GlcNAc6P induction component and method for promoting synthesis of acetylglucosamine

Abstract

The invention discloses a GlcNAc6P induction component and a method for promoting synthesis of acetylglucosamine. The existing GlcNAc6P sensing element has the problems of serious leakage and small response range, which results in that in the process of regulating and controlling the expression of phosphatase by using the GlcNAc6P sensing element, the phosphatase also has higher expression intensity when intracellular GlcNAc6P is not accumulated, and the supply of precursor substances is influenced. Besides, along with rapid increase of the intracellular GlcNAc6P concentration, the upper limit of the expression quantity of phosphatase is low, the intracellular dephosphorylation requirement cannot be met, and further increase of the GlcNAc yield is limited. Therefore, an existing GlcNAc6P sensing element is optimized, a nagC binding sequence is subjected to directed evolution, expression intensity of repressor protein nagC is optimized, the leakage expression level of the sensor is reduced, and the response range of the sensor is expanded, so that the expression intensity of phosphatase is more effectively and rigorously regulated and controlled to adapt to the change of the concentration of GlcNAc6P in cells in different fermentation periods; and a foundation is laid for producing acetylglucosamine by further transforming recombinant bacteria through metabolic engineering.

Description

Technical Field

[0001] The invention relates to a GlcNAc6P sensing component and a method for promoting acetylglucosamine synthesis, belonging to the technical field of genetic engineering. Background Art

[0002] Acetyl glucosamine is a monosaccharide in organisms, widely found in bacteria, yeast, mold, plants and animals. In the human body, acetylglucosamine is a synthetic precursor of the disaccharide unit of glycosaminoglycans, which plays an important role in repairing and maintaining the function of cartilage and joint tissue. Therefore, acetylglucosamine is widely used as a drug and nutritional dietary supplement to treat and repair joint injuries. In addition, acetylglucosamine also has many applications in the cosmetics and pharmaceutical fields.

[0003] Microbial fermentation is now the main method for producing acetylglucosamine at home and abroad. The genetically engineered bacteria used are mainly Escherichia coli, Corynebacterium glutamicum and Bacillus subtilis. By introducing glucosamine-6-phosphate synthase (glmS) and glucosamine-6-phosphate N-acetyltransferase (GNA1) coding genes in these strains, glucose is used as raw material to carry out microbial synthesis of acetylglucosamine. Wherein the last step of the synthesis reaction is the dephosphorylation reaction of N-acetylglucosamine-6-phosphate (GlcNAc6P), and GlcNAc6P is converted into acetylglucosamine by phosphatase. Studies on the biosynthesis of acetylglucosamine show that in Escherichia coli, because the phosphatase reaction speed of the cell itself is lower than the synthesis speed of GlcNAc6P, GlcNAc6P accumulation is toxic to cells, and cell growth inhibition phenomenon can be caused, resulting in a reduction in production rate, which is not conducive to production economy. Overexpression of phosphatases such as yqaB and BT4131 can effectively alleviate the accumulation of GlcNAc6P and increase the production of acetylglucosamine.

[0004] The active center of phosphatase has a strong binding force with the phosphate group, which leads to the fact that phosphatase generally has a broad substrate spectrum and has dephosphorylation activity on the phosphosugar precursors Glc6P (glucose-6-phosphate), Fru6P (fructose-6-phosphate) and GlcN6P (glucosamine-6-phosphate) in the acetylglucosamine synthesis pathway. Therefore, constitutive expression of phosphatase leads to a decrease in the concentration of intracellular phosphosugar precursors and the production of other byproducts, thereby reducing the conversion rate and affecting production economy. The GlcNAc6P response characteristics of the transcription factor nagC are used to construct a GlcNAc6P sensing component, and the expression of phosphatase is regulated by this sensing component to cope with the different degrees of accumulation of GlcNAc6P at various stages of the production process. Thus, while using phosphatase to strengthen the dephosphorylation reaction of GlcNAc6P, the impact on the precursor substances Glc6P, Fru6P, and GlcN6P is reduced, effectively improving the yield of acetylglucosamine in Escherichia coli and the conversion rate of raw materials. However, the response range of the currently published GlcNAc6P sensing components is small, the leakage expression is serious, and the upper limit of the expression intensity is low. When GlcNAc6P accumulates in large quantities, it is difficult to significantly increase the expression of phosphatase to relieve phosphate sugar pressure. In addition, the high leakage expression intensity will lead to overexpression of phosphatase when GlcNAc6P is not accumulated, thereby affecting the normal metabolism of intracellular phosphorylated substrates and then affecting the synthesis of products. The existing GlcNAc6P sensing components are all designed based on the naturally existing wild-type nagC binding sequence, and the binding sequence has not been artificially modified or directed evolution. There is potential for further optimization of the response range. Summary of the invention

