Method for producing N-acetylneuraminic acid based on artificial double carbon sources

Abstract

The invention discloses a production method of N-acetylneuraminic acid based on artificial double carbon sources, and belongs to the technical field of genetic engineering. Escherichia coli with high yield of N-acetylneuraminic acid as a carbon source is taken as an original strain, 6-phosphofructokinase is knocked out, glycerol kinase is knocked out, a glycerol kinase mutant is introduced, PTS phosphotransferase I is replaced by glucose-promoted diffusion transporter, and a promoter is optimized, so that the N-acetylneuraminic acid with high yield is obtained. The recombinant escherichia coli can grow with glucose and glycerol as double carbon sources, N-acetylneuraminic acid is synthesized, the yield under the pore plate level reaches 9.2 g / L, meanwhile, the generation amount of acetic acid is extremely low, and a new direction is provided for large-scale production of N-acetylneuraminic acid.

Description

Technical Field

[0001] The invention relates to a method for producing N-acetylneuraminic acid based on an artificial double-carbon source, and belongs to the technical field of genetic engineering. Background Art

[0002] N-acetylneuraminic acid (NeuAc), as a precursor for the synthesis of other sialic acids, is the most critical and important functional sugar among many sialic acids. NeuAc has broad application prospects and is an important component of gangliosides, which is essential for the development and function of the nervous system; it can be used as a drug precursor for the synthesis of zanamivir and oseltamivir, which are important inhibitors against influenza viruses A and B; it also plays an important role in cancer targeted therapy, reproduction and immunity, whitening, anti-inflammation, etc.

[0003] Escherichia coli is simple to culture, easy to operate, and has complete genetic manipulation tools. It is a model chassis microorganism that is widely used in metabolic engineering to produce chemicals. At present, glycerol or glucose is mainly used as a carbon source for the production of N-acetylneuraminic acid in Escherichia coli. According to current reports, the production of NeuAc with glycerol as a carbon source is high, but the utilization rate of glycerol is relatively slow, the energy conversion efficiency and metabolic efficiency are low, and the viscosity is high, which is very unfavorable for large-scale industrial applications. Glucose is the preferred carbon source in the industrial fermentation process. The use of glucose can make the strain have a faster growth rate and higher biomass accumulation; the glucose metabolic pathway is thoroughly studied and usually provides higher energy efficiency (ATP generation) and precursor substances; glucose is also a standard carbon source used for a long time. Many fermentation processes have optimized its use conditions, which is conducive to the industrial production and application of products. However, glucose is easily metabolized and overflowed during microbial fermentation to produce acetic acid. There are disadvantages in using both as single carbon sources to produce NeuAc. Therefore, if a method can be found to produce N-acetylneuraminic acid with a composite carbon source while reducing acetic acid production, it may be possible to further increase the N-acetylneuraminic acid production of Escherichia coli. Summary of the invention

[0004] In order to solve the above problems, the present invention knocks out the 6-phosphofructokinase encoding gene pfkA through combined transformation, replaces the glycerol kinase GlpK in situ with the glycerol kinase mutant GlpK22, knocks out the PTS phosphotransferase I ptsI gene and overexpresses the glucose-facilitated diffusion transporter gene glf from Pseudomonas motilus that has been codon-optimized, and also combines promoter optimization, so as to achieve the efficient production of N-acetylneuraminic acid by Escherichia coli using artificial dual carbon sources, namely glucose and glycerol, while greatly reducing the generation of byproduct acetic acid.

[0005] The first object of the present invention is to provide a method for producing N-acetylneuraminic acid, comprising the steps of fermentation production using recombinant Escherichia coli, wherein:

[0006] The recombinant Escherichia coli comprises the following modifications: knocking out the 6-phosphofructokinase encoding gene (pfkA) and the glycerol kinase encoding gene (GlpK), overexpressing the glycerol kinase mutant encoding gene (GlpK22), knocking out the PTS phosphotransferase I encoding gene (ptsI) and integrating and expressing the glucose facilitated diffusion transporter encoding gene (glf) at the site, wherein the glucose facilitated diffusion transporter encoding gene is expressed by a promoter with a nucleotide sequence as shown in SEQ ID NO.10, and the amino acid sequence of the glycerol kinase mutant is shown in SEQ ID NO.1;

[0007] The fermentation production comprises fermentation with a composite carbon source, wherein the composite carbon source contains glucose and glycerol.

[0008] Furthermore, the host bacteria of the recombinant Escherichia coli include Escherichia coli

[0009] BL21(DE3)ΔcheW::P6-glmSA,overexpression ofneuC gene in the temminator locus ofpspG gene under the control ofpromoter Pl,ΔmotA::P1-glmM-P4-glmUglmSA,overexpression ofNemneuB gene in the terminator locus ofyahOgene under the control ofpromoter Pl,ΔwecBΔmanAΔpykA,ΔnagΔnanATEΔmanXYZ.

