Development and application of biosensor for detecting L-threonine

Abstract

The invention discloses development and application of a biosensor for detecting L-threonine, and belongs to the field of genetic engineering. The invention provides a construction method of a novel threonine biosensor and an application method for screening L-threonine high-yield mutants in an auxiliary manner through the biosensor. The biosensor can sensitively detect the concentration change of L-threonine. By combining with a high-throughput screening system, L-threonine high-yield strain mutants with improved production performance can be rapidly screened. By means of the biosensor, an L-threonine high-yield mutant LMTM35 is successfully screened from an L-threonine production strain LMT4 mutant library, 166 g / L of L-threonine is accumulated in a 5L fermentation tank after 45 hours, and the saccharic acid conversion rate is 0.67 g / g.

Description

Technical Field

[0001] The present invention relates to the development and application of a biosensor for detecting L-threonine, belonging to the field of genetic engineering. Background Art

[0002] L-threonine is an important essential amino acid, which is widely used in the fields of medicine, food, and feed. Especially in animal feed, it is used as an important nutritional additive to improve protein utilization rate and promote animal growth, and its market demand continues to grow. However, at present, the production of L-threonine mainly relies on the microbial fermentation method, which mainly uses Escherichia coli as the production host. Although the industrial production process has gradually matured, the fermentation efficiency and the yield of L-threonine are still limited, and further optimization is urgently needed to meet the market demand.

[0003] Escherichia coli is an important industrial microorganism, which is widely used in the fields of metabolic engineering and industrial fermentation due to its clear genetic background, fast growth rate, and easy gene manipulation. In recent years, the rapid development of synthetic biology and systems metabolic engineering has provided effective means for the rational transformation of Escherichia coli, and engineering strains with high-yield L-threonine can be obtained quickly. However, the L-threonine synthesis pathway of Escherichia coli is restricted by a complex metabolic network and unknown multi-level regulation. Using biosensors combined with high-throughput screening technology to screen high-yield mutants of chemicals has become an available strategy to further improve the product yield. Therefore, developing a sensitive L-threonine biosensor to assist in screening high-yield L-threonine mutants is expected to further improve the L-threonine production level of strains. Summary of the Invention

[0004] The technical content involved in the present invention is to develop a biosensor for detecting L-threonine in Escherichia coli.

[0005] The present invention provides a CysB protein mutant, which is obtained by mutating the amino acid at position 138 from valine to alanine on the basis of the amino acid sequence corresponding to the parental CysB protein, and is named CysB V138A ;

[0006] The amino acid sequence of the parental CysB protein has at least 95% identity with the amino acid shown in SEQ ID NO.6 and has CysB protein activity, and at the same time, the amino acid at position 138 is valine.

[0007] The amino acid sequence of the parental CysB protein is as shown in SEQ ID NO.6.

[0008] SEQ ID NO.6:

[0009] MKLQQLRYIVEVVNHNLNVSSTAEGLYTSQPGISKQVRMLEDELGIQIFSRSGKHLTQVTPA

[0010] GQEIIRIAREVLSKVDAIKSVAGEHTWPDKGSLYIATTHTQARYALPNVIKGFIERYPRVSLH

[0011] MHQGSPTQIADAVSKGNADFAIATEALHLYEDLVMLPCYHWNRAIVVTPDHPLAGKKAITI

[0012] EELAQYPLVTYTFGFTGRSELDTAFNRAGLTPRIVFTATDADVIKTYVRLGLGVGVIASMAV

[0013] DPVADPDLVRVDAHDIFSHSTTKIGFRRSTFLRSYMYDFIQRFAPHLTRDVVDAAVALRSNEE

[0014] IEVMFKDIKLPEK

[0015] In one embodiment, the CysB original nucleotide is as shown in SEQ ID NO.5:

[0016] SEQ ID NO.5:

[0017] atgaaattacaacaacttcgctatattgttgaggtggtcaatcataacctgaatgtctcatcaacagcggaaggactttacacatcacaacccgggatcagtaaacaagtcagaatgctggaagacgagctaggcattcaaattttttcccgaagcggcaagcacctgacgcaggtaacgccagcagggcaagaaataattcgtatcgctcgcgaagtcctgtcgaaagtcgatgccataaaatcggttgccggagagcacacctggccggataaaggttcactgtatatcgccaccacgcatacccaggcacgctacgcattaccaaacgtcatcaaaggctttattgagcgttatcctcgcgtttctttgcatatgcaccagggctcgccgacacaaattgctgatgccgtctctaaaggcaatgctgatttcgctatcgccacagaagcgctgcatctgtatgaagatttagtgatgttaccgtgctaccactggaatcgggctattgtagtcactccggatcacccgctggcaggcaaaaaagccattaccattgaagaactggcgcaatatccgttggtgacatataccttcggctttaccggacgttcagaactggatactgcctttaatcgcgcagggttaacgccgcgtatcgttttcacggcaacggatgctgacgtcattaaaacttacgtccggttagggctgggggtaggggtcattgccagcatggcggtggatccggtcgccgatcccgaccttgtgcgtgttgatgctcacgatatcttcagccacagtacaaccaaaattggttttcgccgtagtactttcttgcgcagttatatgtatgatttcattcagcgttttgcaccgcatttaacgcgtgatgtcgttgatgcggctgtcgcattgcgctctaatgaagaaattgaggtcatgtttaaagatataaaactgccggaaaaataa

[0018] The present invention also provides a polynucleotide encoding the above-mentioned CysB protein mutant, a vector carrying the polynucleotide, or a cell expressing the above-mentioned CysB protein mutant.

