BmoR mutant capable of absolutely and specifically responding to isoamyl alcohol and application of BmoR mutant

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a BmoR protein mutant with absolute specificity response to isoamyl alcohol and application of the BmoR protein mutant in isoamyl alcohol detection or biosensors. The BmoR protein mutant is obtained by carrying out M94V / W128R / F272L mutation on the basis of wild type BmoR protein as shown in SEQ ID NO.1 in a sequence table, and the M94V / W128R / F272L mutant with an amino acid sequence as shown in SEQ ID NO.3 is only absolutely sensitive to isoamyl alcohol and has no response to n-amyl alcohol, n-butyl alcohol and isobutyl alcohol, so that the problem that the wild type BmoR protein cannot distinguish an isomeride from a structural analogue is solved; meanwhile, the detection range of the isoamyl alcohol reaches 0-60 mM, the response saturation of the BmoR protein to the isoamyl alcohol is improved, and the BmoR protein can be used for screening and application of strains with higher yield.

Description

Technical field:

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a BmoR mutant specifically responding to n-pentanol absolute alcohol, and application of the mutant in n-pentanol detection or biosensor. Background technology:

[0002] Isoamyl alcohol synthesized by microorganisms is one of the important transportation fuels and flavor compounds. The synthesis of isoamyl alcohol by metabolic engineering is achieved in many microbial hosts, and the transformation of host strains and the screening of high-yield hosts are the basis and key to the industrial production of alcohol. Biosensors can specifically respond to target compounds and output protein signals that are easy to detect, so they have been widely used in high-throughput screening. However, the wild-type transcription factor BmoR cannot be widely used in biosensors due to its poor response specificity and narrow detection range. The BmoR protein obtained by protein engineering in the present invention can not only achieve an absolute specific response to isoamyl alcohol, but also improve its substrate detection upper limit and response intensity. The most important thing is that it can be used to screen high-yield strains to meet industrial needs.

[0003] Isoamyl alcohol has long been considered by the food industry as one of the many flavor compounds produced during fermentation. Studies have shown that isoamyl alcohol and its related aldehydes, acids, and esters are the most abundant odor and flavor components in fermented beverages, and have been biosynthesized in multiple microbial hosts, but specific screening for isoamyl alcohol is still a cumbersome project. The wild-type transcription factor BmoR responds to both the substrates of pentanol (n-pentanol and isoamyl alcohol) and butanol (n-butanol and isobutanol), and is unable to specifically respond to one of the alcohols to meet industrial needs. Therefore, achieving a specific response to alcohol has become a major problem.

[0004] Biosensors consist of molecular recognition elements and signal converters. When the molecular recognition element binds to the object to be measured, the generated signal can be converted into an optical signal or an electrical signal by the converter, and the object to be measured can be detected and analyzed. As an emerging tool in synthetic biology, biosensors can be designed and constructed to dynamically respond to changes in the concentration of signal molecules. At the same time, biosensors are designed to promote the optimization of microbial cell factories and the production of a series of natural products widely used in industry, such as itaconic acid, fatty acids, n-pentanol, isopentanol, isobutanol, n-butanol and alkaloids. Biosensors mainly include RNA nucleic acid switches, transcription factor-regulated biosensors, G protein-coupled receptors and fluorescent protein biosensors. However, the low dynamic range of fluorescent protein biosensors, the difficulty of RNA nucleic acid switches in vitro, and the fact that G protein-coupled receptors can only be performed outside the cell have hindered the development of biosensors in the biological world.

[0005] Biosensors based on transcription factors (TFs) are the most widely used. The most commonly used transcription factors are bacterial transcription factors, which include a ligand binding domain (LBD) or a metabolic binding domain (MBD) and a DNA binding domain (DBD). BmoR is a transcription factor of the n-alkane metabolic pathway of Pseudomonas and a member of the bEBP. The σ 54 P-dependent promoter bmo The signal molecule is a straight-chain or branched alcohol of C2-C5. BmoR-based biosensors can be used to screen strains that produce high-yield n-butanol or isobutanol, but the wild-type BmoR biosensor has poor response specificity and a narrow detection range (0-40mM) and cannot be used in industrial production. Therefore, the modification of the biosensor provides a solution for the efficient detection of isopentanol production and the rapid screening of high-yield strains. Summary of the invention:

[0006] The purpose of the present invention is to provide a BmoR protein and biosensor that can distinguish between pentanol and butanol. By analyzing the transcriptional working mechanism and functional areas of natural BmoR, protein engineering of BmoR is carried out by combining irrational modification and computer-assisted semi-rational design, and finally a BmoR mutant protein that responds absolutely specifically to isopentanol only is obtained.