[0010] Furthermore, the GeneID of the 6-phosphofructokinase encoding gene is 948412; the GeneID of the glycerol kinase encoding gene is 948423; the nucleotide sequence of the glycerol kinase mutant encoding gene is shown in SEQ ID NO.2; the Gene ID of the PTS phosphotransferase I encoding gene is 946879; the amino acid sequence of the glucose facilitated diffusion transporter is shown in SEQ ID NO.3; and the nucleotide sequence of the glucose facilitated diffusion transporter encoding gene is shown in SEQ ID NO.4.

[0011] Furthermore, the overexpression is integrated expression and / or free expression. Preferably, the glycerol kinase encoding gene is knocked out and the glycerol kinase mutant encoding gene is integrated and expressed at the site; the promoter used is the glpk in situ promoter.

[0012] Furthermore, in the composite carbon source, the mass ratio of glycerol to glucose is 1:(8-16).

[0013] Furthermore, the fermentation medium used in the fermentation production contains: 20-40 g / L complex carbon source, 3-9 g / L urea, 1-6 mg / L zinc sulfate heptahydrate, 0.01-1 g / L manganese sulfate monohydrate, 1-10 g / L iron sulfate heptahydrate, 0.01-1 g / L copper sulfate pentahydrate, 0.01-1 g / L cobalt chloride hexahydrate, 1-10 g / L yeast powder, 1-5 g / L tryptone, 1-10 g / L potassium dihydrogen phosphate, 1-10 g / L dipotassium hydrogen phosphate trihydrate, 1-10 g / L citric acid monohydrate, 1-10 g / L magnesium sulfate heptahydrate, and 1-10 g / L ammonium sulfate.

[0014] Furthermore, the fermentation production includes culturing at 35-37° C. and 180-280 rpm.

[0015] The second object of the present invention is to provide a recombinant Escherichia coli, which comprises the following modifications: knocking out the 6-phosphofructokinase encoding gene (pfkA) and the glycerol kinase encoding gene (GlpK), overexpressing the glycerol kinase mutant encoding gene (GlpK22), knocking out the PTS phosphotransferase I encoding gene (ptsI) and integrating and expressing the glucose facilitated diffusion transporter encoding gene (glf) at the site, wherein the glucose facilitated diffusion transporter encoding gene is expressed by a promoter with a nucleotide sequence as shown in SEQ ID NO.10, and the amino acid sequence of the glycerol kinase mutant is shown in SEQ ID NO.1,

[0016] The host bacteria of the recombinant Escherichia coli include Escherichia coli

[0017] BL21(DE3)ΔcheW::P6-glmSA,overexpression ofneuC gene in the temminator locus ofpspG gene under the control ofpromoter Pl,ΔmotA::P1-glmM-P4-glmUglmSA,overexpression ofNemneuB gene in the terminator locus ofyahOgene under the control ofpromoter Pl,ΔwecBΔmanAΔpykA,ΔnagΔnanATEΔmanXYZ.

[0018] The third object of the present invention is to provide a method for constructing the recombinant Escherichia coli, comprising the following steps:

[0019] Using Escherichia coli BL21 (DE3) ΔcheW::P6-glmSA, overexpression of neuC gene in the terminator locus of pspG gene under the control of promoter Pl, ΔmotA::P1-glmM-P4-glmUglmSA, overexpression of NemneuB gene in the terminator locus of yahO gene under the control of promoter Pl, ΔwecBΔmanAΔpykA, ΔnagΔnanATEΔmanXYZ as the host bacteria, the 6-phosphofructokinase encoding gene (pfkA) was knocked out, the glycerol kinase encoding gene (GlpK) was knocked out and the glycerol kinase mutant encoding gene (GlpK22) with the amino acid sequence shown in SEQ ID NO.1 was integrated into the site by homologous recombination, the PTS phosphotransferase I encoding gene (ptsI) was knocked out and the glucose facilitated diffusion transporter encoding gene (glf) containing the promoter shown in SEQ ID NO.10 was integrated into the site by homologous recombination.

[0020] The fourth object of the present invention is to provide the use of the recombinant Escherichia coli in the preparation of N-acetylneuraminic acid or its derivatives.

[0021] Furthermore, derivative products include but are not limited to medicines or health products.

[0022] Beneficial effects of the present invention:

[0023] The present invention inhibits the glycolysis pathway by knocking out the 6-phosphofructokinase encoding gene pfkA, replaces the original glycerol kinase with a glycerol kinase mutant to relieve the inhibitory effect of glucose carbon source metabolic decomposition, replaces the PTS glucose transport system with a glucose-facilitated diffusion transporter, increases PEP accumulation, enhances the strain's ability to absorb and utilize glucose and the ability to utilize glycerol in late fermentation, and reduces the generation of overflow metabolic byproduct acetic acid. Furthermore, the present invention reasonably regulates the integrated expression of the glucose-facilitated diffusion transporter and optimizes the carbon source ratio of glycerol and glucose through a promoter, so that the N-acetylneuraminic acid yield of the recombinant Escherichia coli is increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the dual-carbon source metabolism flow chart of N-acetylneuraminic acid.

