Bacitracin fluorescence response system based on two-component system and application of bacitracin fluorescence response system
By constructing a bacitracin fluorescence response system based on a two-component system, and utilizing the promoter PbceR and the signal amplification device T7 RNA polymerase, efficient detection and high-throughput screening of bacitracin were achieved. This solved the problem of high detection costs in the bacitracin production process and enabled the application of a highly efficient whole-cell biosensor for bacitracin.
Patent Information
- Application Number
- CN202511256563.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-04
- Publication Date
- 2025-11-28
AI Technical Summary
Existing technologies make it difficult to construct and screen large-scale, multi-parallel libraries in the efficient production process of bacitracin, and traditional detection methods are costly and difficult to achieve high-throughput screening.
A two-component bacitracin fluorescence response system was constructed. The bceR and bceS proteins were expressed using the promoter PbceR. Combined with the signal amplification device T7 RNA polymerase and T7 promoter, the efficient detection of bacitracin was achieved through fluorescent protein expression. A whole-cell bacitracin biosensor was constructed and integrated into Bacillus subtilis for expression.
It achieves ultrasensitive detection and wide-range quantification of bacitracin, with a detection throughput of up to 106 samples/day and a reduction in detection cost of over 90%. It provides a solution for high-throughput screening of high-yield bacitracin strains and intelligent monitoring of the fermentation process.
Smart Images
Figure CN121022897A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of bioengineering technology, in particular to a bacitracin fluorescent response system based on a two-component system and application thereof. BACKGROUND
[0002] Bacitracin, as a broad-spectrum polypeptide antibiotic synthesized by non-ribosomal peptide synthesis enzyme (NRPS), is a secondary metabolite produced by fermentation of Bacillus subtilis, Bacillus licheniformis and Bacillus sonorensis.
[0003] Bacitracin has strong bactericidal effect on gram-positive bacteria and most gram-negative bacteria. Its mechanism of action is that it can combine with isoprene pyrophosphate to form a specific inhibitor of lipid pyrophosphate, resulting in the inability to supply muropeptide to the cell wall scaffold, thereby inhibiting the synthesis of the cell wall. It also binds to the cytoplasmic membrane of sensitive bacteria, leading to impaired cell membrane permeability and the outflow of important substances, ultimately disrupting the normal metabolism of microbial cells and causing cell death.
[0004] Current research on bacitracin biosynthesis is mainly focused on Bacillus licheniformis, including central metabolic reconstruction, enhancement of precursor amino acid supply, modification of transcriptional regulatory factors, and energy metabolism engineering. Currently, bacitracin production is mainly detected by high-performance liquid chromatography, which makes it difficult to achieve large-scale multi-parallel library construction and screening in the process of constructing high-efficiency bacitracin production chassis microorganisms. SUMMARY
[0005] To solve the above technical problems, the present application constructs a bacitracin fluorescent response system, which uses promoter P bceR expresses bceR and bceS proteins, uses promoter P bceA expresses fluorescent proteins, and adds a signal amplifier device upstream of the fluorescent proteins, which contains T7 RNA polymerase and T7 promoter. bceA The structure of the bacitracin fluorescent response system is optimized by replacing the RBS sequence and truncating the promoter P
[0006] The first object of the present application is to provide a bacitracin fluorescent response system based on a two-component system, which uses promoter P bceR expresses bceR and bceS proteins, uses promoter P bceA expresses fluorescent proteins.
[0007] Histidine protein kinases (HPKS) in two-component system as a sensor, can recognize bacitracin. After recognizing the target, HPKS will bind ATP to phosphorylate itself to form HPKS-P protein. After binding with downstream transcription factor (TF) R, HPKS-P protein transfers phosphate group to obtain TFR-P protein, and mediates the expression of genes corresponding to promoters regulated by the TF downstream.
[0008] Further, the nucleotide sequence of the promoter P bceR is shown as SEQ ID NO. 5.
[0009] Further, the nucleotide sequence of the bceS is shown as SEQ ID NO. 6.
[0010] Further, the nucleotide sequence of the bceR is shown as SEQ ID NO. 7.
[0011] Further, the nucleotide sequence of the promoter P bceA is shown as SEQ ID NO. 11. Further, the RBS sequence exists upstream of the fluorescent protein.
[0012] Further, the bacitracin fluorescent response system further comprises a signal amplifier device, wherein the signal amplifier device comprises T7 RNA polymerase and T7 promoter, and the signal amplifier device is located upstream of the fluorescent protein.
[0013] Bacitracin activates the BceRS two-component system, causes the regulatory protein BceR to bind and start the P bceA promoter, and drives the expression of T7 RNA polymerase; then T7 RNA polymerase specifically recognizes the multiple copies of T7 promoter and efficiently transcribes the fluorescent protein.
