Isobutyraldehyde biosensor and use thereof

Abstract

The application relates to establishment and application of an isobutyraldehyde biosensor and belongs to the technical field of bioengineering. yqhD The RFP report system responding to isobutyraldehyde is established, the yqhC gene is rationally designed, a mutant library is constructed through random mutation by using error-prone PCR, isobutyraldehyde is used as a substrate, and through screening of response concentrations and response thresholds, a YqhC mutant without background fluorescence response, with a wider substrate detection range (0-5 g / L) and higher response intensity (24 times) is obtained, and the YqhC mutant is named YqhCm9. The isobutyraldehyde response element (YqhCm9) and the fluorescent protein report element (RFP) are used to construct a high-efficiency and specific isobutyraldehyde biosensor, real-time monitoring of an isobutyraldehyde production process and screening of an isobutyraldehyde production strain can be carried out.

Description

Technical fields:

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

[0002] As is well known, the production and utilization of petroleum-based chemicals are major sources of carbon dioxide emissions, and climate change caused by carbon emissions is a global problem threatening human survival and development. Producing renewable bio-based products through green biomanufacturing technology to replace petroleum-based products is an important means of achieving carbon neutrality. Petroleum-based butyraldehyde (IBAL), composed of isobutyraldehyde and its isomer n-butyraldehyde, already accounts for 75% of the global petroleum-based aldehyde market, with an annual output exceeding 7.5 million tons. Furthermore, with increasing demand for downstream isobutyraldehyde products in solvents, plasticizers, paints, fertilizers, and pharmaceuticals, the global market for isobutyraldehyde is experiencing a supply shortage. Therefore, achieving efficient and green synthesis of isobutyraldehyde has broad prospects.

[0003] Traditionally, isobutyraldehyde is produced through chemical synthesis, using raw materials from petrochemical or coal chemical processes via high-temperature, high-pressure fractionation. This is currently the main method for isobutyraldehyde production in my country, but it is energy-intensive and causes significant environmental pollution. In recent years, advancements in synthetic biology and metabolic engineering have spurred the microbial synthesis of bio-based isobutyraldehyde in hosts such as *E. coli*. This synthesis utilizes the de novo synthesis of isobutanol, eliminating the aldehyde decarboxylase, the final step in the isobutanol synthesis pathway, to achieve isobutyraldehyde production. Specifically, this pathway employs the branched amino acid metabolic pathway of *E. coli*, using an exogenously introduced keto acid decarboxylase (KDC) to convert endogenous keto acid intermediates in *E. coli* into isobutyraldehyde.

[0004] In the microbial production of isobutyraldehyde, since isobutyraldehyde cannot be detected directly or indirectly through color or fluorescence reactions, high-throughput screening methods based on biosensors can serve as an alternative strategy. Biosensors consist of sensing elements and reporter elements. The sensing element identifies specific intracellular metabolites, and after passing through a series of gene circuits, the reporter element (such as fluorescent proteins RFP, GFP, YFP, etc.) generates a quantitative signal. Metabolite-based biosensor fluorescence response systems can serve as a high-throughput screening tool to rapidly perceive and characterize metabolites, thereby screening for high-yielding strains of the corresponding metabolites.

[0005] Transcription factors (TFs) are frequently used to modify metabolite response elements and establish response systems. They primarily work by sensing corresponding metabolites to bind to and dissociate from upstream regulatory sequences of target genes, thereby activating or inhibiting the transcription of the target gene. Currently, transcription factor-based biosensors are widely used in screening or enriching high-yielding bacterial strains, high-throughput screening of enzymes in metabolic pathways, and regulation of metabolic pathways in biosynthetic processes.