[0025] Figure 2 Comparison of fermentation yield and acetic acid production of different engineered strains.

[0026] Figure 3 Optimize results for carbon sources.

[0027] Figure 4 It is a complex carbon source fermentation process.

[0028] Figure 5 These are the fermentation results of different genetically engineered strains in the comparative example using glucose as the sole carbon source. DETAILED DESCRIPTION

[0029] 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.

[0030] 1. The genes and sequences involved in the present invention are as follows:

[0031] The amino acid sequence of the glycerol kinase mutant GlpK22 is shown in SEQ ID NO.1, encoding GlpK22

[0032] The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0033] The amino acid sequence of the glucose facilitated diffusion transporter Glf is shown in SEQ ID NO.3, and the codon-optimized nucleotide sequence encoding the Glf gene is shown in SEQ ID NO.4.

[0034] The P1 promoter nucleotide sequence is shown in SEQ ID NO.5; the P2 promoter nucleotide sequence is shown in SEQ ID NO.6; the P3 promoter nucleotide sequence is shown in SEQ ID NO.7; the P4 promoter nucleotide sequence is shown in SEQ ID NO.8; the P5 promoter nucleotide sequence is shown in SEQ ID NO.9; and the P6 promoter nucleotide sequence is shown in SEQ ID NO.10.

[0035] The specific sequence information is as follows:

[0036] SEQ ID NO.1: GlpK22 amino acid sequence

[0037] MTEKKYIVALDQGTTSSRAVVMDHDANIISVSQREFEQIYPKPGWVEHDPMEIWATQSSTLVEVLAKADISSDQIAAIGITNQRETTIVWEKETGKPIYNA IVWQCRRTAEICEHLKRDGLEDYIRSNTGLVIDPYFSGTKVKWILDHVEGSRERARRGELLFGTVDTWLIWKMTQGRVHVTDYTNASRTMLFNIHTLDWDDKMLEVLDIPREMLPEVRRSSEVYGQTNIGGKGGTRIPISGIAGDQQAALFGQLCVKEGMAKNTYGTGCFMLMNTGEKAVKSENGLLTTIACGPTGEVNY ALESAVFMAGASIQWLRDEMKLINDAYDSEYFATKVQNTNGVYVVPAFTGLGAPYWDPYARGAIFGLTRGVNANHIIRATLESIAYQTRDVLEAMQADSGIRLHALRVDGGAVANNFLMQFQSDILGTRVERPEVREVTALGAAYLAGLAVGFWQNLDELQEKAVIEREFRPGIETTERNYRYAGWKKAVKRAMAWEEHDE

[0038] SEQ ID NO.2: GlpK22 nucleotide sequence

[0039] ATGACTGAAAAAAAATATATCGTTGCGCTCGACCAGGGCACCACCAGCTCCCGCGCGGTCGTAATGGATCACGATGCCAATATCATTAGCGTGTCGCAGCGCGAATTTGAGCAAATCTACCCAAAACCAGGTTGGGTAGAACACGACCCAATGGAAATCTGGGCCACCCAAAGCTCCACGCTGGTAGAAGTGCTGGCGAAAGCCGATATCAGTTCCGATCAAATTGCAGCTATCGGTATTACGAACCAGCGTGAAACCACTATTGTCTGGGAAAAAGAAACCGGCAAGCCTATCTATAACGCCATTGTCTGGCAGTGCCGTCGTACCGCAGAAATCTGCGAGCATTTAAAACGTGACGGTTTAGAAGATTATATCCGCAGCAATACCGGTCTGGTGATTGACCCGTACTTTTCTGGCACCAAAGTGAAGTGGATCCTCGACCATGTGGAAGGCTCTCGCGAGCGTGCACGTCGTGGTGAATTGCTGTTTGGTACGGTTGATACGTGGCTTATCTGGAAAATGACTCAGGGCCGTGTCCATGTGACCGATTACACCAACGCCTCTCGTACCATGTTGTTCAACATCCATACCCTGGACTGGGACGACAAAATGCTGGAAGTGCTGGATATTCCGCGCGAGATGCTGCCAGAAGTGCGTCGTTCTTCCGAAGTATACGGTCAGACTAACATTGGCGGCAAAGGCGGCACGCGTATTCCAATCTCCGGGATCGCCGGTGACCAGCAGGCCGCGCTGTTTGGTCAGTTGTGCGTGAAAGAAGGGATGGCGAAGAACACCTATGGCACTGGCTGCTTTATGCTGATGAACACTGGCGAGAAAGCGGTGAAATCAGAAAACGGCCTGCTGACCACCATCGCCTGCGGCCCGACTGGCGAAGTGAACTATGCGTTGGAAAGTGCGGTGTTTATGGCAGGCGCATCCATTCAGTGGCTGCGCGATGAAATGAAGTTGATTAACGACGCCTACGATTCCGAATATTTCGCCACCAAAGTGCAAAACACCAATGGTGTGTATGTGGTTCCGGCATTTACCGGGCTGGGTGCGCCGTACTGGGACCCGTATGCGCGCGGGGCGATTTTCGGTCTGACTCGTGGGGTGAACGCTAACCACATTATACGCGCGACGCTGGAGTCTATTGCTTATCAGACGCGTGACGTGCTGGAAGCGATGCAGGCCGACTCTGGTATCCGTCTGCACGCCCTGCGCGTGGATGGTGGCGCAGTAGCAAACAATTTCCTGATGCAGTTCCAGTCCGATATTCTCGGCACCCGCGTTGAGCGCCCGGAAGTGCGCGAAGTCACCGCATTGGGTGCGGCCTATCTCGCAGGCCTGGCGGTTGGCTTCTGGCAGAACCTCGACGAGCTGCAAGAGAAAGCGGTGATTGAGCGCGAGTTCCGTCCAGGCATCGAAACCACTGAGCGTAATTACCGTTACGCAGGCTGGAAAAAAGCGGTTAAACGCGCGATGGCGTGGGAAGAACACGACGAA

