Genetically engineered bacterium for producing N-acetylneuraminic acid as well as construction and application of genetically engineered bacterium

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered bacterium for producing N-acetylneuraminic acid as well as construction and application of the genetically engineered bacterium. Accumulation of acetic acid is controlled by combining and applying different strategies: rational transformation is performed on an escherichia coli metabolic pathway, and acetic acid synthesis pathway related genes poxB, ackA and pta are knocked out to reduce acetic acid synthesis; the glyoxylic acid cycle is activated by knocking out the coding gene iclR of the glyoxylic acid cycle transcriptional regulation factor, and acetic acid overflow is reduced; the fermentation temperature is adjusted to control overall metabolism of cells to reduce acetic acid accumulation. Through comprehensive application of the strategy, the Neu5Ac production method capable of effectively controlling acetic acid accumulation is obtained, and the method has a good industrial application prospect.

Description

Technical Field The present invention belongs to the field of genetic engineering technology, and in particular relates to a genetically engineered bacterium for producing N-acetylneuraminic acid and its construction and application. Background Art N-acetylneuraminic acid (Neu5Ac), composed of α-ketoaldonic acid and a nine-carbon skeleton, is the most important sialic acid in the family of various natural sialic acids found in mammals.

[0001] Neu5Ac is usually fused to the non-reducing end of glycolipids and glycoproteins on the cell membrane and participates in cell recognition events and the regulation of various biological processes, including virus invasion, fertilization, inflammation, tumorigenesis, cell differentiation and cell adhesion. The N-acetyl group of N-acetylneuraminic acid is located at the 5 position and the chemical formula is C 11 H 19 NO9, usually located in the form of glycosides at the non-reducing end of glycoconjugates such as glycoproteins or glycolipids, is the basis for the structural and functional diversity of glycoconjugates. N-acetylneuraminic acid is widely used in the pharmaceutical and food industries as an intestinal antibacterial drug, antiviral drug, antioxidant, food ingredient, detoxifier, etc., and the market demand for it continues to grow. Studies have found that Neu5Ac can be safely used in infant formula and food ingredients to promote infant brain and bone development. N-acetylneuraminic acid has potential drug application prospects due to its anti-inflammatory, anticancer, antiviral, anti-adhesion properties and its function as a modified drug carrier.

[0002] Neu5Ac is widely distributed in animals, plants, and microorganisms. Methods for producing Neu5Ac primarily include extraction from natural raw materials, chemical synthesis, enzymatic synthesis, whole-cell catalysis, and microbial fermentation. Natural raw material extraction methods are limited by the extremely low Neu5Ac content in natural products, the complex extraction and processing process, low recovery rates, and the requirement for specialized equipment, making large-scale industrial production difficult. Chemical synthesis requires demanding reaction conditions and relies on expensive and hazardous metal catalysts, increasing production costs and significantly impacting the environment. Furthermore, the yield of this reaction is typically low, a factor that collectively hinders the large-scale industrial production of food-grade Neu5Ac. In enzymatic synthesis, the cumbersome enzyme preparation process, the requirement for expensive raw materials GlcNAc and pyruvate, and the necessary high-cost cofactor ATP significantly increase overall production costs. Therefore, while this method is suitable for producing high-purity Neu5Ac, its application in large-scale Neu5Ac synthesis is constrained by cost. Compared to enzymatic methods, whole-cell catalysis eliminates the complex cell lysis and enzyme purification processes. Furthermore, with the widespread application of genetic engineering techniques, the reaction rate and product yield during the catalytic process have been greatly improved. However, whole-cell catalytic production of Neu5Ac also has significant drawbacks, such as the high cost of substrates, difficulty in product isolation, and the need for multiple steps such as cell culture, collection, and resuspension, which increases production costs. These factors have collectively limited the application and promotion of this technology in the industrial production of Neu5Ac.