[0006] The DNA-binding 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. Previous research teams have successfully modified the transcription factor YqhC into a biosensor, increasing its response intensity to glycolal aldehyde by 70-fold and enabling it to simultaneously respond to multiple aldehydes such as phenylacetaldehyde and vanillin. As the first reported aldehyde-responsive element, YqhC showed almost no response to isobutyraldehyde (5 mM), and its concentration response range and specific response threshold were unclear, thus requiring further modification. Summary of the Invention:

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

[0008] This invention utilizes the aldehyde-responsive transcription factor YqhC derived from Escherichia coli and its regulated promoter element P. yqhD A fluorescent protein reporter element (RFP) reporter system responsive to isobutyraldehyde was established. An irrational design was employed for the yqhC gene, and a mutant library was constructed using error-prone PCR for random mutation. Using isobutyraldehyde as a substrate, a YqhC mutant with background-free fluorescence response, a wider substrate detection range (0-5 g / L), and a 24-fold higher response intensity was obtained through screening based on response concentration and threshold. This mutant was named YqhCm9. Furthermore, a biosynthetic pathway from glucose to isobutyraldehyde was constructed using *E. coli* as the host. Real-time monitoring of the isobutyraldehyde synthesis process was achieved using a biosensor containing an isobutyraldehyde response element (YqhCm9) and a fluorescent protein reporter element (RFP). Finally, a mutagenic strain library was constructed using heavy ion radiation, and the biosensor system was used to monitor the isobutyraldehyde synthesis process in real time, screening for high-yielding isobutyraldehyde strains.

[0009] To achieve the above objectives, the technical approach adopted by the present invention is as follows:

[0010] One of the technical solutions provided by this invention is a YqhC mutant, which is obtained by M65T, V122G, R184H, S204R and L227R mutations on the basis of wild-type transcription factor YqhC shown in SEQ ID NO.1. 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, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0013] The second technical solution provided by this invention is the application of the YqhC mutant or its encoding gene described in the first technical solution, particularly in the detection of samples containing isobutyraldehyde or the screening of isobutyraldehyde-producing strains, and more particularly in the construction of a biosensor for the detection of isobutyraldehyde.

[0014] The third technical solution provided by this invention is a biosensor containing the yqhCm9 gene. The biosensor comprises the following elements: a promoter regulating the yqhCm9 gene, the yqhCm9 gene, and the promoter P regulated by the yqhCm9 gene. yqhD By promoter P yqhD Reporter genes driven by [the virus / organization];

[0015] Furthermore, the promoters regulating the yqhCm9 gene include, but are not limited to, P. rrnB P j23100 P j23108 P j23119 wait;

[0016] Furthermore, the reporter genes include, but are not limited to: red fluorescent protein gene rfp, green fluorescent protein gene gfp, yellow fluorescent protein gene yfp, etc.

[0017] Furthermore, the biosensor also includes a replicon gene for controlling the initiation of replication of the entire biosensor;

[0018] Furthermore, the biosensor also includes an resistance gene for screening transformants containing the biosensor;

[0019] Furthermore, the replicons include, but are 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 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, particularly in the detection of samples containing isobutyraldehyde or in the screening of isobutyraldehyde-producing strains, and more particularly in the screening of key genes or enzymes that affect isobutyraldehyde production. The key genes or enzymes include, but are not limited to, key genes or enzymes in the isobutyraldehyde production metabolic pathway, such as acetylhydroxy acid synthase (AlsS), acetyllactate isomer reductase (IlvC), dihydroxy acid dehydratase (IlvD), 2-ketoisovalerate decarboxylase (KivD), etc.

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

[0029] Beneficial effects:

[0030] This invention provides a highly efficient isobutyraldehyde biosensor system based on the aldehyde-responsive transcription factor YqhCm9. Compared to the wild-type YqhC, which exhibits virtually no fluorescence response to isobutyraldehyde, YqhCm9 demonstrates advantages such as background fluorescence, a wide dynamic detection range (0-5 g / L), and a 24-fold higher response intensity. Furthermore, this invention combines the biosensor with the isobutyraldehyde synthesis pathway, enabling dynamic real-time monitoring of the isobutyraldehyde synthesis process. Based on this, a high-yielding isobutyraldehyde strain (36h: 7.3 g / L) with a 2.8-fold increase in yield compared to the wild-type strain (36h: 2.6 g / L) was screened from a heavy-ion radiation mutagenesis strain library. Attached image description:

[0031] Figure 1 Isobutyraldehyde biosensor system based on 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 A biosensor system for real-time monitoring of isobutyraldehyde synthesis.