[0040] SEQ ID NO.3: Glf amino acid sequence

[0041] MSSESSQGLVTRLALIAAIGGLLFGYDSAVIAAIGTPVDIHFIAPRHLSATAAASLSGMVVVAVLVGCVTGSLLSGWIGIRFGRRGGLLMSSICFVAAGFGAALTEKLFGTGGSALQIFCFFRFLAGLGIGVVSTLTPTYIAEIAPPDKRGQMVSGQQMAIVTGALTGYIFTWLLAHFGSIDWVNASGWCWSPASEGLIGIAFLLLLLTAPDTPHWLVMKGRHSEASKILARLEPQADPNLTIQKIKAGFDKAMDKSSAGLFAFGITVVFAGVSVAAFQQLVGINAVLYYAPQMFQNLGFGADTALLQTISIGVVNFIFTMIASRVVDRFGRKPLLIWGALGMAAMMAVLGCCFWFKVGGVLPLASVLLYIAVFGMSWGPVCWVVLSEMFPSSIKGAAMPIAVTGQWLANILVNFLFKVADGSPALNQTFNHGFSYLVFAALSILGGLIVARFVPETKGRSLDEIEEMWRSQK

[0042] SEQ ID NO.4: Glf nucleotide sequence

[0043]

[0044] SEQ ID NO.5: P1 promoter nucleotide sequence

[0045] TTTACGGCTAGCTCAGTCCTAGGTACTATGCTAGC

[0046] SEQ ID NO.6: P2 promoter nucleotide sequence

[0047] TTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGC

[0048] SEQ ID NO.7: P3 promoter nucleotide sequence

[0049] TTGACAGCTAGCTCAGTCCTAGGTACTGTGCTAGC

[0050] SEQ ID NO.8: P4 promoter nucleotide sequence

[0051] GATTGATGACAATGTGAGTGCTTCCCTTGAAACCCTGAAACTGATC CCCATAATAAGCGAAGTTAGCGAGATGAATGCGAAAAAAACG

[0052] SEQ ID NO.9: P5 promoter nucleotide sequence

[0053] TTGAGGAATCATAGAATTTTTAATTTAAATTTTATTTGACAAAAATGG GCTCGTGTTGTATAATCTAAGCTAGTGTATTT

[0054] SEQ ID NO.10: P6 promoter nucleotide sequence

[0055] CGTTACAGGAAAAGCCAAAGCTGAATCGATTTTATGATTTGGGTTCAATTCTTCCTTTAGCGGCATAATGTTTAATGACGTACGAAACGTCAGCGGTCAACACC

[0056] 2. The materials and methods of the present invention are as follows:

[0057] Starting strain NBC45: recorded in "Metabolic Engineering of Escherichia coli for Increased Bioproduction of N-Acetylneuraminic Acid", the genotype is Escherichia coli BL21 (DE3) ΔcheW::P6-glmSA, overexpression ofneuC gene in the temminator locus ofpspG gene under the control of promoter Pl, ΔmotA::P1-glmM-P4-glmUglmSA, overexpression ofNemneuB gene in the terminator locus ofyahO gene under the control of promoter Pl, ΔwecBΔmanAΔpykA, ΔnagΔnanATEΔmanXYZ.

[0058] The formula of seed liquid culture medium is: 10g / L tryptone, 10g / L sodium chloride, 5g / L yeast powder.

