Isobutyraldehyde biosensor and application thereof

Abstract

The invention relates to establishment and application of an isobutyraldehyde biosensor, 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 utilized to establish an RFP report system of isobutyraldehyde response, irrational design is carried out on a yqhC gene, random mutation is carried out through error-prone PCR to construct a mutation library, isobutyraldehyde is taken as a substrate, and through screening of response concentration and response threshold value, a non-background fluorescent response, a non-background fluorescent response function and a non-background fluorescent response function are obtained. The YqhC mutant has a wider substrate detection range (0-5g / L) and higher response strength (24 times), and is named as YqhCm9. An isobutyraldehyde response element (YqhCm9) and a fluorescent protein report element (RFP) are utilized to construct an efficient and specific isobutyraldehyde biosensor, and the isobutyraldehyde biosensor can be used for real-time monitoring of an isobutyraldehyde production process and screening of isobutyraldehyde production strains.

Description

Technical field:

[0001] The invention relates to the establishment and application of an isobutyraldehyde biosensor, belonging to the technical field of bioengineering. Background technology:

[0002] As we all know, the production and use of petroleum-based chemicals are the main source of carbon dioxide emissions, and climate change caused by carbon emissions is a global problem that threatens human survival and development. Producing renewable bio-based products to replace petroleum-based products through green biomanufacturing technology is an important means to achieve carbon neutrality. The market share of petroleum-based butyraldehyde composed of isobutyraldehyde (Isobutyraldehyde, IBAL) and its isomer n-butyraldehyde has occupied 75% of the world's petroleum-based aldehyde market, with an annual output of more than 7.5 million tons. With the increasing demand for isobutyraldehyde downstream products in the fields of solvents, plasticizers, paints and coatings, fertilizers and medicines, the global market for isobutyraldehyde has also been in short supply. Therefore, the realization of efficient and green synthesis of isobutyraldehyde not only has broad prospects.

[0003] The traditional production method of isobutyraldehyde is chemical synthesis, which is made from raw materials such as petrochemicals or coal chemicals through high-temperature and high-pressure distillation. It is also the main method for producing isobutyraldehyde in my country at present, but this chemical synthesis method has high energy consumption and serious environmental pollution. In recent years, the development of synthetic biology and metabolic engineering technology has promoted the microbial synthesis of bio-based isobutyraldehyde in hosts such as Escherichia coli. This synthesis process borrows the de novo synthesis process of isobutanol and achieves the production of isobutyraldehyde by removing the aldehyde decarboxylase (aldehyde reductase) in the last step of the isobutanol synthesis pathway. Specifically, this pathway uses the branched amino acid metabolic pathway of Escherichia coli, and converts the endogenous ketoacid intermediate of Escherichia coli into isobutyraldehyde through the exogenously introduced ketoacid decarboxylase (KDC).

[0004] In the microbial production of isobutyraldehyde, since isobutyraldehyde cannot be detected by direct or indirect color or fluorescence reactions, high-throughput screening methods based on biosensors can be used as an alternative strategy. Biosensors are composed of sensing elements and reporter elements. The sensing element recognizes specific intracellular metabolites and generates quantitative signals through a series of gene circuits by the reporter element (such as fluorescent proteins RFP, GFP, YFP, etc.). Metabolite-based biosensor fluorescence response systems can be used as a high-throughput screening tool to quickly realize the perception and characterization of metabolites, thereby screening high-yield strains of the corresponding metabolites.

[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 a transcription factor for aldehydes and belongs 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. A research team has successfully transformed the transcription factor YqhC into a biosensor, which has increased its response intensity to ethanolaldehyde by 70 times, and can simultaneously respond to a variety of aldehydes such as phenylacetaldehyde and vanillin. As the first reported aldehyde response element, YqhC has almost no response to isobutyraldehyde (5mM), and the concentration response range and specific response threshold are unclear, so it is urgent to transform it. Summary of the invention:

[0007] The purpose of the present invention is to provide an efficient isobutyraldehyde biosensor system: no background fluorescence response, a wider substrate detection range (0-5g / L), and a higher response intensity (24 times), and to apply it to the screening of isobutyraldehyde high-yielding strains.

