BmoR mutant 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 specifically responding to isoamyl alcohol / n-butyl alcohol and application of the BmoR protein mutant in isoamyl alcohol / n-butyl alcohol detection or biosensors. The BmoR protein mutant is obtained by mutation containing W128R on the basis of wild type BmoR protein as shown in SEQ ID NO.1 in a sequence table, the W128R mutant with an amino acid sequence as shown in SEQ ID NO.3 has specific response to isoamyl alcohol / n-butyl alcohol, and the W128R mutant is only sensitive to isoamyl alcohol / n-butyl alcohol and has no response to n-amyl alcohol / isobutyl alcohol. The problem that the wild type BmoR protein cannot distinguish the isomeride and the structural analogue is solved; meanwhile, the detection range of the W128R to isoamyl alcohol reaches 0-60 mM, the detection range of the W128R to n-butyl alcohol reaches 0-100 mM, the response saturation of the BmoR protein to isoamyl alcohol / n-butyl alcohol is improved, and the W128R 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 protein mutant specifically responding to isopentanol / n-butanol, and application of the BmoR protein mutant in isopentanol / n-butanol detection or biosensor. Background technology:

[0002] Isoamyl alcohol / n-butanol synthesized by microorganisms is one of the important transportation fuels and flavor compounds. The synthesis of isoamyl alcohol / n-butanol 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, and have therefore 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 a specific response to isoamyl alcohol / n-butanol, but also improve its substrate detection upper limit and response intensity, and most importantly, it can be used to screen high-yield strains to meet industrial needs.

[0003] Isoamyl alcohol / n-butanol 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 screening for isoamyl alcohol or n-butanol is still a cumbersome process. The wild-type transcription factor BmoR has a low response to the substrates isoamyl alcohol and n-butanol, which cannot 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 bEBP, which is used to regulate the σ 54 P-dependent promoter bmo , the signal molecule is a straight-chain or branched alcohol of C2-C5. However, the wild-type transcription factor BmoR has poor response specificity. It responds to a variety of higher alcohols such as n-pentanol, isopentanol, n-butanol, isobutanol, etc., but cannot specifically respond to one of the alcohols and produce a high response value to meet industrial needs; and the detection range is narrow (0-40mM), so it cannot be widely used in biosensors. Therefore, achieving a specific response to alcohol and improving the detection range has become an urgent problem to be solved.

[0006] The BmoR protein mutant obtained by protein modification in the present invention can not only achieve a specific response to isopentanol / n-butanol, but also improve its substrate detection upper limit and response intensity, providing a solution for efficient detection of isopentanol / n-butanol and rapid screening of high-yield strains. Summary of the invention:

[0007] The purpose of the present invention is to provide a BmoR protein and a biosensor and applications that can distinguish between isopentanol / n-butanol and other higher alcohols. By analyzing the transcriptional working mechanism and functional areas of natural BmoR, protein engineering of BmoR is carried out by combining irrational transformation methods and computer-assisted semi-rational design methods, and a random mutation library is constructed using error-prone PCR technology. The mutation library is screened and analyzed by exogenously adding n-pentanol, n-butanol, isopentanol, and isobutanol, and finally a BmoR mutant protein that responds highly to isopentanol and responds to n-butanol is obtained.

[0008] Furthermore, the BmoR mutant protein having a high response to isoamyl alcohol and a response to n-butanol is obtained by subjecting the wild-type BmoR protein shown in SEQ ID NO.1 to a W128R mutation, hereinafter referred to as the W128R mutant, and the mutant protein is specifically:

[0009] (1) the amino acid sequence shown in SEQ ID NO.3 in the sequence listing; or

[0010] (2) an amino acid sequence with a homology of more than 75% to SEQ ID NO. 3; or

[0011] (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.

[0012] Furthermore, the present invention also provides a gene encoding a W128R mutant;

[0013] Furthermore, the coding gene is shown in the sequence listing SEQ ID NO.4.