[0007] Furthermore, the BmoR mutant protein that responds absolutely and specifically only to isoamyl alcohol is obtained by subjecting the wild-type BmoR protein shown in SEQ ID NO.1 to M94V / W128R / F272L mutation, hereinafter referred to as M94V / W128R / F272L mutant, and the mutant protein is specifically:

[0008] (1) the amino acid sequence shown in SEQ ID NO.3 in the sequence listing; or

[0009] (2) an amino acid sequence with a homology of more than 75% to SEQ ID NO. 3; or

[0010] (3) An amino acid sequence having the same function as SEQ ID NO. 3 obtained by substituting, deleting, and / or adding one or more amino acids to SEQ ID NO. 3.

[0011] Furthermore, the present invention also provides a gene encoding the M94V / W128R / F272L mutant;

[0012] Furthermore, the coding gene is shown in the sequence listing SEQ ID NO.4.

[0013] Another object of the present invention is to provide an application of the M94V / W128R / F272L mutant, particularly an application in a sensor for detecting a sample containing isopentanol or screening an isopentanol-producing strain, and more particularly, an application in constructing a biosensor for detecting isopentanol.

[0014] Furthermore, the biosensor is a biosensor based on the M94V / W128R / F272L mutant, and the biosensor comprises a gene encoding the M94V / W128R / F272L mutant and its promoter, a promoter P bmo and reporter gene expression elements; the promoter activates the expression of the bmoR gene, and the BmoR protein binds to the alcohol molecule to form a hexamer, which in turn activates the downstream promoter P bmo , thereby expressing the reporter gene and generating signals such as fluorescence; the biosensor can achieve specific response and screening for isoamyl alcohol at a concentration of 0-60mM, and is further applied in industrial production to achieve specific screening of samples containing isoamyl alcohol and isoamyl alcohol-producing strains;

[0015] Furthermore, the promoter of the mutant encoding gene includes but is not limited to P bmoR , P tac , P T7 , P LlacO1 wait;

[0016] Furthermore, the reporter gene includes but is not limited to genes such as gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp, etc.;

[0017] Preferably, the biosensor comprises a gene encoding the M94V / W128R / F272L mutant and its promoter P bmoR , promoter P bmo and a recombinant plasmid of a gfp reporter gene; further, the expression vector that can be used for the recombinant plasmid includes but is not limited to the expression vector commonly used in the art;

[0018] More preferably, the biosensor is obtained by replacing the wild-type BmoR protein encoding gene on the plasmid pYH1 with the M94V / W128R / F272L mutant encoding gene, that is, replacing the wild-type BmoR protein encoding gene on the plasmid pYH1 with the M94V / W128R / F272L mutant encoding gene. bmoR The gene encoding the M94V / W128R / F272L mutant was linked to the colE1 replication origin, amp r and P bmo The driven gfp gene is obtained;

[0019] Furthermore, the strains include but are not limited to Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc.;

[0020] Furthermore, the promoter PbmoR The nucleotide sequence is shown in the sequence listing SEQ ID NO.5;

[0021] Furthermore, the promoter P bmo The nucleotide sequence is shown in the sequence listing SEQ ID NO.6;

[0022] Furthermore, the nucleotide sequence of the gfp reporter gene is shown in the sequence listing SEQ ID NO.7.

[0023] The present invention also provides the application of the above-mentioned biosensor in isoamyl alcohol detection, especially in the screening of isoamyl alcohol producing strains, by introducing the above-mentioned plasmid into production strains, such as Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc., to detect the production of isoamyl alcohol.

[0024] Beneficial effects:

[0025] 1. The BmoR-based biosensor can be used to screen pentanol or butanol high-yield strains, but the wild-type BmoR responds to both pentanol and butanol and cannot distinguish them, with poor specificity. The M94V / W128R / F272L mutant provided by the present invention is only sensitive to isopentanol, and has no response to n-pentanol, n-butanol and isobutanol, thus solving the problem that the wild-type BmoR protein cannot distinguish between isomers and structural analogs.

