Hexanal biosensor

Abstract

The invention relates to a biosensor capable of efficiently responding to hexanal, and belongs to the technical field of bioengineering. An aldehyde response transcription factor YqhC derived from Escherichia coli and a promoter element PyqhD regulated by the aldehyde response transcription factor YqhC are utilized to establish an RFP report system of hexaldehyde response, a yqhC gene is subjected to semi-rational design, a mutation library is constructed through saturated mutation, hexaldehyde is taken 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 (0-10mM) and higher response strength (36 times), and is named as YqhC-H7. The biosensor containing a hexanal response element (YqhC-H7) and a fluorescent protein report element (RFP) is further obtained and can be applied to detection of a sample containing hexanal, or screening of a hexanal production strain, or regulation and control of a hexanal production process.

Description

Technical field:

[0001] The invention relates to a biosensor capable of efficiently responding to hexanal, belonging to the technical field of bioengineering. Background technology:

[0002] Hexanal, also known as n-hexanal, is a colorless transparent liquid with a pungent odor. Its chemical formula is C6H12O and its molecular weight is 100.16. It is insoluble in water, but soluble in benzene and acetone, and easily soluble in ethanol and ether. As an important organic synthetic raw material, hexanal is widely used in many industries, especially in the fields of flavors and fragrances, pharmaceutical intermediates, polymer modifiers, etc.

[0003] At present, hexanal on the market mainly comes from two methods: chemical synthesis and biosynthesis. The cost of hexanal produced by chemical synthesis is relatively low, and the selling price is about 15 US dollars / kg, but it has the disadvantages of expensive raw materials, poor product purity, and non-natural, which cannot meet the market's demand for the safety of natural flavors. Natural hexanal is mainly obtained by plant extraction and biosynthesis. Plant extraction is expensive due to the scarce supply of raw materials, which is about 1,200 US dollars / kg, and cannot meet market demand. Therefore, the use of microbial cell factories to develop a low-cost raw material hexanal biosynthesis process can effectively improve the shortcomings of plant extraction and chemical synthesis, and the price of the obtained hexanal is moderate, about 700 US dollars / kg, with broad industrial prospects and market application potential, and has become a research hotspot at home and abroad.

[0004] With the rise of bio-based chemicals, the technology of synthesizing hexanal through biofermentation and enzyme catalysis has attracted attention, which not only reduces the dependence on petroleum resources, but also reduces the environmental impact during the production process. At the same time, the modification research on hexanal, such as the introduction of functional groups or synthetic derivatives, has also broadened its application in high value-added products. In the future, the production of hexanal will pay more attention to sustainability and the development of bio-based routes. The large-scale production of bio-based hexanal will rely on the progress of enzyme engineering and metabolic engineering to improve yield and selectivity and reduce costs.

[0005] 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.

[0006] The DNA-bound transcription activator YqhC is an aldehyde transcription factor belonging to the AraC / XylS family of proteins. It activates the transcriptional response of the downstream gene cluster yqhD-dkgA by binding to aldehydes, thereby upregulating 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, increasing the response intensity of the response element to ethanolaldehyde by 70 times. As the first reported aldehyde response element, the wild-type YqhC has almost no response to hexanal, so it is urgently needed to be transformed. Summary of the invention:

[0007] The purpose of the present invention is to provide an efficient hexanal biosensor system: no background fluorescence, linear response in the range of 0-10mM substrate concentration, and the response intensity reaches 36 times.

[0008] The present invention utilizes the aldehyde-responsive transcription factor YqhC from Escherichia coli and the promoter element P regulated by it yqhD The hexanal-responsive RFP reporter system was established, the yqhC gene was semi-rationally designed, a mutant library was constructed by saturation mutagenesis, and hexanal was used as a substrate. Through screening of response concentration and response threshold, a YqhC mutant with no background fluorescence response, a wider substrate detection range (0-10mM), and a higher response intensity (36 times) was obtained, which was named YqhC-H7. A biosensor containing a hexanal-responsive element (YqhC-H7) and a fluorescent protein reporter element (RFP) was then obtained.

[0009] In order to achieve the above purpose, the technical route adopted by the present invention is as follows:

[0010] One of the technical solutions provided by the present invention is a YqhC mutant, which is obtained by mutation of T62D, P63S, V64E, S204R, and L227R on the wild-type transcription factor YqhC shown in SEQ ID NO.1, and the mutant is named YqhC-H7 mutant;

[0011] Furthermore, the amino acid sequence of the YqhC-H7 mutant is shown in SEQ ID NO.3;

[0012] Furthermore, the encoding gene of the YqhC-H7 mutant is the YqhC-H7 gene, and the nucleotide sequence is shown in SEQ ID NO.4.