[0059] The fermentation medium formula is: 30g / L carbon source (glycerol and glucose), 6g / L urea, 3.8mg / L zinc sulfate heptahydrate, 0.33g / L manganese sulfate monohydrate, 5g / L ferric sulfate heptahydrate, 0.1g / L copper sulfate pentahydrate, 0.1g / L cobalt chloride hexahydrate, 4.8g / L yeast powder, 2.4g / L tryptone, 5.336g / L potassium dihydrogen phosphate, 3.284g / L potassium hydrogen phosphate trihydrate, 2.84g / L citric acid monohydrate, 2g / L magnesium sulfate heptahydrate, 4g / L ammonium sulfate, and the pH is adjusted to 7.

[0060] Culture conditions: The recombinant E. coli was inoculated into LB medium, cultured at 37°C and 220 rpm for 12-14 h to obtain seed solution, and then transferred to fermentation medium at an inoculum of 1-2% for fermentation, and reacted at 37°C and 220 rpm for 48 h.

[0061] Sample detection method: N-acetylneuraminic acid was detected by Waters liquid chromatography, the chromatographic column was Aminex HPX-87H column (300×7.8mm), the absorption peak was detected at UV 210nm, the mobile phase was 10mM sulfuric acid, the flow rate was 0.5mL / min, and the elution time of N-acetylneuraminic acid was about 9.8 minutes.

[0062] Example 1 Construction of recombinant strains

[0063] (1) Knockout of pfkA and glpK

[0064] The CRISPER / Cpf1 gene editing technology was used to knock out the 6-phosphofructokinase encoding gene pfkA (GeneID: 948412) and the glycerol kinase encoding gene glpK (GeneID: 948423) in the genome of the starting Escherichia coli strain. The N23 sequence of pfkA is

[0065] TGACATGATCAACCGTGGCGGTA, the N23 sequence of glpK is

[0066] TACGCGACAACCTG CTTCAGGAT. First, the Cpf1 plasmid containing the Cas12 cleavage protein was transformed into the Escherichia coli host bacteria by chemical transformation. Using Escherichia coli BL21 (DE3) as a template, PCR amplified the upstream and downstream fragments of the knockout site, each about 800 bp. After the fragments were obtained by PCR amplification, they were connected to the targeted cutting plasmid pcrEG vector in the CRISPER / Cpf1 system through the Gibson assembly kit. After the plasmid was constructed and sequenced, Escherichia coli competent cells containing Cpf1 were made to achieve the knockout of the target gene. After transformation, the transformants were spread on the resistant plate, and single colonies were picked for colony PCR verification of positive transformants and sequencing. The strain with pfkA knocked out was named DC1, and the strain with glpK knocked out on the basis of DC1 was named DC2.

[0067] (2) Construction of genomic recombinant integration glpK22 fragment

[0068] A glycerol kinase mutant enzyme GlpK22 (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) was selected, and the integration site of glpK22 was selected at the position where glpK was located.

[0069] Using Escherichia coli BL21 (DE3) as a template, primers BL-glpK-3F and BL-glpK-3R were designed to amplify the recombinant integrated NeuC left arm gene and the first half of the glpK mutation fragment.

[0070] BL-glpK-3F:GTTAGCTTGGCCAATCGTTACCGTCAGTTCGCTCAAC

[0071] BL-glpK-3R:TATGCGTTGGAAAGTGCGGTGTT

[0072] Using Escherichia coli BL21 (DE3) as a template, primers PCREG-glpK-4F-2k and BL-glpK-4R-1.5k were designed to amplify the recombinant integrated NeuC right arm gene fragment and the second half of the glpK mutation fragment.

[0073] BL-glpK-4F:ACACCGCACTTTCCAACGCATAG

[0074] BL-glpK-4R:TCGACTTCGAGCAGACTCATCACATTG

[0075] The amplified glpK22 left arm gene fragment, the glpK mutation front half fragment, the glpK mutation second half fragment, and the right arm fragment were constructed by fusion PCR technology to obtain a glpK22 gene fragment with introduced mutations.

[0076] (3) Construction of genomic recombinant integrated glf fragment

[0077] According to the glucose-facilitated diffusion transporter gene glf of Pseudomonas mobilis (amino acid sequence as shown in SEQ ID NO.3), a nucleotide sequence encoding the gene glf after codon optimization was synthesized (as shown in SEQ ID NO.4), and the integration site of glf was selected at the location of the PTS phosphotransferase I ptsI gene (Gene ID: 946879) and the end of the dadX gene (Gene ID: 945754) for knock-in integration.

[0078] Using Escherichia coli BL21 (DE3) as a template, primers BL-glf-ptsI-3F and BL-glf-ptsI-3R were designed to amplify and recombinantly integrate the glf left arm gene.