[0008] The present invention utilizes the aldehyde-responsive transcription factor YqhC from Escherichia coli and the promoter element P regulated by it yqhD , an RFP reporter system for isobutyraldehyde response was established, the yqhC gene was irrationally designed, and a mutant library was constructed by random mutation using error-prone PCR. Using isobutyraldehyde as a substrate, a YqhC mutant with no background fluorescence response, a wider substrate detection range (0-5g / L), and a higher response intensity (24 times) was obtained by screening the response concentration and response threshold, and it was named YqhCm9. Then, the biosynthetic pathway from glucose to isobutyraldehyde was constructed using Escherichia coli as a host, and the real-time monitoring of the isobutyraldehyde synthesis process was achieved by a biosensor containing an isobutyraldehyde response element (YqhCm9) and a fluorescent protein reporter element (RFP). Finally, a mutagenic strain library was constructed by heavy ion radiation, and the isobutyraldehyde synthesis process was monitored in real time using a biosensor system to screen for isobutyraldehyde high-yield strains.

[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 causing M65T, V122G, R184H, S204R and L227R mutations on the wild-type transcription factor YqhC shown in SEQ ID NO.1, and the mutant is named YqhCm9 mutant;

[0011] Furthermore, the amino acid sequence of the YqhCm9 mutant is shown in SEQ ID NO.3;

[0012] Furthermore, the encoding gene of the YqhCm9 mutant is the yqhCm9 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 isobutyraldehyde or screening isobutyraldehyde-producing strains, and more particularly, in constructing a biosensor for detecting isobutyraldehyde.

[0014] The third technical solution provided by the present invention is a biosensor comprising the yqhCm9 gene, wherein the biosensor comprises the following elements: a promoter regulating the yqhCm9 gene, the yqhCm9 gene, and a promoter P regulated by the yqhCm9 gene. yqhD , by promoter P yqhD driven reporter gene;

[0015] Furthermore, the promoter regulating the yqhCm9 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 PrrnB 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 、yqhCm9、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 the application in detecting samples containing isobutyraldehyde or screening isobutyraldehyde producing strains, and more particularly the application in screening key genes or enzymes affecting isobutyraldehyde production, wherein the key genes or enzymes include but are not limited to key genes or enzymes in the isobutyraldehyde production metabolic pathway, such as acetohydroxyacid synthase (AlsS), acetolactate isomeroreductase (IlvC), dihydroxyacid dehydratase (IlvD), 2-ketoisovalerate decarboxylase (KivD), etc.;

[0028] Furthermore, the isobutyraldehyde producing strains include but are not limited to Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis and the like.

[0029] Beneficial effects:

[0030] The present invention provides an efficient isobutyraldehyde biosensor system based on aldehyde-responsive transcription factor YqhCm9. Compared with wild-type YqhC that has basically no fluorescence response to isobutyraldehyde, YqhCm9 has the advantages of no background fluorescence response, wide dynamic detection range (0-5g / L) and higher response intensity (24 times) when responding to isobutyraldehyde. In addition, the present invention combines the biosensor with the isobutyraldehyde synthesis pathway to achieve dynamic real-time monitoring of the isobutyraldehyde synthesis process, and based on this, screened out an isobutyraldehyde high-yield strain (36h: 7.3g / L) with a yield 2.8 times higher than that of the wild type (36h: 2.6g / L) from a strain library induced by heavy ion radiation. Description of the drawings:

[0031] Figure 1 Isobutyraldehyde biosensor system based on the aldehyde-responsive transcription factor YqhC.

[0032] Figure 2 Fluorescence response curves of wild-type YqhC and mutant YqhCm9 biosensor systems to 0-5 g / L isobutyraldehyde.

[0033] Figure 3 Biosensor system for real-time monitoring of isobutyraldehyde synthesis process.

[0034] Figure 4 Monitoring the isobutyraldehyde production process using the isobutyraldehyde sensor system.

