CN112779278A - 6-glucosamine phosphate biosensor and application thereof in strain screening - Google Patents
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Abstract
The invention relates to a glucosamine-6-phosphate biosensor and application thereof in strain screening, wherein the glucosamine-6-phosphate biosensor is integrated and expressed on the genome of bacillus subtilis and comprises a transcription factor GamR, a promoter comprising a GamR binding site, T7 RNA polymerase expressed by the promoter comprising the GamR binding site and fluorescent protein expressed by a T7 promoter. The invention directly realizes the integration and expression of the 6-phosphoglucose biosensor on the genome, and the fluorescence expression intensity reaches 3 times of that of direct integration through a signal amplification loop based on T7 RNA polymerase. The biosensor is used for high-throughput screening of microbial strains introduced with a mutation library, and the yield of N-acetylglucosamine is improved by 31.6%.
Description
Technical Field
The invention belongs to the technical field of metabolic engineering, and particularly relates to a 6-glucosamine phosphate biosensor and application thereof in strain screening.
Background
The microbial cell factory constructed by artificially controlling the metabolic pathway of the microbial cells can be used for green sustainable production of various high value-added products including food, medicines, chemicals and the like. However, due to the complex metabolic regulation mechanism of the cell itself, in order to obtain a high-yield microbial strain, a great deal of modification and optimization of relevant metabolic pathways are often required. However, the measuring speed and flux of the conventional measuring method such as liquid chromatography or mass spectrometry limit the testing process of the microbial strains, so that the high-flux screening of the microbial strains can be realized by constructing a biosensor which can respond to related metabolites and directly converting the concentrations of the metabolites into the intensity signals of fluorescent proteins.
Glucosamine-6-phosphate is an important precursor for synthesis of various products such as glucosamine, neuraminic acid, hyaluronic acid and the like, but cannot be accumulated in a large amount in cells due to the own metabolic regulation mechanism of the cells, so that the related products and the synthesis process are limited. Therefore, the biosensor can be used for high-throughput screening of microbial strains with excessive accumulation of glucosamine 6-phosphate, and the synthesis capacity of products such as glucosamine, neuraminic acid, hyaluronic acid and the like can be further improved.
In patent application 201911174644.1, we utilized the action mechanism of transcription factor GamR (i.e. GamR binds to the corresponding promoter to inhibit its expression when the concentration of glucosamine-6-phosphate is low, GamR drops from the promoter to recover its expression when the concentration of glucosamine-6-phosphate is high), constructed a series of glucosamine-6-phosphate activated promoters with different expression intensities in Bacillus subtilis, and obtained a glucosamine-6-phosphate biosensor by controlling the expression of Green Fluorescent Protein (GFP) using the above promoters.
The related gene elements of the existing biosensor for 6-phosphoglucosamine are expressed by using plasmids, so that the stability is poor; furthermore, if it is directly integrated into the genome, the expression of the fluorescent protein will be weakened, resulting in a decrease in sensitivity.
Disclosure of Invention
In order to solve the technical problems, the invention constructs a biosensor of 6-glucosamine phosphate with good stability in bacillus subtilis through the integrated expression of a transcription factor GamR and related gene elements, and the biosensor can be used for high-throughput screening of 6-glucosamine phosphate high-yield microorganism strains. In order to avoid weakening of a fluorescence signal due to low copy number on a genome, the sensitivity of the biosensor is enhanced after the fluorescence signal is amplified by using a signal amplification gene loop based on T7 RNA polymerase.
The first object of the present invention is to provide a glucosamine 6-phosphate biosensor, which is integrally expressed on the genome of Bacillus subtilis, and comprises a transcription factor GamR, a promoter including a GamR binding site, T7 RNA polymerase expressed using the promoter including the GamR binding site, and a fluorescent protein expressed using a T7 promoter.
Further, the glucosamine-6-phosphate biosensor also comprises a RBS which adopts a nucleotide sequence shown as SEQ ID NO.4 to regulate the expression intensity of T7 RNA polymerase.
Further, the glucosamine-6-phosphate biosensor further comprises a binding site lacO for adding the repressor LacI to the promoter including the binding site of GamR.
Further, the glucosamine 6-phosphate biosensor also comprises a binding site gamO for adding a transcription factor GamR at the downstream of the T7 promoter.