[0003] Compared to the four aforementioned methods, microbial fermentation allows for the de novo synthesis of Neu5Ac using lower-cost carbon sources (such as glucose and glycerol), avoiding the need for additional precursors and offering lower production costs. Furthermore, due to its environmentally friendly fermentation process, it exhibits significant potential for industrialization. Furthermore, microbial fermentation offers advantages such as mild reaction conditions and high purity, and the relatively mature fermentation process using low-cost raw materials holds promise for addressing social challenges such as resource shortages and environmental pollution. Therefore, the de novo synthesis of Neu5Ac by microbial fermentation is highly likely to meet the needs of large-scale industrial production.

[0004] During E. coli fermentation, the accumulation of acetic acid, a byproduct, significantly harms cell growth and product synthesis. High concentrations of acetic acid lower the intracellular pH, disrupt cell membrane integrity, and inhibit the activity of key enzymes, thereby affecting cell growth and metabolism. Furthermore, acetic acid interferes with protein synthesis and DNA replication, reducing cell viability and ultimately leading to decreased fermentation efficiency. When using E. coli to ferment Neu5Ac, large amounts of acetic acid, a byproduct, tend to accumulate. This not only has a serious toxic effect on E. coli growth but also leads to carbon source loss, affecting Neu5Ac production and conversion, thereby restricting the continued stability of the fermentation process. The problem of acetic acid accumulation is a bottleneck limiting the efficient synthesis of Neu5Ac in E. coli. Summary of the Invention To address the above problems, this application controls the accumulation of acetic acid by combining different strategies: rationally transforming the metabolic pathway of Escherichia coli and knocking out genes related to the acetic acid synthesis pathway. poxB , ackA , pta Reduce acetate synthesis; Knockout genes encoding glyoxylate cycle transcriptional regulators iclR By activating the glyoxylate cycle to reduce acetate overflow and adjusting the fermentation temperature to control overall cell metabolism to reduce acetate accumulation, the combined application of these strategies resulted in a Neu5Ac production method that effectively controls acetate accumulation and has promising industrial application prospects.

[0005] One of the technical solutions provided by the present invention is a method for improving the ability of Escherichia coli to produce Neu5Ac, wherein the method is to weaken the acetic acid synthesis related genes in the Neu5Ac production strain. poxB , ackA , pta and glyoxylate cycle transcriptional regulator genes iclR to achieve the expression; described poxB Gene encoding pyruvate oxidase; pta Gene encoding phosphotransacetylase; ackA Gene encoding acetate kinase; iclR Gene encoding a glyoxylate cycle transcriptional regulator; Furthermore, the methods of reducing the expression of the gene include but are not limited to knocking out, inhibiting or inactivating the gene; preferably, the gene is knocked out; Furthermore, the production of Neu5Ac by the gene-edited strain above at 30°C can further increase the yield.

[0006] The second technical solution provided by the present invention is an engineered Escherichia coli strain that produces Neu5Ac. The engineered strain is a strain of Neu5Ac-producing strains. poxB ,ackA , pta as well as iclR Gene deletion expression is obtained; Furthermore, the pyruvate oxidase encoding gene poxB , the nucleotide sequence is shown in SEQ ID NO.1.

[0007] Furthermore, the phosphotransacetylase encoding gene pta , the nucleotide sequence is shown in SEQ ID NO.2.

[0008] Furthermore, the gene encoding acetate kinase ackA , the nucleotide sequence is shown in SEQ ID NO.3.

[0009] Furthermore, genes encoding glyoxylate cycle transcriptional regulators iclR , the nucleotide sequence is shown in SEQ ID NO.4.