[0019] In one embodiment, the cell uses bacteria or fungi as an expression host.

[0020] The present invention also provides a method for increasing the response threshold of an L-threonine biosensor to L-threonine, wherein the biosensor is a vector containing the above-mentioned CysB protein mutant CysB V138A , the PcysB promoter, the complete non-coding region sequence upstream of the cysK gene, and a fluorescent protein;

[0021] In one embodiment, the nucleotide sequence encoding the CysB protein mutant is as shown in SEQ ID NO.2;

[0022] In one embodiment, the nucleotide sequence of the complete non-coding region upstream of the cysK gene is as shown in SEQ ID NO.1;

[0023] In one embodiment, the nucleotide sequence of the PcysB promoter is as shown in SEQ ID NO.3;

[0024] In one embodiment, the fluorescent protein is a green fluorescent protein;

[0025] In one embodiment, the nucleotide sequence encoding the green fluorescent protein is as shown in SEQ ID NO.4.

[0026] The present invention provides an L-threonine biosensor, which contains the above-mentioned CysB protein mutant CysB V138A .

[0027] In one embodiment, the biosensor is a vector carrying the PcysB promoter, the CysB protein mutant, the complete non-coding region sequence upstream of the cysK gene, and a fluorescent protein.

[0028] In one embodiment, the complete non-coding region sequence (PcysK) upstream of the cysK gene includes the promoter of the cysK gene and the binding site of the CysB protein upstream thereof.

[0029] In one embodiment, the nucleotide sequence encoding the CysB protein mutant is as shown in SEQ ID NO.2;

[0030] In one embodiment, the nucleotide sequence of the complete non-coding region upstream of the cysK gene is as shown in SEQ ID NO.1;

[0031] In one embodiment, the nucleotide sequence of the PcysB promoter is as shown in SEQ ID NO.3;

[0032] In one embodiment, the fluorescent protein is green fluorescent protein;

[0033] In one embodiment, the nucleotide sequence encoding the green fluorescent protein is as shown in SEQ ID NO.4.

[0034] In one embodiment, the CysB protein mutant is driven by the PcysB promoter, and the fluorescent protein is driven by the complete non-coding region sequence upstream of the cysK gene.

[0035] In one embodiment, the vector is the pTrc99A plasmid.

[0036] The present invention also provides a method for increasing the response threshold of a recombinant strain to L-threonine, wherein the recombinant strain contains the above-mentioned L-threonine biosensor.

[0037] The present invention provides a method for constructing an L-threonine biosensor, comprising the following steps:

[0038] (1) Integrate the complete non-coding region sequence upstream of the cysK gene (SEQ ID NO.1) and the green fluorescent protein encoding gene eGFP (SEQ ID NO.4) into the pTrc99A plasmid backbone to obtain pTrc99A-cysK 非编码区 -eGFP;

[0039] (2) Further integrate the CysB mutant driven by the PcysB promoter into the above plasmid to obtain the L-threonine biosensor: pTrc99A-cysK 非编码区 -eGFP-PcysB-CysB V138A .

[0040] The present invention also provides a method for detecting the concentration of L-threonine prepared from a recombinant strain using the above-mentioned biosensor.

[0041] In one embodiment, the method comprises the following steps: transforming the biosensor into a strain producing L-threonine to construct a recombinant strain; after fermenting and culturing the recombinant strain, detecting the fluorescence intensity in the fermentation broth, and determining the concentration of L-threonine according to the fluorescence intensity.

[0042] In one embodiment, the method is to inoculate the Escherichia coli model strain MG1655 containing the L-threonine biosensor into LB liquid medium, and add L-threonine with final concentrations of 0, 1, 2, 3, and 4 g / L respectively. After culturing for a period of time, the fluorescence intensity is measured.

[0043] The present invention also provides an L-threonine producing strain containing the biosensor.

[0044] In one embodiment, the L-threonine producing strain is Escherichia coli LMT4, which is described in the invention patent with the publication number CN115011620B.

[0045] The L-threonine producing strain containing the biosensor in the present invention refers to the transformation of the biosensor into Escherichia coli LMT4.

[0046] The present invention also provides a method for screening high-yield mutants of L-threonine production. A recombinant plasmid containing a mutation element is introduced into a recombinant strain containing the biosensor, the expression of the mutation element is induced, and random mutations are carried out to obtain mutants of the L-threonine producing strain; the mutant strain is inoculated into a fermentation medium, and after culturing, the fluorescence intensity is measured; high-yield L-threonine strains are screened through the fluorescence intensity.