Further, the promoter including the GamR binding site is Pvg6、PgamAOr Psg2. Preferably Psg2See patent application 201911174644.1.
Further, the site of integration expression is the aprE site of the Bacillus subtilis genome.
The second purpose of the invention is to provide the application of the glucosamine 6-phosphate biosensor in high-throughput screening of microbial strains.
Further, the application is to screen the bacillus subtilis with high yield of 6-phosphoglucosamine, N-acetylglucosamine, neuraminic acid or hyaluronic acid.
Further, in the application process, genes of enzymes for converting 6-glucosamine phosphate into N-acetylglucosamine, neuraminic acid or hyaluronic acid in the bacillus subtilis are eliminated, strains with high 6-glucosamine phosphate yield are obtained through screening, and then related genes are transferred.
Further, in the process of screening the bacillus subtilis with high N-acetylglucosamine yield, the phosphoglucosamine acetylase gene is eliminated.
By the scheme, the invention at least has the following advantages:
the invention directly realizes the integration and expression of the 6-phosphoglucose biosensor on the genome, and the fluorescence expression intensity reaches 3 times of that of direct integration through a signal amplification loop based on T7 RNA polymerase. The biosensor is used for high-throughput screening of microbial strains introduced with a mutation library, and the yield of N-acetylglucosamine is improved by 31.6%.
The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a preferred embodiment of the present invention and is described in detail below.
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FIG. 1 is a schematic diagram showing the construction of the gene elements of a glucosamine-6-phosphate biosensor;
FIG. 2 shows the functional verification of glucosamine-6-phosphate;
FIG. 3 shows the application of glucosamine-6-phosphate biosensor in high throughput screening (A) high throughput screening using glucosamine-6-phosphate biosensor using flow cytometer (B) measurement of fluorescence intensity of screened strains using 96 well plate (C) the change of yield before and after shake flask fermentation compared to high throughput screening;
FIG. 4 is a comparison of the effect of the signal amplification gene circuit.
Detailed Description
Seed medium (g/L): tryptone 10, yeast powder 5 and NaCl 10.
Shake flask fermentation medium (g/L): tryptone 6, yeast powder 12, urea 6, K2HPO4·3H2O 12.5，KH2PO42.5，CaCO 35, trace elements 10 ml/L; the trace element solution contains in g/L: MnSO4·5H2O 1.0，CoCl2·6H2O 0.4，NaMoO4·2H2O 0.2，ZnSO4·7H2O 0.2，AlCl3·6H2O 0.1，CuCl2·H2O 0.1，H3BO40.05, 5M HCl.
The method for measuring N-acetylglucosamine comprises the following steps: high Performance Liquid Chromatography (HPLC) detection: agilent 1260, RID detector, HPX-87H column (Bio-Rad Hercules, Calif.), mobile phase: 5mM H2SO4The flow rate is 0.6mL/min, the column temperature is 40 ℃, and the injection volume is 10 mu L.
Green fluorescent protein expression assay: the cells were cultured in a 96-well plate with a black transparent bottom, and after 20 hours, the relative fluorescence intensity of GFP was measured using a multi-functional microplate reader. The fluorescence measurement used an excitation wavelength of 490nm, an emission wavelength of 530nm, a gain of 60, and a measurement of the cell optical density at a wavelength of 600 nm. And finally, dividing the fluorescence intensity by the optical density of the cells to obtain the relative fluorescence intensity.
Example 1: design construction of glucosamine 6-phosphate biosensor containing signal amplification loop
The 6-phosphoglucose biosensor is integrally expressed and functionally verified by using the strain BS03 (obtained by knocking out gamR, nagB and gamA from the Bacillus subtilis 168 strain, so that the concentration of the 6-phosphoglucose in the cell can be controlled by adding glucosamine with different concentrations extracellularly) described in the patent CN110713966A by using a CRISPR/Cpf1 gene editing system (reference: Wu, Y., Liu, Y., Lv, X., Li, J., Du, G., Liu, L.,2020.CAMERS-B: CRISPR/Cpf1 assisted multiple-genes editing and regulation system for Bacillus subtilis and Bioengineering 117,1817 and 1825). The plasmid pHT-XCR6 was first transformed and the biosensor was integrated into the aprE site of the Bacillus subtilis genome by transforming the plasmid pcrF11-SR4T7 containing the crRNA of the targeting gene aprE with the corresponding homology arms.
