Genetic coding VB12 whole-cell biosensor applicable to screening of industrially produced strains and preparation method of genetic coding VB12 whole-cell biosensor

Abstract

The invention provides a genetic coding VB12 whole-cell biosensor suitable for screening industrially produced strains and a preparation method, and belongs to the technical field of bioengineering, the genetic coding VB12 whole-cell biosensor comprises a sensing element EutR, the nucleic acid sequence of the sensing element EutR is as shown in SEQ ID NO: 1, and the amino acid sequence of the sensing element EutR is as shown in SEQ ID NO: 2. The response range of the obtained sensor with the expanded response range is expanded to 0.1-100 mg / L and is higher than the response range of an existing reported sensor, and the requirement for industrial strain breeding is met.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a genetic coding VB12 whole cell biosensor suitable for industrial strain screening and a preparation method. BACKGROUND

[0002] Industrial biotechnology is usually based on microbial cell factories to produce many important products, such as enzymes, antibiotics, etc. However, few naturally occurring microorganisms can be directly used for large-scale production because the yield of the naturally occurring microorganisms is low and the tolerance to the industrial production environment is poor. In order to overcome the above series of problems, physical and chemical mutagenesis, adaptive laboratory evolution, metabolic engineering and many other strategies are used to construct large mutant libraries, and then a small number of desired phenotypes are screened.

[0003] In the mutant library, the probability of beneficial mutations is very low, and traditional screening methods have problems such as low throughput, large workload, high cost, etc., such as plate transparent circle screening, microplate screening technology, etc. High-throughput screening technology is urgently needed. Droplet microfluidic technology is a high-throughput screening technology that has emerged in recent years and has shown great application potential in related fields such as synthetic biology, but because microfluidic, flow and other related instruments require fluorescence signals as the basis for sorting, high-throughput screening methods based on fluorescence signals have become a research hotspot.

[0004] Vitamin B12 is one of the most complex substances in nature, which cannot be synthesized by the human body itself and will cause a series of diseases in the absence of it, and is widely used in medicine, cosmetics, food, feed and other aspects. However, due to its structural complexity, it cannot be mass-produced by chemical synthesis, and at present it is mainly obtained by fermentation of non-model microorganisms, with low yield and high price. Due to the difficulty of genetic modification, the method of random mutation combined with high-throughput screening to select high-yield strains has become an effective way to improve yield.

[0005] However, in the currently reported screening methods, the response range of the sensor is relatively low, generally below 40 mg / L, while the yield of industrial production bacteria is generally above 50 mg / L, and the existing sensor cannot meet the needs of high-throughput screening of industrial strains. SUMMARY

[0006] The purpose of this section is to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments.

[0007] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0008] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art, and to provide a genetic coding VB12 whole cell biosensor suitable for industrial strain screening.

[0009] Another object of the present application is to overcome the deficiencies in the prior art and provide a full-cell biosensor for screening of high-yield VB12 strains in industrial production.

[0010] The present application has the following advantages:

[0011] (1) The sensor of the present application has an expanded response range of 0.1-100 mg / L, which is higher than the response range of the reported sensors and is suitable for the requirements of industrial strain breeding.

[0012] (2) The sensor of the present application has a wider response range and a higher upper limit, which is suitable for screening of high-yield strains in industrial production; the sensor has good compatibility and has a good and similar response range and signal strength for different forms of VB12 in the fermentation broth in the production process.

[0013] (3) The full-cell biosensor of the present application is based on Escherichia coli and does not need to genetically manipulate the production strains, which is convenient to use. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The figure is a schematic diagram of the sensor in the embodiment of the present application.

[0015] Figure 2 The figure is a result diagram of the response range of the sensor in the embodiment of the present application.

[0016] Figure 3 The figure is a result diagram of the response of the sensor to methylcobalamin, adenosylcobalamin and cyanocobalamin in the embodiment of the present application.

[0017] Figure 4 The figure is a response range diagram of different sensors to VB12, wherein CK represents a sensor formed by the sequence of the reported sensor element, and pBL1031 represents the sensor constructed in Example 1.

[0018] Figure 5 The figure is a response range diagram of different sensors to VB12, wherein Mutant2 represents a sensor constructed by mutant 2, and pBL1031 represents the sensor constructed in Example 1. DETAILED DESCRIPTION

[0019] In order to make the above-mentioned objects, features and advantages of the present application more apparent and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the description and examples.

