A vanillin biosensor

Abstract

The present application relates to a kind of high efficiency response vanillin biosensor, belong to the field of bioengineering technology.The present application utilizes aldehyde response transcription factor YqhC of escherichia coli ( Escherichia coli ) origin and the promoter element of the regulated P yqhD Establishment of RFP report system of vanillin response, to yqhC Gene semi-rational design is carried out, and mutant library is constructed by saturation mutation, with vanillin as substrate, by screening response concentration and response threshold, obtain no background fluorescence response, with wider substrate detection range (0-10 mM), and response intensity is higher (167 times) YqhC mutant, it is named YqhC-V18.Further, the biosensor containing vanillin response element (YqhC-V18) and fluorescent protein report element (RFP) is obtained, which can be applied to detect vanillin-containing sample, or screen vanillin production strain, or regulate vanillin production process.

Description

TECHNICAL FIELD

[0001] The present application relates to a high-efficiency vanillin-responsive biosensor, and belongs to the technical field of bioengineering. BACKGROUND

[0002] Vanillin (3-methoxy-4-hydroxybenzaldehyde) is also known as vanillic aldehyde, has a rich vanilla flavor and milk aroma, is the largest and most widely used variety in the world food flavor industry, and is widely used in food, cosmetics, medicine, chemical industry, agriculture and other industries.

[0003] At present, vanillin on the market includes natural vanillin and chemically synthesized vanillin, wherein the vanillin synthesized by chemical method has a relatively low price (15 US dollars / kg), but has the disadvantages of expensive raw materials, poor product purity, non-natural product, etc., and cannot meet the edible safety demand of people on natural essence. Natural vanillin is mainly obtained by plant extraction method and biosynthesis method, wherein the plant extraction method is obtained by extraction from vanilla orchid beans of Rutaceae, the method cannot meet the current market demand due to the scarcity of vanilla orchid beans and high price (1200 US dollars / kg); the biosynthesis process of vanillin developed by using microbial cell factories can effectively improve the shortcomings of the plant extraction method and the chemical synthesis method, and the obtained vanillin has a moderate price (700 US dollars / kg), has broad industrial prospect and market application potential, and has become a research hotspot at home and abroad.

[0004] Transcription factors (TF) are often used for the modification of metabolite-responsive elements and the establishment of response systems, mainly through sensing the corresponding metabolites to realize the binding and dissociation with the upstream regulatory sequence of the target gene, and then realize the transcription activation or inhibition of the target gene. At present, the transcription factor-based biosensor has 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 biosynthesis process.

[0005] DNA-binding transcriptional activator YqhC is a transcription factor of aldehyde, belongs to the AraC / XylS family protein, and activates the transcription response of downstream gene cluster yqhD-dkgA by binding aldehyde, and then up-regulates the expression of aldehyde reductase YqhD and DkgA. Based on the regulation mechanism of YqhC transcription factor, a research team has successfully transformed it into a biosensor, so that the response intensity of the response element to ethanol aldehyde is increased by 70 times. As the first reported aldehyde-responsive element, the wild-type YqhC has almost no response to vanillin, and therefore needs to be transformed. SUMMARY

[0006] The present application aims to provide a highly efficient vanillin biosensor system: no background fluorescence, linear response in the range of 0-10 mM substrate concentration, and response intensity reaching 167 times.

[0007] The present application utilizes aldehyde-responsive transcription factor YqhC from Escherichia coli Escherichia coli ) and the promoter element regulated by YqhC P yqhD Establish a RFP reporter system responsive to vanillin, semi-rational design of the gene, construct a mutant library by saturation mutation, use vanillin as a substrate, screen the response concentration and response threshold, obtain a YqhC mutant with no background fluorescence response, a wider substrate detection range (0-10 mM), and a higher response intensity (167 times), which is named YqhC-V18. Further, a biosensor containing a vanillin response element (YqhC-V18) and a fluorescent protein reporter element (RFP) is obtained. yqhC

[0008] In order to achieve the above-mentioned purpose, the technical route adopted by the present application is as follows:

[0009] One of the technical solutions provided by the present application is a YqhC mutant, which is obtained by F59T and A60T mutations on the basis of the wild-type transcription factor YqhC shown in SEQ ID NO. 1, and the mutant is named YqhC-V18 mutant.

[0010] Further, the amino acid sequence of the YqhC-V18 mutant is shown in SEQ ID NO. 3.