The construction of the integrated genome biosensor is shown in FIG. 1, in which the glucosamine-6-phosphate-activated promoter Psg2The expression of T7 RNA polymerase is realized, and GFP is expressed by using a T7 promoter, so that the high-efficiency transcription of T7 RNA polymerase can be utilized to improve the expression intensity of GFP. In order to avoid toxicity of the over-expressed T7 RNA polymerase to cells, the expression intensity is finely adjusted through RBS; and a binding site lacO of the repressor LacI is added to construct a logical AND gate to further reduce the toxicity, so that the T7 RNA polymerase can be expressed only when two inducers, namely IPTG and 6-phosphoglucose, exist simultaneously; also, to further reduce the leakage expression of the whole system (i.e., the expression of GFP when glucosamine-6-phosphate is in the initial state), the promoter was placed in the T7The downstream of the gene is also added with a binding site gamO of a repressor protein gamR, thereby realizing double repression of T7 RNA polymerase and GFP.
To verify the function of the biosensor, 1mM IPTG and 10mM glucosamine (which can be directly transferred into cells to produce glucosamine-6-phosphate) were added, and then the expression level of GFP was measured. As shown in Table 1, a strong fluorescent signal was detected only when IPTG and glucosamine were added simultaneously, indicating that the designed logical AND gate could exert its effect. And then glucosamine with different concentrations is added outside the cells to control the intracellular glucosamine-6-phosphate concentration, and the fluorescence intensity of the glucosamine-6-phosphate is measured, so that the result shows that the biosensor can well respond to the change of the intracellular glucosamine-6-phosphate concentration, and the dynamic range of the glucosamine-6-phosphate biosensor can reach 45.8 (figure 2).
TABLE 1 response of biosensors with different inducers
Example 2: application of glucosamine 6-phosphate biosensor in high-throughput screening
Based on Bacillus subtilis 168(BS168) in CN107699533A, the genotype was modified as follows:. DELTA.nagP. DELTA.gamP. DELTA.gamA. DELTA.nagA. DELTA.nagB. delta. ldh. DELTA.pta. glcK. DELTA.pckA. DELTA. pyk. DELTA.gamR:: lox72, and the promoter P was usedvegRegulated expression of the phosphatase yqaB from E.coli with the glmS of Bacillus subtilis 168 itself, on a plasmid with promoter P43Regulating and controlling recombinant expression of GNA 1) as starting strain, and plasmid P contained in the starting strain43Elimination of GNA1 (the plasmid contains the phosphoglucosamine acetylase gene and converts 6-phosphoglucosamine into 6-acetylglucosamine phosphate, which is further converted into N-acetylglucosamine by its own phosphatase and secreted extracellularly) allows intracellular accumulation of 6-phosphoglucosamine. Then, RBS calculator is utilized to design a key enzyme 6-phosphoglucosamine synthetase group in the synthetic pathwayThe translational strength of glmS-derived RBS libraries (AMGGNSGYAHATAAGA, where M ═ a or C, N ═ a or T or C or G, S ═ C or G, Y ═ C or T, H ═ a or C or T) was optimized to promote the accumulation of intracellular 6-phosphoglucosamine in excess and replaced into the genome using the CRISPR/Cpf1 system. Then, 0.1% of 1000000 cells, which showed the strongest fluorescence, was selected by flow cytometry (FIG. 3A), and applied to a plate containing chloramphenicol, kanamycin, and xylose. The fluorescence intensity of individual colonies on the plate was then measured by 96-well plates (FIG. 3B), and two strains with the strongest fluorescence were selected, and the plasmid pP43-GNA1 was transformed to convert the intracellular excess accumulation of glucosamine-6-phosphate into extracellular N-acetylglucosamine. Finally, the yield changes before and after screening are compared through shake flask fermentation, as shown in fig. 3C, the yields of N-acetylglucosamine of the screened mutant strains P2E12 and P3D6 are respectively increased by 23.9% and 31.6% as compared with BNDR000, which indicates that the biosensor can be used for high-throughput screening of microbial strains.