[0005] To solve the above problems, the present invention first optimizes the sensing components to address the defects of the existing GlcNAc6P sensing components such as limited response range and high leakage expression, and constructs

[0006] pACYC-Duet1-P nagBM -BT4131 / I49Q / L129Q / G172L-P J23118 -nagC plasmid, greatly reducing the leakage expression of phosphatase and expanding the response range. The present invention applies its sensing components in the production of acetylglucosamine, and it is found that the product synthesis yield and production intensity are further improved.

[0007] The first object of the present invention is to provide an N-acetylglucosamine-6-phosphate sensing component, comprising:

[0008] a phosphatase whose expression is regulated by a first promoter;

[0009] the transcription factor nagC, whose expression is regulated by the second promoter;

[0010] The nucleotide sequence of the first promoter is shown in SEQ ID NO.2, and the nucleotide sequence of the second promoter is P J23118 Promoter.

[0011] Furthermore, the phosphatase is acetylglucosamine-6-phosphate phosphatase, which may be a wild type or a mutant, including but not limited to the amino acid sequence shown in SEQ ID NO.5 or a mutant thereof.

[0012] Furthermore, the mutants include but are not limited to:

[0013] Mutation of leucine at position 129 to glutamine (L129Q); or

[0014] Mutation of isoleucine at position 49 to glutamine and mutation of leucine at position 129 to glutamine (I49Q / L129Q); or

[0015] Isoleucine at position 49 was mutated to glutamine, leucine at position 129 was mutated to glutamine, and glycine at position 172 was mutated to leucine (I49Q / L129Q / G172L, SEQ ID NO. 6).

[0016] Furthermore, the P J23118 The sequence of the promoter is shown in SEQ ID NO.3.

[0017] Furthermore, the amino acid sequence of the transcription factor nagC is shown in SEQ ID NO.4.

[0018] The second object of the present invention is to provide a promoter for phosphatase regulated expression, the sequence of the promoter is shown in SEQ ID NO.2.

[0019] The third object of the present invention is to provide a dynamic regulation expression system, which includes the N-acetylglucosamine-6-phosphate sensing element or the promoter.

[0020] Furthermore, the dynamic regulation expression system also includes N-acetylglucosamine-6-phosphate.

[0021] The fourth object of the present invention is to provide a nucleic acid molecule encoding the N-acetylglucosamine-6-phosphate sensing element, promoter or dynamic regulation expression system.

[0022] The fifth object of the present invention is to provide a gene expression cassette or a recombinant plasmid carrying the nucleic acid molecule.

[0023] Furthermore, the vector backbone of the recombinant plasmid includes pACYC-Duet1.

[0024] The sixth object of the present invention is to provide a recombinant cell containing the gene expression cassette or recombinant plasmid.

[0025] Furthermore, the host cell of the recombinant cell includes a microorganism, such as bacteria or fungi.

[0026] The seventh object of the present invention is to provide the N-acetylglucosamine-6-phosphate sensing component, promoter, dynamic regulation expression system, nucleic acid molecule, gene expression frame or recombinant plasmid, and recombinant cell for use in dynamically regulating the dephosphorylation reaction of N-acetylglucosamine-6-phosphate (GlcNAc6P) or in the synthesis of acetylglucosamine.

