Engineering strain for producing glycine as well as construction method and application of engineering strain

Abstract

The invention discloses an engineering strain for producing glycine. The engineering strain is obtained by modifying the following steps: (i) respectively introducing an exogenous acetyl coenzyme A synthetase gene acs, an isocitrate lyase gene aceA and an isocitrate dehydrogenase kinase gene aceK; (ii) introducing a glutamic acid glyoxylate transaminase gene cgaT from quinoa; and (iii) respectively knocking out a DNA (Deoxyribose Nucleic Acid) binding transcription inhibition factor gene iclR, a glycine dehydrogenase gene gcvP, an alanine transaminase gene alaA, a malate dehydrogenase gene maeA and a malate synthetase G gene glcB. According to the invention, an engineering strain capable of efficiently synthesizing glycine is constructed by transforming a glycine biosynthesis metabolic pathway, and the strain can produce glycine by fermentation with glucose as a carbon source and has a relatively high conversion rate.

Description

Technical Field

[0001] The invention relates to the technical field of industrial microorganisms, and in particular to an engineering strain for efficiently producing glycine, a construction method and application thereof. Background Art

[0002] Glycine, also known as aminoacetic acid, is one of the most important basic amino acids and is widely used in the fields of pesticides, medicine, food, feed and cosmetics. In the field of pesticides, glycine can be used to produce organophosphorus herbicides glyphosate, and can also be used to prepare plant growth regulators (such as glyphosate). In the field of medicine, glycine can be used to synthesize antihypertensive drugs (such as delapril hydrochloride) and stomach medicines (such as calcium carbonate preparations). In the field of food, glycine can be used as a food preservative to effectively extend the shelf life of food, and can also be used as a seasoning to enhance the flavor of food. In the field of feed, glycine is mainly used as an additive for chicken, cattle and sheep feed. It not only provides important nutrients for animals, but also prevents feed from oxidation and deterioration, thereby extending the shelf life of feed. In the field of daily chemicals, glycine can be used as a skin conditioner, and its derivative capryloyl glycine is used as a moisturizer, detergent and surfactant in cosmetics and has an antibacterial effect. The amino acid surfactants sodium cocoyl glycinate and potassium cocoyl glycinate synthesized with glycine as raw material are widely used in cleansing and bathing products. In recent years, with the deepening of the concept of green and sustainable development, consumers have paid more attention to the green sources of raw materials in the fields of daily chemicals and food. The traditional glycine synthesis process mainly uses raw materials from petrochemical sources. In order to better meet the general trend of green development, it is of great practical significance to develop a method for producing glycine from natural raw materials.

[0003] There are three main methods for synthesizing traditional glycine, namely protein hydrolysis, chemical synthesis and biosynthesis. Protein hydrolysis is an early method used in the industrial field to prepare glycine. Due to limited raw materials, this method has been basically eliminated. At present, glycine is mainly obtained through chemical synthesis and biosynthesis. Chemical synthesis methods mainly include chloroacetic acid aminolysis, Streck method, aminoacetonitrile method, direct hyne method, alcohol ammonia oxidation method and methyl glycolate method. The biosynthesis method includes using ethanolamine as a substrate, and converting glycine under the action of microorganisms such as aerobic Agrobacterium, Brevibacterium, and Corynebacterium; there are also bacteria such as Pseudomonas, Casein, and Alcaligenes to hydrolyze glycineamide to produce glycine; the typical biosynthesis method for preparing glycine is to use aminoacetonitrile (also known as glycine nitrile) as a raw material, based on microbial enzymes, to catalyze the hydrolysis of aminoacetonitrile to produce glycine. Although the above methods can achieve the production of glycine, there are problems such as high raw material cost and mostly petrochemical sources, low enzyme activity, and low synthesis efficiency. Bioconversion still needs to establish a cheaper raw material route.

[0004] As a model microorganism with a clear genetic background and simple genetic manipulation, Escherichia coli is often used for metabolic engineering to produce metabolites with high added value. In Escherichia coli, glycine can be obtained from precursors such as serine and threonine, but there are few studies on synthesizing glycine through metabolic engineering using glucose as a substrate. Based on the above, providing a strain capable of efficiently fermenting and producing glycine and a method for constructing the strain are of great significance for increasing the yield of glycine, reducing production costs, and developing raw materials from natural sources. Summary of the invention

[0005] In view of the problem that the existing glycine synthesis technology uses petrochemical raw materials or natural raw materials but has low yield / conversion rate, the purpose of this study is to provide an engineered strain for efficiently producing glycine and its construction method and application, and to construct an engineered strain that can efficiently synthesize glycine by modifying the glycine biosynthetic metabolic pathway. It can ferment glycine using glucose as a carbon source and has a high conversion rate.

[0006] To this end, the present invention provides the following technical solutions.

[0007] The first aspect of the present invention provides an engineered strain for producing glycine, wherein the engineered strain is obtained by the following modification:

[0008] (i) introducing exogenous acetyl-CoA synthetase gene acs, isocitrate lyase gene aceA and isocitrate dehydrogenase kinase gene aceK respectively;

[0009] (ii) introducing the glutamate glyoxylate aminotransferase gene cgaT from quinoa;

[0010] (iii) The DNA-binding transcriptional repressor gene iclR, glycine dehydrogenase gene gcvP, alanine aminotransferase gene alaA, malate dehydrogenase gene maeA, and malate synthase G gene glcB were knocked out respectively.