[0047] In one embodiment, the method includes: introducing the Mp6ts plasmid (public literature: Significantly enhancing production of trans-4-hydroxy-l-proline by integrated system engineering in Escherichia coli) into the L-threonine producing strain containing the biosensor constructed in the present invention. The Mp6ts plasmid is used to randomly mutate the L-threonine producing strain through arabinose induction, thereby obtaining mutants of the L-threonine producing strain. The mutant strain is inoculated into a 96-well plate containing a fermentation medium and cultured for a period of time, and then the fluorescence intensity is measured.

[0048] Beneficial effects

[0049] The biosensor provided by the present invention has good response ability to fluorescence intensity in the range of L-threonine concentration from 0 to 4 g / L. Thus, it lays a foundation for further screening L-threonine producing strains. The method for constructing the biosensor provided by the present invention is simple, easy to use, and has good application prospects. Description of the drawings

[0050] Figure 1 The linear relationship between the fluorescence intensity generated by adding different concentrations of L-threonine and the cells per unit OD.

[0051] Figure 2 It is a structural diagram of an L-threonine biosensor.

[0052] Figure 3 It is the response ability of the biosensor to endogenous L-threonine.

[0053] Figure 4 They are the fermentation parameters of the L-threonine-producing mutant strain LMTM35. Detailed implementation manners

[0054] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0055] Some definitions or terms related to the present invention:

[0056] Corresponds to: As used herein, the term "corresponds to" refers to a way of determining a specific amino acid in a sequence (where a specific amino acid sequence is referenced). For example, for the purposes of the present invention, when referring to a specific amino acid position, one skilled in the art can align another amino acid sequence with the amino acid sequence that has been referenced, so as to determine which specific amino acid may be of interest in the other amino acid sequence. Alternative alignment methods can be used, and these methods are well known to those skilled in the art.

[0057] Mutant: As used herein, when referring to the use of variants of the present invention, the term "mutant" means a polypeptide having CysB protein mutant activity and containing alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "parent" CysB protein. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a certain position. In describing the variants of the present invention, for ease of reference, the following nomenclature has been adapted. The accepted IUPAC single-letter or three-letter amino acid abbreviations are used.

[0058] Parent or parental CysB protein: As used herein, the term "parental CysB protein" means the CysB protein that has been altered to produce the CysB protein mutants of the present invention. This term also refers to the polypeptide to which the mutants of the present invention are compared. The parent can be a naturally occurring (wild-type) polypeptide, or it can even be a variant thereof prepared by any suitable means. For example, the parent protein can be a variant of a naturally occurring polypeptide that has been modified or altered in its amino acid sequence. Thus, the CysB protein can have one or more (or one or several) amino acid substitutions, deletions, and / or insertions. Thus, the CysB protein can be a variant of the parental CysB protein. The parent can also be an allelic variant, which is a polypeptide encoded by any one of two or more alternative forms of a gene that occupy the same chromosomal locus.

[0059] Wild-type enzyme: When referring to an amino acid sequence or a nucleic acid sequence, the term "wild-type" means that the amino acid sequence or the nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, an amino acid, or a nucleic acid sequence). In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in the laboratory, or modifications of wild-type sequences). When the parental enzyme is not a variant enzyme, the terms "wild-type enzyme" and "parental enzyme" can be used interchangeably.

[0060] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0061] It will be recognized from the foregoing that the present disclosure can be embodied in various ways, including but not limited to the following:

[0062] Example 1: A CysB protein mutant, which is obtained by mutating the amino acid at position 138 from valine to alanine on the basis of the amino acid sequence corresponding to the parental CysB protein, and is named CysB V138A ;

[0063] The amino acid sequence of the parental CysB protein has at least 95% identity with the amino acid shown in SEQ ID NO.6 and has CysB protein activity, and the amino acid at position 138 is valine.

[0064] Example 2: A polynucleotide encoding the CysB protein mutant CysB V138A of.

[0065] Example 3: A recombinant vector carrying the polynucleotide encoding the CysB protein mutant CysB V138A of.

[0066] Example 4: A recombinant cell expressing the CysB protein mutant CysB V138A .

[0067] Example 5: The recombinant cell according to Example 4, wherein the recombinant cell uses bacteria or fungi as an expression host.

[0068] Example 6: A method for increasing the response threshold of an L-threonine biosensor to L-threonine. The biosensor contains the CysB protein mutant of Example 1, and also contains the PcysB promoter, the complete non-coding region sequence upstream of the cysK gene, and a fluorescent protein; the nucleotide sequence encoding the CysB protein mutant is as shown in SEQ ID NO.2; the nucleotide sequence of the complete non-coding region upstream of the cysK gene is as shown in SEQ ID NO.1; the nucleotide sequence of the PcysB promoter is as shown in SEQ ID NO.3; the fluorescent protein is a green fluorescent protein; the nucleotide sequence encoding the green fluorescent protein is as shown in SEQ ID NO.4.

[0069] Example 7: An L-threonine biosensor, which contains the above-mentioned CysB protein mutant CysB V138A .

