Vanillin biosensor

Abstract

The invention relates to a biosensor capable of efficiently responding to vanillin, and belongs to the technical field of bioengineering. An aldehyde response transcription factor YqhC from Escherichia coli and a promoter element PyqhD regulated by the aldehyde response transcription factor YqhC are used for establishing a vanillin response RFP report system, a yqhC gene is subjected to semi-rational design, a mutation library is constructed through saturated mutation, vanillin is used as a substrate, and a non-background fluorescence response, a non-background fluorescence response function and a non-background fluorescence response function are obtained by screening response concentration and response threshold values. The YqhC mutant has a wider substrate detection range (0-10 mM) and higher response strength (167 times), and is named as YqhC-V18. The biosensor containing a vanillin response element (YqhC-V18) and a fluorescent protein report element (RFP) is further obtained, and can be applied to detection of a sample containing vanillin, or screening of a vanillin production strain, or regulation and control of the production process of vanillin.

Description

Technical field: The invention relates to a biosensor capable of efficiently responding to vanillin, belonging to the technical field of bioengineering. Background technology: Vanillin (3-methoxy-4-hydroxybenzaldehyde, vanillin), also known as vanillin, has a strong vanilla flavor and milky aroma. It is the largest and most widely used variety in the world's food and flavoring industry. It is widely used in food, cosmetics, medicine, chemical industry, agriculture and other industries.

[0003] At present, the vanillin on the market includes natural vanillin and chemically synthesized vanillin. Among them, the vanillin synthesized by chemical method has a relatively low price (15 US dollars / kg), but it has disadvantages such as expensive raw materials, poor product purity, and non-natural products, which cannot meet people's demand for edible safety of natural flavors. Natural vanillin mainly comes from plant extraction and biosynthesis. Among them, the plant extraction method is obtained by extracting vanilla beans from the Rutaceae plant. This method is expensive (1200 US dollars / kg) due to the scarce supply of vanilla beans, which cannot meet the current market demand; the use of microbial cell factories to develop a low-cost raw material vanillin biosynthesis process can effectively improve the shortcomings of plant extraction and chemical synthesis, and the obtained vanillin is moderately priced (700 US dollars / kg), with broad industrial prospects and market application potential, and has become a research hotspot at home and abroad.

[0004] Transcription factors (TFs) are often used to modify metabolite response elements and establish response systems. They mainly sense the corresponding metabolites to achieve binding and dissociation with the upstream regulatory sequences of target genes, thereby achieving transcriptional activation or inhibition of target genes. Currently, biosensors based on transcription factors have been widely used in the screening or enrichment of high-yield strains, high-throughput screening of related enzymes in metabolic pathways, and metabolic pathway regulation in biosynthetic processes.

[0005] The DNA-bound transcription activator YqhC is an aldehyde-mediated transcription factor belonging to the AraC / XylS family of proteins. It activates downstream gene clusters by binding to aldehydes. yqhD-dkgA The YqhC transcription factor has a transcriptional response, which in turn upregulates the expression of aldehyde reductases YqhD and DkgA. Based on the regulatory mechanism of the YqhC transcription factor, a research team has successfully transformed it into a biosensor, which has increased the response intensity of the response element to ethanolaldehyde by 70 times. As the first reported aldehyde response element, wild-type YqhC has almost no response to vanillin, so it is urgently needed to be modified. Summary of the invention: The purpose of the present invention is to provide an efficient vanillin biosensor system: no background fluorescence, linear response in the range of 0-10mM substrate concentration, and the response intensity reaches 167 times.

[0007] The present invention utilizes Escherichia coli ( Escherichia coli )-derived aldehyde-responsive transcription factor YqhC and its regulated promoter elements P yqhD Establish a RFP reporting system for vanillin responses. yqhC The gene was semi-rationally designed, and a mutant library was constructed through saturation mutagenesis. Using vanillin as a substrate, a YqhC mutant with no background fluorescence response, a wider substrate detection range (0-10 mM), and a higher response intensity (167 times) was obtained through screening of response concentration and response threshold, and it was named YqhC-V18. A biosensor containing a vanillin response element (YqhC-V18) and a fluorescent protein reporter element (RFP) was then obtained.

[0008] In order to achieve the above purpose, the technical route adopted by the present invention is as follows: One of the technical solutions provided by the present invention is a YqhC mutant, which is obtained by mutation of F59T and A60T on the basis of the wild-type transcription factor YqhC shown in SEQ ID NO.1, and the mutant is named YqhC-V18 mutant; Furthermore, the amino acid sequence of the YqhC-V18 mutant is shown in SEQ ID NO.3; Furthermore, the coding gene of the YqhC-V18 mutant is YqhC-V18 Gene, nucleotide sequence is shown as SEQ ID NO.4.

