BmoR mutant biosensor capable of specifically responding to erythritol

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a BmoR protein mutant specifically responding to erythritol and application of the BmoR protein mutant in erythritol detection or biosensors. The BmoR protein mutant is obtained by carrying out mutation containing I96T / I183F / G188D / F204L on the basis of a wild type BmoR protein as shown in SEQ ID NO. 1 in a sequence table. The I96T / I183F / G188D / F204L mutant is only sensitive to erythritol and has no response to sucrose, so that the problem that the erythritol and the sucrose cannot be distinguished by wild type BmoR protein is solved; meanwhile, the detection range of the I96T / I183F / G188D / F204L mutant on the erythritol reaches 0-100mM, the response saturation of the BmoR protein on the erythritol is improved, and the mutant can be used for screening and applying high-level production strains of the erythritol.

Description

Technical field:

[0001] The invention belongs to the field of bioengineering technology, and specifically relates to a BmoR mutant with a wider detection range (0-200 mM) for erythritol, and application of the mutant in erythritol detection or biosensor. Background technology:

[0002] Erythritol is a naturally occurring four-carbon sugar alcohol, widely used as a low-calorie sweetener in food and beverages. It was originally extracted from some naturally fermented foods, such as fruits, vegetables and fermented foods, but in industrial production it is mainly obtained by glucose fermentation. The synthesis of erythritol 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 to 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 narrow detection range. The BmoR protein obtained by protein engineering in the present invention improves its detection upper limit and response intensity for erythritol, and can significantly distinguish erythritol from sucrose. Most importantly, it can be used to screen erythritol high-yield strains to meet industrial needs.

[0003] Erythritol has very low calories, about 0.2 kcal / g, compared to about 4 kcal / g for regular sugar. Its energy is hardly metabolized by the human body, so it helps with weight loss and blood sugar control. At the same time, the sweetness of erythritol is about 60%-70% of sucrose, providing sweetness without high calories. The wild-type transcription factor BmoR can bind to erythritol and sucrose at the same time and output corresponding detection signals, but cannot specifically distinguish between erythritol and sucrose. At the same time, the wild-type BmoR's response concentration to erythritol reaches saturation at 60mM, which cannot meet industrial needs.

[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 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 high-yield strains of high-value compounds. However, the detection range of wild-type BmoR biosensors for erythritol is too narrow (0-60mM). At the same time, wild-type BmoR responds to both erythritol and sucrose, and cannot achieve specific detection of erythritol, making it unusable in industrial production. Therefore, the modification of biosensors provides a solution for efficient detection of erythritol production and rapid screening of high-yield strains. Summary of the invention:

[0006] The purpose of the present invention is to provide a BmoR protein and a biosensor that can distinguish between erythritol and sucrose. By non-rational modification of natural BmoR and combining high-throughput screening technology to perform protein engineering on BmoR, a BmoR mutant protein that specifically responds to erythritol is finally obtained.

[0007] Furthermore, the BmoR mutant protein capable of specifically responding to erythritol is obtained by subjecting the wild-type BmoR protein shown in the sequence table SEQ ID NO.1 to I96T / I183F / G188D / F204L mutation, hereinafter referred to as I96T / I183F / G188D / F204L mutant, and the mutant protein specifically has the amino acid sequence shown; or

[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 75% or more 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 an I96T / I183F / G188D / F204L 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 I96T / I183F / G188D / F204L mutant, particularly an application in a sensor for detecting samples containing erythritol or screening erythritol-producing strains, and more particularly, an application in constructing a biosensor for detecting erythritol;

[0014] Furthermore, the biosensor is a biosensor based on the I96T / I183F / G188D / F204L mutant, and the biosensor comprises a gene encoding the I96T / I183F / G188D / F204L 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 response and screening of 0-100mM erythritol and 0-1mM trace erythritol, and is further applied in industrial production to achieve the screening of samples containing erythritol, the screening of erythritol high-yield strains or the detection of trace erythritol;

[0015] Furthermore, the promoter of the mutant encoding gene includes but is not limited to P bmoR , P tac , P T7 , P L lacO1, etc.