[0035] Figure 5 The real-time monitoring system was used to screen high-yielding strains of isobutyraldehyde.

[0036] Figure 6 Shake flask fermentation of isobutyraldehyde high-producing strains. Specific implementation method:

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] The following examples are provided to further illustrate the present invention and are not intended to limit the essential contents of the present invention.

[0040] 1. The following definitions are adopted in the present invention:

[0041] (1) Nomenclature of amino acid and DNA sequences

[0042] 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.

[0043] (2) Identification of YqhC mutants

[0044] The mutated amino acid in the YqhCm9 mutant is represented by "original amino acid + position + replaced amino acid". For example, M65T means that the 65th amino acid is mutated from methionine to threonine, and the position number corresponds to the amino acid sequence number of the wild-type YqhC in SEQ ID NO.1.

[0045] In the present invention, lowercase italic yqhC represents the coding gene of the wild-type aldehyde-responsive transcription factor YqhC, and lowercase italic yqhCm9 represents the coding gene of the mutant YqhCm9. The specific information is shown in the following table.

[0046]

[0047] 2. Some of the primers involved in the present invention and embodiments are shown in the following table:

[0048]

[0049]

[0050] 3. Some strains and plasmids involved in the present invention and embodiments are shown in the following table:

[0051] Bacteria or plasmid describe E.coli strains JM109 BWΔ4 Laboratory preserved strains YJ022 JM109 carries pYJ12 YJ027 JM109 carries pYJ18 sYJ028 JM109 carries pYJ16 YJ029 BWΔ4 carries pYJ16 sYJ030 BWΔ4 carries pYJ16 and pYJ18 sYJ031 Isobutyraldehyde mutagenesis strain library-100Gy sYJ032 Isobutyraldehyde mutagenesis strain library-150Gy BWΔ4-G9 Isobutyraldehyde high-yielding strain G9 Plasmids oeLh <![CDATA[P rrnB -yqhC;P yqhD -rfp-Cm r ;p15A]]> oeLh <![CDATA[P L lacO1-alsS-ilvC-ilvD-kivd-rrnB T1 terminator;ori;amp r ]]> oeLh <![CDATA[P rrnB -yqhCm9;P yqhD -rfp-Cm r ;p15A]]>

[0052] The present invention is further explained below by means of specific examples.

[0053] Example 1: Screening of mutant YqhCm9

[0054] (1) In order to obtain a biosensor that efficiently responds to isobutyraldehyde, we first used YqhC and its regulated promoter element PyqhD to establish an aldehyde-responsive RFP reporter system. Then, a random mutation library of the YqhC fragment was constructed by error-prone PCR. High-throughput screening was performed by flow cytometry to obtain the aldehyde-responsive transcription factor YqhC that efficiently responds to isobutyraldehyde.

[0055] The mechanism of action of this biosensor is Figure 1 As shown, the promoter P rrnB P composed of yqhC rrnB -yqhC acts as a sensor module to drive the constitutive expression of the yqhC gene, which is driven by the promoter P yqhD and the red fluorescent protein gene rfp yqhD -RFP as a reporter gene module driven by P yqhD When the sensor system is exposed to aldehydes, the transcription factor YqhC activates the downstream gene cluster P by binding to aldehydes. yqhD -rfp transcriptional response, thereby upregulating the expression of red fluorescent protein, and the expression level of red fluorescent protein RFP is used to characterize the concentration response of the sensor to the aldehyde in the environment.

[0056] (2) Construction of a biosensor system based on the wild-type transcription factor YqhC: Using the genome MG1655 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.

[0057] (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. 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.

[0058] 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.

[0059] Table 1 PCR reaction system

[0060]

[0061] Table 2 PCR reaction program

[0062]

[0063] Table 3 PCR reaction system

[0064]

[0065] Table 4 PCR reaction program

[0066]

[0067] (4) Construction of YqhC random mutation library and high-throughput screening: First, using wild-type pYJ12 as a template, plasmid backbone fragment 12G was amplified by PCR, and the yqhC gene fragment was amplified by error-prone PCR to obtain the yqhC mutation library. The PCR reaction system is shown in Table 5, the PCR reaction program of the yqhC mutation library is shown in Table 6, and the PCR reaction program of the backbone fragment 12G is shown in Table 7.

