctttatcgac cgcaatgagg gggcattt 108
<210>14
<211>117
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
atgaacaata acgatctctt tcaggcatca cgtcggcgtt ttctggcaca actcggcggc 60
ttaaccgtcg ccgggatgct ggggccgtca ttgttaacgc cgcgacgtgc gactgcg 117
<210>15
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
gaattcctgt tataaaaaaa ggatc 25
<210>16
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
tctagaagct tgggcaaagc gtttt 25
<210>17
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
gtttattatc cataccctta c 21
<210>18
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
cagatttcgt gatgcttgtc 20
Claims (8)
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.
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