[0011] The first aspect of the present invention provides a method for constructing an engineered strain for producing glycine, the method comprising:

[0012] Constructing a host strain: replacing the pyruvate oxidase gene poxB in the Escherichia coli genome with the acetyl-CoA synthetase gene acs, and replacing the malate synthase gene aceB in the Escherichia coli genome with the isocitrate lyase gene aceA and the isocitrate dehydrogenase kinase gene aceK;

[0013] The DNA binding transcription repressor gene iclR, the glycine dehydrogenase gene gcvP, the alanine aminotransferase gene alaA, the malate dehydrogenase gene maeA, and the malate synthase G gene glcB were knocked out to obtain the mutant Escherichia coli HG07;

[0014] Construction of recombinant vector: inserting the exogenous glutamate glyoxylate aminotransferase gene cgaT into the NcoI and EcoRI sites of the plasmid pRB1k to obtain the recombinant vector pG01;

[0015] Construction of engineered strain: The above recombinant vector pG01 was introduced into the above mutant Escherichia coli HG07 to obtain the recombinant engineered strain SG0701.

[0016] The third aspect of the present invention provides a use of the engineered strain as described above in the preparation of glycine.

[0017] The fourth aspect of the present invention provides a method for preparing glycine according to the engineered strain as described above, the method comprising:

[0018] S1: Strain expansion and induction: The recombinant engineered strain SG0701 was inoculated onto a LB solid plate containing kanamycin and placed in a 37°C incubator for 12-16 hours. A single colony was picked and inoculated into a LB liquid medium containing kanamycin for expansion. Subsequently, the colony was transferred to a ZYM autoinduction medium containing kanamycin at a volume ratio of 0.5-2% for autoinduction overnight. Finally, the OD was measured. 600 The light absorption value was collected and 3×10 10 -4×10 10 / mL bacteria;

[0019] S2: Whole-cell catalytic transformation: The collected cells were centrifuged, washed with physiological saline, and the supernatant was discarded and the resulting cells were resuspended to obtain a final concentration of 3×10 10 -4×10 10 / mL of bacterial suspension;

[0020] The bacterial suspension is mixed with 50-80 mM glucose, 40-64 mM NH4Cl, 2-4 mM MgSO4, 10-15 mM sodium glutamate and 1×M9 Salts to carry out a conversion reaction to obtain glycine.

[0021] In a preferred embodiment of the present invention, in step S1, the expansion culture conditions are: temperature 25-42°C, rotation speed 100-300 rpm, time 8-12h.

[0022] In a preferred embodiment of the present invention, in step S1, the auto-induction culture conditions are: temperature 25-42°C, rotation speed 100-300 rpm.

[0023] In a preferred embodiment of the present invention, in step S2, the centrifugation conditions are: temperature 4-15°C, rotation speed 4000-6000 rpm, time 5-10 min.

[0024] In a preferred embodiment of the present invention, in step S2, the conversion reaction conditions are: temperature 25-42°C, rotation speed 100-300 rpm, time 2-24 h.

[0025] In a preferred embodiment of the present invention, the kanamycin content is 50 μg / mL.

[0026] By means of the above technical solution, the present invention has at least the following advantages:

[0027] Compared with the traditional glycine synthesis method, the raw material used in the present invention is glucose, a sustainable carbon source, which is more in line with the general trend of green sustainability, and its natural source index can reach 100%, while the natural source index of glycine synthesized from petrochemical raw materials is 0. The biological method for producing glycine adopted by the present invention replaces the traditional chemical process, replaces the non-renewable petrochemical raw materials with renewable green raw materials, further reduces the production cost, and the production process is more green and environmentally friendly, and the market competitive advantage is obvious. Compared with the reported fermentation process for producing glycine, the strain of the present invention has the advantage of high conversion rate (0.33g / g), and has more industrialization prospects.

[0028] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the plasmid map of pRB1k;

[0030] Figure 2 The glycine production of the recombinant E. coli engineered strain using glucose as raw material at different time points;

[0031] Figure 3 Comparison of glycine production at different time points by other recombinant E. coli engineered strains using glucose as raw material. DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0034] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to final concentrations, which refer to the percentage of the added component in the system after the addition of the component.

[0035] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0036] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] The quantitative experiments in the embodiments of the present invention were repeated three times, and the results were averaged.

[0039] In the embodiments of the present invention, unless otherwise specified, the sequencing verification processes involved are all completed by a third-party testing agency, which is Suzhou Genewise Biotechnology Co., Ltd.

[0040] In the embodiment of the present invention, Escherichia coli K12 is recorded in the document "Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H: Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006, 2: 2006.0008." It is a non-pathogenic bacterium with a clear genetic background, a short generation time, easy cultivation and low-cost culture medium raw materials. The GenBank Accession of the whole genome sequence of Escherichia coli K12 is U00096.3 (GI: 545778205, update date is AUG 01, 2014, version is 3). The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0041] In an embodiment of the present invention, the coding sequence of the glutamate glyoxylate aminotransferase gene cgaT (Cqu) from Chenopodium quinoa is shown in SEQ ID NO.2.The coding sequence of the pyruvate oxidase gene (poxB) is shown in GeneID: 946132 (consisting of 1719 nucleotides), encoding the pyruvate oxidase shown in Acession No. NP_415392 (consisting of 572 amino acid residues); the coding sequence of the acetyl-CoA synthetase gene (acs) is shown in GeneID: 948572 (consisting of 1959 nucleotides), encoding the acetyl-CoA synthetase shown in Acession No. NP_418493 (consisting of 622 amino acid residues); the coding sequence of the DNA-binding transcription repressor gene (iclR) is shown in Gene ID: 948524 (consisting of 825 nucleotides), encoding the DNA-binding transcription repressor shown in Acession No. NP_418442 (consisting of 274 amino acid residues); the coding sequence of the isocitrate lyase gene (aceA) is shown in Gene ID: The coding sequence of the isocitrate dehydrogenase kinase gene (aceK) is shown in Gene ID: 944197 (consisting of 1737 nucleotides), encoding the isocitrate dehydrogenase kinase shown in Accession No. NP_418440 (consisting of 578 amino acid residues); the coding sequence of the malate synthase gene (aceB) is shown in Gene ID: 948512 (consisting of 1602 nucleotides), encoding the malate synthase shown in Accession No. NP_418438 (consisting of 533 amino acid residues); the coding sequence of the glycine dehydrogenase gene (gcvP) is shown in Gene ID: The coding sequence of the gene for alanine aminotransferase (alaA) is shown in Gene ID: 946772 (consisting of 1218 nucleotides), encoding the gene for alanine aminotransferase (NP_416793 (consisting of 405 amino acid residues) as shown in Gene ID: 947394 (consisting of 2874 nucleotides), encoding the gene for glycine dehydrogenase (NP_417379 (consisting of 957 amino acid residues) as shown in Gene ID: 946772 (consisting of 1218 nucleotides), encoding the gene for alanine aminotransferase (NP_416793 (consisting of 405 amino acid residues) as shown in Gene ID: The coding sequence of malate synthase G gene (glcB) is shown in GeneID:946031 (consisting of 1698 nucleotides), encoding the malate dehydrogenase (requiring NAD) shown in Acession No. NP_415996 (consisting of 565 amino acid residues); the coding sequence of malate synthase G gene (glcB) is shown in GeneID:948857 (consisting of 2172 nucleotides), encoding the malate synthase G shown in Acession No. NP_417450 (consisting of 723 amino acid residues).