[0068] The backbone fragment 12G and the error-prone PCR fragment were purified and recovered for Gibson ligation. All ligation products were transformed into 100 μL JM109 chemical competent medium, and 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), and cultured at 37°C, 220 rpm in a shaker until OD 600 The concentration of isobutyraldehyde was about 0.6, 500 μL of 50 mM isobutyraldehyde (final concentration of 5 mM) was added for induction for 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 isobutyraldehyde was obtained, which was named sYJ027, the sensor plasmid it carried was named pYJ18, and the YqhC mutant on the plasmid was named YqhCm9. The amino acid sequence of the YqhCm9 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.

[0069] Table 5 PCR reaction system

[0070]

[0071] Table 6 PCR reaction program

[0072]

[0073] Table 7 PCR reaction program

[0074]

[0075] Example 2: Fluorescence response of mutant YqhCm9

[0076] In order to further verify the performance of mutant YqhCm9, a gradient exogenous addition experiment is needed to determine its fluorescence response curve. The mutant strain sYJ027 and wild-type strain sYJ022 obtained by sorting were inoculated into 5mL LB (25μg / mL Cm) liquid culture medium, respectively, and cultured at 37°C and 220rpm for 12 hours as seed liquid. The seed liquid was incubated, and then the fluorescence response curves of the mutant and wild type to gradient concentrations of isobutyraldehyde were determined using a 2mL96 deep-well plate.

[0077] 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, repeat three times for each tube of seed solution, seal 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 0-25 g / L gradient concentration of isobutyraldehyde (final concentration of 0-5 g / L), seal the sealing film, and continue to put it back in the shaker for 4-8 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 580nm and an emission wavelength of 610nm, with a gain value of 80. The average value of three parallel data is calculated. The bacterial density is measured at a wavelength of 600nm, and the RFP / OD of each well is calculated on this basis. 600 As relative fluorescence intensity values.

[0078] The RFP / OD of strain sYJ022 600 The results were analyzed as wild-type controls, with RFP / OD 600 The vertical axis was the concentration of isobutyraldehyde and the horizontal axis was the concentration of isobutyraldehyde. The graph was drawn using GraphPad Prism 8.0 software and the curve was fitted using LOWESS.

[0079] The results are as follows Figure 2 As shown, compared with the wild-type YqhC which has basically no response to isobutyraldehyde, the mutant YqhCm9 can respond linearly in the range of 0-5g / L isobutyraldehyde, and there is basically no background fluorescence response when no isobutyraldehyde is added (0g / L). When the concentration of isobutyraldehyde added is 5g / L, the response intensity is increased by 24 times compared with the case when no isobutyraldehyde is added (0g / L), indicating that the mutant can respond to isobutyraldehyde efficiently, and has the advantages of no background leakage, a wide dynamic detection range (0-5g / L), and a higher response intensity (24 times).

[0080] Example 3: Real-time monitoring application of YqhCm9 biosensor system

[0081] This example is used to illustrate the application of the isobutyraldehyde biosensor based on the mutant YqhCm9 obtained by the above screening in real-time monitoring of the isobutyraldehyde synthesis process.

[0082] (1) Construction of isobutyraldehyde production plasmid: The isobutyraldehyde synthesis pathway refers to the use of glucose as a substrate, first generating pyruvate through the glycolysis pathway, then generating α-ketoisovalerate under the catalysis of acetohydroxyacid synthase (AlsS) (SEQ ID NO.11), acetolactate isomeroreductase (IlvC) (SEQ ID NO.12), and dihydroxyacid dehydratase (IlvD) (SEQ ID NO.13), and then catalyzing 2-ketoisovalerate decarboxylase (KivD) (SEQ ID NO.14) to generate isobutyraldehyde. The synthesis pathway consists of two operons, alsS-ilvC / D and kivd. P L lacO1-alsS-ilvC-ilvD gene fragment (SEQ ID NO.15, including promoter P L lacO1 and alsS, ilvC, ilvD gene fragments); kivD-rrnB T1terminator gene fragment (SEQ ID NO.16, including kivD and rrnB T1 terminator gene fragments) and ori-Amp gene fragment (SEQ ID NO.17, including ori and Amp gene fragments) were amplified by gene synthesis. The PCR reaction system is shown in Table 8, and the PCR reaction procedure is shown in Table 9.