[0027] The eighth object of the present invention is to provide a recombinant Escherichia coli, wherein the recombinant Escherichia coli contains the N-acetylglucosamine-6-phosphate sensing element.

[0028] Furthermore, the recombinant Escherichia coli also includes but is not limited to the following modifications: knocking out mannose-specific EIIAB component-related genes manXYZ, N-acetylglucosamine-6-phosphate deacetylase gene nagA, glucosamine-6-phosphate deaminase gene nagB, N-acetylglucosamine-specific EIICBA component gene nagE, L-fucose isomerase gene fucI, L-fucokinase gene fucK, lactate dehydrogenase gene ldhA and pyruvate oxidase gene poxB, and enhancing the expression of glucosamine 6-phosphate N-acetyltransferase gene GNA1 and glucosamine-6-phosphate synthase gene glmS.

[0029] Furthermore, the enhanced expression is overexpression (integrated expression in the embodiment of the present invention) of the GNA1 gene controlled by at least two T7lac promoters and the glmS gene controlled by at least one T7lac promoter.

[0030] Further, the NCBI number of the mannose-specific EIIAB component-related gene manX is 946334, the NCBI number of manY is 946332, and the NCBI number of manZ is 946342, the NCBI number of the N-acetylglucosamine-6-phosphate deacetylase gene nagA is 945289, the NCBI number of the glucosamine-6-phosphate deaminase gene nagB is 945290, the NCBI number of the N-acetylglucosamine-specific EIICBA component gene nagE is 945292, the NCBI number of the L-fucose isomerase gene fucI is 946195, the NCBI number of the L-fucokinase gene fucK is 946022, the NCBI number of the lactate dehydrogenase gene ldhA is 946315, the NCBI number of the pyruvate oxidase gene poxB is 946132, and the sequence of the glucosamine 6-phosphate N-acetyltransferase gene GNA1 is as shown in SEQ ID As shown in NO.7, the NCBI number of the glucosamine-6-phosphate synthase gene glmS is 948241.

[0031] Furthermore, the starting strains include E. coli ATCC25947 (DE3), E. coli K12

[0032] MG1655(DE3), E. coli W3110(DE3). E. coli ATCC25947(DE3) was used in the examples.

[0033] The ninth object of the present invention is to provide a method for producing acetylglucosamine, comprising the following steps: using the recombinant Escherichia coli for fermentation production.

[0034] Furthermore, the fermentation comprises the following steps: inoculating the strain into a seed culture medium for cultivation to obtain a seed solution, inoculating the seed solution into a fermentation culture medium, fermenting at a temperature of 30-37°C, a pH of 6.0-8.0, and a dissolved oxygen content of 30%-40%, adding glucose after the initial glucose is exhausted, controlling the glucose concentration to be below 1 g / L, and inducing expression after culturing to an OD of 20-30.

[0035] Furthermore, the concentration of glucose added is 50%-90% (w / v).

[0036] Furthermore, the conditions for culturing in the seed culture medium are: 30-37° C. at a rotation speed of 180-260 rpm for 6-10 hours.

[0037] Furthermore, the components of the seed culture medium include: 5-15 g / L tryptone, 1-10 g / L yeast powder, and 5-15 g / L NaCl.

[0038] Furthermore, the inoculation amount of the fermentation medium is 1%-10%.

[0039] Furthermore, the components of the fermentation medium include: 10-15 g / L tryptone, 20-30 g / L yeast powder, 10-15 g / L dipotassium hydrogen phosphate, 1-5 g / L potassium dihydrogen phosphate, 2-8 g / L glycerol, and 1-10 g / L glucose.