[0042] The nucleotide sequence of the pRB1k vector in the following example is shown in SEQ ID NO.1, which includes the following fragments: (1) araC-araBAD-MCS fragment (containing arabinose-inducible promoter and multiple cloning site); (2) MCSTrrnB fragment (containing multiple cloning site and terminator TrrnB); (3) RSF1030 replication initiation site fragment; (4) kanamycin resistance gene Kan fragment. The map of the pRB1k vector is shown in Figure 1 shown.

[0043] SEQ ID NO.1:

[0044]

[0045] SEQ ID NO.2:

[0046]

[0047] 1. Culture medium

[0048] LB medium formula: 1% tryptone, 0.5% yeast powder, 1% NaCl.

[0049] ZMY self-induction medium formula: 100mL A+2mL B+2mL C+200μL D+100μL E (the following are all mass percentage concentrations); among which,

[0050] A.ZY: 1% tryptone, 0.5% yeast powder;

[0051] B. 50×M: 1.25M Na2HPO4, 1.25M KH2PO4, 2.5M NH4Cl and NaSO4;

[0052] C.50×5052: 25% glycerol, 2.5% glucose, 10% L-arabinose;

[0053] D. 500×MgSO4: 1M MgSO4;

[0054] E.1000× trace elements: 50mM FeCl3, 20mM CaCl2, 10mM MnCl2, 10mM ZnSO4, 2mM each of CoCl2, NiCl2, Na2Mo4, Na2SeO3 and H3BO3.

[0055] M9 Salts formula: 10g / L NH4Cl, 12.8g / L Na2HPO4·7H2O, 3g / L KH2PO4, 0.5g / L NaCl.

[0056] 2. Use Agilent-1200 HPLC to detect glycine

[0057] The collected samples were centrifuged at 4°C and 12000rpm for 2min, and the supernatant was taken. The components in the fermentation broth were determined using an Agilent (Agilent-1200) high performance liquid chromatograph: the sample to be tested was appropriately diluted and derivatized with 2,4-dinitrofluorobenzene (DNFB), 100μL of the sample was added with 50μL of 10g / L DNFB in acetonitrile solution and 100μL of 0.5M NaHCO3 solution, mixed thoroughly, reacted at 60°C in the dark for 1h, cooled, added with 750μL of 0.01M KH2PO4 solution, mixed, filtered with a 0.22μm filter membrane, and then detected by high performance liquid chromatography. The chromatographic column was a ZORBAX Eclipse XDB-C18 column (4.6×150 mm, 5 μm; Agilent), the column temperature was 30° C., the mobile phase was a 35% acetonitrile-formic acid (1 / 1000) aqueous solution, the flow rate was 1 mL / min, and the detection wavelength was 360 nm.

[0058] Example 1: Construction of a recombinant plasmid pG01 expressing the glutamate glyoxylate aminotransferase gene cgaT (Cqu) from Chenopodium quinoa

[0059] The synthetic glutamate glyoxylate aminotransferase gene plasmid pUC-cgaT(Cqu) was used as a template and primers cgaT(Cqu)-F and cgaT(Cqu)-R were used for PCR amplification to obtain the glutamate glyoxylate aminotransferase gene cgaT(Cqu) from Chenopodium quinoa.

[0060] The primer sequence information used in the above PCR amplification is:

[0061] cgaT(Cqu)-F SEQ NO.3 5'-ggaattaaccatggcgatgagcaagggtctggactac-3'

[0062] cgaT(Cqu)-R SEQ NO.4 5'-cgagctcaccgaattcttacatacggctataacccgc-3'

[0063] The PCR amplification system was as follows: 5X SF Buffer 10 μL, dNTP Mix (10 mM each) 1 μL, template pUC-cgaT (Cqu) 20 ng, primers (10 μM) 2 μL each, Phanta Super-Fidelity DNA Polymerase (Nanjing Novezan Biotechnology Co., Ltd., product catalog P501) 1 μL, distilled water 34 μL, and the total volume was 50 μL.

[0064] PCR amplification conditions were as follows: pre-denaturation at 95°C for 2 minutes (1 cycle); denaturation at 95°C for 10 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 1.5 minutes (30 cycles); and extension at 72°C for 5 minutes (1 cycle).

[0065] The pRB1k vector was double-digested with NcoI and EcoRI to recover the large vector fragment of about 3450 bp. The cgaT (Cqu) gene fragment obtained above was ligated with the large vector fragment using the Gibson method (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009, 6: 343-345.) to obtain a ligation product. The ligation product was transformed into Fast-T1 competent cells (Nanjing Novizan Biotechnology Co., Ltd., product catalog number C505), coated on LB solid plates containing kanamycin (50 μg / mL), and incubated at 37°C overnight. A single clone was picked to extract the plasmid, and a pair of primers pBAD-F and pBAD-R were designed for PCR verification. The correct recombinant vector plasmid was screened for sequencing; the fragment between the NcoI and EcoRI sites of the pRB1k vector was replaced with the glutamate glyoxylate aminotransferase gene shown in SEQ ID NO.2, and the recombinant vector obtained was named pG01. In the glutamate glyoxylate aminotransferase gene expression cassette, the promoter that initiates the transcription of the glutamate glyoxylate aminotransferase gene is the pBAD promoter.