[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 expression element is a gene encoding the I96T / I183F / G188D / F204L 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 I96T / I183F / G188D / F204L mutant encoding gene, that is, replacing the wild-type BmoR protein encoding gene on the plasmid pYH1 with the I96T / I183F / G188D / F204L mutant encoding gene. bmoR The gene encoding the I96T / I183F / G188D / F204L 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 P bmoR 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 erythritol detection, especially in the screening of high-yield erythritol strains or the detection of trace erythritol, by introducing the above-mentioned plasmid into production strains, such as Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc., to detect the production of erythritol.

[0024] Beneficial effects:

[0025] 1. The BmoR-based biosensor can be used to detect erythritol, but the wild-type BmoR responds to both erythritol and sucrose and cannot distinguish them, with poor specificity. The I96T / I183F / G188D / F204L mutant provided by the present invention is only sensitive to erythritol and has no response to sucrose, thus solving the problem that the wild-type BmoR protein cannot distinguish between erythritol and sucrose.

[0026] 2. The detection range of wild-type BmoR is too narrow, and the detection range for erythritol is 0-60mM. When the substrate concentration reaches 60mM, the response has reached saturation, so it cannot be used to identify strains that produce erythritol with an output higher than 60mM. The I96T / I183F / G188D / F204L mutant provided by the present invention has a detection range of 0-100mM for erythritol, which improves the response saturation of BmoR protein to erythritol and can be used for the screening and application of high-level erythritol production strains. Description of the drawings:

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

[0028] First, the first 1000bp of the N-terminus of the wild-type bmoR was randomly mutated by error-prone PCR to obtain a random mutation library of BmoR; GFP fluorescent protein was added downstream of the bmoR gene, and the response of the mutant BmoR to erythritol was reflected by detecting the fluorescence intensity. The response of the mutant BmoR to erythritol was detected by adding different concentrations of erythritol and sucrose.

[0029] Figure 2 The response of wild-type BmoR to 0-100 mM erythritol and sucrose;

[0030] Figure 3 The response of BmoR mutant to 0-100 mM erythritol and sucrose;

[0031] Figure 4 The response of BmoR mutant to 0-1 mM erythritol;

[0032] Figure 5 The molecular docking of I96T / I183F / G188D / F204L mutant and erythritol; 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 I96T / I183F / G188D / F204L mutant, wherein the biosensor comprises a gene encoding the I96T / I183F / G188D / F204L 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.

[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 Screening of BmoR mutant I96T / I183F / G188D / F204L

[0038] Construction and high-throughput screening of a random mutation library for the transcription factor BmoR

[0039] (1) Plasmid pYH1 (construction see DOI: https: / / doi.org / 10.1016 / j.ymben.2019.08.015; https: / / doi.org / 10.1186 / s12934-019-1084-2) containing the wild-type BmoR encoding gene (shown in SEQ ID NO. 2) was used as a template to obtain the bmoR mutant gene by error-prone PCR amplification (by adding Mn2+ , increase the Mg content in the PCR system 2+ The concentration was adjusted and the ratio of dNTPs was adjusted to introduce random mutations. A 10× unbalanced dNTPs mixture was prepared, in which the concentrations of dCTP and dTTP were four times that of dATP and dGTP. The PCR program was set as follows: 94°C pre-denaturation for 2 minutes, 30 amplification cycles including: 95°C denaturation for 1 minute, 55-68°C annealing for 1 minute, and the appropriate extension time was determined according to the amplification rate of 1 kb per minute, and the extension temperature was 72°C. The storage temperature was set to 16°C. ); The PCR product was confirmed by gel electrophoresis and recovered and purified; the purified product was placed in a 37°C water bath and digested with DpnI (1μL / 50μL purified product) for 1-2h. Take 2μL of the bmoR mutant fragment, 3μL of the pYH1 backbone (a construct with P bmoR , P bmo , gfp fluorescent protein gene plasmid as the backbone, equivalent to the pYH1 backbone) and 5μL Gibson Assemble Mix, after mixing, place in a 50℃ water bath for ligation for 1h. Take 5-10μL of the ligation product and transfer it into 50μL E. coli XL10-Gold transfection competent cells. After the transformation incubation, directly transfer it to 4mL LB, incubate it at 37℃, 220rpm constant temperature shaker for 1h, add 5μL 50mg / mL carbenicillin after 1h, and culture overnight. After obtaining the seed solution, take 50μL and transfer it to 5mL fresh LB liquid culture medium (50μg / mL), add 10mM erythritol at the same time, cover the tube tightly, and induce it at 30℃, 220rpm constant temperature shaker for 8-10h to obtain the library induced sample S1. The library induced sample S1 was screened continuously for two rounds by flow cytometry to obtain the enriched excellent BmoR mutants.