[0014] Further, the RBS sequence exists upstream of the T7 RNA polymerase.
[0015] Further, the RBS sequence is shown as SEQ ID NO. 22.
[0016] Further, the P bceA promoter is truncated.
[0017] Further, the P bceA promoter with the nucleotide sequence shown as SEQ ID NO. 11 has the first to 200th nucleotides truncated.
[0018] The second objective of this invention is to provide a bacitracin whole-cell biosensor based on a two-component system, wherein the bacitracin whole-cell biosensor includes the aforementioned bacitracin fluorescence response system, and the chassis strain is Bacillus subtilis.
[0019] Furthermore, the bacitracin fluorescent response system is integrated and expressed in Bacillus subtilis.
[0020] Furthermore, the site of integration expression is aprE.
[0021] A third objective of this invention is to provide the application of the above-mentioned bacitracin whole-cell fluorescence response system or the above-mentioned bacitracin whole-cell biosensor in high-throughput screening of high-yield bacitracin strains.
[0022] The fourth objective of this invention is a high-throughput screening method for high-yield bacitracin-producing strains, characterized by the following steps: co-culturing the fermentation supernatant of the bacitracin-producing strain to be screened with a strain containing the above-mentioned bacitracin fluorescence response system, and characterizing the bacitracin yield by the intensity of the fluorescent protein.
[0023] The beneficial effects of this invention are:
[0024] The bacitracin fluorescence response system and bacitracin whole-cell biosensor constructed in this invention achieve ultrasensitive detection and wide-range quantification of bacitracin through a three-stage cascade amplification mechanism, with a detection throughput of up to 10. 6 Samples / day. Compared to traditional detection methods, the detection cost is reduced by more than 90%, providing a breakthrough solution for high-throughput screening of high-yield bacitracin strains and intelligent monitoring of the fermentation process. The resulting whole-cell bacitracin biosensor has a detection range of 0.05-150 μg / mL. Attached Figure Description
[0025] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:
[0026] Figure 1 This is the construction of the bacitracin fluorescence response system based on a two-component system in Example 1 of the present invention, where A is a schematic diagram of the structure of different fluorescence response systems and B is the response result of different fluorescence response systems;
[0027] Figure 2 This is a schematic diagram of the construction of the bacitracin-responsive sensor signal amplification device in Embodiment 1 of the present invention;
[0028] Figure 3 The fluorescence response result after signal amplification of the bacitracin-responsive sensor in Example 1 of this invention;
[0029] Figure 4This illustrates the effect of different intensities of RBS sequences on the dynamic range of the bacitracin whole-cell sensor response in Example 2 of the present invention.
[0030] Figure 5 P in Embodiment 2 of the present invention bceA The effect of promoter truncation on the dynamic range of the bacitracin whole-cell sensor response;
[0031] Figure 6 The ARTP mutagenesis mortality curve of Sonora desert Bacillus in Example 3 of this invention;
[0032] Figure 7 This is a schematic diagram of the process for screening high-yield bacitracin strains using a bacitracin whole-cell sensor in Example 3 of the present invention;
[0033] Figure 8 The results of screening high-yield strains of Sonora desert Bacillus ATP mutagenesis library for bacitracin in Example 3 of this invention. Detailed Implementation
[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.
[0035] The reagents involved in the examples are as follows:
[0036] DNA polymerase was purchased from Takara, plasmid extraction kit from Sangon Biotech (Shanghai) Co., Ltd., and PCR product nucleic acid purification kit from Thermo Scientific.
[0037] Bacillus subtilis was cultured on LB medium containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl. Sonora desert Bacillus fermented bacitracin on mM medium containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 10 g / L KH₂PO₄, adjusted to pH 6.5.
[0038] Example 1: Construction of a bacitracin whole-cell sensor
[0039] The two-component systems BceRS and LiaRS were chosen as the basic models for the bacitracin biosensor. The sensor operates as follows: Histidine protein kinases (HPKs) in the two-component system act as sensors, recognizing bacitracin. Upon recognition, HPKs binds to ATP and undergoes autophosphorylation to form the HPKS-P protein. The HPKS-P protein then binds to a downstream transcription factor (TF) R, undergoing phosphate group transfer to obtain the TFR-P protein, which mediates the expression of the gene corresponding to the promoter regulated by that TF.