[0066] The primer sequence information used in the above PCR verification is:

[0067] pBAD-F SEQ ID NO.5 5'-cggcgtcacactttgctatg-3'

[0068] pBAD-R SEQ ID NO.6 5'-cgtttcacttctgagttcggc-3'.

[0069] Example 2: Construction of mutant Escherichia coli HG07

[0070] The Escherichia coli mutant HG07 was generated using CRISPR technology (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015,81:2506-2514.) The acetyl-CoA synthetase gene (acs) of Escherichia coli K-12MG1655 replaced the pyruvate oxidase gene (poxB) in the Escherichia coli genome, and the malate synthase gene (aceB) in the Escherichia coli genome was replaced with the isocitrate lyase gene (aceA) and the isocitrate dehydrogenase kinase gene (aceK); and the DNA binding transcription inhibitor gene (iclR), glycine dehydrogenase gene (gcvP), alanine aminotransferase gene (alaA), malate dehydrogenase (NAD required) gene (maeA), and malate synthase G gene (glcB) were simultaneously knocked out to obtain a mutant Escherichia coli, referred to as HG07 in this application, whose genotype is E. coli BW25113,ΔpoxB::119-acs,ΔiclR,ΔaceB::tac-aceAK,ΔgcvP,ΔalaA,ΔmaeA,ΔglcB.

[0071] Specifically, in this embodiment, the Escherichia coli mutant HG07 is obtained by replacing the pyruvate oxidase gene (poxB) in the Escherichia coli genome with the acetyl-CoA synthetase gene (acs) shown in Gene ID: 948572 of Escherichia coli K12 MG1655, replacing the malate synthase gene (aceB) in the Escherichia coli genome with the isocitrate lyase gene (aceA) shown in Gene ID: 948517 and the isocitrate dehydrogenase kinase gene (aceK) shown in Gene ID: 944197; and simultaneously knocking out the DNA-binding transcriptional repressor gene (iclR), the glycine dehydrogenase gene (gcvP), the alanine aminotransferase gene (alaA), the malate dehydrogenase (NAD-requiring) gene (maeA), and the malate synthase G gene (glcB).

[0072] The specific construction steps of the above-mentioned E. coli mutant HG07 are as follows:

[0073] (1) Preparation of electrocompetent cells: The pCas plasmid (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81: 2506-2514.) was transformed into Escherichia coli K12 by chemical transformation, and positive clones were screened on LB plates containing kanamycin (kanamycin concentration was 50 μg / mL) at 30°C. Positive clones were inoculated in LB liquid medium containing 2 g / L arabinose and cultured at 30°C until OD 600 After the concentration is about 0.6, prepare the electrocompetent cells.

[0074] (2) Construction of pTarget plasmid: Use the website https: / / crispy.secondarymetabolites.org to select the knockout site N20 and design primers to construct the pTarget plasmid. Using pTargetF (Jiang Y, Chen B, Duan C, SunB, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81: 2506-2514.) as a template, PCR amplification was performed using primer pairs pTarget-poxB-F and pTarget-poxB-R, pTarget-iclR-F and pTarget-iclR-R, pTarget-aceB-F and pTarget-aceB-R, pTarget-gcvP-F and pTarget-gcvP-R, pTarget-alaA-F and pTarget-alaA-R, pTarget-maeA-F and pTarget-maeA-R, pTarget-glcB-F and pTarget-glcB-R, respectively, to obtain fragments of about 2100 bp in size.

[0075] The above PCR amplification primer sequence information is:

[0076] pTarget-poxB-F SEQ ID NO.7 5'-cagcaaggtggatatggcacgttttagagctagaaatagc-3'

[0077] pTarget-poxB-R SEQ ID NO.8 5'-gtgccatatccaccttgctgactagtattatacctactaggac-3'

[0078] pTarget-aceB-F SEQ ID NO.9 5'-cctggcaggaagcggcctgggttttagagctagaaatagc-3'

[0079] pTarget-aceB-R SEQ ID NO.10 5'-ccaggccgcttcctgccaggcagactagtattatacctaggact-3'

[0080] pTarget-iclR-F SEQ ID NO.11 5'-gctaaccacgatgcaacagcgttttagagctagaaatagc-3'

[0081] pTarget-iclR-R SEQ ID NO.12 5'-gctgttgcatcgtggttagcactagtattatacctaggac-3'

[0082] pTarget-gcvP-F SEQ ID NO.13 5'-gggctacggtggcccacacggttttagagctagaatagc-3'

[0083] pTarget-gcvP-R SEQ ID NO.14 5'-cgtgtgggccaccgtagcccactagtattatacctaggac-3'

[0084] pTarget-alaA-F SEQ ID NO.15 5'-gggccgaaaaacacgccaagttttagagctagaaatagc-3'

[0085] pTarget-alaA-R SEQ ID NO.16 5'-ttggcgtgtttttcggcccactagtattacctactaggac-3'

[0086] pTarget-maeA-F SEQ ID NO.17 5'-gtctaacccgacgtcacgcggttttagagctagaaatagc-3'

[0087] pTarget-maeA-R SEQ ID NO.18 5'-cgcgtgacgtcgggttagaccagactagtattatacctaggact-3'

[0088] pTarget-glcB-F SEQ ID NO.19 5'-cctcgatgaatctctaccgcgttttagagctagaaatagc-3'

[0089] pTarget-glcB-R SEQ ID NO.20 5'-gcggtagagattcatcgaggactagtattatacctaggac-3'

[0090] pTarget-cexu-F SEQ ID NO. 21 5'-ctttcctgcgttatcccctg-3'.