[0040] (2) First, by analyzing the samples induced by the wild-type BmoR sensor system and the control system, determine the location of the fluorescence peak (P1) and the appropriate sorting voltage; based on this, sort the mutant library samples, collect the first 1% of the bacteria in the P3 region (about 10,000) in the first round, and collect them in 1 mL of fresh antibiotic-free LB liquid culture medium. After the collection is completed, add 4 mL of fresh antibiotic-free LB liquid culture medium to the collected bacterial liquid, incubate at 37°C 220 rpm constant temperature shaker for 1 hour, add 5 μL 50 mg / mL carbenicillin after 1 hour, and culture overnight at 37°C 220 rpm constant temperature shaker. After obtaining the BmoR mutation library after the first round of enrichment, the same induction method was used for the second round of induction S2 and FACS sorting. The second round of sorting was based on the fluorescence peak offset of the first round of sorting. The first 1% of the bacteria in the P3 area (about 10,000) were collected. After the collection was completed, 4 mL of anti-free liquid LB medium was added to the collected bacterial solution, and the culture was incubated at 37 ° C for 1 hour. After the incubation, 200 μL of bacterial solution was first applied to the plate containing carbobenzyl antibiotics, the sealing film was sealed, and it was inverted and placed in a 37 ° C constant temperature incubator for overnight culture. Then, the corresponding amount of carbobenzyl antibiotics was added to the remaining liquid sample, and it was placed in a 37 ° C constant temperature incubator for overnight culture. Ten single colonies were randomly selected from the single colonies obtained after the second round of sorting for colony PCR, and the PCR products were sent to Jinweizhi Company for sequencing to verify the mutation efficiency of the library and the FACS screening effect.

[0041] (3) The sequenced mutant I96T / I183F / G188D / F204L (amino acid sequence as shown in the sequence list SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4) was verified in a 96-deep well plate. 950 μL of fresh LB (100 μg / mL Amp) medium was first added to each well; erythritol or sucrose was added to the wells to make the final concentration 0-100 mM; finally, 50 μL of seed solution was inoculated into each well. After sealing the sealing film, the deep well plate was placed in a shaker at 30°C and 220 rpm for 16 h.

[0042] (3) 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, 510 nm emission wavelength, and 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.

[0043] (4) GFP / OD of I96T / I183F / G188D / F204L mutant 600 like Figure 3 As shown, it can be seen that compared with the wild-type BmoR ( Figure 2 ), the I96T / I183F / G188D / F204L mutant was able to respond to 100 mM erythritol but was unresponsive to sucrose.

[0044] Example 2 Model Analysis

[0045] The I96T / I183F / G188D / F204L mutants were sequenced and the changes in amino acids at the mutation sites were analyzed. At the same time, the BmoR mutants were modeled using software such as AUTODOCK and ChimeraX. The mutants were docked with the substrate small molecule erythritol to analyze the binding sites and hydrogen bond formation between the mutants and erythritol.

[0046] Using the wild-type BmoR protein three-dimensional structure as a template, homology modeling was performed on the I96T / I183F / G188D / F204L mutant, with a homology rate of 99.9%. The mutant structure was further docked with the substrate molecule erythritol. The results showed that in the complex, the mutant interacted with erythritol through a total of 4 hydrogen bonds (Tyr171, Met48, Arg250), indicating that erythritol can tightly bind to the mutant ( Figure 5 ).

[0047] 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 I96T / I183F / G188D / F204L mutations on the basis of 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 erythritol or in screening erythritol-producing strains.

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

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 erythritol, and in screening strains producing erythritol.

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