[0009] The second technical solution provided by the present invention is the application of the YqhC mutant or its encoding gene described in the first technical solution, especially in the detection of samples containing vanillin or the screening of vanillin producing strains, and more particularly, in the construction of a biosensor for detecting vanillin.

[0010] The third technical solution provided by the present invention is a yqhC-V18 A biosensor for a gene, the biosensor comprising the following elements: a regulator yqhC-V18 Gene promoter, yqhC-V18 Gene, yqhC-V18 Gene promoter P yqhD , by promoter P yqhD driven reporter gene; Furthermore, the regulation yqhC-V18Gene promoters include but are not limited to P rrnB , P j23100 , P j23108 , P j23119 wait; Furthermore, the reporter gene includes but is not limited to: red fluorescent protein gene rfp , green fluorescent protein gene gfp , yellow fluorescent protein gene yfp wait; Furthermore, the biosensor further comprises a replicon gene for controlling the initial replication of the entire biosensor; Furthermore, the biosensor further comprises a resistance gene for screening transformants comprising the biosensor; Furthermore, the replicon includes but is not limited to p15A, ori, ColE1 wait; Furthermore, the resistance genes include but are not limited to Cm, Amp, Kan wait; Furthermore, the promoter P rrnB The nucleotide sequence is shown in SEQ ID NO.5; Furthermore, the promoter P yqhD The nucleotide sequence is shown in SEQ ID NO.6; Furthermore, the rfp The nucleotide sequence of the coding gene is shown in SEQ ID NO.7; Furthermore, the replicon gene p15A The nucleotide sequence is shown in SEQ ID NO.8; Furthermore, the resistance gene Cm The nucleotide sequence is shown in SEQ ID NO.9; Preferably, the biosensor comprises P rrnB , yqhC-V18, P yqhD , rfp, p15A, Cm , more preferably, the nucleotide sequence of the biosensor is as shown in SEQ ID NO.10.

[0011] The fourth technical solution provided by the present invention is the application of the biosensor described in the third technical solution, especially in the detection of samples containing vanillin or the screening of vanillin producing strains.

[0012] Beneficial effects: The present invention provides an efficient aldehyde-responsive transcription factor YqhC-V18 and a vanillin biosensor system constructed therefrom, which is regulated by yqhC-V18 Gene promoter, yqhC-V18 Gene, yqhC-V18 Gene promoter P yqhD , by promoter P yqhD The biosensor is composed of a reporter gene for driving, a replicon gene for controlling the initial replication of the entire biosensor, and a resistance gene for selecting transformants containing the biosensor.

[0013] Compared with the wild-type YqhC which has basically no fluorescence response to vanillin, YqhC-V18 has the advantages of no background fluorescence response, linear response in the substrate concentration range of 0-10mM, and a response intensity of 167 times when responding to vanillin. Description of the drawings: Figure 1 Figure 2 shows the fluorescence response curves of the wild-type YqhC and mutant YqhC-V18 biosensor systems to 0-10 mM vanillin. Specific implementation method: Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0016] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0017] The following examples are provided to further illustrate the present invention and are not intended to limit the essential contents of the present invention.

[0018] 1. The following definitions are adopted in the present invention: (1) Nomenclature of amino acid and DNA sequences The generally accepted IUPAC nomenclature for amino acid residues is used, either as a single letter or three letter code. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0019] (2) Identification of YqhC mutants The mutated amino acid in the YqhC-V18 mutant is represented by "original amino acid + position + replaced amino acid". For example, F59T means that the 59th amino acid is mutated from phenylalanine to threonine, and the position number corresponds to the amino acid sequence number of the wild-type YqhC in SEQ ID NO. 1.

[0020] In the present invention, lowercase italics yqhC Indicates the gene encoding the wild-type aldehyde-responsive transcription factor YqhC, in lowercase italics YqhC-V18It represents the coding gene of mutant YqhC-V18. The specific information is shown in the following table.

[0021]

[0022] In the present invention, the amino acid sequence of the YqhC-V18 mutant is shown in SEQ ID NO.3: MLQNCAQSNCRIIPKKLRDMKREEICRLLADKVNKLKNKENSLSGLLPDVRLLYGETPTTRTPVMYEPGIIILFSGHKIGYINERVFRYDANEYLLLTVPLPFECETYATSEVPLAGLRLNVDILQLQELLMDIGEDEHFQPSMAASGINSATLSEEIL CAAERLLDVMERPLDARILGKQIIREILYYVLTGPCGGALLALVSRQTHFSLISRVLKRIENKYTENLSVEQLAAEANMSVSAFHHNFKSVTSTSPLQYLKNYRLHKARMMIIHDGMKASAAAMRVGYESASQFSREFKRYFGVTPGEDAARMRAMQGN.