[0011] Further, the coding gene of the YqhC-V18 mutant is YqhC-V18 gene, and the nucleotide sequence is shown in SEQ ID NO. 4.

[0012] The second technical solution provided by the present application is the application of the YqhC mutant or the coding gene thereof in technical solution one, in particular, the application in detecting vanillin-containing samples or screening vanillin-producing strains, and more particularly, the application in constructing a biosensor for detecting vanillin.

[0013] The third technical solution provided by the present application is a biosensor containing yqhC-V18 gene, the biosensor contains the following elements: a promoter regulating yqhC-V18 gene, yqhC-V18 gene, yqhC-V18 the promoter regulated by the gene P yqhD , and a reporter gene driven by the promoter P yqhD ;​

[0014] Further, the regulation of the expression of the target gene is achieved by regulating the expression of the reporter gene. yqhC-V18 The promoter of the target gene includes but is not limited to P rrnB , P j23100 , P j23108 , P j23119 , etc.

[0015] Further, the reporter gene includes but is not limited to: red fluorescent protein gene rfp , green fluorescent protein gene gfp , yellow fluorescent protein gene yfp , etc.

[0016] Further, the biosensor further comprises a replicon gene for controlling the initiation of replication of the entire biosensor.

[0017] Further, the biosensor further comprises a resistance gene for screening the transformant comprising the biosensor.

[0018] Further, the replicon includes but is not limited to p15A, ori, ColE1 , etc.

[0019] Further, the resistance gene includes but is not limited to Cm, Amp, Kan , etc.

[0020] Further, the nucleotide sequence of the promoter P rrnB is shown as SEQ ID NO. 5;

[0021] Further, the nucleotide sequence of the promoter P yqhD is shown as SEQ ID NO. 6;

[0022] Further, the nucleotide sequence of the rfp coding gene is shown as SEQ ID NO. 7;

[0023] Further, the nucleotide sequence of the replicon gene p15A is shown as SEQ ID NO. 8;

[0024] Further, the nucleotide sequence of the resistance gene Cm is shown as SEQ ID NO. 9;

[0025] Preferably, the biosensor comprises P rrnB , yqhC-V18, P yqhD, rfp, p15A, Cm More preferably, the nucleotide sequence of the biosensor is shown as SEQ ID NO. 10.

[0026] The fourth technical solution of the present application provides an application of the biosensor of the third technical solution, in particular, in detecting a sample containing vanillin or screening a vanillin-producing strain.

[0027] Beneficial effects:

[0028] The present application provides an efficient aldehyde response-based transcription factor YqhC-V18 and a vanillin biosensor system constructed therefrom, which is composed of a promoter regulating yqhC-V18 a gene, yqhC-V18 a gene, yqhC-V18 a promoter regulated by the gene P yqhD a reporter gene driven by the promoter, P yqhD a replicon gene for controlling the initial replication of the entire biosensor, and a resistance gene for screening a transformant containing the biosensor.

[0029] Compared with the wild-type YqhC which has substantially no fluorescent response to vanillin, YqhC-V18 has the advantages of no background fluorescence response, linear response in the range of 0-10 mM substrate concentration, and response intensity reaching 167 times when responding to vanillin. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is the fluorescence response curve of the wild-type YqhC and the mutant YqhC-V18 biosensor system to 0-10 mM vanillin. DETAILED DESCRIPTION

[0031] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0032] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0033] The following examples are further illustrations of the present application and do not limit the essential content of the present application.

[0034] 1. In the present application, the following definitions are adopted:

[0035] (1) Nomenclature of amino acid and DNA nucleic acid sequences

[0036] The recognized IUPAC nomenclature of amino acid residues is used in the form of single-letter or three-letter code. The recognized IUPAC nomenclature of DNA nucleic acid sequences is used.

[0037] (2) Identification of YqhC mutants

[0038] The mutated amino acid in YqhC-V18 mutant is represented by "original amino acid + position + substituted amino acid". For example, F59T, means the 59th amino acid is mutated from phenylalanine to threonine, and the position number corresponds to the amino acid sequence number of wild type YqhC in SEQ ID NO. 1.

[0039] In the present application, the lower case italic yqhC represents the coding gene of wild type aldehyde-responsive transcriptional factor YqhC, and the lower case italic YqhC-V18 represents the coding gene of mutant YqhC-V18, and the specific information is shown in the following table.