[0040] Using pHT01 plasmid as the backbone, promoter P was selected. liaG (nucleotide sequences as shown in SEQ ID NO.1) Expression of liaS (nucleotide sequence as shown in SEQ ID NO.2), liaR (nucleotide sequence as shown in SEQ ID NO.3), and liaF (nucleotide sequence as shown in SEQ ID NO.4), with promoter P selected. bceR (nucleotide sequences are shown in SEQ ID NO. 5) Expression of bceS (nucleotide sequence is shown in SEQ ID NO. 6) and bceR (nucleotide sequence is shown in SEQ ID NO. 7). The promoters P corresponding to the LiaR-P regulated operons liaI, ydhE, yhcYZ-yhdA and the BceR-P regulated operon bceA were selected. liaI (nucleotide sequence as shown in SEQ ID NO.8), P ydhE (nucleotide sequence as shown in SEQ ID NO.9), P yhcYZ-yhdA (nucleotide sequence as shown in SEQ ID NO.10) and P bceA (The nucleotide sequence is shown in SEQ ID NO. 11) These promoters regulate the expression of the reporter gene egfp. They contain a TFR-P binding site, which, upon binding, activates the expression of downstream green fluorescent protein. Simultaneously, TFR-P can also bind to HDKS, causing its phosphate group to dissociate, thus completing the detection function of the biosensor in one step. Four corresponding sensor plasmids were constructed based on the selected downstream promoters, and P... yhcYZ-yhdA and P bceA The RBS sequence was replaced (the replaced RBS sequence is shown in SEQ ID NO.12), denoted as (opt), and the specific construction method is as follows: Figure 1 As shown in Figure A, a total of six groups of sensor plasmids with different structures were obtained.
[0041] Six sensor groups were subjected to experiments to induce the expression of the effector bacitracin. Seed culture was incubated at 37℃ and 220 rpm for 12 h, then transferred 10% (v / v) to 24-well plates containing LB medium. Bacitracin zinc was added in a gradient to a final concentration of 0-150 μg / mL for induction culture. After 12 h, the green fluorescence value (OD) was detected using a multi-functional microplate reader. 600 The induction results are as follows: Figure 1 As shown in B. BceRS-P bceA The highest induction fold was 11.7 times. After replacing RBS, the induction range increased, but the induction fold decreased.
[0042] A bio-amplifier scheme is chosen to amplify the fluorescence signal. The principle is as follows: Figure 2 As shown, a three-stage signal amplification system, "BceRS-T7 RNA polymerase-eGFP," was constructed. Bacitracin activates the BceRS two-component system, prompting the regulatory protein BceR to bind and initiate P... bceA The promoter drives T7 RNA polymerase expression; subsequently, T7 RNA polymerase specifically recognizes multiple copies of the T7 promoter, efficiently transcribing the eGFP reporter gene, corresponding to the aforementioned P... bceA and P bceA(opt) , respectively denoted as FD-P bceA and FD-P bceA(opt) Bacitracin zinc was added in a gradient to a final concentration of 0-150 μg / mL for induction culture. After 12 h, the green fluorescence value (OD) was detected using a multi-functional microplate reader. 600 The result is as follows Figure 3 As shown, introducing an amplifier through genomic integration expression at the aprE site in the genome can significantly improve the dynamic range and fluorescence response value of the sensor. At the same time, the results show that different RBS have a significant impact on the sensor response range and response fold.
[0043] Example 2: Structural optimization of bacitracin whole-cell sensor
[0044] Next, the amplifier elements were optimized. Ten RBS elements of different intensities were calculated using the RBS Calculator (RBS Library Calculator | DeNovo DNA Docs) and constructed. The RBS sequence intensities corresponding to different strains are shown in Table 1, and the fluorescence response results are as follows: Figure 4 As shown, the FD-10 has the best response range and multiplier.
[0045] Table 1 RBS Strength
[0046] Strains RBS sequence RBS strength Strains RBS sequence RBS strength FD-1 SEQ ID NO. 13 130.06 FD-2 SEQ ID NO. 14 1412.61 FD-3 SEQ ID NO. 15 2413.30 FD-4 SEQ ID NO. 16 22974.16 FD-5 SEQ ID NO. 17 126230.00 FD-6 SEQ ID NO. 18 136.23 FD-7 SEQ ID NO. 19 375.00 FD-8 SEQ ID NO. 20 4091.44 FD-9 SEQ ID NO. 21 11623.19 FD-10 SEQ ID NO. 22 23349.41
[0047] The next step in sensor modification will be carried out using FD-10 as the starting strain. Promoter P was selected. bceA The element's nucleotide sequence was truncated, removing positions 1-174 to obtain promoter P. bceA126 (The nucleotide sequence is shown in SEQ ID NO.23). After removing positions 1-150, the promoter P is obtained. bceA150 (The nucleotide sequence is shown in SEQ ID NO.24). After removing positions 1-100, the promoter P is obtained. bceA200 (The nucleotide sequence is shown in SEQ ID NO.25). After removing positions 1-50, the promoter P is obtained. bceA250 (The nucleotide sequence is shown in SEQ ID NO.26). Strains FD10-126, FD10-150, FD10-200 and FD10-250 were constructed respectively. FD10(300) is the previous round of screening strain FD-10. The fluorescence response was detected by bacitracin zinc induction.