[0083] (2) Using 2K PlusⅡ as a DNA marker, the target fragment of the correct size was obtained by agarose gel electrophoresis. The purified target fragment was obtained by DNA recovery and the P was assembled by Gibson assembly technology. L The lacO1-alsS-ilvC-ilvD gene fragment, kivD-rrnB T1 terminator gene fragment, and ori-Amp gene fragment were connected, and all the connection products were transformed into 100μL JM109 chemical competent state, placed on ice, and ice bathed 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 culture medium was added to the centrifuge tube, placed in a 37℃ shaker, and revived at 220rpm for 45min before applying ampicillin resistance plate. The plate was placed in a 37℃ constant temperature incubator for overnight growth.

[0084] The next day, a single colony was picked for PCR to verify whether the plasmid was successfully constructed. The primers were 16-F / R, the target fragment was cPCR-16, and the size was 1206 bp. The PCR reaction system is shown in Table 10, and the PCR reaction procedure is shown in Table 11. After the PCR reaction, agarose gel electrophoresis was performed, and the bands were compared with the DNA Marker of 2K PlusⅡ to obtain the target band of the correct size, that is, the isobutyraldehyde production plasmid was successfully constructed. The plasmid was named pYJ16, and the strain carrying the plasmid was named sYJ028.

[0085] Table 8 PCR reaction system

[0086]

[0087]

[0088] Table 9 PCR reaction program

[0089]

[0090] Table 10 PCR reaction system

[0091]

[0092] Table 11 PCR reaction program

[0093]

[0094] (3) Construction of isobutyraldehyde production strain: It was obtained by transferring the isobutyraldehyde synthesis pathway plasmid pYJ16 into Escherichia coli BWΔ4 (laboratory collection strain). The host cell BWΔ4 was prepared into a competent cell using a competent cell preparation kit, and 1 μL of isobutyraldehyde production plasmid pYJ16 was transferred into 50 μL of BWΔ4 competent cells, placed on ice, and ice-bathed for 25 minutes. After the ice-bath, the centrifuge tube was placed in a 42°C water bath for heat shock for 1 minute 30 seconds. After the heat shock, the centrifuge tube was placed on ice again for an ice bath for 2 minutes. After the ice bath, 900 μL of LB medium was added to the centrifuge tube, which was placed in a 37°C shaker, and revived at 220 rpm for 45 minutes before being coated with an ampicillin resistance plate. The plate was placed in a 37°C constant temperature incubator for overnight growth. The next day, a single colony was picked and transferred to 5 mL LB (100 μg / mL Amp) medium, cultured at 37°C, 220 rpm for 12 hours, and then preserved and stored at -80°C to obtain an isobutyraldehyde-producing strain, which was named sYJ029.

[0095] (4) Construction of a dual-plasmid system to achieve real-time monitoring of the isobutyraldehyde production process: Figure 3As shown in the figure, the isobutyraldehyde biosensor plasmid pYJ18 and the isobutyraldehyde production plasmid pYJ16 were simultaneously introduced into the host cell BWΔ4. During the fermentation process, as isobutyraldehyde was biosynthesized and accumulated, the aldehyde-responsive transcription factor mutant YqhCm9 could sense the concentration of isobutyraldehyde in real time in the cell, and then the promoter P regulated by it was activated. yqhD By driving the expression of the reporter gene rfp, chemical signals are converted into light signals, thus achieving dynamic real-time monitoring of the isobutyraldehyde production process.