[0079] BL-glf-ptsI-3F:gttAGCTTGGCCattgattcagcctgtcggaactgg

[0080] BL-glf-ptsI-3R:aaccctaccttacttgtgactgatttttaaaagaac

[0081] Using Escherichia coli BL21 (DE3) as a template, primers BL-glf-ptsI-4F and BL-glf-ptsI-4R were designed to amplify the recombinant integrated glf right arm gene fragment.

[0082] BL-glf-ptsI-4F:GCCAGAAGTAATAAtccacgagatgcggcccaattta

[0083] BL-glf-ptsI-4R:agatctaaccagcagcatgagagcgatgaattg

[0084] The glf gene fragment with a nucleotide sequence as shown in SEQ ID NO.4 and the P1 promoter fragment with a nucleotide sequence as shown in SEQ ID NO.5 were synthesized.

[0085] The amplified glf left arm gene fragment, glf right arm gene fragment, P1 promoter fragment and glf gene were constructed and integrated by fusion PCR technology to obtain the recombinant integrated glf gene fragment P1-ptsI-glf.

[0086] Using Escherichia coli BL21 (DE3) as a template, primers BL-glf-dadX-3F and BL-glf-dadX-3R were designed to amplify and recombinantly integrate the left arm gene of glf-dadX.

[0087] BL-glf-dadX-3F:ggcattattttgtatggcgcttcgc

[0088] BL-glf-dadX-3R:gcgtaagcgttttatgtagaacgcc

[0089] Using Escherichia coli BL21 (DE3) as a template, primers BL-glf-dadX-4F and BL-glf-dadX-4R were designed to amplify and recombinantly integrate the glf-dadX right arm gene fragment.

[0090] BL-glf-dadX-4F:cacgctttacccttgatggagcct

[0091] BL-glf-dadX-4R:cgcgtattgcggcactgtttcgt

[0092] The synthetic nucleotide sequences are as shown in SEQ ID NO.4 for the glf gene fragment and SEQ ID NO.5 for the P1 promoter fragment.

[0093] The amplified glf-dadX left arm gene fragment, glf-dadX right arm gene fragment, P1 promoter fragment and glf gene were constructed and integrated by fusion PCR technology to obtain the recombinant integrated glf gene fragment P1-dadX-glf.

[0094] (4) Gene integration

[0095] The fusion fragment glpK22 constructed in step (2) was connected to the targeted cutting plasmid pcrEG vector containing the N23 recognition sequence (gggactaccggatgcggcataaa) in the CRISPER / Cpf1 system through the Gibson assembly kit, and then the constructed plasmid was transformed into the Escherichia coli DC2 competent cells constructed in step (1) containing the Cpf1 plasmid by chemical transformation to obtain a recombinant strain in which glpK22 was integrated at Δglpk, which was named DC3 after verification.

[0096] The fusion fragment P1-ptsI-glf constructed in step (3) was connected to the targeted cutting plasmid pcrEG vector containing the N23 recognition sequence (acgcagtacactctggcagttga) in the CRISPER / Cpf1 system through the Gibson assembly kit, and then the constructed plasmid was transformed into wild-type Escherichia coli BL21 (DE3) competent cells and Escherichia coli DC1, DC3 competent cells by chemical transformation to obtain a recombinant strain in which P1-ptsI-glf was integrated at ΔptsI. The transformants were taken for colony PCR verification, and the strains after successful integration were named DC4, DC5 and DC6-1 respectively. Similarly, after P1-dadX-glf was connected to the targeted cutting plasmid pcrEG vector containing the N23 recognition sequence (tctgaagtcgagttcgagtcaac) in the CRISPER / Cpf1 system, the constructed plasmid was transformed into DC3 competent cells using the chemical transformation method to obtain a recombinant strain with P1-dadX-glf integrated at the end of dadX, which was named DC7-1 after verification.

[0097] Example 2: Construction of Escherichia coli with glf gene regulated by constitutive promoters of different strengths

[0098] The promoter fragments P2 to P6 shown in SEQ ID NOs. 6 to 10 were synthesized.

[0099] The glf left arm gene fragment, glf right arm fragment, and glf gene obtained in step (3) of Example 1 were fused with the synthetic promoter fragment to obtain Promoter2-6-ptsI-glf fusion fragment.

[0100] The constructed Promoter2~6-ptsI-glf fusion fragment was connected to the targeted cutting plasmid pcrEG vector containing the N23 recognition sequence (acgcagtacactctggcagttga) in the CRISPER / Cpf1 system using the Gibson assembly kit, and then the constructed plasmid was transformed into DC3 using the chemical transformation method to obtain DC6-2~DC6-6.