[0023] 2. Some of the primers involved in the present invention and embodiments are shown in the following table:

[0024] 3. Some strains and plasmids involved in the present invention and embodiments are shown in the following table:

[0025] The present invention is further explained below by means of specific examples.

[0026] Example 1: Screening of mutant YqhC-V18 (1) In order to obtain a biosensor that efficiently responds to vanillin, it is first necessary to use YqhC and the promoter elements it regulates P yqhD An aldehyde-responsive RFP reporter system was established, and then semi-rational design was performed through structural simulation and molecular docking to find the ligand binding cavity. A mutation library of the YqhC fragment was constructed through saturation mutation of the amino acids where the ligand binding cavity is located. Finally, high-throughput screening was performed by flow cytometry to obtain the aldehyde-responsive transcription factor YqhC that efficiently responds to vanillin.

[0027] (2) Construction of a biosensor system based on the wild-type transcription factor YqhC: MG1655 Using the genome as a template to clone an aldehyde-responsive transcription factor from Escherichia coli yqhC(shown in SEQ ID NO.2) and promoter P yqhD (shown in SEQ ID NO.6), promoter element obtained by gene synthesis P rrnB (shown in SEQ ID NO.5), red fluorescent protein gene rfp (shown in SEQ ID NO.7), resistance gene Cm (shown in SEQ ID NO.9), replicon p15A (shown in SEQ ID NO.8), the PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2.

[0028] (3) Using 2KPlusⅡ as a DNA marker, the target fragment of the correct size was obtained by agarose gel electrophoresis, and the purified target fragment was obtained by DNA recovery. P rrnB , yqhC-P yqhD , rfp, p15A, Cm Perform fragment ligation to obtain ligation products P rrnB -yqhC-P yqhD -rfp-p15A-Cm Fragments, all ligation products were transformed into 100 μL JM109 chemical competent cells, placed on ice, and iced for 25 min. After the ice bath, the centrifuge tube was placed in a 42°C water bath for heat shock for 1 min 30 s. After the heat shock, the centrifuge tube was placed on ice again for an ice bath for 2 min. After the ice bath, 900 μL LB medium was added to the centrifuge tube, placed in a 37°C shaker, and revived at 220 rpm for 45 min before coating with chloramphenicol resistance plates. The plates were placed in a 37°C constant temperature incubator for overnight growth.

[0029] The next day, a single colony was picked for PCR to verify whether the plasmid was successfully constructed. The verification primer was sequencing-yqhC-F / R, the target fragment was cPCR-yqhC, and the size was 1510bp. The PCR reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4. After the PCR reaction, agarose gel electrophoresis was performed, and the bands were compared with DNA Marker: 2KPlusⅡ to obtain the target band of the correct size, that is, the wild-type YqhC biosensor plasmid was successfully constructed. The plasmid was named pYJ12, and the E. coli strain carrying the plasmid was named sYJ022.

[0030] Table 1 PCR reaction system

[0031] Table 2 PCR reaction procedure

[0032] Table 3 PCR reaction system

[0033] Table 4 PCR reaction procedure

[0034] (4) Construction of YqhC saturation mutation library and high-throughput screening: A saturation mutation library was constructed by saturating the key amino acids F59 and A60 in the substrate binding pocket of wild-type YqhC.

[0035] First, wild-type pYJ12 was used as a template, and primers 251 and 252 were designed to introduce saturation mutations through loop P in an NNS manner. The saturation mutation PCR product was named yqhC Saturation mutation library. The PCR reaction system is shown in Table 5, and the PCR reaction program is shown in Table 6.

[0036] The digestion system shown in Table 7 was used to yqhC The PCR products of the saturated mutation library were digested and all the digestion products were transformed into 100 μL JM109 chemical competent cells. After 1 hour of recovery, LB was added to 4.5 mL, and 4.5 μL 25 mg / mL chloramphenicol was added (final concentration was 25 μg / mL). The cells were cultured at 37°C and 220 rpm in a shaker until the OD 600 The concentration of vanillin was about 0.6, and 500 μL of 50 mM vanillin (final concentration of 5 mM) was added for 4 hours of induction. Then, the cells in the top 1% of the fluorescence level were sorted by flow cytometry, and finally a mutant strain that efficiently responded to vanillin was obtained, which was named sYJ036, the sensor plasmid it carried was named pYJ24, and the YqhC mutant on the plasmid was named YqhC-V18. The amino acid sequence of the YqhC-V18 mutant was determined by sequencing as shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene was shown in SEQ ID NO.4.