[0040] Beneficial effects of the present invention:

[0041] In order to optimize the GlcNAc6P sensing components, the present invention inserts the green fluorescent protein sfGFP (excitation wavelength 488nm, emission wavelength 516nm) gene and the repressor protein nagC coding gene under the control of the PnagB promoter into the plasmid pACYC-Duet1, and inserts different nagC binding sequences into the -35 region of the PnagB promoter to characterize the response range by fluorescence intensity. In addition, different nagC binding sequences in Escherichia coli are compared, and non-conserved sequences in the binding sequences are randomly mutated and high-throughput screened to obtain the mutant sequence chbM-nagC. The results of the fluorescence detection experiment show that the promoter P obtained by inserting the chbM-nagC binding sequence in the -35 region nagBM Has the largest response range. In order to further reduce P nagBM To determine the degree of leakage, the repressor protein nagC was replaced with promoters of different strengths, including P 118 The promoter most significantly reduced leaky expression without affecting the maximum expression intensity. nagBM Phosphatases under the control of BT4131 / I49Q / L129Q / G172L and P 118 The plasmid vector of the nagC gene under control was transformed into the acetylglucosamine production strain, and the acetylglucosamine conversion rate and production intensity of the recombinant strain were significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the acetylglucosamine synthesis pathway.

[0043] Figure 2 Schematic diagram of the principle of GlcNAc6P sensing components.

[0044] Figure 3 P nagB Schematic diagram of the sequence and its -35 and -10 regions.

[0045] Figure 4 P for inserting different nagC binding sequences nagB The response range of the promoter.

[0046] Figure 5 This is a conservation analysis of the nagC binding sequence.

[0047] Figure 6 The response range of different promoters controlling nagC gene expression.

[0048] Figure 7 This is the fermentation curve of the recombinant Escherichia coli producing acetylglucosamine. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0050] 1. The methods and materials involved in the present invention are as follows:

[0051] (I) Determination method of N-acetylglucosamine:

[0052] High performance liquid chromatography (HPLC) detection method: Agilent 1260, RID detector, HPX-87H column (Bio-Rad Hercules, CA), mobile phase: 5 mM H 2 S0 4 , flow rate 0.6mL / min, column temperature 35℃, injection volume 10μL.

[0053] (II) PCR method

[0054] The PCR reaction system was: 25 μL PrimeSTAR Max (purchased from Takara), 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 1 μL template DNA, and 22 μL double distilled water. The PCR amplification conditions were: 98°C pre-denaturation for 5 min; followed by 30 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 3 min, and finally 72°C for 5 min.

[0055] (III) Method for preparing competent E. coli cells

[0056] E. coli competent cells were prepared using a Competent Cell Preparation Kit (purchased from Takara). The preparation method is described in the kit instructions.

[0057] (IV) Plasmid chemical transformation of E. coli competent cells

[0058] The competent E. coli cells stored in the ultra-low temperature refrigerator were placed on ice to melt; 6 μL of PCR product was added and gently mixed, and ice bathed for 45 minutes. The centrifuge tube containing the competent cells was placed in a 42°C water bath for heat shock for 90 seconds, and the centrifuge tube was transferred to an ice bath to cool the cells for 2 minutes; 800 μL of LB culture medium was added and cultured at 37°C for 1 hour; a small amount of supernatant was removed by centrifugation, and the bacteria were resuspended, and the culture solution was spread on the LB plate containing the corresponding antibiotics, and inverted at 37°C for 10-12 hours. The colonies were observed, and a single colony was picked to verify the positive clone using colony PCR. The positive single colony was picked and cultured in LB liquid culture medium overnight, and the plasmid was extracted and sequenced for verification.