[0101] The glf-dadX left arm gene fragment, glf-dadX right arm fragment, and glf gene obtained in step (3) of Example 1 were fused with the synthetic promoter fragment to obtain Promoter2~6-dadX-glf fusion fragments. The constructed Promoter2~6-dadX-glf fusion fragments were connected to the targeted cutting plasmid pcrEG vector containing the N23 recognition sequence (tctgaagtcgagttcgagtcaac) in the CRISPER / Cpf1 system through the Gibson assembly kit, and the constructed plasmid was transformed into DC3 by chemical transformation to obtain DC7-2~DC7-6.

[0102] After eliminating the tool plasmid in the gene editing system, the culture was cultured in a fermentation medium (glycerol: glucose = 1:12) at 37°C and 220 rpm for 48 h, and the concentration of the product in the supernatant was determined by high performance liquid chromatography. Figure 2 The strain in which the ptsI system was knocked out and regulated by the P6 promoter showed the highest N-acetylneuraminic acid (NeuAc) production of 8.2 g / L, and the acetic acid production of less than 0.2 g / L. This strain was named DC6.

[0103] Example 3: Optimization of the ratio of glycerol and glucose as fermentation carbon sources

[0104] The fermentation medium formula is: 30g / L carbon source (glycerol and glucose), 6g / L urea, 3.8mg / L zinc sulfate heptahydrate, 0.33g / L manganese sulfate monohydrate, 5g / L iron sulfate heptahydrate, 0.1g / L copper sulfate pentahydrate, 0.1g / L cobalt chloride hexahydrate, 4.8g / L yeast powder, 2.4g / L tryptone, 5.336g / L potassium dihydrogen phosphate, 3.284g / L potassium hydrogen phosphate trihydrate, 2.84g / L citric acid monohydrate, 2g / L magnesium sulfate heptahydrate, 4g / L ammonium sulfate, pH adjusted to 7. Conditions were optimized by controlling the relative ratio of glycerol and glucose.

[0105] Inoculate DC6 in LB medium, culture at 37℃ and 220rpm for 12-14h to obtain seed solution, then transfer 1-2% of the inoculum to fermentation medium with glycerol:glucose = 4:1, 2:1, 1:0, 0:1, 1:1, 1:2, 1:4, 1:8, 1:12, 1:16, and ferment at 37℃ and 220rpm for 48h. Figure 3 As shown, when glycerol: glucose = 1:12, the yield is the highest, which is 9.2 g / L (slightly different from the yield in Example 2. After multiple fermentation verifications, the yield fluctuated between 8-10 g. The reason for the difference may be batch difference. Therefore, the fermentation yield of the batch to which the strain belongs is recorded throughout the text). Figure 4As shown, DC6 achieves simultaneous utilization of glycerol and glucose.

[0106] The starting strain NBC45 was fermented in the same manner as above, with glycerol:glucose = 0:1, 1:0, 1:1. The results are shown in Figure 3 It can be seen that the effect of NBC45 in producing NeuAc is not good when glucose exists in the carbon source. Therefore, the strain constructed by the present invention increases the yield by 76.33% (the starting strain uses glucose as the carbon source) / 11.9% (the starting strain uses glycerol as the carbon source) by using the composite carbon source.

[0107] Comparative Example

[0108] According to the method described in the example, knockout or integration was performed to construct the corresponding engineering strain (see Figure 5 , wherein √ indicates that the starting strain has been modified in this way), and the production of N-acetylneuraminic acid was carried out by the fermentation method described in the embodiment, the only difference being that the carbon source was 30 g / L glucose.

[0109] from Figure 5 It can be seen that when glucose is the only carbon source, no target product is generated whether the starting strain is subjected to single knockout of pfkA, integration of p6-glf at the ptsI knockout site, integration of glpK22 at the glpK knockout site, or a combination of two modifications. This may be because such modifications affect the gene network and its metabolism. When the three-step modification is combined, efficient synthesis of N-acetylneuraminic acid is achieved. It is speculated that this may be because: ΔptsI::glf increases the glucose uptake rate, alleviates metabolic overflow, enables higher strain growth levels, reduces PEP consumption, and reduces byproduct levels; ΔpfkA increases the growth limit (30%) and alleviates metabolic overflow; ΔglpK::glpK22 is speculated to reduce the glucose utilization rate and promote the flow of carbon sources to the NeuAc metabolic pathway under the glycerol pathway, thereby meeting the carbon source requirements for growth and production of NeuAc. Its combined modification maintains the balance of each metabolic pathway and achieves high yield of the target product.

[0110] 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. A method for producing N-acetylneuraminic acid, characterized in that: The method comprises the steps of using recombinant Escherichia coli for fermentation production, wherein: The recombinant Escherichia coli comprises the following modifications: knocking out the 6-phosphofructokinase encoding gene and the glycerol kinase encoding gene, overexpressing the glycerol kinase mutant encoding gene, knocking out the PTS phosphotransferase I encoding gene and integrating and expressing the glucose-facilitated diffusion transporter encoding gene at the site, wherein the glucose-facilitated diffusion transporter encoding gene is expressed by a promoter with a nucleotide sequence as shown in SEQ ID NO.10, and the amino acid sequence of the glycerol kinase mutant is shown in SEQ ID NO.1; The fermentation production comprises fermentation with a composite carbon source, wherein the composite carbon source contains glucose and glycerol.