[0091] The PCR amplification system was as follows: 5X SF Buffer 10 μL, dNTP Mix (10 mM each) 1 μL, template pTargetF 20 ng, primers (10 μM) 2 μL each, Phanta Super-Fidelity DNA Polymerase (Nanjing Novezan Biotechnology Co., Ltd., product catalog P501) 1 μL, distilled water 34 μL, and the total volume was 50 μL.

[0092] PCR amplification conditions were as follows: pre-denaturation at 95°C for 2 minutes (1 cycle); denaturation at 95°C for 10 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 1.5 minutes (30 cycles); and extension at 72°C for 10 minutes (1 cycle).

[0093] After about 3 hours of reaction with DpnI methylase, the competent Escherichia coli Fast-T1 was directly transformed by chemical transformation, and positive clones were screened on LB plates containing streptomycin (streptomycin concentration was 100 μg / mL), and sequenced and verified with primer pTarget-cexu-F. After correct sequencing, they were named pTarget-poxB, pTarget-iclR, pTarget-aceB, pTarget-gcvP, pTarget-alaA, pTarget-maeA, and pTarget-glcB, respectively.

[0094] (3) Amplification of the target fragment: PCR amplification was performed using primer pairs poxB-up800-F and poxB-up500-R, acs-F and acs-R, and poxB-down500-F and poxB-down800-R to obtain fragments of approximately 800 bp, 2000 bp, and 800 bp, respectively. The mixture of the three fragments was used as a template and PCR amplification was performed using primer pairs poxB-up500-F and poxB-down500-R to obtain a poxB::acs target fragment of approximately 3000 bp; primer pairs aceB-up800-F and aceB- PCR amplification was performed on aceB-up500-R, aceAK-F and aceAK-R, aceB-down500-F and aceB-down800-R to obtain fragments of approximately 800 bp, 3000 bp and 800 bp, respectively. The mixture of the three fragments was used as a template and PCR amplification was performed on aceB-up500-F and aceB-down500-R to obtain an aceB::aceAK targeting fragment of approximately 4000 bp; primer pairs iclR-up800-F and iclR-up500-R, iclR-down500-F and iclR- PCR amplification was performed with primers iclR-up500-F and iclR-down500-R to obtain fragments of approximately 800 bp and 800 bp, respectively. The mixture of the two fragments was used as a template and PCR amplification was performed with primers iclR-up500-F and iclR-down500-R to obtain a ΔiclR targeting fragment of approximately 1000 bp. PCR amplification was performed with primers gcvP-up800-F and gcvP-up500-R, gcvP-down500-F and gcvP-down800-R to obtain fragments of approximately 800 bp and 800 bp, respectively. The mixture of the two fragments was used as a template and PCR amplification was performed with primers gcvP-up500-F and gcvP-down500-R were PCR amplified to obtain a ΔgcvP target fragment of about 1000 bp; primer pairs alaA-up800-F and alaA-up500-R, alaA-down500-F and alaA-down800-R were used to PCR amplify to obtain fragments of about 800 bp and 800 bp, respectively; a mixture of the two fragments was used as a template, and primer pairs alaA-up500-F and alaA-down500-R were used to PCR amplify to obtain a ΔalaA target fragment of about 1000 bp;PCR amplification was performed with primer pairs maeA-up800-F and maeA-up500-R, maeA-down500-F and maeA-down800-R to obtain fragments of approximately 800 bp and 800 bp, respectively. Using the mixture of the two fragments as a template, PCR amplification was performed with primer pairs maeA-up500-F and maeA-down500-R to obtain a ΔmaeA targeting fragment of approximately 1000 bp. PCR amplification was performed with primer pairs glcB-up800-F and glcB-up500-R, glcB-down500-F and glcB-down800-R to obtain fragments of approximately 800 bp and 800 bp, respectively. Using the mixture of the two fragments as a template, PCR amplification was performed with primer pairs glcB-up500-F and glcB-down500-R to obtain a ΔglcB targeting fragment of approximately 1000 bp. ;

[0095] The primer sequences used are as follows:

[0096] poxB-up800-F SEQ ID NO.22 5'-tacctgcgacgataaagcag-3'

[0097] poxB-up500-R SEQ ID NO.23 5'-gacgacgtggtgttagctgtgcatggttctccatctcctgaatg-3'

[0098] acs-F SEQ ID NO.24 5'-atggaaagccctactccaca-3'

[0099] acs-R SEQ ID NO.25 5'-ttatataatcgccatcactttcc-3'

[0100] poxB-down500-F SEQ ID NO.26 5'-cgatatcgcaggtgagtactaaaacaaactggctaaggta-3'poxB-down800-R SEQ ID NO.27 5'-tgtcgtggtcttcctgcaag-3'

[0101] poxB-up500-F SEQ ID NO.28 5'-tcacgtaccgtgatgacctg-3'