[0070] Example 8: An L-threonine biosensor, which is a vector carrying the PcysB promoter, the CysB protein mutant, the complete non-coding region sequence upstream of the cysK gene, and a fluorescent protein.

[0071] Principle of the biosensor of the present invention: After overexpressing the CysB protein, cysK 非编码区 initiates the expression of the fluorescent protein (preferably the gfp protein), resulting in an increase in the fluorescence level of the fluorescent protein (compared with before overexpressing the CysB protein);

[0072] After mutating the CysB protein, the expression level of the fluorescent protein is further increased.

[0073] Example 9: The L-threonine biosensor according to Example 8, wherein the complete non-coding region sequence (PcysK) upstream of the cysK gene includes the promoter of the cysK gene and the binding site of the CysB protein upstream thereof;

[0074] The nucleotide sequence encoding the CysB protein mutant is as shown in SEQ ID NO.2;

[0075] The nucleotide sequence of the complete non-coding region upstream of the cysK gene is as shown in SEQ ID NO.1;

[0076] The nucleotide sequence of the PcysB promoter is shown as SEQ ID NO.3;

[0077] The fluorescent protein is green fluorescent protein;

[0078] The nucleotide sequence encoding the green fluorescent protein is shown as SEQ ID NO.4.

[0079] The CysB protein mutant is driven by the PcysB promoter, and the fluorescent protein is driven by the complete non-coding region sequence upstream of the cysK gene.

[0080] In a preferred embodiment, the vector is the pTrc99A plasmid.

[0081] Example 10: A method for constructing an L-threonine biosensor, comprising the following steps:

[0082] (1) Integrate the complete non-coding region sequence upstream of the cysK gene (SEQ ID NO.1) and the green fluorescent protein encoding gene eGFP (SEQ ID NO.4) into the pTrc99A plasmid backbone to obtain pTrc99A-cysK 非编码区 -eGFP;

[0083] (2) Further integrate the CysB mutant driven by the PcysB promoter into the above plasmid to obtain the L-threonine biosensor: pTrc99A-cysK 非编码区 -eGFP-PcysB-CysB V138A .

[0084] Example 11: A method for detecting the concentration of L-threonine prepared by a recombinant strain using the L-threonine biosensor; the method comprises the following steps: transforming the L-threonine biosensor into a strain producing L-threonine to construct a recombinant strain; after fermenting and culturing the recombinant strain, detecting the fluorescence intensity in the fermentation broth, and determining the concentration of L-threonine according to the fluorescence intensity.

[0085] Example 12: A method for detecting the concentration of L-threonine prepared by a recombinant strain using the L-threonine biosensor, the method is to introduce the L-threonine biosensor into the Escherichia coli model strain MG1655 to obtain a recombinant strain, inoculate the recombinant strain into an LB liquid medium, and add L-threonine with final concentrations of 0, 1, 2, 3, and 4 g / L respectively, and measure the fluorescence intensity after culturing for a period of time.

[0086] Example 13: An L-threonine-producing strain containing the L-threonine biosensor; the L-threonine-producing strain is Escherichia coli LMT4, which is described in the invention patent with the publication number CN115011620B.

[0087] Example 14: A method for screening high-yield mutants of L-threonine-producing strains. A recombinant plasmid containing a mutation element is introduced into a recombinant strain containing a biosensor, the expression of the mutation element is induced, and random mutations are carried out to obtain mutants of L-threonine-producing strains. The mutant strains are inoculated into a fermentation medium, and the fluorescence intensity is measured after cultivation. High-yield L-threonine strains are screened through the fluorescence intensity; strains with high fluorescence intensity correspond to high-yield L-threonine strains.

[0088] Example 15: A method for screening high-yield mutants of L-threonine-producing strains, the method comprising:

[0089] (1) Introduce the L-threonine biosensor of the present invention into the L-threonine-producing strain Escherichia coli LMT4 to prepare a recombinant strain;

[0090] (2) Introduce the Mp6ts plasmid (public literature: Significantly enhancing production of trans-4-hydroxy-l-proline by integrated system engineering in Escherichia coli) into the recombinant strain obtained in step (1);

[0091] (3) Randomly mutate the recombinant strain obtained in step (2) by inducing the Mp6ts plasmid with arabinose to obtain mutants of L-threonine-producing strains. The mutant strains are inoculated into 96-well plates containing a fermentation medium and cultivated for a period of time, and then the fluorescence intensity is measured. High-yield L-threonine strains are screened through the fluorescence intensity; strains with high fluorescence intensity correspond to high-yield L-threonine strains.

[0092] Example 16: A method for increasing the response threshold of a recombinant strain to L-threonine, wherein the recombinant strain contains the L-threonine biosensor described in Example 8 or 9. The recombinant strain can be an L-threonine-producing strain, or the L-threonine-producing strain LMT4; it can also be Escherichia coli MG1655.

[0093] The media involved in the following examples are as follows:

[0094] Fermentation medium (g / L): glucose 10, tryptone 5, yeast extract 5, corn steep liquor 3, potassium dihydrogen phosphate 1, ammonium sulfate 2, urea 1, dipotassium hydrogen phosphate 0.5.