[0034] Figure 4 The isobutyraldehyde production process was monitored using an isobutyraldehyde sensor system.

[0035] Figure 5 High-yielding isobutyraldehyde strains were screened using a real-time monitoring system.

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

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

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] The following examples are further illustrations of the present invention and do not constitute a limitation on the substantive content of the present invention.

[0040] 1. The following definitions are used in this invention:

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

[0042] The IUPAC nomenclature, a recognized system for naming amino acid residues, is used, employing single-letter or three-letter codes. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0043] (2) Identification of YqhC mutants

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

[0045] In this invention, lowercase italic yqhC represents the encoding gene of the wild-type aldehyde-responsive transcription factor YqhC, and lowercase italic yqhCm9 represents the encoding gene of the mutant YqhCm9. Specific information is shown in the table below.

[0046]

[0047] 2. Some of the primers involved in this invention and its embodiments are shown in the table below:

[0048]

[0049]

[0050] 3. Some of the strains and plasmids involved in this invention and its embodiments are shown in the table below:

[0051] strain or plasmid describe E.coli strains JM109 BWΔ4 Laboratory-preserved strains sYJ022 JM109 carries pYJ12 sYJ027 JM109 carries pYJ18 sYJ028 JM109 carries pYJ16 sYJ029 BWΔ4 carries pYJ16 sYJ030 BWΔ4 carries pYJ16 and pYJ18 sYJ031 Isobutyraldehyde Mutagenic Strains Library - 100Gy sYJ032 Isobutyraldehyde Mutagenic Strains Library - 150Gy BWΔ4-G9 High-yield isobutyraldehyde strain G9 plasmid pYJ12 <![CDATA[P rrnB -yqhC;P yqhD -rfp-Cm r ;p15A]]> pYJ16 <![CDATA[P L lacO1-alsS-ilvC-ilvD-kivd-rrnB T1 terminator;ori;amp r ]]> pYJ18 <![CDATA[P rrnB -yqhCm9;P yqhD -rfp-Cm r ;p15A]]>

[0052] The present invention will be further explained and illustrated below through specific embodiments.

[0053] Example 1: Screening of mutant YqhCm9

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

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

[0056] (2) Construction of a biosensor system based on wild-type transcription factor YqhC: Using the genome MG1655 as a template, clone the aldehyde-responsive transcription factor yqhC (SEQ ID NO.2) and promoter P from Escherichia coli. yqhD (As shown in SEQ ID NO.6), gene synthesis yields promoter element P rrnBThe PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2. The red fluorescent protein gene rfp (SEQ ID NO. 7), the resistance gene Cm (SEQ ID NO. 9), and the replicon p15A (SEQ ID NO. 8) are shown in SEQ ID NO. 5.

[0057] (3) Using 2KPlus II 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 then assembled using Gibson assembly technology. rrnB yqhC-P yqhD Fragment ligation was performed using rfp, p15A, and Cm to obtain the ligation product P. rrnB -yqhC-P yqhD The -rfp-p15A-Cm fragment was used to transform the ligation product into 100 μL of JM109 chemocompetent cells. The cells were placed on ice and incubated for 25 min. After the ice bath, the centrifuge tubes were placed at 42°C for 1 min 30 s heat shock. After the heat shock, the centrifuge tubes were returned to ice for 2 min. After the ice bath, 900 μL of LB medium was added to the centrifuge tubes, and the cells were placed in a shaker at 37°C and 220 rpm for 45 min to recover. The recovered medium was then plated onto chloramphenicol-resistant plates. The plates were incubated overnight at 37°C.