[0040]

[0041] In the present application, the amino acid sequence of YqhC-V18 mutant is shown in SEQ ID NO. 3:

[0042] MLQNCAQSNCRIIPKKLRDMKREEICRLLADKVNKLKNKENSLSGLLPDVRLLYGETPTTRTPVMYEPGIIILFSGHKIGYINERVFRYDANEYLLLTVPLPFECETYATSEVPLAGLRLNVDILQLQELLMDIGEDEHFQPSMAASGINSATLSEEILCAAERLLDVMERPLDARILGKQIIREILYYVLTGPCGGALLALVSRQTHFSLISRVLKRIENKYTENLSVEQLAAEANMSVSAFHHNFKSVTSTSPLQYLKNYRLHKARMMIIHDGMKASAAAMRVGYESASQFSREFKRYFGVTPGEDAARMRAMQGN.

[0043] 2. Some primers involved in the present application and examples are shown in the following table:

[0044]

[0045] 3. Some strains and plasmids involved in the present application and examples are shown in the following table:

[0046]

[0047] The present application is further explained and described by specific examples.

[0048] Example 1: Screening of mutant YqhC-V18

[0049] (1) In order to obtain a high-efficiency vanillin-responsive biosensor, YqhC and its regulated promoter element P yqhD An RFP reporter system responsive to aldehyde was established, and then a ligand binding cavity was found through semi-rational design of structure simulation and molecular docking. A mutant library of YqhC fragments was constructed by saturation mutation of the amino acids in the ligand binding cavity, and finally a high-efficiency vanillin-responsive aldehyde-responsive transcription factor YqhC was obtained through high-throughput screening by flow cytometry.

[0050] (2) Construction of a biosensor system based on wild-type transcription factor YqhC: with MG1655 The aldehyde-responsive transcription factor YqhC (SEQ ID NO. 2) and the promoter (SEQ ID NO. 6) from Escherichia coli were cloned as templates, and the promoter element yqhC P yqhD (SEQ ID NO. 5), the red fluorescent protein gene (SEQ ID NO. 7), the resistance gene (SEQ ID NO. 9), and the replicon (SEQ ID NO. 8) were obtained by gene synthesis. The PCR reaction system is shown in Table 1, and the PCR reaction program is shown in Table 2. P rrnB rfp Cm p15A

[0051] (3) The correct size of the target fragment was obtained by agarose gel electrophoresis with 2KPlus II as the DNA marker, and the purified target fragment was obtained by DNA recovery. The YqhC gene P rrnB , yqhC-P yqhD , rfp, p15A, Cm The fragment was connected to obtain the connection product P rrnB - yqhC-P yqhD - rfp-p15A-Cm The fragment was connected to obtain the connection product

[0052] ​​​​​The following day, single colonies were picked for PCR to verify successful plasmid construction. The verification primers were sequencing-yqhC-F / R, and the target fragment was cPCR-yqhC, 1510 bp in size. 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. A target band of the correct size was obtained, indicating successful construction of the wild-type YqhC biosensor plasmid. This plasmid was named pYJ12, and the *E. coli* strain carrying this plasmid was named sYJ022.

[0053] Table 1 PCR reaction system

[0054]

[0055] Table 2 PCR reaction procedure

[0056]

[0057] Table 3 PCR reaction system

[0058]

[0059] Table 4 PCR reaction procedure

[0060]

[0061] (4) Construction of YqhC saturated mutant library and high-throughput screening: A saturated mutant library was constructed by saturating the key amino acids F59 and A60 in the substrate binding pocket of wild-type YqhC.

[0062] First, using wild-type pYJ12 as a template, primers 251 and 252 were designed to introduce a saturation mutation via loop P using the NNS approach. The PCR product of the saturation mutation was named... yqhC Saturated mutant library. The PCR reaction system is shown in Table 5, and the PCR reaction procedure is shown in Table 6.

[0063] Using the digestion system shown in Table 7, Dpn1 was used for... yqhC The PCR products of the saturated mutant library were digested, and the entire digested product was transformed into 100 μL of JM109 chemocompetent cells. After 1 hour of recovery, LB was added to a final volume of 4.5 mL, followed by 4.5 μL of 25 mg / mL chloramphenicol (final concentration 25 μg / mL). The cells were incubated at 37°C and 220 rpm in a shaker until OD500. 600about 0.6, 500 μL of 50 mM vanillin (5 mM final concentration) was added to induce 4H, and then the cells with fluorescence level in the top 1% were sorted by flow cytometry. Finally, a mutant strain with high response to vanillin was obtained, which was named sYJ036. The sensor plasmid carried by sYJ036 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, which is shown in SEQ ID NO. 3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 4.