[0095] The detection methods involved in the following examples are as follows:

[0096] Determination method of L-threonine:

[0097] Sample treatment: Take 1 mL of the fermentation broth after 48 h of fermentation, centrifuge at 12,000 rpm for 10 min to remove the thalli, and take the supernatant. Dilute the supernatant appropriately with deionized water and then filter it through a filter membrane with a pore size of 0.22 μm.

[0098] Analysis method: OPA pre-column derivation; Chromatographic conditions: (1) Chromatographic column: Chromatographic column C18 (250×4.6) mm; (2) Column temperature: 40 °C; (3) Mobile phase A: Weigh 3.01 g of anhydrous sodium acetate into a beaker, dissolve it with ultrapure water and make up the volume to 1 L, then add 200 μL of triethylamine, adjust the pH to 7.20±0.05 with 5% acetic acid; After suction filtration, add 5 mL of tetrahydrofuran, mix and then filter through a 0.22 μm inorganic filter membrane, and then put it into an ultrasonic cleaning pot to exhaust for 20 min for standby. Mobile phase B: Weigh 3.01 g of anhydrous sodium acetate into a beaker; dissolve it with ultrapure water and make up the volume to 200 mL; adjust the pH to 7.20±0.05 with 5% acetic acid; Then add 400 mL of acetonitrile and 400 mL of methanol to this solution, mix and filter, and put it into an ultrasonic cleaning pot to exhaust for 20 min for standby. (4) Flow rate: 1.0 ml / min; (5) UV detector: 338 nm; (6) Column temperature: 40 °C.

[0099] Detection method of fluorescence threshold:

[0100] Centrifuge to remove the supernatant in the culture medium, wash the culture twice with phosphate buffer, and then resuspend the thalli with phosphate buffer to an OD 600 of about 0.5, and detect the fluorescence value of GFP at an excitation wavelength of 490 nm and an emission wavelength of 530 nm.

[0101] Example 1: Construction of pTrc99A-cysK 非编码区 -gfp-P cysB -CysB

[0102] The specific steps are as follows:

[0103] (1) Using the pTrc99A plasmid as a template, amplify the plasmid with primers FP-99A-R (5’-CGGTTTACAAAATAACCGGTAGATTGAGCAACGACTGTTTGCCCG-3’) and FP-99A-F (5’-GATTACACATGGCATGGATGAACTATACAAATAACCTGCAGGCATGCAAGCTT-3’) to obtain a linearized pTrc99A plasmid.

[0104] (2) Using the Escherichia coli MG1655 genome as a template, the genome was amplified using primers PcysK-F (5’-CAATCTACCGGTTATTTTGTAAACCGT-3’) and PcysK-R (5’-GGCCTGTCCTTAACTGTATGAAA-3’) to obtain the complete upstream non-coding region sequence of the cysK gene (cysK 非编码区 , SEQ ID NO.1).

[0105] (3) Using the complete upstream non-coding region sequence of the cysK gene (cysK 非编码区 ) and the green fluorescent protein DNA fragment (SEQ ID NO.4) as templates, amplification was performed using primers PcysK-F and gfp-R (5’-TTATTTGTATAGTTCATCCATGCCATGTGTAATC-3’) to obtain the fusion fragment of the complete upstream non-coding region sequence of the cysK gene (cysK 非编码区 ) and the green fluorescent protein.

[0106] (4) Using a seamless cloning kit, the linearized pTrc99A plasmid was fused with the fusion fragment of the complete upstream non-coding region sequence of the cysK gene (cysK 非编码区 ) and the green fluorescent protein, and introduced into Escherichia coli JM109 to obtain a positive clone containing the pTrc99A-cysK 非编码区 -gfp plasmid. The pTrc99A-cysK 非编码区 -gfp plasmid was obtained using a plasmid extraction kit.

[0107] (5) Using the pTrc99A-cysK 非编码区 -gfp plasmid as a template, and cysK 非编码区 -gfp-F (5’-GTCATGTTTAAAGATATAAAACTGCCGGAAAAATAACTTATCAGACCGTTTCCCG C-3’) and cysK 非编码区 -gfp-R (5’-CCTTTGTTTATAAAAACCTGACAGAATTAAAGGAGACACCGGCATACTCTGC-3’) were used to amplify the pTrc99A-cysK 非编码区 -gfp plasmid to obtain the linearized pTrc99A-cysK 非编码区 -gfp plasmid.

[0108] (6) Using the Escherichia coli MG1655 genome as a template, the genome was amplified with primers cysB-F (5’-CCTTTAATTCTGTCAGGTTTTTATAAACAAAGGG-3’) and cysB-R (5’-TTATTTTTCCGGCAGTTTTATATCTTTAAACATGACC-3’) to obtain a DNA fragment of CysB protein containing the P cysB promoter: P cysB

[0109] (7) Use a seamless cloning kit to linearize the pTrc99A-cysK 非编码区 -gfp plasmid obtained in step (5) and the DNA fragment containing the P cysB promoter of CysB protein (P cysB -CysB) for fusion, and introduce it into Escherichia coli JM109 to obtain a positive clone containing the pTrc99A-cysK 非编码区 -gfp-P cysB -CysB plasmid. Use a plasmid extraction kit to obtain the pTrc99A-cysK 非编码区 -gfp-P cysB -CysB plasmid (the plasmid containing the wild-type WT enzyme of CysB).