[0010] Furthermore, the Neu5Ac producing strain is Escherichia coli; Furthermore, the Neu5Ac-producing strains include but are not limited to: E.coli W3110 、E.coli W3110NEA-1, E.coli MG1655, etc. Furthermore, the strain E. coli W3110 NEA-1 is based on Escherichia coli and has a xylose-inducible promoter P xylF The controlled RNA polymerase from T7 phage can activate the strong expression system through xylose induction, achieving controllable enhancement of the target gene; sugar metabolism transcription repressor mutant mlc* Gene replacement mlc gene; on this basis, knocked out nagA, nagB, nagC, nagE, manX, manY, manZ Gene, blocking the GlcNAc catabolic pathway; single copy of the glucosamine-6-phosphate N-acetyltransferase gene in the genome Sc-gna1 , two copies of the fructose-6-phosphate aminotransferase gene glmS , a single-copy phosphotransferase gene yqaB , enhances the synthesis of ManNAc, the precursor required for Neu5Ac; single copy N-acetylglucosamine 2-epimerase gene bAGE , two copies of N-acetylneuraminic acid synthetase gene neuB , construct the Neu5Ac synthesis pathway; and knock out Neu5Ac catabolism-related genes nanATEK , achieved the de novo synthesis of Neu5Ac in E. coli; knocked out genes related to the PTS system ptsG, eliminating the glucose-specific PTS system and integrating the glucose facilitator transporter gene glf , Escherichia coli glucokinase gene glk , strengthen the supply of phosphoenolpyruvate, the precursor required for the synthesis of Neu5Ac; on this basis, knock out pyruvate kinase pykA , further strengthening the accumulation of phosphoenolpyruvate. E. coli The W3110 NEA-1 strain and its construction method have been disclosed in Example 1 of Chinese Patent ZL202110973426.5 and are designated as W3110 NEA-1 in this application. E.coli W3110 NEA-1.

[0011] The third technical solution provided by the present invention is the use of the engineered bacteria described in the second technical solution in the production of Neu5Ac; Furthermore, the engineered bacteria were used to produce and accumulate Neu5Ac at 30°C; Furthermore, the shake flask fermentation method for producing Neu5Ac using the engineered bacteria is as follows: Shake flask fermentation: Inoculate the seed liquid into the fermentation medium at an inoculum rate of 8-15%, and shake the culture at 28-37 ° C and 180-240 r / min. During the fermentation process, add ammonia water to maintain the pH at 6.8-7.2. When the pH does not decrease slowly or even increases, it indicates that the bacteria are lacking sugar. Glucose solution is added to maintain the fermentation. The fermentation cycle is 24-36 hours. Further, using E.coli When W3110 NEA-1 was used as the base strain, the Neu5Ac yield reached 12.5-18.1 g / L, and the production intensity could reach 0.44-0.58 g / (L×h); Furthermore, the engineered bacteria has the best effect in producing Neu5Ac when fermented at 30°C. Furthermore, the fermentation method for producing Neu5Ac using the engineered bacteria is as follows: Fermentation culture in fermenter: OD of seed culture 600 When the value increases to between 10 and 18, 10-20% of the volume is transferred to the fermentation medium for subsequent fermentation experiments. During the fermentation process, the pH is maintained at 7.0-7.2, the temperature is maintained at 28°C-37°C, and the dissolved oxygen is controlled between 25% and 45%. When the glucose in the fermentation tank is completely consumed, glucose solution is added at a certain rate. During this period, the glucose concentration in the tank is controlled between 0.1-5 g / L. The fermentation cycle is about 36h-58h. Furthermore, the engineered bacteria has the best effect in producing Neu5Ac when fermented at 30°C. After 36-58 h of fermentation, the fermentation yield of Neu5Ac in a 5 L fermenter reached 28-70 g / L, and the production intensity could reach 0.7-1.2 g / (L×h).