[0013] 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 detecting samples containing hexanal or screening hexanal-producing strains, and more particularly, in constructing a biosensor for detecting hexanal.

[0014] The third technical solution provided by the present invention is a biosensor comprising the YqhC-H7 gene, wherein the biosensor comprises the following elements: a promoter regulating the YqhC-H7 gene, the YqhC-H7 gene, a promoter P regulated by the YqhC-H7 gene, yqhD , by promoter P yqhD driven reporter gene;

[0015] Furthermore, the promoter regulating the YqhC-H7 gene includes but is not limited to P rrnB , P j23100 , P j23108 , P j23119 wait;

[0016] 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, etc.;

[0017] Furthermore, the biosensor further comprises a replicon gene for controlling the initial replication of the entire biosensor;

[0018] Furthermore, the biosensor further comprises a resistance gene for screening transformants comprising the biosensor;

[0019] Furthermore, the replicon includes but is not limited to p15A, ori, ColE1, etc.;

[0020] Furthermore, the resistance genes include but are not limited to Cm, Amp, Kan, etc.;

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

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

[0023] Furthermore, the nucleotide sequence of the rfp encoding gene is shown in SEQ ID NO.7;

[0024] Furthermore, the nucleotide sequence of the replicon gene p15A is shown in SEQ ID NO.8;

[0025] Furthermore, the nucleotide sequence of the resistance gene Cm is shown in SEQ ID NO.9;

[0026] Preferably, the biosensor comprises P rrnB 、YqhC-H7、P yqhD , rfp, p15A, Cm, more preferably, the nucleotide sequence of the biosensor is as shown in SEQ ID NO.10.

[0027] 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 hexanal or the screening of hexanal-producing strains.

[0028] Beneficial effects:

[0029] The present invention provides an efficient transcription factor YqhC-H7 based on aldehyde response and a hexanal biosensor system constructed by the same, comprising a promoter regulating the YqhC-H7 gene, the YqhC-H7 gene, and a promoter P regulated by the YqhC-H7 gene. 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.

[0030] Compared with the wild-type YqhC which has basically no fluorescence response to hexanal, YqhC-H7 has the advantages of no background fluorescence response, linear response in the substrate concentration range of 0-10mM, and a response intensity of 36 times when responding to hexanal. Description of the drawings:

[0031] Figure 1 The fluorescence response curves of the wild-type YqhC and mutant YqhC-H7 biosensor systems to 0-10 mM hexanal. Specific implementation method:

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] The following examples are provided to further illustrate the present invention and are not intended to limit the essential contents of the present invention.

[0035] 1. The following definitions are adopted in the present invention:

[0036] (1) Nomenclature of amino acid and DNA sequences

[0037] 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.

[0038] (2) Identification of YqhC mutants

[0039] The mutated amino acid in the YqhC-H7 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.

[0040] In the present invention, lowercase italic yqhC represents the coding gene of the wild-type aldehyde-responsive transcription factor YqhC, and lowercase italic YqhC-H7 represents the coding gene of the mutant YqhC-H7. The specific information is shown in the following table.

[0041]

[0042] 2. Some of the primers involved in the present invention and embodiments are shown in the following table:

[0043]

[0044] 3. Some strains and plasmids involved in the present invention and embodiments are shown in the following table:

[0045] Bacteria or plasmid describe E.coli strains JM109 YJ022 JM109 carries pYJ12 YJ037 JM109 carries pYJ25 Plasmids oeLh <![CDATA[P rrnB -yqhC;P yqhD -rfp-Cm r ;p15A]]> oeLh <![CDATA[P rrnB -YqhC-H7;P yqhD -rfp-Cm r ;p15A]]>

[0046] The present invention is further explained below by means of specific examples.

[0047] Example 1: Screening of mutant YqhC-H7

[0048] (1) In order to obtain a biosensor that efficiently responds to hexanal, it is first necessary to use YqhC and its regulated promoter element 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 hexanal.

[0049] (2) Construction of a biosensor system based on the wild-type transcription factor YqhC: Using the MG1655 genome as a template, the aldehyde-responsive transcription factor yqhC (shown in SEQ ID NO. 2) and the promoter P from Escherichia coli were cloned. yqhD (shown in SEQ ID NO.6), promoter element P was obtained by gene synthesis 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.