[0110] Example 2: Construction of a biosensor

[0111] The specific steps are as follows:

[0112] (1) Using the pTrc99A-cysK 非编码区 -gfp-P cysB -CysB plasmid as a template, use different primer sequences to perform site-directed mutagenesis on the CysB protein on the pTrc99A-cysK 非编码区 -gfp-P cysB -CysB plasmid, and introduce it into Escherichia coli JM109 to obtain cells containing the pTrc99A-cysK 非编码区 -gfp-P cysB -CysBmut plasmid. Use a plasmid extraction kit to obtain the pTrc99A-cysK 非编码区 -gfp-P cysB -CysBmut plasmid;

[0113] Respectively prepare recombinant vectors: pTrc99A-cysK 非编码区 -gfp-P cysB -CysB V138A 、pTrc99A-cysK 非编码区 -gfp-P cysB -CysB T149A 、pTrc99A-cysK 非编码区 -gfp-P cysB -CysB E150A 、pTrc99A-cysK 非编码区 -gfp-P cysB -CysB T202A 、pTrc99A-cysK 非编码区-gfp-P cysB -CysB T102A 。

[0114] The primer sequences involved are as follows:

[0115] CysB138-F: 5’-GCCGCCTCTAAAGGCAATGCTG-3’;

[0116] CysB138-R: 5’-AGAGGCGGCATCAGCAATTTGTG-3’

[0117] CysB 149- F: 5’- TATCGCCGCTGAAGCGCTG -3’;

[0118] CysB1 49 -R: 5’- CAGCGCTTCAGCGGCGATA -3’

[0119] CysB 150- F: 5’- ATCGCCACAGCTGCGCTGCAT -3’;

[0120] CysB1 50 -R: 5’- ATGCAGCGCAGCTGTGGCGAT -3’

[0121] CysB 202- F: 5’- TTCGGCTTTGCCGGACGTTCAG -3’;

[0122] CysB 202 -R: 5’- CTGAACGTCCGGCAAAGCCGAA -3’

[0123] CysB 102- F: 5’-ACCACGCATGCCCAGGCACGC-3’;

[0124] CysB 102-R : 5’-GCGTGCCTGGGCATGCGTGGT-3’

[0125] (2) Verification of the mutant effect

[0126] The recombinant plasmid pTrc99A-cysK containing WT (CysB) 非编码区 -gfp-P cysB -CysB, and the above-mentioned L-threonine biosensor containing the mutant (pTrc99A-cysK 非编码区 -gfp-P cysB -CysB V138A 、pTrc99A-cysK 非编码区 -gfp-P cysB-CysB T149A 、pTrc99A-cysK 非编码区 -gfp-P cysB -CysB E150A 、pTrc99A-cysK 非编码区 -gfp-P cysB -CysB T202A 、pTrc99A-cysK 非编码区 -gfp-P cysB -CysB T102A ) were separately introduced into the Escherichia coli model strain MG1655 to obtain recombinant strains;

[0127] Single colonies of the prepared recombinant Escherichia coli were picked and inoculated into 10 mL of LB liquid medium, and cultured at 37 °C and 220 rpm for 10 h; a seed solution was prepared.

[0128] The seed solution was taken and inoculated into LB liquid media containing 0, 1, 2, 3, and 4 g / L L-threonine at an inoculation amount of 2% (v / v), and after culturing at 37 °C and 220 rpm for 10 h, the fluorescence intensity and the corresponding OD600 were measured, and the effects of different mutants on the fluorescence threshold were calculated. The results are shown in Table 1.

[0129] Table 1: Fluorescence intensity (a.u.) / OD600 of recombinant strains containing different cysB in the presence of different concentrations of L-threonine

[0130]

[0131] The values shown in the table are the means.

[0132] Calculation of the multiple of the increase in the fluorescence threshold:

[0133] (Fluorescence intensity (a.u.) / OD600 value of the strain containing the cysB mutant with 4 g / L L-threonine added - Fluorescence intensity (a.u.) / OD600 value of the strain containing the cysB mutant with 0 g / L L-threonine added) / (Fluorescence intensity (a.u.) / OD600 value of the strain containing the wild-type cysB with 4 g / L L-threonine added - Fluorescence intensity (a.u.) / OD600 value of the strain containing the wild-type cysB with 0 g / L L-threonine added)

[0134] Table 2: Multiple of the increase in the fluorescence threshold

[0135]

[0136]

[0137] The results show that:

[0138] The L-threonine biosensor constructed with the V138A mutant in the present invention can increase the fluorescence threshold of the L-threonine biosensor constructed with the wild-type CysB by 3.07 times in the presence of 0-4 g / L L-threonine in the recombinant strain.