[0058] 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.

[0059] Table 1 PCR reaction system

[0060]

[0061] Table 2 PCR reaction procedures

[0062]

[0063] Table 3 PCR reaction system

[0064]

[0065] Table 4 PCR reaction procedures

[0066]

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

[0068] The 12G backbone fragment and the error-prone PCR fragment were purified and recovered, then ligated using Gibson ligation. All ligation products were 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. 600 At a concentration of approximately 0.6, 500 μL of 50 mM isobutyraldehyde (final concentration 5 mM) was added to induce a 4-hour induction period. Cells in the top 1% of fluorescence levels were then sorted by flow cytometry, ultimately yielding a mutant strain that highly responded to isobutyraldehyde. This strain was named sYJ027, its sensor plasmid 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 is shown in SEQ ID NO.4.

[0069] Table 5 PCR reaction system

[0070]

[0071] Table 6 PCR reaction procedure

[0072]

[0073] Table 7 PCR reaction procedure

[0074]

[0075] Example 2: Fluorescence response of mutant YqhCm9

[0076] To further verify the performance of the mutant YqhCm9, a gradient exogenous addition experiment was needed to determine its fluorescence response curve. The sorted mutant strain sYJ027 and wild-type strain sYJ022 were inoculated into 5 mL of LB (25 μg / mL Cm) liquid medium and cultured at 37°C and 220 rpm for 12 hours as seed cultures. The seed cultures were then preserved, and the fluorescence response curves of the mutant and wild-type strains to gradient concentrations of isobutyraldehyde were measured using 2 mL 96-well plates.

[0077] Add 800 μL of fresh M9 medium (25 μg / mL cm) using a pipette, followed by 10 μL of seed culture to each well. Perform three replicates per well. After sealing with a sealing film, incubate the deep-well plate at 37°C and 220 rpm for 4 hours. Then, remove the plate and add 200 μL of medium (25 μg / mL) containing a gradient concentration of isobutyraldehyde (0-25 g / L, final concentration 0-5 g / L). Seal the plate again and incubate for another 4-8 hours. Pipette 200 μL of bacterial culture into each well of a black 96-well plate and measure the fluorescence intensity and OD using a BioTek Cytation 3 microplate reader. 600 Quantitative detection of red fluorescent protein (RFP) was performed using an excitation wavelength of 580 nm and an emission wavelength of 610 nm, with a gain of 80. The average of three parallel datasets was calculated. Cell density was measured at a wavelength of 600 nm. Based on this, the RFP / OD ratio for each well was calculated. 600 As a relative fluorescence intensity value.

[0078] RFP / OD of strain sYJ022 600 Results were analyzed as a wild-type control, using RFP / OD 600 The graph is plotted using GraphPad Prism 8.0 software with isobutyraldehyde concentration on the x-axis and fitted with LOWESS.

[0079] The results are as follows Figure 2 As shown, compared with the wild-type YqhC which is basically unresponsive to isobutyraldehyde, the mutant YqhCm9 can respond linearly in the range of 0-5 g / L isobutyraldehyde. There is basically no background fluorescence response when isobutyraldehyde is not added (0 g / L). When the concentration of isobutyraldehyde is added at 5 g / L, the response intensity is increased by 24 times compared with that without isobutyraldehyde (0 g / L). This indicates that the mutant can respond to isobutyraldehyde efficiently and has the advantages of no background leakage, wide dynamic detection range (0-5 g / L), and higher response intensity (24 times).