[0102] <h2 style=";text-align:left;direction:ltr">poxB-down500-R SEQ ID NO.29 5'-ctcgcgggtaaattcccatg-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0103] <h2 style=";text-align:left;direction:ltr"> aceB-up800-F SEQ ID NO.30 5'-gctgtgctttggggatttt-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0104] <h2 style=";text-align:left;direction:ltr"> aceB-up500-R SEQ ID NO.31 5'-tgtggagtagggctttccataagactttctccagtgatgttga-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0105] <h2 style=";text-align:left;direction:ltr"> aceAK-F SEQ ID NO.32 5'-atggaaagccctactccaca-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0106] <h2 style=";text-align:left;direction:ltr"> aceAK-R SEQ ID NO.33 5'-ttatataatcgccatcactttcc-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0107] <h2 style=";text-align:left;direction:ltr"> aceB-down500-F SEQ ID NO.34 5'-aagtgatggcgattatataaccgaacgaactggtttaatcttg-3' aceB-down800-R SEQ ID NO.35 5'-gatgcgagcagcgttttggg-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0108] <h2 style=";text-align:left;direction:ltr"> aceB-up500-F SEQ ID NO.36 5'-aattcttttggggagtgcg-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0109] <h2 style=";text-align:left;direction:ltr"> aceB-down500-R SEQ ID NO.37 5'-ccgccagcttataagttaat-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0110] <h2 style=";text-align:left;direction:ltr"> iclR-up800-F SEQ ID NO.38 5'-cgttttcaccgcaaataccg-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0111] <h2 style=";text-align:left;direction:ltr"> iclR-up500-R SEQ ID NO.39 5'-caccgtacgccatgacagtctcttttttctg-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0112] <h2 style=";text-align:left;direction:ltr"> iclR-down500-F SEQ ID NO.40 5'-gactgtcatggcgtacggtggaatgcgctg-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0113] iclR-down800-R SEQ ID NO.41 5'-gcagcaatgtgtcggcatac-3'

[0114] iclR-up500-F SEQ ID NO.42 5'-cgcaaagttgatttccgccg-3'

[0115] iclR-down500-R SEQ ID NO.43 5'-cagcttgtaagacggacgtg-3'

[0116] gcvP-up800-F SEQ ID NO.44 5'-ccgcgcgattacttaacc-3'

[0117] gcvP-up500-R SEQ ID NO.45 5'-cagcccaggccatagcgggatatctgtcga-3'

[0118] gcvP-down500-F SEQ ID NO.46 5'-tcccgctatggcctgggctgaaagtcacta-3'

[0119] gcvP-down800-R SEQ ID NO.47 5'-cgcgcaggctatttctaat-3'

[0120] gcvP-up500-F SEQ ID NO.48 5'-ctgtctggcgcagatgga-3'

[0121] gcvP-down500-R SEQ ID NO.49 5'-cccgtattcgtgtcattaaag-3'

[0122] alaA-up800-F SEQ ID NO.50 5'-aaagcgactcacattgttcc-3'

[0123] alaA-up500-R SEQ ID NO.51 5'-gataaccagacatgaatcttgaccttttttgctg-3'

[0124] alaA-down500-F SEQ ID NO.52 5'-aagattcatgtctggttatcatcagctgtaatct-3'

[0125] alaA-down800-R SEQ ID NO.53 5'-tcaccattatcgtgatggcg-3'

[0126] alaA-up500-F SEQ ID NO.54 5'-gcgaattatctgatgcaaagt-3

[0127] alaA-down500-R SEQ ID NO.55 5'-tgcatctgcctgtttcacca-3'

[0128] maeA-up800-F SEQ ID NO.56 5'-cggatcataccaatcacaatc-3'.

[0129] maeA-up500-R SEQ ID NO.57 5'-cggcggtacatgtcactcactctttttgaat-3'

[0130] maeA-down500-F SEQ ID NO.58 5′-gtgagtgacatgtaccgccgtacctccatcta-3′.

[0131] maeA-down800-R SEQ ID NO.59 5'-aatttttggcggcgtcttc-3'

[0132] maeA-up500-F SEQ ID NO.60 5'-gggttgaagcttctgataacg-3'

[0133] maeA-down500-R SEQ ID NO.61 5′-gatgcactcttccgccat-3′;

[0134] glcB-up800-F SEQ ID NO.62 5′-gaggaaccgctggaaaaact-3′

[0135] glcB-up500-R SEQ ID NO.63 5'-atgactttctttttcgcgcattgtttatctcctcgttttcg-3'

[0136] glcB-down500-F SEQ ID NO.64 5'-catgcgcgaaaaagaagtcattaatagc-3'

[0137] glcB-down800-R SEQ ID NO.65 5'-caacgcaccaaaggagaaac-3'

[0138] glcB-up500-F SEQ ID NO.66 5'-tcgtacctctgtgcgtcact-3'

[0139] glcB-down500-R SEQ ID NO. 67 5'-taagggttattgctccagcg-3'.

[0140] The PCR amplification system was as follows: 5X SF Buffer 10 μL, dNTP Mix (10 mM each) 1 μL, template 5-20 ng, primer (10 μM) 2 μL each, Phanta Super-Fidelity DNA Polymerase (Nanjing Novezan Biotechnology Co., Ltd., product catalog P501) 1 μL, distilled water 34 μL, and the total volume was 50 μL.

[0141] PCR amplification conditions were as follows: pre-denaturation at 95°C for 2 minutes (1 cycle); denaturation at 95°C for 10 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 1.5 minutes (30 cycles); and extension at 72°C for 10 minutes (1 cycle).

[0142] The targeting fragments poxB::acs, aceB::aceAK, ΔiclR, ΔgcvP, ΔalaA, ΔmaeA and ΔglcB were recovered respectively. The targeting fragments sequentially comprised a 500 bp upstream homology arm, a replacement gene expression cassette and a 500 bp downstream homology arm from upstream to downstream.

[0143] (4) Electroporation: 200 ng of pTarget-poxB plasmid, 400 ng of the targeting fragment poxB::acs and 100 μL of the electroporation competent cells prepared in step (1) were mixed and placed in a 2 mm electroporation cuvette, electroporated at 2.5 kV, and 1 mL of LB liquid culture medium was added to resuscitate at 30°C. The mixture was then spread on an LB plate containing kanamycin and streptomycin (50 μg / mL of kanamycin and 100 μg / mL of streptomycin), cultured at 30°C, positive clones were screened, and PCR amplification was performed using primers poxB-up800-F and poxB-down800-R. The amplified fragments were sequenced and verified. The PCR amplification system was: Green Taq Mix 10μL (Nanjing Novezan Biotechnology Co., Ltd., product catalog P131), 0.8μL of primers (10μM), 8.2μL of distilled water, 0.2μL of template bacterial solution, the total volume is 20μL; the PCR amplification conditions are: 95℃ pre-denaturation for 3 minutes (1 cycle); 95℃ denaturation for 15 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 1-5 minutes (60 seconds / kb) (30 cycles); 72℃ extension for 5 minutes (1 cycle).