Comparative example 1: comparison of Effect of Signal amplification Gene circuits
To compare the effects of the signal amplification loop, P will be usedsg2The directly expressed GFP was also integrated into the aprE site of the BS3 genome, and the RBSs of T7 RNA polymerase in the signal amplification gene loop were optimized to control its expression using 6 RBSs as shown in Table 2. The amplification loop containing RBS1 and RBS2 has toxicity to cells due to high expression intensity, so that integrated strains cannot be obtained smoothly, and the amplification loop containing RBS3-RBS6 can successfully realize genome integration. As shown in FIG. 4, when the signal amplification circuit comprising RBS3 and RBS4 was used, the expression intensity of GFP was directly Psg23.6-fold and 3.0-fold when expressed, but leaky expression was more pronounced with RBS 3; while the amplification effect was not ideal with RBS5, the fluorescence signal appeared to be significantly reduced with RBS 6. The above results indicate that the use of a signal amplification gene circuit based on T7 RNA polymerase can improve the sensitivity of the biosensor, and that the expression intensity of T7 RNA polymerase is also crucial for signal amplification.
TABLE 2 RBS for T7 RNA polymerase optimization and its translational strength
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university in south of the Yangtze river, Shandong Rundd Biotech Co., Ltd
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Claims (10)
1. A glucosamine 6-phosphate biosensor, wherein the glucosamine 6-phosphate biosensor is integrated and expressed on the genome of Bacillus subtilis, and comprises a transcription factor GamR, a promoter comprising a GamR binding site, T7 RNA polymerase expressed by the promoter comprising the GamR binding site, and a fluorescent protein expressed by a T7 promoter.
2. The glucosamine 6-phosphate biosensor according to claim 1, wherein said glucosamine 6-phosphate biosensor further comprises RBS-regulated T7 RNA polymerase expression strength as represented by SEQ ID No. 4.
3. The glucosamine 6-phosphate biosensor of claim 1, further comprising a binding site lacO for adding repressor LacI to a promoter comprising a binding site of GamR.
4. The glucosamine 6-phosphate biosensor according to claim 1, further comprising a binding site gamO for adding transcription factor GamR downstream of T7 promoter.
5. The glucosamine-6-phosphate biosensor of claim 1, wherein said promoter comprising a GamR binding site is Pvg6、PgamAOr Psg2。
6. The glucosamine 6-phosphate biosensor according to claim 1, wherein the site of integration expression is aprE site of Bacillus subtilis genome.
7. Use of the glucosamine 6-phosphate biosensor according to any one of claims 1 to 6 for high throughput screening of microbial strains.
8. The use of claim 7, wherein the use is screening of Bacillus subtilis for high yields of glucosamine-6-phosphate, N-acetylglucosamine, neuraminic acid or hyaluronic acid.
9. The use of claim 8, wherein in the use process, the gene of the enzyme that converts glucosamine-6-phosphate into N-acetylglucosamine, neuraminic acid or hyaluronic acid in Bacillus subtilis is eliminated, and after the strain with high glucosamine-6-phosphate yield is obtained by screening, the relevant gene is transferred.
10. The use according to claim 9, wherein the glucosamine phosphate acetylase gene is eliminated in the screening of Bacillus subtilis which produces N-acetylglucosamine at a high yield.
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Cited By (4)
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| CN115820633A (en) * | 2022-08-15 | 2023-03-21 | 中国科学院天津工业生物技术研究所 | Biosensor for positively responding glucose absorption rate and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337573A (en) * | 2021-05-31 | 2021-09-03 | 江南大学 | Construction method and application of micro-droplet biosensor |
| CN113416685A (en) * | 2021-07-05 | 2021-09-21 | 青岛农业大学 | Biosensor with signal amplification effect and capable of visually detecting explosive molecules and preparation method and application of biosensor |
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| CN117645985A (en) * | 2023-11-27 | 2024-03-05 | 山东润德生物科技有限公司 | Acetylglucosamine-6 phosphate phosphatase mutant and application thereof |
| CN117645985B (en) * | 2023-11-27 | 2024-06-04 | 山东润德生物科技有限公司 | Acetylglucosamine-6 phosphate phosphatase mutant and application thereof |
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