[0012] The fermentation medium comprises: 15-30 g / L glucose, 5-20 g / L xylose, 2-5 g / L yeast extract, 2-10 g / L (NH4)2SO4, 4-10 g / L KH2PO4, 2-8 g / L MgSO4·7H2O, 0.5-3 g / L NaCl, 5-30 mg / L FeSO4·7H2O, 1-5 mg / L MnSO4·7H2O, 15-30 mg / L CaCl2·2H2O, V H 0.05-2 mg / L, V B1 0.1-2 mg / L, trace element mixture 1-3 mL / L, phenol red indicator 1-3%, defoamer 1-2 drops, the remainder is water, pH 6.8-7.2. Autoclave at 121°C for 20 min. The composition of the trace element mixture is: Na2MoO4·2H2O 1-3 g / L, NiCl2·6H2O 0.5-1.5 g / L, CaCl2·2H2O 2-8 g / L, CuSO4·5H2O 0.1-0.5 g / L, Al2(SO4)3·18H2O 1-1.5 g / L, CoCl2·6H2O 0.5-1.5 g / L, ZnSO4·2H2O 0.1-0.5 g / L, H3BO30.05-0.2 g / L, and the rest is water.

[0013] Beneficial effects: poxB The gene encodes pyruvate oxidase, which catalyzes pyruvate to produce acetate; pta Gene encoding phosphotransacetylase and ackA The gene encodes acetate kinase, which converts acetyl-CoA into acetate in two steps. E.coli W3110 NEA-1 For the starting strain, knockout pta After knocking out the gene, the amount of acetate accumulated decreased by 21.8%. poxB and ackA The acetic acid content of the strain decreased significantly compared with pta knockout strain, decreased by 72.8%. poxB Gene, pta Gene, ackA Genes can effectively reduce acetic acid accumulation. At the same time, in order to dredge the downstream carbon flux and avoid carbon source overflow, by knocking out iclRThe gene activated the glyoxylate cycle, which enhanced growth and productivity. A fermentation process protocol was developed by adjusting the fermentation temperature between 28°C and 37°C, ultimately determining the optimal fermentation temperature at 30°C. This resulted in a high-yielding strain of N-acetylneuraminic acid (N-acetylneuraminic acid). After 52 hours of fermentation in a 2-L fermentor, Neu5Ac production reached a maximum of 40 g / L. At this temperature, after 58 hours of fermentation in a 5-L fermentor, Neu5Ac production reached a maximum of 70 g / L, demonstrating promising industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : pta Construction of gene knockout fragments and electrophoresis verification Wherein: M: 1kb DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: overlapping fragment; 4: positive bacteria identification fragment; 5: original bacteria genome PCR fragment.

[0015] Figure 2 : iclR Construction of gene knockout fragments and electrophoresis verification Wherein: M: 1kb DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: overlapping fragment; 4: positive bacteria identification fragment; 5: original bacteria genome PCR fragment. DETAILED DESCRIPTION

[0016] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention.

[0017] In a first aspect, the present invention provides a method for improving the ability of Escherichia coli to produce Neu5Ac, wherein the method is to knock out the acetic acid synthesis-related genes in the Neu5Ac production strain. poxB , ackA , ​ ; Simultaneously knock out the glyoxylate cycle transcriptional regulator gene ​ To activate the glyoxylate cycle, regulate the metabolism of the central carbon metabolic pathway, and adjust the fermentation temperature to finally determine the optimal fermentation temperature as 30℃.

[0018] In a second aspect, the present invention provides a strain of genetically engineered Escherichia coli with high Neu5Ac production, wherein the engineered strain is a strain in which genes related to acetic acid synthesis in Escherichia coli with the ability to produce Neu5Ac are transformed.​ 、 ​ Knockout, and further knock out the glyoxylate cycle transcriptional regulator gene ​ Obtained by knockout.