[0050] (3) Using 2KPlusⅡ as 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. The P rrnB 、yqhC-P yqhD , rfp, p15A, Cm to obtain the ligation product P rrnB -yqhC-P yqhD -rfp-p15A-Cm fragment, all the ligation products were transformed into 100μL JM109 chemical competent state, placed on ice, ice bath for 25min, after the ice bath, the centrifuge tube was placed in a water bath at 42℃ for heat shock for 1min 30s, after the heat shock, the centrifuge tube was placed on ice again for ice bath for 2min. After the ice bath, 900μL LB medium was added to the centrifuge tube, placed in a 37℃ shaker, revived at 220rpm for 45min, and then coated with chloramphenicol resistance plate. The plate was placed in a 37℃ constant temperature incubator for overnight growth.

[0051] 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.

[0052] Table 1 PCR reaction system

[0053]

[0054] Table 2 PCR reaction program

[0055]

[0056] Table 3 PCR reaction system

[0057]

[0058] Table 4 PCR reaction program

[0059]

[0060] (4) Construction of YqhC saturation mutation library and high-throughput screening: A saturation mutation library was constructed by performing saturation mutations on the key amino acids T62, P63 and V64 in the substrate binding pocket of wild-type YqhC.

[0061] First, using wild-type pYJ12 as a template, primers 253 and 252 were designed to introduce saturation mutations through loop P in an NNS manner, and 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.

[0062] The PCR products of the yqhC saturation mutation library were digested by Dpn1 using the digestion system shown in Table 7. All digestion products were transformed into 100 μL JM109 chemically 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 OD 600 About 0.6, 500 μL of 50 mM hexanal (final concentration of 5 mM) was added to induce 4 hours, and 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 hexanal was obtained, which was named sYJ037, the sensor plasmid it carried was named pYJ25, and the YqhC mutant on the plasmid was named YqhC-H7. The amino acid sequence of the YqhC-H7 mutant was determined by sequencing as shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0063] Table 5 PCR reaction system

[0064]

[0065] Table 6 PCR reaction program

[0066]

[0067] Table 7 Dpn1 digestion system

[0068]

[0069]

[0070] Example 2: Verification of the fluorescence response of mutant YqhC-H7

[0071] In order to further verify the performance of mutant YqhC-H7, a gradient hexanal exogenous addition experiment is needed to determine its fluorescence response curve.

[0072] The mutant strain sYJ037 and wild-type strain sYJ022 obtained by sorting were inoculated into 5 mL LB (25 μg / mL Cm) liquid medium, and cultured at 37°C, 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 hexanal were measured using a 2 mL 96 deep-well plate.

[0073] 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, do three replicates for each tube of seed solution, seal the sealing film, place the deep-well plate at 37°C, 220 rpm shaker for 4 hours, then take it out, add 200 μL of culture medium (25 μg / mL) containing 0-50 mM gradient concentration of hexanal (final concentration of 0-10 mM), seal the sealing film, and continue to put it back on 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 Cytation3) 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.

[0074] The RFP / OD of strain sYJ022 600 The results were analyzed as wild-type controls, with RFP / OD 600 The vertical axis is the concentration of hexanal, and the horizontal axis is the concentration of hexanal. The graph was drawn using GraphPad Prism8.0 software, and the curve was fitted using LOWESS. Figure 1 As shown, compared with the wild-type YqhC which has basically no response to hexanal, the mutant YqhC-H7 can respond linearly in the range of 0-10 mM hexanal, and there is no background fluorescence response when no hexanal is added (0 g / L). When the hexanal concentration is 10 mM, the response intensity is increased by 36 times compared with when no hexanal is added (0 mM), indicating that the mutant can respond to hexanal efficiently, and has the advantages of no background leakage, linear response in the range of 0-10 mM hexanal, and response intensity can reach 36 times.

[0075] 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-H7 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 hexanal, in screening strains with high hexanal production, or in constructing a biosensor for detecting hexanal.

4. A biosensor, characterized in that: The biosensor comprises the following elements: a promoter regulating the YqhC-H7 gene, the YqhC-H7 gene, a promoter P regulated by the YqhC-H7 gene, yqhD , by promoter P yqhD driven reporter gene.

5. The biosensor according to claim 4, wherein: The promoter regulating the YqhC-H7 gene includes but is not limited to P rrnB , P j23100 , P j23108 , P j23119 ; The reporter genes include but are not limited to: red fluorescent protein gene rfp, green fluorescent protein gene gfp, and 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, and ColE1; the resistance genes include but are not limited to Cm, Amp, and Kan.

8. The biosensor according to claim 4, wherein: The biosensor comprises P rrnB 、YqhC-H7、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: The invention is used in detecting samples containing hexanal, in screening strains with high hexanal production, or in regulating the production process of hexanal.

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