[0144] (5) Elimination of pTarget plasmid: The positive clones verified by sequencing were inoculated into LB liquid medium containing 0.1 mM IPTG and kanamycin (kanamycin 50 μg / mL) and cultured at 30°C overnight to eliminate the pTarget plasmid. The strains after overnight culture were streaked on LB solid plates containing kanamycin and cultured at 30°C overnight to obtain the E. coli mutant BW25113ΔpoxB::acs containing the pCas plasmid, named HG01;

[0145] (6) Prepare electrocompetent cells of the E. coli mutant HG01 containing the pCas plasmid, mix with the pTarget-iclR plasmid and the ΔiclR targeting fragment, repeat the above steps (4)-(5), and sequence and verify with the primers iclR-up800-F and iclR-down800-R to obtain the E. coli mutant BW25113ΔpoxB::acs, ΔiclR containing the pCas plasmid, named HG02;

[0146] (7) Prepare electrocompetent cells from the E. coli mutant HG02 containing the pCas plasmid, mix with the pTarget-aceB plasmid and the aceB::aceAK targeting fragment, repeat the above steps (4)-(5), and sequence and verify with the primers aceB-up800-F and aceB-down800-R to obtain the E. coli mutant BW25113ΔpoxB::acs, ΔiclR, ΔaceB::aceAK containing the pCas plasmid, named HG03;

[0147] (8) Prepare electrocompetent cells from the E. coli mutant HG03 containing the pCas plasmid, mix with the pTarget-gcvP plasmid and the ΔgcvP targeting fragment, repeat the above steps (4)-(5), and sequence and verify with the primers gcvP-up800-F and gcvP-down800-R to obtain the E. coli mutant BW25113ΔpoxB::acs, ΔiclR, ΔaceB::aceAK, ΔgcvP containing the pCas plasmid, named HG04;

[0148] (9) Prepare electrocompetent cells from the E. coli mutant HG04 containing the pCas plasmid, mix with the pTarget-alaA plasmid and the ΔalaA targeting fragment, repeat the above steps (4)-(5), and sequence and verify with primers alaA-up800-F and alaA-down800-R to obtain the E. coli mutant BW25113ΔpoxB::acs, ΔiclR, ΔaceB::aceAK, ΔgcvP, ΔalaA containing the pCas plasmid, named HG05;

[0149] (10) Prepare electrocompetent cells from the E. coli mutant HG05 containing the pCas plasmid, mix with the pTarget-maeA plasmid and the ΔmaeA targeting fragment, repeat the above steps (4)-(5), and sequence and verify with the primers maeA-up800-F and maeA-down800-R to obtain the E. coli mutant BW25113ΔpoxB::acs, ΔiclR, ΔaceB::aceAK, ΔgcvP, ΔalaA, ΔmaeA containing the pCas plasmid, named HG06;

[0150] (11) Prepare electrocompetent cells from the E. coli mutant HG06 containing the pCas plasmid, mix with the pTarget-glcB plasmid and the ΔglcB targeting fragment, repeat the above steps (4)-(5), and sequence and verify with the primers glcB-up800-F and glcB-down800-R to obtain the E. coli mutant BW25113ΔpoxB::acs, ΔiclR, ΔaceB::aceAK, ΔgcvP, ΔalaA, ΔmaeA, ΔglcB containing the pCas plasmid, named HG07;

[0151] (12) Elimination of pCas plasmid: The E. coli mutant BW25113ΔpoxB::acs, ΔaceB::aceAK, ΔiclR, ΔgcvP, ΔalaA, ΔmaeA, ΔglcB containing the pCas plasmid verified by sequencing was inoculated into LB liquid medium and cultured at 42°C overnight to eliminate the pCas plasmid. The strain after overnight culture was streaked on an LB solid plate and cultured at 42°C overnight to obtain the E. coli mutant BW25113ΔpoxB::acs, ΔiclR, ΔaceB::aceAK, ΔgcvP, ΔalaA, ΔmaeA, ΔglcB without plasmid, referred to as HG07.

[0152] Example 3: Construction of an engineered strain HG0701 that produces high levels of glycine

[0153] The expression vector pG01 constructed in Example 1 was used to transform the Escherichia coli mutant HG07 by chemical transformation, and positive clones were screened on LB plates containing kanamycin (the concentration of kanamycin was 50 μg / mL). The obtained clone strain was named SG0701.

[0154] According to the same operation as above, pG01 was transformed into Escherichia coli mutants HG01, HG02, HG03, HG04, HG05, and HG06 to obtain the corresponding cloning strains SG0101, SG0201, SG0301, SG0401, SG0501, and SG0601.

[0155] Example 4: Preparation of glycine using glucose as raw material (I)

[0156] (1) Bacterial culture and enzyme induction: The mutant E. coli SG0701 strain was streaked from the glycerol storage tube onto a LB solid plate containing kanamycin (50 μg / mL). The next day, a single colony was picked and inoculated into LB liquid medium containing kanamycin (50 μg / mL), cultured at 37°C and 220 rpm for 8 h, and then transferred to ZYM autoinduction medium containing kanamycin (50 μg / mL) at a volume ratio of 1%. After culturing overnight at 30°C and 220 rpm, the OD was measured. 600 The light absorption value was collected and 3×10 10 / mL of bacteria.

[0157] This embodiment only provides a specific implementation scheme. As an alternative implementation scheme of this embodiment, the culture conditions can be replaced with any value within the following range: the culture temperature is 25-42°C, and the shaking speed is 100-300 rpm.