2. The production method according to claim 1, characterized in that The host bacteria of the recombinant Escherichia coli include Escherichia coli BL21 (DE3) ΔcheW::P6-glmSA, overexpression of neuC gene in the temminator locus of pspG gene under the control of promoter Pl, ΔmotA::P1-glmM-P4-glmUglmSA, overexpression of NemneuB gene in the terminator locus of yahO gene under the control of promoter Pl, ΔwecBΔmanAΔpykA, ΔnagΔnanATEΔmanXYZ.

3. The production method according to claim 1, characterized in that The GeneID of the 6-phosphofructokinase encoding gene is 948412; the GeneID of the glycerol kinase encoding gene is 948423; the nucleotide sequence of the glycerol kinase mutant encoding gene is shown in SEQ ID NO.2; the GeneID of the PTS phosphotransferase I encoding gene is 946879; the amino acid sequence of the glucose facilitated diffusion transporter is shown in SEQ ID NO.3; and the nucleotide sequence of the glucose facilitated diffusion transporter encoding gene is shown in SEQ ID NO.

4.

4. The production method according to claim 1, characterized in that The overexpression is integrated expression and / or free expression; preferably, the glycerol kinase encoding gene is knocked out and the glycerol kinase mutant encoding gene is integrated and expressed at the site.

5. The production method according to claim 1, characterized in that: In the composite carbon source, the mass ratio of glycerol to glucose is 1:(8-16).

6. The production method according to claim 1, characterized in that: The fermentation medium used in the fermentation production contains: 20-40 g / L composite carbon source, 3-9 g / L urea, 1-6 mg / L zinc sulfate heptahydrate, 0.01-1 g / L manganese sulfate monohydrate, 1-10 g / L iron sulfate heptahydrate, 0.01-1 g / L copper sulfate pentahydrate, 0.01-1 g / L cobalt chloride hexahydrate, 1-10 g / L yeast powder, 1-5 g / L tryptone, 1-10 g / L potassium dihydrogen phosphate, 1-10 g / L potassium hydrogen phosphate trihydrate, 1-10 g / L citric acid monohydrate, 1-10 g / L magnesium sulfate heptahydrate, and 1-10 g / L ammonium sulfate.

7. The production method according to claim 1, characterized in that: The fermentation production includes culturing at 35-37°C and 180-280 rpm.

8. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli comprises the following modifications: knocking out the 6-phosphofructokinase encoding gene and the glycerol kinase encoding gene, overexpressing the glycerol kinase mutant encoding gene, knocking out the PTS phosphotransferase I encoding gene and integrating and expressing the glucose-facilitated diffusion transporter encoding gene at the site, wherein the glucose-facilitated diffusion transporter encoding gene is expressed by a promoter with a nucleotide sequence as shown in SEQ ID NO.10, and the amino acid sequence of the glycerol kinase mutant is shown in SEQ ID NO.1, The host bacteria of the recombinant Escherichia coli include Escherichia coli BL21(DE3)ΔcheW::P6-glmSA,overexpression ofneuC gene in the temminator locus ofpspG gene under the control of promoter Pl,ΔmotA::P1-glmM-P4-glmUglmSA, overexpression ofNemneuB gene in the terminator locus ofyahO gene under the control of promoter Pl,ΔwecBΔmanAΔpykA,ΔnagΔnanATEΔmanXYZ.

9. The method for constructing the recombinant Escherichia coli according to claim 8, characterized in that: The following steps are involved: Using Escherichia coli BL21 (DE3) ΔcheW::P6-glmSA, overexpression ofneuC gene in the terminator locus of pspG gene under the control of promoter Pl, ΔmotA::P1-glmM-P4-glmUglmSA, overexpression of NemneuB gene in the terminator locus of yahO gene under the control of promoter Pl, ΔwecBΔmanAΔpykA, ΔnagΔnanATEΔmanXYZ as the host bacteria, the 6-phosphofructokinase encoding gene was knocked out, the glycerol kinase encoding gene was knocked out and the glycerol kinase mutant encoding gene with the amino acid sequence shown in SEQ ID NO.1 was integrated into the site by homologous recombination, the PTS phosphotransferase I encoding gene was knocked out and the glucose facilitated diffusion transporter encoding gene expression cassette containing the promoter shown in SEQ ID NO.10 was integrated into the site by homologous recombination.

10. Use of the recombinant Escherichia coli according to claim 8 or the recombinant Escherichia coli obtained by the construction method according to claim 9 in the preparation of N-acetylneuraminic acid or its derivatives.

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