[0048] Fluorescence response characterization results are as follows Figure 5 As shown, the FD10-200 exhibits the optimal response range and fold increase of 53.5 times, with a fluorescence response value 64.6% higher than that of FD-10. Based on the above modifications, a Bacillus subtilis bacitracin whole-cell sensing strain with high bacitracin response fold increase and almost no background was finally obtained, and this sensor was named BacTCS.
[0049] Example 3: Screening of Sonora desert BacTCS strains with high bacitracin synthesis efficiency using ARTP mutagenesis combined with the BacTCS sensor
[0050] Bacillus sonorae seed culture was cultured at 220 rpm for 12 h in a 37°C incubator, then transferred 1% (v / v) to LB medium and cultured at 220 rpm for 6 h in a 37°C incubator to reach the logarithmic growth phase. OD... 600 Diluted to 1, the mixture was inducing mutagenesis using atmospheric and room temperature plasma (ARTP) and then plated onto LB solid culture plates. The lethality rate was calculated, and a power of 100W was ultimately selected. The mutagenesis duration was 55s, and the lethality rate was approximately 80%. Figure 6 ).
[0051] The main flowchart is as follows Figure 7As shown, single colonies were picked and cultured in 96-well plates containing 150 μL of LB liquid medium at 37°C with shaking at 700 rpm for 12 h. Then, 3% (v / v) of the culture was transferred to 96-well plates containing mM medium and cultured at 37°C with shaking at 700 rpm for 40 h. The supernatant was centrifuged and divided into two parts. One part was used for bacitracin production detection by HPLC, and the other part was added as an inducer at 2% (v / v) to 96-well plates containing a whole-cell sensor for co-culture, and the final fluorescence and OD were detected. 600 The sensor usage and detection scheme are the same as in Example 1. 400 strains were initially screened using 96-well plates, and then the 8 mutant strains with the highest fluorescence were selected for yield determination using HPLC. The yield and fluorescence results are shown below. Figure 8 As shown, the results indicate that the fluorescence response intensity of the BacTCS sensor correlates with the yield trend, with induced strain 2 exhibiting the highest yield at 5.14 g / L, a 46.7% increase compared to the starting strain. This experiment significantly improved throughput and reduced costs by 98% compared to conventional HPLC detection of 400 mutagenic strains.
[0052] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A bacitracin fluorescence response system based on a two-component system, characterized in that: The bacitracin fluorescence response system uses promoter P bceR Expressing bceR and bceS proteins using promoter P bceA Express fluorescent proteins.
2. The bacitracin fluorescence response system according to claim 1, characterized in that: An RBS sequence is present upstream of the fluorescent protein.
3. The bacitracin fluorescence response system according to claim 1, characterized in that: The bacitracin fluorescence response system further includes a signal amplification device comprising T7 RNA polymerase and a T7 promoter, and the signal amplification device is located upstream of the fluorescent protein.
4. The bacitracin fluorescence response system according to claim 3, characterized in that: An RBS sequence exists upstream of the T7 RNA polymerase.
5. The fluorescence response system according to claim 4, characterized in that: The P bceA The promoter is truncated.
6. The fluorescence response system according to claim 5, characterized in that: The nucleotide sequence is as shown in SEQ ID NO.
11. bceA Nucleotides 1-200 of the promoter were truncated.
7. A bacitracin whole-cell biosensor based on a two-component system, characterized in that: The bacitracin whole-cell biosensor comprises the bacitracin fluorescence response system according to any one of claims 1-6, and the chassis strain is Bacillus subtilis.
8. The Bacillus subtilis according to claim 7, characterized in that: The bacitracin fluorescent response system is integrated and expressed in Bacillus subtilis.
9. The application of the bacitracin whole-cell fluorescence response system according to any one of claims 1-6 or the bacitracin whole-cell biosensor according to claim 7 or 8 in high-throughput screening of high-yield bacitracin strains.
10. A method for high-throughput screening of bacitracin-producing strains, characterized in that: The steps include co-culturing the bacitracin-producing strain to be screened with a strain containing the bacitracin fluorescence response system of any one of claims 1-6, and characterizing the bacitracin yield by the intensity of the fluorescent protein.