[0139] The fluorescence value per unit cell (Fluorescence level / OD 600 ) shows an almost linear increase with the increase of L-threonine concentration in the range of 0-4 g / L L-threonine.

[0140] As Figure 1 shown, the recombinant Escherichia coli MG1655 / pTrc99A-cysK 非编码区 -eGFP-PcysB-CysB V138A The fluorescence value per unit cell (Fluorescence level / OD 600 ) shows an almost linear increase with the increase of L-threonine concentration in the range of 0-4 g / L L-threonine.

[0141] Example 3: Characterization of the response ability of the biosensor to endogenously produced L-threonine in cells

[0142] The specific steps are as follows:

[0143] (1) The pTrc99A-cysK 非编码区 -eGFP-PcysB-CysB V138A plasmid ( Figure 2 ) was introduced into the L-threonine producing strain LMT4 (described in the patent with the publication number CN 115011620 B) to construct the strain LMT4 / pTrc99A-cysK 非编码区 -eGFP-PcysB-CysB V138A .

[0144] (2) A single colony was picked and inoculated into 10 mL of LB liquid medium and cultured at 37 °C and 220 rpm for 10 h; a seed solution was prepared.

[0145] (2) The seed solution obtained in step (1) was inoculated into the fermentation medium at an inoculation amount of 2% (v / v) and continuously cultured at 37 °C and 220 rpm for 12 h, and the fluorescence intensity of the fermentation product was measured every 2 h.

[0146] At the same time, the strain containing the pTrc99A-cysK-gfp-CysB plasmid (MG1655 / pTrc99A-cysK 非编码区-eGFP-PcysB-CysB V138A ) was used as a fluorescence control (the Escherichia coli model strain MG1655 cannot accumulate L-threonine).

[0147] The results were as Figure 3 shown. As the fermentation time progressed, L-threonine gradually accumulated in the strain LMT4 / pTrc99A-cysK 非编码区 -eGFP-PcysB-CysB V138A , and the fluorescence value gradually increased. However, since the MG1655 strain could not accumulate L-threonine, its fluorescence value did not change significantly. This result indicates that the biosensor can detect endogenously produced L-threonine.

[0148] Example 4: Screening for high L-threonine-producing mutants using a biosensor

[0149] The specific steps are as follows:

[0150] (1) Transfer the MP6 ts mutant plasmid (disclosed in the paper "Significantly enhancing production of trans-4-hydroxy-l-proline by integrated system engineering in Escherichia coli") into the L-threonine-producing strain LMT4 / pTrc99A-cysK containing the biosensor constructed in Example 2 非编码区 -eGFP-PcysB-CysB V138A to obtain the recombinant strain: LMT4 / pTrc99A-cysK 非编码区 -eGFP-PcysB-CysB V138A / MP6 ts .

[0151] (2) Select a single positive clone and inoculate it into 10 mL of LB medium and culture for 20 h (30 °C, 220 rpm) to obtain a seed solution;

[0152] Then, take 2 mL of the seed solution and inoculate it into a liquid medium containing 50 mL of LB. After culturing at 30 °C, 220 rpm for 4 h, add 0.1 mM arabinose to induce the expression of the mutant element in the MP6s plasmid, and continue to culture (30 °C, 220 rpm) for 20 h to generate a mutant library.

[0153] (3) Transfer the culture medium obtained in step (2) to a 42°C constant temperature shaker (220 rpm) and continue culturing for 20 h to remove the MP6s plasmid in the mutants (the plasmid is a temperature-sensitive plasmid. When cultured at 42°C for 20 h, the strains with the plasmid will die; removing the MP6s plasmid under stress conditions). Subsequently, spread the culture medium without the plasmid on an LB solid plate medium and continue culturing (30°C, 220 rpm) for 20 h. Then, inoculate the single colonies on the plate into 96-well plates containing 200 μL of shake flask fermentation medium (fermentation medium) respectively, and place the 96-well plates in a shaker at 37°C and 220 rpm for 12 h to prepare the fermentation broth.

[0154] (4) Subsequently, use a plate centrifuge to remove the supernatant in the fermentation broth, take the precipitate, wash the precipitate 2 times with phosphate buffer, and resuspend the cells with phosphate buffer, and measure the fluorescence value in the cells.

[0155] The above operation steps (1) to (4) can be repeated for multiple rounds of mutagenesis. Subsequently, perform shake flask fermentation on the screened high-fluorescent cells to further verify the L-threonine production performance of the mutant strains.

[0156] Based on the above operations, a L-threonine-producing mutant strain LMTM35 was successfully screened from the mutant library of the L-threonine-producing strain LMT4.

[0157] Example 5: Verify the production performance of the L-threonine-producing mutant strain LMTM35 in a 5 L fermenter

[0158] The specific steps are as follows:

[0159] (1) Take out the preservation tube of the LMTM35 strain stored in a -80°C ultra-low temperature freezer to thaw, pipette 10 μL of the bacterial liquid and streak it for activation on an LB solid plate, and invert it in a 37°C constant temperature incubator for 20 h.