[0080] Example 3: Application of the YqhCm9 biosensor system in real-time monitoring

[0081] This embodiment illustrates the application of the isobutyraldehyde biosensor based on the mutant YqhCm9 obtained through 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 process using glucose as a substrate, first generating pyruvate through glycolysis, then generating α-ketoisovalerate under the catalysis of acetylhydroxy acid synthase (AlsS) (SEQ ID NO.11), acetyllactate isomer reductase (IlvC) (SEQ ID NO.12), and dihydroxy acid dehydratase (IlvD) (SEQ ID NO.13), and finally generating isobutyraldehyde under the catalysis of 2-ketoisovalerate decarboxylase (KivD) (SEQ ID NO.14). This synthesis pathway consists of two operons, alsS-ilvC / D and kivd, and is constructed by gene synthesis amplification of P... L The lacO1-alsS-ilvC-ilvD gene fragment (SEQ ID NO. 15, including promoter P) L The lacO1, alsS, ilvC, and ilvD gene fragments were amplified; the kivD-rrnB T1 terminator gene fragment (SEQ ID NO.16, including the kivD and rrnB T1 terminator gene fragments) and the ori-Amp gene fragment (SEQ ID NO.17, including the 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 Plus II 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 then assembled using Gibson assembly technology. L The lacO1-alsS-ilvC-ilvD gene fragment, kivD-rrnB T1 terminator gene fragment, and ori-Amp gene fragment were ligated. All ligation products were transformed into 100 μL of JM109 chemocompetent cells and placed on ice for 25 min. After the ice bath, the centrifuge tubes were placed at 42°C for 1 min 30 s heat shock. After the heat shock, the centrifuge tubes were returned to ice for 2 min. After the ice bath, 900 μL of LB medium was added to the centrifuge tubes, and the tubes were placed in a shaker at 37°C and 220 rpm for 45 min to recover. The recovered medium was then plated onto ampicillin-resistant plates and incubated overnight at 37°C.

[0084] The following day, a single colony was selected for PCR to verify successful plasmid construction. The verification primers were 16-F / R, and the target fragment was cPCR-16, 1206 bp in size. 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 Plus II. A target band of the correct size was obtained, indicating successful construction of the isobutyraldehyde production plasmid. This plasmid was named pYJ16, and the strain carrying this plasmid was named sYJ028.

[0085] Table 8 PCR Reaction System

[0086]

[0087]

[0088] Table 9 PCR reaction procedure

[0089]

[0090] Table 10 PCR Reaction System

[0091]

[0092] Table 11 PCR reaction procedure

[0093]

[0094] (3) Construction of isobutyraldehyde producing strain: The isobutyraldehyde synthesis pathway plasmid pYJ16 was obtained by transforming *Escherichia coli* BWΔ4 (a laboratory-preserved strain) into the strain. The host cells BWΔ4 were prepared into competent cells using a competent cell preparation kit. 1 μL of the isobutyraldehyde production plasmid pYJ16 was transformed into 50 μL of BWΔ4 competent cells and placed on ice for 25 min. After the ice bath, the centrifuge tubes were placed at 42℃ for 1 min 30 s heat shock. After the heat shock, the centrifuge tubes were placed back on ice for 2 min. After the ice bath, 900 μL of LB medium was added to the centrifuge tubes, and the tubes were placed in a shaker at 37℃ and thawed at 220 rpm for 45 min. The mixture was then plated on ampicillin-resistant plates. The plates were incubated overnight at 37℃. The next day, a single colony was picked and transferred to 5 mL of LB (100 μg / mL Amp) medium. After culturing at 37°C and 220 rpm for 12 hours, the bacteria were preserved and stored at -80°C to obtain the isobutyraldehyde producing strain, which was named sYJ029.

[0095] (4) Construct a dual-plasmid system to achieve real-time monitoring of the isobutyraldehyde production process: such as Figure 3As shown, when isobutyraldehyde biosensor plasmid pYJ18 and isobutyraldehyde production plasmid pYJ16 were simultaneously introduced into host cells BWΔ4, during fermentation, as isobutyraldehyde was biosynthesized and accumulated, the aldehyde-responsive transcription factor mutant YqhCm9 could sense the concentration of isobutyraldehyde in real time within the cell, and then, through its regulated promoter P... yqhD The expression of the reporter gene rfp is driven to convert chemical signals into light signals, enabling dynamic real-time monitoring of the isobutyraldehyde production process.