[0059] (V) Fluorescence characterization method of the response range of GlcNAc6P sensing components

[0060] The recombinant strain was inoculated into a 96-well plate containing LB liquid medium (containing 50 mg / L kanamycin) and cultured at 900 rpm and 37°C for 8 h. Then, the inoculation volume was transferred to another 96-well plate containing LB liquid medium (containing the corresponding antibiotic) at a volume of 1%, and after culturing at 900 rpm and 37°C for 4 h, a final concentration of 20 g / L acetylglucosamine was added (acetylglucosamine will be phosphorylated to GlcNAc6P during transport into the cell). After further culturing for 4 h, the cell growth (OD600) and the green fluorescent protein sfGFP fluorescence intensity (excitation wavelength 488 nm; emission wavelength 516 nm) were detected using a Cytation microplate reader (BioTek, USA). The relative fluorescence intensity is defined as the ratio of the fluorescence intensity without background fluorescence to the OD600 without background absorption. The lower limit of the fluorescence response range is defined as the fluorescence intensity when no GlcNAc is added, and the upper limit is defined as the fluorescence intensity when 20 g / L GlcNAc is added.

[0061] (VI) Fed-batch fermentation

[0062] Seed culture medium (g / L): tryptone 10, yeast powder 5, NaCl 10.

[0063] Fermentation medium (g / L): tryptone 12, yeast powder 24, dipotassium hydrogen phosphate 12.54, potassium dihydrogen phosphate 2.31, glycerol 4, glucose 5.

[0064] Sterilization conditions: 115℃, 20min.

[0065] Pick a single colony of the target strain into which the recombinant plasmid has been transferred and cultured in a 10L conical flask containing 1.5L seed culture medium, culture at 37°C and 220rpm for 9 hours, inoculate the cultured seed solution into a 30L fermentation tank containing 15L fermentation medium according to a 10% inoculation amount, and ferment at 37°C and pH=7. After the glucose is exhausted, start to add 800g / L glucose solution, maintain the glucose concentration below 1g / L, and maintain the DO value between 30% and 40%. The DO value is adjusted by appropriately adjusting the glucose flow acceleration. When the OD is 26.6, add 0.4mmol / L IPTG to induce the expression of the target gene. Continue to culture for 72 hours after induction.

[0066] 2. The sequence involved in the present invention is as follows:

[0067] Table 1

[0068]

[0069] P nagB Promoter sequence (SEQ ID NO.1):

[0070] TTACTGGCTAAACCAGAAAACTTATTTTATCATTCAAAAAATCAGGTCGGATTGACGCCTGTC

[0071] P nagBM Promoter sequence (SEQ ID NO.2):

[0072] AATAATTCGCAAAGTTAATTAAGTTACTGGCTAAACCAGAAAACTTA TTTTATCATTCAAAAAATCAGGTCGGATTGACGCCTGTC

[0073] P J23118 Promoter sequence (SEQ ID NO.3):

[0074] TTGACGGCTAGCTCAGTCCTAGGTATTGTGCTAGC

[0075] Amino acid sequence of transcription factor nagC (SEQ ID NO.4):

[0076] MTPGGQAQIGNVDLVKQLNSAAVYRLIDQYGPISRIQIAEQSQLAPASVTKITRQLIERGLIKEVDQQASTGGRRAISIVTETRNFHAIGVRLGRHDATITLFDLSSKVLAEEHYPLPERTQQTLEHALLNAIAQFIDSYQRKLRELIAISVILPGLVDPDSGKIHYMPHIQVENWGLVEALEERFKVTCFVGHDIRSLALAEHYFGASQDCEDSILVRVHRGTGAGIISNGRIFIGRNGNVGEIGHIQVEPLGERCHCGNFGCLETIAANAAIEQRVLNLLKQGYQSRVPLDDCTIKTICKAANKGDSLASEVIEYVGRHLGKTIAIAINLFNPQKIVIAGEITEADKVLLPAIESCINTQALKAFRTNLPVVRSELDHRSAIGAFALVKRAMLNGILLQHLLEN

[0077] Amino acid sequence of wild-type phosphatase (SEQ ID NO.5):

[0078] MTKALFFDIDGTLVSFETHRIPSSTIEALEAAHAKGLKIFIATGRPKAIINNLSELQDRNLIDGYITMNGAYCFVGEEVIYKSAIPQEEVKAMAAFCEKKGVPCIFVEEHNISVCQPNEMVKKIFYDFLHVNVIPTVSFEEASNKEVIQMTPFITEEEEKEVLPSIPTCEIGRWYPAFADVTAKGDTKQKGIDEIIRHFGIKLEETMSFGDGGNDISMLRHAAIGVAMGQAKEDVKAAADYVTAPIDEDGISKAMKHFGII