[0064] Table 5 PCR reaction system

[0065]

[0066] Table 6 PCR reaction program

[0067]

[0068] Table 7 Dpn1 digestion system

[0069]

[0070] Example 2: Verification of the fluorescence response of mutant YqhC-V18

[0071] In order to further verify the performance of mutant YqhC-V18, a gradient vanillin exogenous addition experiment was needed to determine its fluorescence response curve.

[0072] The mutant strain sYJ036 and the wild type strain sYJ022 obtained by sorting were inoculated into 5 mL of LB (25 μg / mL Cm) liquid medium, respectively, and incubated at 37 °C, 220 rpm for 12 hours as seed liquid. The seed liquid was preserved, and then 2 mL of 96 deep well plates were used to determine the fluorescence response curve of the mutant and the wild type to gradient concentration vanillin.

[0073] 800 μL of fresh M9 medium (25 μg / mL Cm) was added with a pipette, and then 10 μL of seed liquid was added to the medium, with three repeats for each seed liquid. After sealing the sealing film, the deep well plate was placed in a shaking incubator at 37 °C, 220 rpm for 4 h, then taken out, and 200 μL of medium containing 0-50 mM gradient concentration vanillin (final concentration 0-10 mM) (25 μg / mL) was added, the sealing film was sealed, and it was put back into the shaking incubator for 12 h. 200 μL of bacterial liquid was taken into a black 96-well plate, and the fluorescence intensity and OD 600The quantitative detection of red fluorescent protein RFP uses an excitation wavelength of 580 nm and an emission wavelength of 610 nm, the gain value is 80, each well is read three times to obtain an 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 a relative fluorescence intensity value.

[0074] The RFP / OD of strain sYJ022 is 600 The results are analyzed as a wild type control, and the RFP / OD 600 is taken as the vertical coordinate, the vanillin concentration is taken as the horizontal coordinate, and the graph is drawn using GraphPad Prism 8.0 software, and the LOWESS fitting curve is used.

[0075] The results are shown in Table 1. Figure 1 Compared with the wild type YqhC which has no response to vanillin, the mutant YqhC-V18 can linearly respond in the range of 0-10 mM vanillin, has no background fluorescence response when no vanillin (0 g / L) is added, and the response intensity is increased by 167 times when the vanillin concentration is 10 mM compared with that when no vanillin (0 mM) is added, which indicates that the mutant can efficiently respond to vanillin, has no background leakage, can linearly respond in the range of 0-10 mM vanillin, and the response intensity can reach 167 times.

[0076] Although the present application has been disclosed as above with preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various forms and details of changes, modifications, replacements and variations to these embodiments without departing from the spirit and principles of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A YqhC mutant, characterized in that, The mutant is obtained by F59T and A60T mutations on the basis of wild-type transcription factor YqhC shown in SEQ ID NO. 1, and the mutant is named YqhC-V18 mutant, and the amino acid sequence is shown in SEQ ID NO.

3.

2. A coding gene of the YqhC mutant of claim 1.

3. Use of a YqhC mutant according to claim 1 or of a gene encoding according to claim 2, characterized in that, The application is applied in detecting samples containing vanillin, or in screening vanillin high-yield strains, or in constructing a biosensor for detecting vanillin.

4. A biosensor characterized by, The biosensor comprises the following elements: a regulatory YqhC-V18 gene, YqhC-V18 gene, YqhC-V18 gene, P yqhD reporter gene driven by the promoter P yqhD ​ The YqhC-V18 Gene is the coding gene of the YqhC mutant of claim 2.

5. The biosensor of claim 4, wherein, The regulation YqhC-V18 The promoter of a gene comprises: P rrnB , P j23100 , P j23108 , P j23119 ; The reporter gene includes: a red fluorescent protein gene rfp , a green fluorescent protein gene gfp , a yellow fluorescent protein gene yfp .

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

7. The biosensor of claim 6, wherein, The replicon comprises: p15A, ori, ColE1 ; the resistance gene comprises: Cm, Amp, Kan .

8. The biosensor of claim 4, wherein, The biosensor comprises P rrnB YqhC-V18, P yqhD rfp, p15A, Cm The nucleotide sequence of the biosensor is shown as SEQ ID NO.

10.

9. Use of a biosensor according to claim 4, characterized in that The application is applied in detecting samples containing vanillin, or in screening vanillin high-yield strains.

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