[0096] In order to verify the performance of the dual plasmid real-time monitoring system in practical application, this example measured the yield and fluorescence value (RFP / OD) of isobutyraldehyde in isobutyraldehyde fermentation production. 600 ), analyze the correlation between the two. First, prepare the host cell BWΔ4 into a competent state, transfer 1 μL of isobutyraldehyde production plasmid pYJ16 and 1 μL of isobutyraldehyde biosensor plasmid pYJ18 into 100 μL BWΔ4 competent cells, place on ice, and ice bath for 25 minutes. After the ice bath, place the centrifuge tube in a water bath at 42°C for heat shock for 1min 30s. After the heat shock, place the centrifuge tube back on ice for an ice bath of 2 minutes. After the ice bath, add 900 μL LB medium to the centrifuge tube, place it in a 37°C shaker, and apply ampicillin-chloramphenicol double-resistance plate after 45 minutes of recovery at 220rpm. The plate was placed in a 37°C constant temperature incubator for overnight growth to obtain the isobutyraldehyde production strain sYJ030 containing a dual-plasmid real-time monitoring system.

[0097] The next day, three single colonies (parallel control) were picked and transferred to 5 mL LB (100 μg / mL Amp + 25 μg / mL Cm) medium and cultured at 37°C, 220 rpm for 12 h as seed solution. Subsequently, a 250 mL screw-cap conical flask was used for fermentation. First, 20 mL of the prepared sterile M9 medium was added to the sterilized shake flask, and then the corresponding antibiotics and 2 μL IPTG stock solution (final concentration 0.1 mM, inducing promoter P L lacO1 expression), and finally inoculated with 200 μL of fresh seed solution. Fermentation was carried out at 30°C in a shaking incubator at 220 rpm, and 1 mL of sample was taken at 4, 8, 12, 24 and 36 hours.

[0098] The formula of fermentation medium M9: 6 g / L NaH2PO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 1 mM MgSO4, 0.1 mM CaCl2, 10 mg / L VB1 and 40 g / L glucose.

[0099] M9 salt solution: weigh 6.0g NaH2PO4, 3.0g KH2PO4, 0.5g NaCl, 1.0g NH4Cl and 4g yeast powder, dissolve in distilled water to 900mL, sterilize at 121℃ for 20 minutes, and cool to room temperature for later use.

[0100] Preparation of fermentation medium M9: Take 100 mL of sterile 20% glucose solution, 1 mL of 1M MgSO4 solution, 1 mL of 0.1M CaCl2 solution and 1 mL of 10 mg / mL VB1 solution, add them to 900 mL of sterile M9 salt solution and mix well to obtain fermentation medium M9.

[0101] Sample processing: 200 μL of bacterial solution was taken from each sample and RFP and OD were measured using an ELISA reader. 600 For the determination, the remaining bacterial solution was centrifuged at 12000rpm for 10 minutes, 100μL of the supernatant was aspirated and added to the inner liner of the gas chromatography vial, and then 100μL of the prepared 2g / L isovaleraldehyde (final concentration of 1g / L) was added as the internal standard for gas chromatography detection, the lid of the gas chromatography vial was screwed on, and it was fully shaken and evenly mixed.

[0102] Gas chromatography detection procedure: A91 gas chromatograph (GC) was used with a DB-FFAP capillary column (30 m × 0.32 mm × 0.25 μm) for quantitative determination of isobutyraldehyde. The GC oven temperature was initially maintained at 80 °C for 3 minutes, then increased to 230 °C at a gradient of 115 °C per minute and maintained for 1 minute. The carrier gas was nitrogen, and the injector and detector temperatures were maintained at 250 °C and 280 °C, respectively. The injector injection volume was 0.2 μL, and the split ratio was 30:1. Using isovaleraldehyde as the internal standard, the isobutyraldehyde content of each sample was calculated by the internal standard method with the standard as the reference.