[0079] Amino acid sequence of phosphatase mutant BT4131 / I49Q / L129Q / G172L (SEQ ID NO.6):

[0080] MTKALFFDIDGTLVSFETHRIPSSTIEALEAAHAKGLKIFIATGRPKAQINNLSELQDRNLIDGYITMNGAYCFVGEEVIYKSAIPQEEVKAMAAFCEKKGVPCIFVEEHNISVCQPNEMVKKIFYDFQH VNVIPTVSFEEASNKEVIQMTPFITEEEEKEVLPSIPTCEILRWYPAFADVTAKGDTKQKGIDEIIRHFGIKLEETMSFGDGGNDISMLRHAAIGVAMGQAKEDVKAAADYVTAPIDEDGISKAMKHFGII

[0081] NCBI accession number of manX gene: 946334

[0082] NCBI accession number of manY gene: 946332

[0083] NCBI accession number of manZ gene: 946342

[0084] NCBI accession number of nagA gene: 945289

[0085] NCBI accession number of nagB gene: 945290

[0086] NCBI accession number of nagE gene: 945292

[0087] NCBI accession number of fucI gene: 946195

[0088] NCBI accession number of fucK gene: 946022

[0089] NCBI accession number of ldhA gene: 946315

[0090] NCBI accession number of poxB gene: 946132

[0091] Sequence of GNA1 gene (SEQ ID NO.7):

[0092] ATGAGCTTACCCGATGGATTTTATAAGGCGAATGGAAGAGGGGGATTTGGAACAGGTCACTGAGACGCTAAAGGTTTTGACCACCGTGGGCACTATTACCCCCGAATCCTTCAGCAAACTCATAAAATACTGGAATGAAGCCACAGTATGGAATGATAACGAAGATAAAAAAATAATGCAATATAACCCCATGGTGATTGTGGACAAGCGCACCGAGACGGTTGCCGCTACGGGGAAT ATCATCATCGAAAGAAAGATCATTCATGAACTGGGGCTATGTGGCCACATCGAGGACATTGCAGTAAACTCCAAGTATCAGGGCCAAGGTTTGGGCAAGCTCTTGATTGATCAATTGGTAACTATCGGCTTTGACTACGGTTGTTATAAGATTATTTTAGATTGCGATGAGAAAAATGTCAAATTCTATGAAAAATGTGGGTTTAGCAACGCAGGCGTGGAAATGCAAATTAGAAAATAG

[0093] NCBI accession number of glmS gene: 948241

[0094] Example 1 Construction and verification of GlcNAc6P sensing components

[0095] GlcNAc6P sensing device principle Figure 2 As shown. nagC is a transcription factor that responds to GlcNAc6P. When the concentration of GlcNAc6P in the environment is low, nagC binds to a specific DNA sequence, preventing the binding of RNA polymerase and inhibiting the transcription of downstream genes. When the concentration of GlcNAc6P in the environment increases, GlcNAc6P binds to the nagC protein, causing the structure of nagC to change, thereby eliminating its DNA binding activity, allowing RNA polymerase to bind to the promoter and initiate the transcription of downstream genes. In addition, nagC not only regulates the nagABE gene, but also has nagC binding sequences of varying strengths in the promoter regions of gene clusters such as manXYZ, chb, and glmUS.

[0096] Based on the GlcNAc6P-responsive properties of nagC and combined with promoter engineering strategies, GlcNAc6P-sensing components were constructed. nagBThe -35 region upstream of the promoter ( Figure 3 ), and obtained hybrid promoters P with different dynamic response ranges nagB1 , P nagB2 , P nagB3 ( Figure 4 The above promoters were placed upstream of the sfGFP gene and transformed into Escherichia coli DH5α using the pACYC-Duet1 vector for verification, and their response range was evaluated by fluorescence intensity. nagB1 , P nagB2 , and P nagB3 The response ranges were 9602-31944, 16486-56155 and 7992-31040, respectively, including the P of the chb-nagC binding sequence. nagB3 It has a lower leakage level and has a significant response after the addition of GlcNAc.