[0160] Pick a single colony from the plate and inoculate it into 10 mL of LB medium, and culture it in a constant temperature shaker at 37°C and 220 rpm for 12 h to prepare the primary seed liquid.

[0161] (2) Subsequently, take 2 mL of the prepared seed liquid and transfer it to a 500 mL round-bottom flask containing 100 mL of LB medium (4 flasks), and continue culturing under the same culture conditions (37°C, 220 rpm) for 10 h; prepare the secondary seed liquid.

[0162] (3) The prepared secondary seed liquid was inoculated into a 5 L bioreactor containing 1.6 L of fermentation medium (fermentation medium) at an inoculation amount of 20%. The initial rotation speed was 300 rpm. By correlating the stirring with the dissolved oxygen (DO) in the fermentation broth to 30 ± 2%, the stirring speed was automatically adjusted, and the aeration rate was 2 vvm.

[0163] Fermentation process temperature: It was controlled at 36 °C from 0 to 20 h and at 37 °C from 20 h to the end of fermentation. The pH value was maintained at pH 7.0 ± 0.02 by automatically adding ammonia water. Samples were taken every 3 h to measure the biomass (OD 600 ) of the strain and the glucose content in the fermentation broth. When the initial glucose was exhausted, the fed-batch feeding system was started to maintain a specific glucose concentration.

[0164] The results were as Figure 4 shown. After 45 h of fermentation, 166 g / L of L-threonine could be accumulated, and the sugar-acid conversion rate reached 0.67 g / g of glucose.

[0165] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A CysB protein mutant, characterized in that, The CysB protein mutant is obtained by mutating the amino acid at position 138 from valine to alanine on the basis of the amino acid sequence corresponding to the parental CysB protein; The amino acid sequence of the parental CysB protein has at least 98% identity with the amino acid shown in SEQ ID NO.6 and has CysB protein activity, and the amino acid at position 138 is valine.

2. A polynucleotide encoding the CysB protein mutant according to claim 1, a vector carrying the polynucleotide, or a cell expressing the CysB protein mutant according to claim 1.

3. A method for increasing the response threshold of an L-threonine biosensor to L-threonine, characterized in that, The method is to construct an L-threonine biosensor using a vector containing the CysB protein mutant according to claim 1; the biosensor is a vector containing the CysB protein mutant according to claim 1, the PcysB promoter, the complete non-coding region sequence upstream of the cysK gene, and a fluorescent protein; Preferably, the nucleotide sequence encoding the CysB protein mutant is as shown in SEQ ID NO.2; Preferably, the nucleotide sequence of the complete non-coding region upstream of the cysK gene is as shown in SEQ ID NO.1; Preferably, the nucleotide sequence of the PcysB promoter is as shown in SEQ ID NO.3; Preferably, the fluorescent protein is green fluorescent protein; Preferably, the nucleotide sequence encoding the green fluorescent protein is as shown in SEQ ID NO.

4.

4. An L-threonine biosensor, characterized in that, The biosensor contains the CysB protein mutant according to claim 1.

5. The L-threonine biosensor according to claim 4, wherein The biosensor is a vector carrying the PcysB promoter, the CysB protein mutant, the complete non-coding region sequence upstream of the cysK gene, and a fluorescent protein; Preferably, the nucleotide sequence encoding the CysB protein mutant is as shown in SEQ ID NO.2; Preferably, the nucleotide sequence of the complete non-coding region upstream of the cysK gene is as shown in SEQ ID NO.1; Preferably, the nucleotide sequence of the PcysB promoter is as shown in SEQ ID NO.3; Preferably, the fluorescent protein is green fluorescent protein; Preferably, the nucleotide sequence encoding the green fluorescent protein is as shown in SEQ ID NO.4; Preferably, the CysB protein mutant is driven by the PcysB promoter, and the fluorescent protein is driven by the complete non-coding region sequence upstream of the cysK gene.

6. A recombinant strain, characterized in that, The recombinant strain contains the L-threonine biosensor of claim 4 or 5.

7. A method for screening a high L-threonine-producing strain, characterized in that the application is to introduce a recombinant plasmid containing a mutant element into the recombinant strain according to claim 6, induce the expression of the mutant element, perform random mutation, so as to obtain a mutant of the L-threonine-producing strain; inoculate the mutant strain into a fermentation medium, and measure the fluorescence intensity after cultivation; screen a high L-threonine-producing strain through the fluorescence intensity.

8. Use of the L-threonine biosensor according to claim 4 or 5 in detecting the concentration of L-threonine prepared from a recombinant strain.

9. The application according to claim 8, wherein The recombinant strain is constructed by transforming the biosensor into a strain that produces L-threonine; The application is that after the recombinant strain is fermented and cultured, the fluorescence intensity in the fermentation broth is detected, and the concentration of L-threonine is determined according to the fluorescence intensity.

10. Use of the L-threonine biosensor according to claim 4 or 5 in the screening of high-yield L-threonine strains.

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