[0014] Another object of the present invention is to provide an application of the W128R mutant, particularly in detecting samples containing isoamyl alcohol / n-butanol or screening isoamyl alcohol / n-butanol producing strains, and more particularly in constructing a biosensor for detecting isoamyl alcohol / n-butanol.

[0015] Furthermore, the biosensor is a biosensor based on the W128R mutant, and the biosensor comprises a gene encoding the W128R 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 biosensor can achieve high fluorescence response and screening for isoamyl alcohol at a concentration of 0-60mM and high fluorescence response and screening for n-butanol at a concentration of 0-100mM, and is further applied in industrial production to achieve high fluorescence response value screening for samples containing isoamyl alcohol / n-butanol and isoamyl alcohol / n-butanol producing strains;

[0016] Furthermore, the promoter of the mutant encoding gene includes but is not limited to P bmoR , P tac , P T7 , P LlacO1 wait;

[0017] Furthermore, the reporter gene includes but is not limited to genes such as gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp, etc.;

[0018] Preferably, the biosensor comprises a gene encoding a W128R 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;

[0019] More preferably, the biosensor is obtained by replacing the wild-type BmoR protein encoding gene on the plasmid pYH1 with the W128R mutant encoding gene, that is, replacing the wild-type BmoR protein encoding gene on the plasmid pYH1 with the W128R mutant encoding gene.bmoR The W128R mutant encoding gene was linked to the colE1 replication origin, amp r and P bmo The driven gfp gene is obtained;

[0020] Furthermore, the isoamyl alcohol / n-butanol producing strains include but are not limited to Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc.;

[0021] Furthermore, the promoter P bmoR The nucleotide sequence is shown in the sequence listing SEQ ID NO.5;

[0022] Furthermore, the promoter P bmo The nucleotide sequence is shown in the sequence listing SEQ ID NO.6;

[0023] Furthermore, the nucleotide sequence of the gfp reporter gene is shown in the sequence listing SEQ ID NO.7.

[0024] The present invention also provides the use of the above-mentioned biosensor in the detection of isoamyl alcohol / n-butanol, especially in the use of environmental, food, medical, and biological samples containing isoamyl alcohol / n-butanol, or in the screening of isoamyl alcohol / n-butanol producing strains, by introducing the above-mentioned plasmid into a production strain, such as Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc., or into a separate host, such as E. coli, etc., to detect the production of isoamyl alcohol / n-butanol.

[0025] Beneficial effects:

[0026] 1. The BmoR-based biosensor can be used to screen high-yield isopentanol / n-butanol strains, but the wild-type BmoR responds to a variety of higher alcohols such as n-pentanol, n-butanol, isopentanol, and isobutanol, but cannot distinguish them, and has poor specificity. The W128R mutant provided by the present invention has a higher response value to isopentanol (0-60mM), responds to n-butanol (0-100mM), and has no response to n-pentanol and isobutanol, which solves the problem that the wild-type BmoR protein cannot distinguish between isopentanol / n-butanol and other higher alcohols.

[0027] 2. The detection range of wild-type BmoR is too narrow, and the detection range for isopentanol is 0-30mM. When the substrate concentration reaches 30mM, the response has reached saturation, so it cannot be used to identify strains with isopentanol production higher than 30mM. The detection range of the W128R mutant provided by the present invention reaches 0-60mM for isopentanol and 0-100mM for n-butanol, which improves the response saturation of BmoR protein to isopentanol / n-butanol, and can be used for the screening and application of high-level production strains of isopentanol / n-butanol. Description of the drawings:

[0028] Figure 1 It is the principle flow chart;

[0029] 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 prediction, 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 specific detection of isopentanol / n-butanol by BmoR was finally successfully achieved.

[0030] Figure 2 Fluorescence microscopy observation of BmoR mutant and wild type to 10 mM n-pentanol, isopentanol, n-butanol and isobutanol;

[0031] Figure 3 The responses of BmoR mutant and wild type to n-pentanol, isopentanol, n-butanol and isobutanol;

[0032] Figure 4 The response of the W128R mutant to the addition of mixed alcohols. Specific implementation method:

[0033] 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.