[0020] Example 1

[0021] (1) Sensor structure

[0022] The sensor structure is shown in Figure 1 The plasmid contains:

[0023] A sensor element EutR, whose nucleic acid sequence is shown as SEQ ID NO: 1, and whose amino acid sequence is shown as SEQ ID NO: 2;

[0024] A pRSFori replicon (functioning as a plasmid ensuring replication in E. coli at a certain copy number), whose nucleic acid sequence is shown as SEQ ID NO: 3;

[0025] A kanamycin resistance tag KanR (functioning as a resistance screening marker after transformation into E. coli), whose nucleic acid sequence is shown as SEQ ID NO: 4;

[0026] A lactose repressor protein (EutR-induced expression system), whose nucleic acid sequence is shown as SEQ ID NO: 5;

[0027] A lactose operon (EutR-induced expression system), whose nucleic acid sequence is shown as SEQ ID NO: 6;

[0028] A T7 promoter (EutR-induced expression system);

[0029] A nucleic acid sequence shown as SEQ ID NO: 7;

[0030] A eutS promoter (EutR binds to different concentrations of VB12, and different changes in conformation will cause different intensities of expression after binding to the downstream eutS promoter), whose nucleic acid sequence is shown as SEQ ID NO: 8;

[0031] An sfGFP fluorescent tag (fluorescent knowledge tag), whose nucleic acid sequence is shown as SEQ ID NO: 9.

[0032] (2) Construction method of sensor:

[0033] The EutR gene fragment synthesized by Geneart was used as a template, and primer pair 1031-1F and 1031-1R were used for PCR to obtain an EutR fragment, wherein the sequence of 1031-1F is shown as SEQ ID NO: 10;

[0034] The sequence of 1031-1R is shown as SEQ ID NO: 11;

[0035] The sensor plasmid 1025 was used as a template, and primer pair 1031-2F and 1031-2R were used for PCR to obtain a vector fragment, wherein the sequence of 1031-2F is shown as SEQ ID NO: 12;

[0036] The sequence of 1031-2R is shown as SEQ ID NO: 13;

[0037] The nucleic acid sequence of plasmid 1025 is shown as SEQ ID NO: 14;

[0038] Then the EutR fragment library and the carrier fragment were mixed for seamless cloning assembly to obtain a sensor plasmid, and the plasmid nucleic acid sequence is shown as SEQ ID NO: 15.

[0039] The concentration of the EutR fragment and the carrier fragment was measured by using an ultramicro spectrophotometer, the concentration ratio of the EutR fragment to the carrier was 3:1, the MultiF Seamless Assembly Mix was 5 μL, the total reaction system was 10 μL, the cloning assembly temperature was 50℃, and the time was 1 hour.

[0040] The source of the raw material and the concentration ratio of the EutR fragment to the carrier are shown in Table 1.

[0041] Table 1

[0042]

[0043] After the plasmid construction was completed, the plasmid was transformed into E. coli BL21 (DE3).

[0044] (3) Sensor response range

[0045] VB12 was dissolved in water to prepare 10 g / L stock solution 1 and 1 g / L stock solution 2, and the screened sensor suitable for industrial strain screening was inoculated in the medium containing different final concentrations of VB12 according to the following table, and was incubated at 37℃, 200 rpm overnight, and the conditions are shown in Table 2. Among them, the sensor inoculation conditions are as follows:

[0046] The sensor colony was inoculated in LB medium and cultured at 37℃ for 12 hours to obtain a seed liquid (LB medium formula g / L: tryptone 10, yeast powder 5, sodium chloride 10, pH = 7.0, prepared according to the formula, and sterilized by using a high-pressure sterilization pot at 121℃ for 20 min), and was inoculated in the medium containing different final concentrations of VB12 according to the inoculation amount of 2% (volume percentage).

[0047] Table 2

[0048] VB12 final concentration (mg / L) 0.1 0.5 1 5 10 30 50 75 100 125 150 200 Stock 1 (μL) 0 0 0 0 0 6 10 15 20 25 30 40 Stock 2 (μL) 0.2 1 2 10 20 0 0 0 0 0 0 0 Water (μL) 49.8 49 48 40 30 44 40 35 30 25 20 10 Culture medium (mL) 2 2 2 2 2 2 2 2 2 2 2 2

[0049] The fluorescence intensity was read by using an enzyme-labeled instrument, the excitation wavelength was set to 488 nm, and the emission wavelength was set to 525 nm. According to the relationship between the fluorescence signal (FI) and the VB12 concentration, the response curve was drawn;

[0050] As shown in Figure 2 , the fluorescence intensity increased with the increase of the VB12 concentration, and the response range was 0.1-100 mg / L.