[0019] Furthermore, the method for improving the Neu5Ac production ability of Escherichia coli provided by the present invention can be used in strains with Neu5Ac production ability; Furthermore, the Neu5Ac-producing strains include but are not limited to: ​ W3110 ​ W3110NEA-1 ​ MG1655, etc. Wherein, the Escherichia coli having the ability to produce Neu5Ac is ​ W3110 NEA-1, which has been disclosed in Example 1 of Chinese Patent ZL202110973426.5, is named in this application ​ ; The strain ​ It is based on Escherichia coli and has a xylose-inducible promoter P xylF The controlled RNA polymerase from T7 phage can activate the strong expression system through xylose induction, achieving controllable enhancement of the target gene; sugar metabolism transcription repressor mutant ​ Gene replacement ​ gene; on this basis, knocked out ​ ​ Gene, blocking the GlcNAc catabolic pathway; single copy of the glucosamine-6-phosphate N-acetyltransferase gene in the genome ​ , two copies of the fructose-6-phosphate aminotransferase gene ​ , a single-copy phosphotransferase gene ​ , enhances the synthesis of ManNAc, the precursor required for Neu5Ac; single copy N-acetylglucosamine 2-epimerase gene ​ , two copies of N-acetylneuraminic acid synthetase gene ​ , construct the Neu5Ac synthesis pathway; and knock out Neu5Ac catabolism-related genes ​ , achieved the de novo synthesis of Neu5Ac in E. coli; knocked out genes related to the PTS system ​ , eliminating the glucose-specific PTS system and integrating the glucose facilitator transporter gene ​ , Escherichia coli glucokinase gene ​ , strengthen the supply of phosphoenolpyruvate, the precursor required for the synthesis of Neu5Ac; on this basis, knock out pyruvate kinase ​ , further enhancing the accumulation of phosphoenolpyruvate.

[0020] In a third aspect, the present invention provides a method for producing Neu5Ac using the genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria in a culture medium to produce Neu5Ac; and using a shake flask method or a fermentation tank method to produce Neu5Ac using the genetically engineered bacteria.

[0021] The present invention will be further explained below through specific examples.

[0022] Example 1: Genetically engineered bacteria with high Neu5Ac production ​ W3110NEA-1 - Δ ​ Δ ​ Δ ​ -Δ ​ Construction CRISPR / Cas9 gene editing technology is used to modify genes. The gene editing method used in this invention is based on the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of ​ ​ using CRISPR–Cas9 meditated genome editing. Metabolic engineering, 2015,31: 13-21.).

[0023] Some of the experimental methods involved are as follows: (1) Construction of pGRB plasmid: CRISPR RGEN Tools were used to design a target sequence (PAM: 5'-NGG-3') for cleaving the target gene. After synthesizing the forward and reverse complementary primers, 10 μL of each was added to a PCR tube. After mixing, single-stranded DNA was annealed to generate a DNA fragment containing the target sequence. Reaction conditions included initial denaturation at 95°C for 5 minutes and annealing at 50°C for 1 minute. The resulting DNA fragment was then ligated with the linearized pGRB vector obtained by inverse PCR via homologous recombination to generate the pGRB plasmid. The homologous recombination kit used was the ClonExpress® II OneStep Cloning Kit.

[0024] (2) Construction of overlapping DNA fragments for replacement: The recombinant DNA fragment required to knock out the target gene is composed of two overlapping fragments: the upstream and downstream homology arms of the target gene. The recombinant fragment required to integrate the target gene is composed of three overlapping fragments: the upstream and downstream homology arms of the integration site gene, and the target gene. Using the upstream and downstream sequences of the gene to be knocked out or the integration site of the target gene as templates, primers for the upstream and downstream homology arms are designed, typically with homology arms around 500 bp in length. Primers for amplifying the integration gene are designed using the gene to be integrated as a template. After amplifying the upstream and downstream homology arms and the target gene fragments separately by PCR, overlapping PCR is performed to prepare the recombinant fragments.

[0025] (3) Preparation of competent cells: At 37°C and 220 rpm, culture the cells in a triangular flask containing 100 mL of 2×YT medium until the OD 600 =0.4-0.6, the competent state was prepared. When the cells carried the pREDCas9 plasmid, the culture temperature was adjusted to 32°C, and when the bacterial OD 600 =0.1-0.2, add 0.1M IPTG. The preparation process refers to conventional standard operation.