[0034] The biosensor provided by the present invention is a biosensor based on the W128R mutant, wherein the biosensor comprises a gene encoding the W128R 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 LlacO1The 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.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] The present invention will be further explained below through specific examples.

[0037] Example 1 Design and screening of BmoR mutant W128R

[0038] (1) Error-prone PCR was performed on the full sequence of the wild-type transcription factor BmoR coding gene (shown in SEQ ID NO.2) to construct a full-sequence mutation library, and each mutant was sequenced to determine the mutation site. The obtained data such as the number of BSH between the BmoR mutant and the signal molecule, ΔG, response value GFP / OD600, the fine domain and secondary structure of each mutation site, and the distance between the mutation site and the SBD were sorted and used as a learning set for machine learning. At the same time, the random forest algorithm was used as the output data, and the response value GFP / OD600 was used as the output data to pre-train the learning set, and a model "Model BT" with an accuracy of 88.5% was obtained. This model shows that the number of BSH is a key factor in determining the intensity of BmoR transcriptional activation.

[0039] (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.

[0040] (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 can specifically respond to isopentanol / n-butanol. The obtained BmoR mutants can effectively distinguish between the isomers of n-pentanol and isopentanol, and respond to n-butanol. This property was further verified by exogenously adding a mixture of signal molecules.

[0041] (4) To further verify the specific response of BmoR to isopentanol / n-butanol, we exogenously added different concentrations of n-butanol, isobutanol, n-pentanol or isopentanol, as well as mixtures of different combinations of signal molecules to verify the response of the combined mutant BmoR to these four signal molecules, and successfully achieved the specific response of BmoR to isopentanol / n-butanol.

[0042] Example 2 Concentration gradient experiment to determine the change in detection limit of wild type and mutant

[0043] 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 W128R mutant to 0-60Mm n-pentanol / isopentanol and 0-100mM n-butanol / isobutanol.

[0044] The strains containing the W128R 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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 ).

[0049] 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 is 7.90 mM, and the K m is 8.75 mM; the K of the W128R mutant to isopentanol m 5.68×10 15 mM, K for n-butanol m 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.

[0050] The wild-type BmoR can respond to 0-30mM n-pentanol and isopentanol. When the concentration is higher than 30mM, the response will reach saturation, so the wild-type BmoR cannot distinguish between n-pentanol or isopentanol above 30mM; at the same time, the wild-type BmoR can respond to 0-40mM n-butanol or isobutanol. When the concentration is higher than 40mM, the response will reach saturation, so the wild-type BmoR cannot distinguish between n-butanol or isobutanol above 40mM; the calculation results show that the mutant has a significantly improved Km value for isopentanol and n-butanol compared with the wild type, proving that the BmoR mutant can achieve specific responses to higher concentrations of isopentanol (0-60mM) and higher concentrations of n-butanol (0-100mM), which is consistent with the experimental results.

[0051] Example 3 Mixed alcohol addition experimental verification

[0052] To further verify that the W128R mutant with a higher response to isopentanol and n-butanol can maintain its specificity under the condition of mixed 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 (10mM:100mM), 1:8 (10mM:80mM), 1:6 (10mM:60mM), 1:4 (10mM:40mM), 1:2 (10mM:20mM), 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. Take 200μL of bacterial solution and measure its OD 600 and GFP, and calculated their relative fluorescence intensities.

[0053] GFP / OD of W128R mutant 600 The value only increased with the increase of isoamyl alcohol / n-butanol concentration, while no obvious fluorescence signal was detected when other signal molecules were added. At the same time, the W128R mutant was further verified to have a specific response to isoamyl alcohol / n-butanol by adding mixed signal molecules. The results showed that the W128R mutant could still maintain its specificity for isoamyl alcohol / n-butanol in the presence of other signal molecules ( Figure 4 ).

[0054] 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 a W128R mutation on the basis of the wild-type BmoR protein shown in SEQ ID NO.1 in the sequence table.

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 / n-butanol, or in screening isoamyl alcohol / n-butanol producing strains.

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

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 / n-butanol, and in screening strains producing isopentanol / n-butanol.

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