[0103] The results are as follows Figure 4 As shown in Figure 2, with the increase of isobutyraldehyde production, the individual fluorescence values ​​of the strains (RFP / OD 600 ) also rose synchronously, using the Pearson correlation coefficient for analysis. The correlation coefficient r represents the statistic of the linear correlation strength and direction between two random variables, ranging from -1 to 1. 0 means no correlation at all, greater than 0.8 means strong correlation, and less than 0.3 means weak correlation. The absolute value of the correlation coefficient is close to 1, and the higher the correlation. We found that the yield of this strain is related to the individual fluorescence value (RFP / OD 600 ) is 0.9056, indicating that the correlation between the two is very high, verifying the real-time monitoring performance of the dual-plasmid system in the actual fermentation process.

[0104] Example 4: Screening of high-yielding strains of isobutyraldehyde using a dual-plasmid real-time monitoring system

[0105] This example is used to illustrate the application of the dual-plasmid real-time monitoring system in screening high-yield isobutyraldehyde strains.

[0106] The host cell BWΔ4 was irradiated with 100Gy and 150Gy by heavy ion accelerator to obtain two strain libraries: sYJ031 and sYJ032. They were prepared into competent cells (100μL / tube), and 1μL isobutyraldehyde production plasmid pYJ16 and 1μL isobutyraldehyde biosensor plasmid pYJ18 were transferred. Ice bath for 25min, heat shock at 42℃ water bath for 1min 30s, and the centrifuge tube was placed on ice again for 2min after the heat shock. After the ice bath, 900μL LB medium was added to the centrifuge tube, placed in a 37℃ shaker, and revived at 220rpm for 45min before applying ampicillin-chloramphenicol dual-resistance plates. The plates were placed in a 37℃ constant temperature incubator for overnight growth. The next day, the single colony on the plate was inoculated into a 96-deep-well plate with 1 ml LB (100 μg / mL Amp + 25 μg / mL Cm) medium and placed in a 37°C shaker for 12 h as seed solution. The seed solution was re-inoculated into a 96-deep-well plate containing 1 ml M9 (100 μg / mL Amp + 25 μg / mL Cm) fermentation medium at a ratio of 1%. After 24 h, the fluorescence value (RFP / OD) was detected by microplate reader. 600 ).

[0107] Using the isobutyraldehyde-producing strain sYJ030 containing a dual-plasmid real-time monitoring system as the control strain, we screened the fluorescence expression level (RFP / OD 600 ) a strain that has increased significantly, such as Figure 5 As shown. Since this strain is a mutant screened from the BWΔ4 mutagenesis strain library carrying a dual plasmid monitoring system, we named it BWΔ4-G9. The strain was verified by 36h shake flask fermentation, and the results are shown Figure 6 As shown, after the fermentation system was increased from 1 mL to 20 mL, the isobutyraldehyde production of strain G9 (7.3 g / L) at 36 h of fermentation increased by 2.8 times compared with the control strain (2.6 g / L).

[0108] In summary, we screened a high-yield isobutyraldehyde strain BWΔ4-G9 through the dual-plasmid real-time monitoring system. This strain can produce 7.3 g / L of isobutyraldehyde after 36 hours of shake flask fermentation, and its ability to produce isobutyraldehyde is 2.8 times higher than that of the control strain sYJ030.

[0109] 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 causing M65T, V122G, R184H, S204R and L227R mutations to the wild-type transcription factor YqhC shown in SEQ ID NO.

1. The mutant is named YqhCm9 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 isobutyraldehyde, in screening isobutyraldehyde-producing strains, or in constructing a biosensor for detecting isobutyraldehyde.

4. A biosensor, characterized in that: The biosensor comprises the following elements: a promoter regulating the yqhCm9 gene, the yqhCm9 gene, and a promoter P regulated by the yqhCm9 gene. yqhD , by promoter P yqhD driven reporter gene.

5. The biosensor according to claim 4, wherein: The promoter for regulating the yqhCm9 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 、yqhCm9、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 isobutyraldehyde, or in screening isobutyraldehyde-producing strains, or in screening key genes or enzymes that affect isobutyraldehyde production.

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