[0051] Example 2

[0052] Sensor compatibility

[0053] VB12 has three main different states in fermentation process, i.e. methylcobalamin, adenosylcobalamin and cyanocobalamin, so it is necessary to further verify the compatibility of the sensor with the three different states of VB12.

[0054] The VB12 standards of the three states were dissolved in water to prepare 10 g / L stock solution 1 and 1 g / L stock solution 2, and the screened sensors suitable for industrial strain screening were inoculated into the medium containing different final concentrations of VB12 according to the following table, and incubated at 37℃, 200 rpm overnight, and the conditions are shown in Table 3. Among them, the sensor inoculation conditions are as follows:

[0055] The sensor colonies were picked and inoculated into LB medium and cultured at 37℃ for 12h to obtain the seed liquid (LB medium formula g / L: tryptone 10, yeast powder 5, sodium chloride 10, pH = 7.0, prepared according to the formula, using a high-pressure sterilization pot at 121℃ for 20min), and inoculated into the medium containing different final concentrations of VB12 according to the inoculation amount of 2% (volume percentage).

[0056] Table 3

[0057] VB12 final concentration (mg / L) 0.1 1 10 50 100 150 200 Stock 1 (μL) 0 0 0 10 20 30 40 Stock 2 (μL) 0.2 2 20 0 0 0 0 Water (μL) 49.8 48 30 40 30 20 10 Culture medium (mL) 2 2 2 2 2 2 2

[0058] The fluorescence intensity was read by the enzyme-labeled instrument, and the excitation wavelength was set to 488nm and the emission wavelength was set to 525nm. According to the relationship between the fluorescence signal (FI) and the concentration of VB12, the response curve was drawn;

[0059] As shown in Figure 3 , the sensor has similar response range and signal intensity to methylcobalamin, adenosylcobalamin and cyanocobalamin, indicating that the sensor is suitable for screening of industrial high-yield VB12 strains.

[0060] Comparative Example 1

[0061] Under the experimental conditions of the sensor constructed in Example 1, the sensing element EutR therein was replaced with the natural EutR sequence (nucleic acid sequence as shown in SEQ ID NO: 16), and the sensor was constructed. The response range of the sensor was detected according to step (3) in Reference Example 1, and the results are shown in Figure 4 , wherein pBL1031 is the sensor in Example 1, and CK is the sensor constructed by the natural EutR sequence. It can be seen that the natural EutR sequence has a narrow response range, while the pBL1031 sensor has a significantly improved response range.

[0062] Comparative Example 2

[0063] Under the experimental conditions of the sensor constructed in Embodiment 1, the sensing element EutR therein is replaced by the nucleic acid sequence of the EutR sequence shown in SEQ ID NO: 17 to construct a sensor, and the response range of the sensor is detected according to step (3) in Embodiment 1, and the results are shown in Table 2. Figure 5 It can be seen that the mutant 2 (the mutation sites are R244S, S308T, and the fluorescence signal intensity and the VB12 concentration have no significant correlation) has a poor effect, and the fluorescence signal intensity and the VB12 concentration have no significant correlation.

[0064] The sequences involved in the embodiments of the present application are as follows:

[0065] SEQ ID NO: 1:

[0066]

[0067] SEQ ID NO: 2:

[0068] MKKTRTANLHHLYHEPLPENLKLTPKVEVDNVHQRQ TT DVYEHALTITAWQQIYDQLHPGKFHGEFTEILLDDIQVFREYTGLALRQSCLVWPNSFWFGIPATRGEQGFIGSQCLGSAEIATRPGGTEFELSTPDDYTILGVVLSEDVITRQANFLHNPDRVLHMLRNQSALEVKLQHKAALWGFVQQALATFCENPENLHQPAVRKVLGDNLLMAMGAMLEEAQPMVTAESISHQSYRRLLSRAREYVLENMSEPVTVLDLCNQLHVSRRTLQNAQHAILGIGPNAWLKRIRLNAVRRELISPWSQSMTVKDAAMQWGFWHLGQFATDYQQLFSEKPSLTLHQRMREWG

[0069] SEQ ID NO: 3:

[0070] CTTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGTGTCAGCTCACTCAAAAGCGGTAATACGGTTATCCACAGAATCAGGGGATAAAGCCGGAAAGAACATGTGAGCAAAAAGCAAAGCACCGGAAGAAGCCAACGCCGCAGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGCCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTTGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCATTGGTAACTGATTTAGAGGACTTTGTCTTGAAGTTATGCACCTGTTAAGGCTAAACTGAAAGAACAGATTTTGGTGAGTGCGGTCCTCCAACCCACTTACCTTGGTTCAAAGAGTTGGTAGCTCAGCGAACCTTGAGAAAACCACCGTTGGTAGCGGTGGTTTTTCTTTATTTATGAGATGATGAATCAATCGGTCTATCAAGTCAACGAACAGCTATTCCGTT

[0071] SEQ ID NO: 4:

[0072] TTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCAT

[0073] SEQ ID NO: 5:

[0074]

[0075] SEQ ID NO: 6:

[0076] GGAATTGTGAGCGGATAACAATTCC

[0077] SEQ ID NO: 7:

[0078] TAATACGACTCACTATAGG

[0079] SEQ ID NO: 8:

[0080] GCGCTTGCCGAATTTTGTTATTTACTCTGACGAAAAATTGTCACGATACACGAAAGTTTTTCACAGGCGGCGACTC

[0081] SEQ ID NO: 9:

[0082] ATGAGCAAAGGTGAAGAACTGTTTACCGGCGTTGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGTCACAAATTCAGCGTGCGTGGTGAAGGTGAAGGCGATGCCACGATTGGCAAACTGACGCTGAAATTTATCTGCACCACCGGCAAACTGCCGGTGCCGTGGCCGACGCTGGTGACCACCCTGACCTATGGCGTTCAGTGTTTTAGTCGCTATCCGGATCACATGAAACGTCACGATTTCTTTAAATCTGCAATGCCGGAAGGCTATGTGCAGGAACGTACGATTAGCTTTAAAGATGATGGCAAATATAAAACGCGCGCCGTTGTGAAATTTGAAGGCGATACCCTGGTGAACCGCATTGAACTGAAAGGCACGGATTTTAAAGAAGATGGCAATATCCTGGGCCATAAACTGGAATACAACTTTAATAGCCATAATGTTTATATTACGGCGGATAAACAGAAAAATGGCATCAAAGCGAATTTTACCGTTCGCCATAACGTTGAAGATGGCAGTGTGCAGCTGGCAGATCATTATCAGCAGAATACCCCGATTGGTGATGGTCCGGTGCTGCTGCCGGATAATCATTATCTGAGCACGCAGACCGTTCTGTCTAAAGATCCGAACGAAAAAGGCACGCGGGACCACATGGTTCTGCACGAATATGTGAATGCGGCAGGTATTACGTGGAGCCATCCGCAGTTCGAAAAATAA

[0083] SEQ ID NO: 10: GACCCGTACAGCCAATTTGCAC

[0084] SEQ ID NO: 11 : TGATGCAGCGTCAGTGACGG

[0085] SEQ ID NO: 12: CCGTCACTGACGCTGCATCA

[0086] SEQ ID NO: 13: GTGCAAATTGGCTGTACGGGTC

[0087] SEQ ID NO: 14:

[0088]

[0089] SEQ ID NO: 15:

[0090]

[0091] SEQ ID NO: 16

[0092]

[0093] SEQ ID NO: 17:

[0094] ATGAAAAAGACCCGTACAGCCAATTTGCACCATCTTTATCATGAACCCTTACCCGAAAACCTGAAGC

[0095] TCACGCCGAAGGTCGAAGTGGATAATGTTCATCAACGACAGACAACGGATGTCTATGAACATGCTT

[0096] TAACGATTACCGCCTGGCAGCAGATTTACGATCAGCTGCATCCGGGCAAGTTTCATGGTGAATTTA

[0097] CGGAAATTCTACTCGATGATATTCAGGTTTTTCGTGAATACACCGGTCTGGCGCTGCGTCAGTCGT

[0098] GCCTGGTCTGGCCGAACTCGTTCTGGTTTGGCATTCCGGCGACGCGCGGTGAGCAGGGATTTATC

[0099] GGTTCGCAATGTCTGGGAAGCGCGGAAATCGCCACCCGCCCTGGTGGCACTGAATTTGAACTGAG

[0100] CACGCCGGATGATTACACGATCCTGGGCGTGGTGCTTTCTGAAGATGTCATCACCCGGCAGGCTAA

[0101] CTTTTTGCATAACCCGGATCGGGTATTACATATGTTGCGTAACCAGTCGGCGCTGGAAGTGAAAGA

[0102] GCAGCATAAAGCCGCGCTGTGGGGCTTTGTCCAACAGGCGCTGGCGACGTTTTGCGAGAATCCGG

[0103] AAAATCTCCATCAGCCAGCAGTGCGAAAAGTGCTGGGGGATAATTTGCTAATGGCGATGGGGGCC

[0104] ATGCTGGAAGAAGCGCAACCAATGGTGACGGCGGAAAGCATCAGTCATCAGAGTTACCGTCGATT

[0105] GCTTTCCTCCGCCCGTGAATATGTGCTGGAAAACATGTCCGAACCGGTGACGGTGCTGGATTTGTG

[0106] TAATCAACTGCATGTCAGCCGCCGCACGCTACAAAACGCGTTTCACGCTATTTTAGGCATTGGCCCG

[0107] AACGCGTGGCTGAAACGCATTCGCCTGAACGCCGTACGCCGCGAACTGATAAGTCCGTGGTCGCAA

[0108] ACGATGACGGTAAAAGACGCCGCCATGCAGTGGGGATTCTGGCATCTGGGGCAATTTGCCACGGA

[0109] TTACCAGCAGCTGTTTTCCGAGAAGCCGTCACTGACGCTGCATCAGCGGATGCGGGAGTGGGGGTGA

Claims

1. A genetically encoded whole-cell biosensor for screening industrial strains of VB12, characterized in that: It includes a sensing element EutR, wherein the nucleic acid sequence of the sensing element EutR is shown in SEQ ID NO:1 and the amino acid sequence is shown in SEQ ID NO:

2.

2. The whole-cell biosensor as described in claim 1, characterized in that: The whole-cell biosensor also includes a copy replicon, an resistance tag, an EuTR-induced expression system, an EuTS promoter, and a fluorescent tag.

3. The whole-cell biosensor as described in claim 2, characterized in that: The copy replicon includes the pRSFori replicon, the nucleic acid sequence of which is shown in SEQ ID NO:

3.

4. The whole-cell biosensor as described in claim 2 or 3, characterized in that: The resistance tag includes the kanamycin resistance tag KanR, whose nucleic acid sequence is shown in SEQ ID NO:

4.

5. The whole-cell biosensor as described in claim 4, characterized in that: The EuTR-induced expression system includes a lactose repressor protein, a lactose operon, and a T7 promoter. The nucleic acid sequence of the lactose repressor protein is shown in SEQ ID NO:5, the nucleic acid sequence of the lactose operon is shown in SEQ ID NO:6, and the nucleic acid sequence of the T7 promoter is shown in SEQ ID NO:

7.

6. The whole-cell biosensor as described in claim 5, characterized in that: The nucleic acid sequence of the eutS promoter is shown in SEQ ID NO:

8.

7. The whole-cell biosensor as described in claim 2, characterized in that: The fluorescent tag includes an sfGFP fluorescent tag, the nucleic acid sequence of which is shown in SEQ ID NO:

9.

8. The method for preparing the whole-cell biosensor according to any one of claims 1 to 7, characterized in that: include, Constructing plasmids; After plasmid construction, it was transformed into E. coli BL21 and cultured to obtain a whole-cell biosensor.

9. The preparation method according to claim 8, characterized in that: The method for constructing the plasmid includes, Using the EutR gene fragment synthesized by Genewiz as a template, the EutR fragment was obtained by PCR using primer pairs 1031-1F and 1031-1R. The sequence of 1031-1F is shown in SEQ ID NO:10; the sequence of 1031-1R is shown in SEQ ID NO:

11. Using sensor plasmid 1025 as a template, PCR was performed with primer pairs 1031-2F and 1031-2R ​​to obtain the vector fragment. The sequence of 1031-2F is shown in SEQ ID NO:12; the sequence of 1031-2R ​​is shown in SEQ ID NO:13; and the nucleic acid sequence of plasmid 1025 is shown in SEQ ID NO:

14. Then, the EutR fragment library and vector fragment were mixed and seamlessly cloned and assembled to obtain the sensor plasmid; The plasmid nucleic acid sequence is shown in SEQ ID NO:

15.

10. The application of the whole-cell biosensor according to any one of claims 1 to 7 in the industrial screening of strains that produce high-yield VB12.

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