[0026] 2. The detection range of wild-type BmoR is too narrow, and the detection range of isopentanol is 0-30mM. When the substrate concentration reaches 30mM, the response has reached saturation, so it cannot be used to identify strains with an isopentanol production of more than 30mM. The M94V / W128R / F272L mutant provided by the present invention has a detection range of 0-60mM for isopentanol, which improves the response saturation of BmoR protein to isopentanol and can be used for the screening and application of high-level isopentanol production strains. Description of the drawings:

[0027] Figure 1 It is the principle flow chart;

[0028] First, computer learning was used to study and train multiple factors that affect the binding energy of BmoR to signal molecules. By building a model and using it for predictions, the key areas that affect the selectivity of BmoR signal molecules were successfully identified. Combined with high-throughput screening technology and rational design methods, the absolute specific detection of isopentanol by BmoR was finally successfully achieved.

[0029] Figure 2 Fluorescence microscopy observation of BmoR mutant and wild type to 10 mM n-pentanol, isopentanol, n-butanol and isobutanol;

[0030] Figure 3The responses of BmoR mutant and wild type to n-pentanol, isopentanol, n-butanol and isobutanol;

[0031] Figure 4 The response of the M94V / W128R / F272L mutant to the addition of mixed alcohols. Specific implementation method:

[0032] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0033] The biosensor provided by the present invention is a biosensor based on the M94V / W128R / F272L mutant, wherein the biosensor comprises a gene encoding the M94V / W128R / F272L mutant and its promoter, a promoter P bmo and reporter gene expression elements; the promoter activates the expression of the bmoR gene, and the BmoR protein binds to the alcohol molecule to form a hexamer, which in turn activates the downstream promoter P bmo , thereby expressing the reporter gene and generating fluorescence and other signals. The skilled person in the art can select a promoter to start the expression of the BmoR mutant gene in the prior art according to the actual situation, such as using P bmoR , P tac , P T7 , P LlacO1 The reporter gene can also be selected in many ways. Protein molecules commonly used in the art that can generate visual detection signals or detectable small molecules, such as fluorescent proteins, color proteins, etc., can achieve the response of the biosensor of the present invention, preferably, such as gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp, etc. The above sensor also contains necessary elements for expression such as replication initiation sites, preferably, such as colE1 replication initiation sites, etc. The above sensor can also contain resistance genes and other markers, such as amp r Those skilled in the art can also add other elements to the above sensor according to actual needs, such as constructing the above elements into expression vectors in the prior art, such as pET, pUC19, pMAL, etc., to obtain a recombinant plasmid that can be used as a sensor.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] The present invention will be further explained below through specific examples.

[0036] Example 1 Design and screening of BmoR mutant M94V / W128R / F272L

[0037] (1) Error-prone PCR was performed on the full sequence of the transcription factor BmoR (shown in SEQ ID NO.2) to construct a full sequence mutation library, and each mutant was sequenced to determine the mutation site. The BSH number, ΔG, and response value GFP / OD between the obtained BmoR mutant and the signal molecule were calculated. 600 , the fine domain and secondary structure of each mutation site, the distance between the mutation site and SBD, and other data are sorted out as a learning set for machine learning. At the same time, the BSH quantity and ΔG data are used as output data, and the response value GFP / OD 600 As output data, the learning set was pre-trained using the random forest algorithm, and a model "Model BT" with an accuracy of 88.5% was obtained. This model shows that the amount of BSH is a key factor in determining the intensity of BmoR transcriptional activation.

[0038] (2) Saturation mutations were designed for 1AA-300AA of BmoR, and the molecular docking models of 5700 BmoR mutants and 4 signal molecules (n-pentanol, isopentanol, n-butanol and isobutanol) were batch processed using the software Discovery Studio, totaling 22,800 models. At the same time, the BSH quantity and ΔG of the model response were output. The BSH quantity and ΔG were assembled into a prediction set with other factors, and the response value of the prediction set was predicted using the model Model BT. Finally, the key area that can significantly affect the signal molecule specificity of BmoR was accurately located, which is 110AA-220AA.

[0039] (3) Two rounds of continuous error-prone PCR were performed on the 110AA-220AA region, and the obtained BmoR mutant library was obtained and high-throughput screening was performed using FACS to obtain BmoR mutants that were able to respond specifically to n-pentanol or isopentanol. The obtained BmoR mutants were able to effectively distinguish between the isomers of n-pentanol and isopentanol, and this property was further verified by adding a mixture of exogenous signal molecules. However, these mutants still retained the ability to respond to their structural analog n-butanol, and were unable to achieve effective differentiation between structural analogs and isomers.