[0096] To verify the performance of this dual-plasmid real-time monitoring system in practical applications, this embodiment measures the yield and fluorescence value (RFP / OD) of isobutyraldehyde during fermentation production. 600 The correlation between the two was analyzed. First, host cells BWΔ4 were prepared as chemocompetent cells. 1 μL of isobutyraldehyde production plasmid pYJ16 and 1 μL of isobutyraldehyde biosensor plasmid pYJ18 were transformed into 100 μL of BWΔ4 competent cells and placed on ice for 25 min. After the ice bath, the centrifuge tubes were placed at 42℃ for 1 min 30 s heat shock. After the heat shock, the centrifuge tubes were placed back on ice for 2 min. After the ice bath, 900 μL of LB medium was added to the centrifuge tubes, and the tubes were placed in a shaker at 37℃ and 220 rpm for 45 min to recover. The recovered cells were then plated on ampicillin-kanamycin-chloramphenicol double antibiotic plates. The plates were incubated overnight at 37℃ to obtain isobutyraldehyde producing strain sYJ030 containing a dual plasmid real-time monitoring system.

[0097] The following day, three single colonies (parallel controls) were picked and transferred to 5 mL of LB medium (100 μg / mL Amp + 25 μg / mL Cm) and cultured at 37°C and 220 rpm for 12 h as seed culture. Fermentation was then carried out in 250 mL screw-cap Erlenmeyer flasks. First, 20 mL of prepared sterile M9 medium was added to a sterile shake flask, followed by the appropriate antibiotic and 2 μL of IPTG stock solution (final concentration 0.1 mM, inducing promoter P). L (lacO1 expression), and finally inoculate 200 μL of fresh seed liquid, ferment in a shaker at 30℃ and 220 rpm, and take 1 mL samples at 4, 8, 12, 24 and 36 hours.

[0098] The formula for fermentation medium M9 is: 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 and bring to a final volume of 900mL. Sterilize at 121℃ for 20 minutes. Cool to room temperature before 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 and add them to 900 mL of sterile M9 salt solution and mix well to obtain fermentation medium M9.

[0101] Sample preparation: Take 200 μL of bacterial culture from each sample and perform RFP and OD analysis using an enzyme-linked immunosorbent assay (ELISA) reader. 600 For the determination, the remaining bacterial solution was centrifuged at 12000 rpm for 10 minutes, and 100 μL of supernatant was added to the inner liner of the gas chromatography vial. Then, 100 μL of 2 g / L isovaleraldehyde (final concentration 1 g / L) was added as the internal standard for gas chromatography detection. The gas chromatography vial was then capped and shaken thoroughly.

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

[0103] The results are as follows Figure 4 As shown, with the increase of isobutyraldehyde production, the individual fluorescence value (RFP / OD) of the strain increased. 600 The yield of this strain also increased synchronously. Pearson correlation coefficient analysis was used. The correlation coefficient r represents the statistical measure of the strength and direction of the linear correlation between two random variables, ranging from -1 to 1. A value of 0 indicates no correlation, a value greater than 0.8 indicates a strong correlation, and a value less than 0.3 indicates a very weak correlation. The absolute value of the correlation coefficient is approximately close to 1; the higher the correlation, the stronger the correlation. We found that the yield of this strain was related to the individual fluorescence value (RFP / OD). 600 The correlation coefficient r between the two was 0.9056, indicating a high correlation between them, which verifies the real-time monitoring performance of the dual plasmid system in the actual fermentation process.

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

[0105] This embodiment illustrates the application of the above-mentioned dual plasmid real-time monitoring system in screening high-yield isobutyraldehyde strains.