[0037] Table 5 PCR reaction system

[0038] Table 6 PCR reaction program

[0039] Table 7 Dpn1 digestion system

[0040] Example 2: Verification of the fluorescence response of mutant YqhC-V18 In order to further verify the performance of mutant YqhC-V18, a gradient vanillin exogenous addition experiment is needed to determine its fluorescence response curve.

[0041] The mutant strain sYJ036 and wild-type strain sYJ022 were inoculated into 5 mL LB (25 μg / mL Cm) liquid medium and cultured at 37 °C and 220 rpm for 12 hours as seed solution. The seed solution was incubated, and then the fluorescence response curves of the mutant and wild-type to gradient concentrations of vanillin were measured using a 2 mL 96-deep-well plate.

[0042] Use a pipette to add 800 μL of fresh M9 culture medium (25 μg / mL Cm), then add 10 μL of seed solution to the culture medium, and repeat three times for each tube of seed solution. After sealing the sealing film, place the deep-well plate in a shaker at 37 °C and 220 rpm for 4 hours, then take it out, add 200 μL of culture medium (25 μg / mL) containing 0-50 mM gradient concentration of vanillin (final concentration of 0-10 mM), seal the sealing film, and continue to put it back in the shaker for 12 hours. Pipette 200 μL of bacterial solution into a black 96-well plate, and measure the fluorescence intensity and OD with an enzyme reader (BioTek Cytation 3). 600 The quantitative detection of red fluorescent protein RFP uses an excitation wavelength of 580 nm and an emission wavelength of 610 nm, with a gain value of 80. Each well is read three times to obtain the average value. The bacterial density is measured at a wavelength of 600 nm, and the RFP / OD of each well is calculated on this basis. 600 As relative fluorescence intensity values.

[0043] The RFP / OD of strain sYJ022 600 The results were analyzed as wild-type controls, with RFP / OD 600 The concentration of vanillin was the vertical axis and the concentration of vanillin was the horizontal axis. GraphPad Prism 8.0 software was used to draw the graph, and the curve was fitted using LOWESS.

[0044] The results are as follows Figure 1 As shown, compared with the wild-type YqhC which has basically no response to vanillin, the mutant YqhC-V18 can respond linearly in the range of 0-10 mM vanillin, and there is no background fluorescence response when no vanillin is added (0 g / L). When the concentration of vanillin added is 10 mM, the response intensity is increased by 167 times compared with the case when no vanillin is added (0 mM), indicating that the mutant can respond to vanillin efficiently, and has the advantages of no background leakage, linear response in the range of 0-10 mM vanillin, and response intensity can reach 167 times.

[0045] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A YqhC mutant, characterized in that The mutant is obtained by mutation of F59T and A60T based on the wild-type transcription factor YqhC shown in SEQ ID NO.

1. The mutant is named YqhC-V18 mutant, and the amino acid sequence is shown in SEQ ID NO.

3.

2. The coding gene of the YqhC mutant according to claim 1.

3. Use of the YqhC mutant according to claim 1 or the encoding gene according to claim 2, characterized in that: The invention is used in detecting samples containing vanillin, in screening strains with high vanillin production, or in constructing a biosensor for detecting vanillin.

4. A biosensor, characterized in that: The biosensor comprises the following elements: YqhC-V18 Gene promoter, YqhC-V18 Gene, YqhC-V18 Gene promoter P yqhD , by promoter P yqhD driven reporter gene.

5. The biosensor according to claim 4, wherein: The regulation YqhC-V18 Gene promoters include but are not limited to P rrnB , P j23100 , P j23108 , P j23119 ; The reporter gene includes but is not limited to: red fluorescent protein gene rfp , green fluorescent protein gene gfp , yellow fluorescent protein gene yfp .

6. The biosensor according to claim 4, wherein: The biosensor further comprises a replicon gene, and / or a resistance gene.

7. The biosensor according to claim 6, wherein The replicons include but are not limited to p15A, ori, ColE1 ; The resistance genes include but are not limited to Cm, Amp, Kan .

8. The biosensor according to claim 4, wherein: The biosensor comprises P rrnB 、YqhC-V18、 P yqhD 、rfp、p15A、Cm .

9. The biosensor according to claim 8, wherein The nucleotide sequence of the biosensor is shown as SEQ ID NO.

10.

10. Use of the biosensor according to claim 4, characterized in that: It is used in detecting samples containing vanillin, in screening high-producing vanillin strains, or in regulating the production process of vanillin.

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