[0040] (4) To further verify the absolute specific response of BmoR to isopentanol, site-directed mutagenesis was performed on the existing BmoR mutants. The artificially introduced mutation sites can make BmoR no longer sensitive to n-butanol. By exogenously adding different concentrations of n-butanol, isobutanol, n-pentanol or isopentanol, as well as different combinations of signal molecule mixtures, the response of the combined mutant BmoR mutant to these four signal molecules was verified, successfully achieving the absolute specific response of BmoR to isopentanol.

[0041] Example 2 Concentration gradient experiment to determine the change in detection limit of wild type and mutant

[0042] The biosensor based on wild-type BmoR can respond to 0-30mM n-pentanol and isopentanol and 0-40mM n-butanol and isobutanol; when the substrate concentration is higher than 30 and 40mM, the response value tends to saturation and cannot respond to higher concentrations of alcohol molecules. The following experiments verify the specific response of the BmoR biosensor based on the M94V / W128R / F272L mutant to 0-60mM n-pentanol / isopentanol and 0-100mM n-butanol / isobutanol.

[0043] The strains containing the M94V / W128R / F272L mutation were subjected to exogenous addition experiments with concentration gradients of n-pentanol, isopentanol, n-butanol or isobutanol, and the response curves were determined and K was calculated. m , response intensity value and other parameters.

[0044] A single clone on the plate was picked and inoculated into 5 mL LB (100 μg / mL Amp) liquid medium, and cultured at 37°C and 220 rpm for 8 h as seed solution.

[0045] The exogenous addition experiment was carried out in a sterilized 2mL 96-deep-well plate. Each well was first added with 950μL of fresh LB (100μg / mL Amp) medium, and then n-pentanol, isopentanol, n-butanol or isobutanol were added to the medium to make their final concentrations 0, 1, 10, 20, 40, 60, 80, 100mM, respectively. Finally, 50μL of seed solution was inoculated into each well, and after sealing the sealing film, the deep-well plate was placed at 30°C and 220rpm for 16h in a shaker.

[0046] Use microplate to measure fluorescence intensity GFP and OD 600 Detection: Mix the bacterial solution by blowing, draw 200 μL into the microplate reader, and perform quantitative detection at 30°C. Set the parameters as follows: 470 nm excitation wavelength and 510 nm emission wavelength, with a gain value of 50. The obtained GFP and OD 600 The background control value was first subtracted from the value, and the GFP / OD value of each well was calculated based on this. 600 As relative fluorescence intensity values.

[0047] GFP / OD 600 The final concentrations of n-pentanol, isopentanol, n-butanol, or isobutanol were used as the ordinate and the graphs were drawn using OriginPro 8.5 or GraphPad Prism 8 software. The data were fitted with Michaelis-Menten and the K values ​​of the BmoR mutants for n-pentanol, isopentanol, n-butanol, and isobutanol were calculated based on the fitting results. m , maximum response intensity and other parameters ( Figure 3 ).

[0048] Under the condition of 0-100 mM gradient concentration, the mutant still maintained a specific response to n-pentanol under different concentrations of n-pentanol, isopentanol, n-butanol and isobutanol, but did not respond to the other three alcohols. The K of wild-type BmoR to n-pentanol was plotted using GraphPad 9.0. m is 5.09 mM, and the K m The K of n-butanol is 3.99 mM. m The K of isobutanol is 7.90 mM. m The K of the M94V / W128R / F272L mutant to isopentanol is 8.75 mM. m About 6.04×10 15 mM.K m As a representation of affinity, K m The larger the value, the smaller the affinity; K m The smaller the value, the greater the affinity.

[0049] The wild-type BmoR can respond to 0-30mM of n-pentanol and isopentanol. When the concentration is higher than 30mM, the response will reach saturation, so the wild-type BmoR cannot distinguish n-pentanol or isopentanol above 30mM; at the same time, the wild-type BmoR can respond to 0-40mM of n-butanol or isobutanol. When the concentration is higher than 40mM, the response will reach saturation, so the wild-type BmoR cannot distinguish n-butanol or isobutanol above 40mM; the calculation results show that the mutant has a lower K for isopentanol than the wild-type. m The values ​​were significantly improved, proving that the BmoR mutant can achieve a specific response to higher concentrations of isopentanol (0-60 mM), which is consistent with the experimental results.