[0097] In-depth analysis of multiple nagC binding sequences revealed that they have some conserved features. First, most sequences are rich in adenine (A) and thymine (T), which are typical features of prokaryotic promoters and regulatory regions. Regions with high AT content are prone to localized unwinding of the DNA double helix, which may promote the binding of transcription factors to DNA. Second, there is a conserved "TTAATT" sequence in multiple sequences, indicating that this motif may be the core sequence recognized or bound by nagC ( Figure 5 ). In addition, there are inverted repeats and palindromic structures in the sequence, such as "GCGA...TCGC". These structures may form secondary structures, such as stem loops, to enhance the interaction between nagC and DNA. Inverted repeat sequences are usually hot spots for protein binding, which help to regulate the recognition and binding of proteins. Although the core motif is conserved, there is diversity in other regions. This diversity may affect the binding affinity and specificity of nagC to different promoters, thereby achieving differentiated fine regulation of the expression of different genes. We selected non-conserved regions and randomly mutated them using degenerate primers to insert P nagB -sfGFP pACYC-Duet1 plasmid as a template, using degenerate primers (5'-3')

[0098] TGCTGCCAACTTACTGATTTAATAATTNNNNNNTTAATTAAGTTACTGGCTAAACCAGAAAACTTATTTTATCATTCAAAAAA and primer (5'-3')AAATCAGTAAGTTGGCAGCATCACC, PCR amplification, the linearized vector was transformed into Escherichia coli DH5α for cyclization, coated with LB plates containing chloramphenicol and cultured for 8-12 hours, and the grown colonies were inoculated into two 96-well plates containing LB liquid culture medium for culture, one of which was added with GlcNAc at a final concentration of 20g / L after 4 hours of culture, and the fluorescence intensity was detected after 6 hours of continuous culture, and the strain with the largest range of fluorescence intensity change was selected for gene sequencing. Finally, P was obtained. nagBM promoter, with a response range of 8379-58228, compared to the P containing the wild-type chb-nagC binding sequence nagB3 Significantly increased ( Figure 4 ). nagBM The mutant nagC binding sequence contained in chbM is shown in Table 1.

[0099] Example 2 Optimization of the response range of the GlcNAc6P sensing element

[0100] P nagBM Although the promoter has the most significant response range, it still has a high degree of leakage expression when GlcNAc is not added, so the leakage degree can be reduced by strengthening the expression intensity of the repressor protein nagC. lac , P J23110 (P 110 ), P J23114 (P 114 ), P J23118 (P 118 ), P AMP , P lacUV5 , P nagBM The promoters were placed upstream of the repressor protein nagC gene, P nagBM The promoter was placed upstream of the sfGFP gene, the two expression frames were connected, and the expression was verified by transforming into Escherichia coli DH5α through the pACYC-Duet1 vector. The results showed that the promoter P J23118 Expression of nagC makes P nagBM The response range of the promoter was further expanded to 295-54986, and the leakage strength was significantly improved ( Figure 6 , where the control group CK was a strain without nagC expression).

[0101] Example 3 GlcNAc6P sensing components dynamically regulate phosphatase expression

[0102] PnagBM Phosphatase mutant BT4131 / I49Q / L129Q / G172L under promoter control and P 118 The nagC under the control of the promoter was inserted into the plasmid pACYC-Duet1 to obtain a recombinant plasmid.