[0158] (2) Whole-cell catalytic transformation: The collected cells were centrifuged at 4000 rpm for 5 min at 4°C, washed once with 0.9% saline, and the supernatant was discarded. The cells were resuspended in the transformation solution and transferred to a test tube to a final bacterial concentration of 3 × 10 10 / mL, whole cell conversion to synthesize glycine, the whole cell conversion system contains a final concentration of 70mM glucose, 56mM NH4Cl, 2mMMgSO4, 10mM sodium glutamate and 1×M9 Salts, the conversion conditions are 37℃ and 220rpm, and samples are taken after 2h, 6h and 24h of conversion to test the glycine content. The results are shown in Figure 2 .

[0159] Those skilled in the art may adjust the above conditions to a certain extent according to actual conditions, which will not affect the realization of the purpose of the present invention.

[0160] Example 5: Preparation of glycine using glucose as raw material (II)

[0161] The only difference between this example and Example 4 is that the strain used is Escherichia coli BW25113, and the rest is consistent with Example 4. Samples were taken 2h, 6h and 24h after transformation to test the glycine content. Figure 2 .

[0162] Results: Figure 2 As shown in the figure, with the extension of reaction time, the glycine content in the transformation liquid of mutant Escherichia coli SG0701 showed an obvious upward trend; the glycine production of mutant Escherichia coli SG0701 strain was 0.66g / L at 2h, 2.23g / L at 6h, and 4.16g / L at 24h. The glycine conversion rate in the whole transformation stage could reach 0.33g / g glucose.

[0163] Example 6: Preparation of glycine using glucose as raw material (III)

[0164] The only difference between this example and example 4 is that the strains used are mutant Escherichia coli SG0101, SG0201, SG0301, SG0401, SG0501, and SG0601, respectively, and the rest is consistent with example 4. Samples were taken 2h, 6h, and 24h after transformation to test the glycine content. The results are shown in Figure 3 .

[0165] like Figure 3As shown, there are obvious differences in the ability of different strains to produce glycine: SG0101 and SG0201 produce almost no glycine, the glycine production of SG0103 and SG0104 strains at 24 hours is 0.77 g / L and 1.22 g / L, respectively, the glycine production of SG0501 and SG0601 strains at 24 hours is 2.26 g / L and 2.32 g / L, respectively, and the glycine production of SG0301 and SG0401 is significantly lower than that of SG0501 and SG0601.

[0166] It can be seen that the method of Example 4 of the present invention can obtain the highest yield of glycine.

[0167] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An engineered strain for producing glycine, characterized in that: The engineered strain is obtained by the following modification: (i) introducing exogenous acetyl-CoA synthetase gene acs, isocitrate lyase gene aceA and isocitrate dehydrogenase kinase gene aceK respectively; (ii) introducing the glutamate glyoxylate aminotransferase gene cgaT from quinoa; (iii) The DNA-binding transcriptional repressor gene iclR, glycine dehydrogenase gene gcvP, alanine aminotransferase gene alaA, malate dehydrogenase gene maeA, and malate synthase G gene glcB were knocked out respectively.

2. The method for constructing an engineered strain for producing glycine according to claim 1, characterized in that: The method construction method comprises: Constructing a host strain: replacing the pyruvate oxidase gene poxB in the Escherichia coli genome with the acetyl-CoA synthetase gene acs, and replacing the malate synthase gene aceB in the Escherichia coli genome with the isocitrate lyase gene aceA and the isocitrate dehydrogenase kinase gene aceK; The DNA binding transcription repressor gene iclR, the glycine dehydrogenase gene gcvP, the alanine aminotransferase gene alaA, the malate dehydrogenase gene maeA, and the malate synthase G gene glcB were knocked out to obtain the mutant Escherichia coli HG07; Construction of recombinant vector: inserting the exogenous glutamate glyoxylate aminotransferase gene cgaT into the NcoI and EcoRI sites of plasmid pRB1k to obtain the recombinant vector pG01; Construction of engineered strain: The above recombinant vector pG01 was introduced into the above mutant Escherichia coli HG07 to obtain the recombinant engineered strain SG0701.

3. Use of the engineered strain according to claim 1 in the preparation of glycine.

4. The method for preparing glycine by an engineered strain according to claim 1, characterized in that: The method comprises: S1: Strain expansion and induction: The recombinant engineered strain SG0701 was inoculated onto a LB solid plate containing kanamycin and placed in a 37°C incubator for 12-16 hours. A single colony was picked and inoculated into a LB liquid medium containing kanamycin for expansion. Subsequently, the colony was transferred to a ZYM autoinduction medium containing kanamycin at a volume ratio of 0.5-2% for autoinduction overnight. Finally, the OD was measured. 600 The light absorption value was collected and 3×10 10 -4×10 10 / mL bacteria; S2: Whole-cell catalytic transformation: The collected cells were centrifuged, washed with physiological saline, and the supernatant was discarded and the resulting cells were resuspended to obtain a final concentration of 3×10 10 -4×10 10 / mL of bacterial suspension; The bacterial suspension is mixed with 50-80 mM glucose, 40-64 mM NH4Cl, 2-4 mM MgSO4, 10-15 mM sodium glutamate and 1×M9 Salts to carry out a conversion reaction to obtain glycine.

5. The method according to claim 4, characterized in that In step S1, the expansion culture conditions are: temperature 25-42°C, rotation speed 100-300 rpm, and time 8-12 hours.

6. The method according to claim 4, characterized in that In step S1, the auto-induction culture conditions are: temperature 25-42°C, rotation speed 100-300 rpm.

7. The method according to claim 4, characterized in that In step S2, the centrifugation conditions are: temperature 4-15°C, rotation speed 4000-6000rpm, time 5-10min.

8. The method according to claim 4, characterized in that In step S2, the conversion reaction conditions are: temperature 25-42°C, rotation speed 100-300 rpm, and time 2 to 24 hours.

9. The method according to claim 4, characterized in that The kanamycin content is 50 μg / mL.

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