[0106] The host cells BWΔ4 were mutagenized using a heavy ion accelerator at irradiation doses of 100 Gy and 150 Gy to obtain two bacterial strain libraries: sYJ031 and sYJ032. These were prepared as competent cells (100 μL / tube) and transfected with 1 μL of isobutyraldehyde production plasmid pYJ16 and 1 μL of isobutyraldehyde biosensor plasmid pYJ18, respectively. The cells were incubated on ice for 25 min, followed by heat shock at 42°C for 1 min 30 s. After heat shock, the centrifuge tubes were returned to ice for another 2 min. Following the ice shock, 900 μL of LB medium was added to the centrifuge tubes, and the tubes were incubated at 37°C in a shaker at 220 rpm for 45 min. The resulting culture was then plated onto ampicillin-chloramphenicol plates and incubated overnight at 37°C. The following day, single colonies from the plate were inoculated into 96-well plates containing 1 ml of LB medium (100 μg / mL Amp + 25 μg / mL Cm) and incubated at 37°C for 12 h as seed culture. The seed culture was then re-inoculated at a ratio of 1% into 96-well plates containing 1 ml of M9 fermentation medium (100 μg / mL Amp + 25 μg / mL Cm). After 24 h, the fluorescence values ​​(RFP / OD) were measured using a microplate reader. 600 ).

[0107] Using the isobutyraldehyde-producing strain sYJ030, which contains a dual-plasmid real-time monitoring system, as a control strain, we screened strains with fluorescence expression levels (RFP / OD) that were significantly higher than those of the control strain. 600 A strain that has seen a significant increase, such as Figure 5 As shown in the figure. Since this strain is a mutant obtained from the BWΔ4 mutagenesis strain library carrying a dual plasmid monitoring system, we named it BWΔ4-G9. The strain was validated by 36 hours of shake-flask fermentation, and the results are shown in the figure. Figure 6 As shown, after the fermentation system was increased from 1 mL to 20 mL, the isobutyraldehyde yield of strain G9 after 36 h of fermentation (7.3 g / L) was 2.8 times higher than that of the control strain (2.6 g / L).

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

[0109] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail 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 was obtained by mutating M65T, V122G, R184H, S204R and L227R on the wild-type transcription factor YqhC shown in SEQ ID NO.

1. The mutant was named YqhCm9 mutant and its amino acid sequence is shown in SEQ ID NO.

3.

2. The encoding gene of the YqhC mutant as described in claim 1.

3. The application of the YqhC mutant of claim 1 or the encoding gene of claim 2, characterized in that, It has applications in detecting samples containing isobutyraldehyde, screening isobutyraldehyde-producing strains, and constructing biosensors for detecting isobutyraldehyde.

4. A biosensor, characterized in that, The biosensor includes the following elements: regulation yqhCm9 Gene promoters yqhCm9 Gene, yqhCm9 promoters regulated by genes P yqhD By the promoter P yqhD Reporter genes driven by the virus; The yqhCm9 The gene is the encoding gene of the YqhC mutant as described in claim 2.

5. The biosensor as described in claim 4, characterized in that, The regulation yqhCm9 Gene promoters include: P rrnB , P j23100 , P j23108 , P j23119 ; The reporter gene includes: red fluorescent protein gene. rfp Green fluorescent protein gene gfp Yellow fluorescent protein gene yfp .

6. The biosensor as described in claim 4, characterized in that, The biosensor also includes a replicon gene and / or a resistance gene.

7. The biosensor as described in claim 6, characterized in that, The replicon includes: p15A, ori, ColE1 The resistance gene includes: Cm, Amp, Kan .

8. The biosensor as described in claim 4, characterized in that, The biosensor includes P rrnB yqhCm9 P yqhD ,rfp,p15A,Cm The nucleotide sequence of the biosensor is shown in SEQ ID NO.

10.

9. The application of the biosensor according to claim 4, characterized in that, It is used in the detection of samples containing isobutyraldehyde or in the screening of isobutyraldehyde-producing strains.

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