[0103] The above recombinant plasmid was transformed into the strain E. coli BL21 (DE3)-ΔmanXYZ-ΔnagABE-ΔfucIK-ΔldhA-ΔpoxB-GNA1-glmS (manXYZ gene, nagABE gene, fucIK gene, ldhA gene and poxB gene were knocked out, and the exogenous GNA1 gene was integrated and the glmS gene expression was enhanced) (the specific construction method of the host bacteria is shown in patent CN202210116458.8) for fermentation verification ( Figure 7 ). After 60 hours of fermentation, the GlcNAc production increased to 224.3g / L, which was 10.5% higher than the control strain (T7 promoter constitutively expressing the phosphatase mutant BT4131 / I49Q / L129Q / G172L), the production intensity reached 3.74g / L / h (36.7% higher than the control strain), and the conversion rate was 0.64g / g glucose (8.9% higher than the control strain). By integrating the GlcNAc6P sensing component, a "switch" was installed for the phosphatase. When GlcNAc6P is not accumulated, it remains in the off state; when GlcNAc6P accumulates, the expression is activated and increases with the increase in the accumulation level. This more effectively reduces the impact of phosphatase expression on precursors such as Glc6P.

[0104] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. An N-acetylglucosamine-6-phosphate sensing component, characterized in that: include: a phosphatase whose expression is regulated by a first promoter; the transcription factor nagC, whose expression is regulated by the second promoter; The nucleotide sequence of the first promoter is shown in SEQ ID NO.2, and the nucleotide sequence of the second promoter is P J23118 Promoter.

2. The N-acetylglucosamine-6-phosphate sensing component according to claim 1, characterized in that: Contain at least one of the following characteristics: (1) The phosphatase comprises acetylglucosamine-6-phosphate phosphatase; (2) The phosphatase comprises the amino acid sequence shown in SEQ ID NO.5 or a mutant thereof; Preferably, the mutant includes any one of the following: Mutate leucine at position 129 to glutamine, or Mutation of isoleucine at position 49 to glutamine and leucine at position 129 to glutamine, or The isoleucine at position 49 was mutated to glutamine, the leucine at position 129 was mutated to glutamine, and the glycine at position 172 was mutated to leucine; (3) P J23118 The sequence of the promoter is shown in SEQ ID NO.3; (4) The amino acid sequence of the transcription factor nagC is shown in SEQ ID NO.

4.

3. A promoter, characterized in that The sequence of the promoter is shown in SEQ ID NO.

2.

4. A dynamic regulation expression system, characterized in that: The dynamic regulation expression system comprises the N-acetylglucosamine-6-phosphate sensing element according to claim 1 or 2 or the promoter according to claim 3.

5. A nucleic acid molecule encoding the N-acetylglucosamine-6-phosphate sensing element according to claim 1 or 2, the promoter according to claim 3, or the dynamic regulation expression system according to claim 4.

6. A gene expression cassette or recombinant plasmid carrying the nucleic acid molecule of claim 5.

7. A recombinant cell containing the gene expression cassette or recombinant plasmid according to claim 6.

8. Use of the N-acetylglucosamine-6-phosphate sensing component of claim 1 or 2, the promoter of claim 3, the dynamic regulation expression system of claim 4, the nucleic acid molecule of claim 5, the gene expression cassette or recombinant plasmid of claim 6, and the recombinant cell of claim 7 in dynamically regulating the dephosphorylation reaction of N-acetylglucosamine-6-phosphate or in the synthesis of acetylglucosamine.

9. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli contains the N-acetylglucosamine-6-phosphate sensing component according to claim 1 or 2.

10. The recombinant Escherichia coli according to claim 9, characterized in that The recombinant Escherichia coli also includes the following modifications: knocking out mannose-specific EIIAB component-related genes manXYZ, N-acetylglucosamine-6-phosphate deacetylase gene nagA, glucosamine-6-phosphate deaminase gene nagB, N-acetylglucosamine-specific EIICBA component gene nagE, L-fucose isomerase gene fucI, L-fucokinase gene fucK, lactate dehydrogenase gene ldhA and pyruvate oxidase gene poxB, and enhancing the expression of glucosamine 6-phosphate N-acetyltransferase gene GNA1 and glucosamine-6-phosphate synthase gene glmS.

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