[0050] Example 3 Mixed alcohol addition experimental verification

[0051] To further verify that the M94V / W128R / F272L mutant was able to maintain its specificity in the presence of multiple signal molecules, the concentration ratios of exogenously added n-butanol, isobutanol, n-pentanol or isopentanol in the culture medium were set to 1:10 (10 mM:100 mM), 1:8 (10 mM:80 mM), 1:6 (10 mM:60 mM), 1:4 (10 mM:40 mM), 1:2 (10 mM:20 mM), 1:1 (10 mM:25 mM), 1:2 (10 mM:30 mM), 1:3 (10 mM:40 mM), 1:4 (10 mM:50 mM), 1:5 (10 mM:60 mM), 1:6 (10 mM:70 mM), 1:8 (10 mM:80 mM), 1:8 (10 mM:80 mM), 1:6 (10 mM:60 mM), 1:4 (10 mM:40 mM), 1:2 (10 mM:25 mM), 1:1 (10 mM:25 mM), 1:1 10mM:10mM), 1:0(10mM:0mM), 0:1(0mM:10mM), 1:1(1mM:10mM), 2:1(20mM:10mM), 4:1(40mM:10mM), 6:1(60mM:10mM), 8:1(80mM:10mM) or 10:1(100mM:10mM), 500mM ethanol was added to the culture medium as background noise. In addition, the 96-well plate contained 950μL of LB (50μg / mL Carb.) liquid culture medium and 50μL of seed solution. Cover the lid and culture at 30℃, 220rpm for 16h. Pipette 200μL of bacterial solution to determine its OD 600 and GFP, and calculated their relative fluorescence intensities.

[0052] GFP / OD of M94V / W128R / F272L mutant 600 The value only increased with the increase of isoamyl alcohol concentration, while no obvious fluorescence signal was detected when other signal molecules were added. At the same time, the absolute specificity of the M94V / W128R / F272L mutant to isoamyl alcohol was further verified by adding mixed signal molecules. The results showed that the M94V / W128R / F272L mutant can still maintain its absolute specificity to isoamyl alcohol in the presence of other signal molecules ( Figure 4 ).

[0053] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A BmoR mutant protein, characterized in that: The mutant is obtained by causing M94V / W128R / F272L mutations to occur on the wild-type BmoR protein shown in SEQ ID NO.

1.

2. The BmoR mutant protein according to claim 1, characterized in that The mutant protein is specifically: (1) the amino acid sequence shown in SEQ ID NO.3 in the sequence listing; or (2) an amino acid sequence with a homology of more than 75% to SEQ ID NO. 3; or (3) An amino acid sequence having the same function as SEQ ID NO. 3 obtained by substituting, deleting, and / or adding one or more amino acids to SEQ ID NO.

3.

3. Use of the BmoR mutant according to claim 1 in detecting samples containing isoamyl alcohol or in screening isoamyl alcohol-producing strains.

4. Use of the BmoR mutant according to claim 1 in constructing a biosensor for detecting isoamyl alcohol.

5. The gene encoding the BmoR mutant of claim 1.

6. The coding gene according to claim 5, characterized in that As shown in the sequence listing SEQ ID NO.

4.

7. A recombinant plasmid or recombinant strain comprising the coding gene according to claim 5.

8. A biosensor, characterized in that: The sensor comprises a gene encoding the mutant of claim 5 and a promoter P activated by the BmoR mutant. bmo , by P bmo Expression elements of the promoter driving the reporter gene and expressing the BmoR mutant.

9. The biosensor according to claim 8, wherein The reporter genes include but are not limited to gfp, rfp, cfp, sfgfp, egfp, yfp, and ecfp genes.

10. The biosensor according to claim 8, wherein The promoters expressing BmoR mutants include, but are not limited to, P bmoR , P tac , P T7 , P LlacO1 .

11. The biosensor according to claim 8, wherein The sensor is to be bmoR The mutant encoding gene was linked to the colE1 replication origin, amp r and P bmo The gfp gene was activated.

12. The biosensor according to claim 8, wherein Promoter P bmo The nucleotide sequence is shown in the sequence listing SEQ ID NO.

6.

13. Use of the biosensor according to claim 8 in detecting environmental, food, medical and biological samples containing isopentanol, and in screening industrial microbial strains that produce isopentanol.

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