[0026] (4) Transformation of pGRB plasmid and recombinant DNA: Simultaneously electroporate the pGRB plasmid and the overlapping DNA fragments into electrocompetent cells containing pREDCas9. Resuscitate the electroporated cells for 2 hours, then plate them onto LB plates containing ampicillin and spectinomycin and incubate them overnight at 32°C. Perform colony PCR verification using an upstream primer for the upstream homology arm and a downstream primer for the downstream homology arm, or design specialized identification primers, to screen for positive recombinants.

[0027] (5) Elimination of plasmids: Inoculate the resulting positive recombinants into LB shake tubes containing 0.2% L-arabinose and spectinomycin resistance and incubate at 32°C for approximately 12 hours. Dip the bacterial solution into three zones on a spectinomycin resistance LB plate and continue incubation. Use a toothpick to pick individual colonies from the three zones and spot them onto LB plates containing spectinomycin resistance and ampicillin resistance, respectively, and continue incubation. Select the colonies that grow on the spectinomycin resistance plate but not on the ampicillin plate to identify the recombinant strains that have eliminated the pGRB plasmid. Transfer the positive recombinants to LB liquid medium without resistance and incubate at 42°C for approximately 12 hours. Dip the bacterial solution into three zones on a plate without resistance and continue incubation. Use a toothpick to pick individual colonies from the three zones and spot them onto LB plates containing spectinomycin resistance and ampicillin resistance, respectively, and continue incubation. Select the colonies that grow on the plate without resistance but not on the spectinomycin plate to identify the recombinant strains that have eliminated the pREDas9 plasmid.

[0028] The specific method for strain construction is as follows: (1) Genes related to acetate synthesis in Neu5Ac-producing Escherichia coli ​ 、 ​ Knock out.

[0029] Escherichia coli ( ​ W3110) genome as a template, according to its ​ The upstream and downstream sequences of the gene were designed with upstream homology arm primers pta-UF and pta-UR and downstream homology arm primers pta-DF and pta-DR. The upstream and downstream homology arms were first obtained by PCR, and then overlapping fragments (pta-U—pta-D) were obtained by overlapping PCR. The appropriate gRNA sequence was found using the gRNA search tool (http: / / www.rgenome.net / cas-designer), and the gRNA-pta-S and gRNA-pta-A sequences were synthesized. The two single-stranded primers were complementary paired by PCR annealing to obtain double-stranded gRNA-pta, which was homologously recombined with the pGRB linearized vector to obtain pGRB-pta. The overlapping fragments and pGRB-pta were electroporated into a pREDCas9 vector. ​ ​ In competent cells, the revived cells after electroporation were plated on LB plates containing ampicillin and spectinomycin. After overnight culture at 32°C, positive recombinants were verified by colony PCR. Then, the pGRB-pta used for gene editing was eliminated, and finally a successful knockout was achieved. ​ Positive strains ​ Δ ​ .

[0030] Knock out in the same way ​ Genes and ​ Gene (related primers are shown in Table 1), blocking the acetate synthesis pathway. ​ and ​ strains ​ Δ ​ Δ ​ -Δ ​

[0031] ​ The construction of gene knockout fragments and electrophoresis verification are shown in the attached figure. ​ Among them: M: 1kb DNA marker; 1: upstream homology arm 459 bp; 2: downstream homology arm 858 bp; 3: overlapping fragment 1314 bp; 4: positive bacteria identification fragment 1314 bp; 5: original bacteria genome PCR fragment 2783 bp.

[0032] (2) Knockout of glyoxylate cycle transcriptional regulator genes ​Activates the glyoxylate cycle and regulates the metabolism of the central carbon metabolism pathway.

[0033] Escherichia coli ( ​ W3110) genome as a template, according to its ​ The upstream homology arm primers iclR-UF and iclR-UR and the downstream homology arm primers iclR-DF and iclR-DR were designed for the upstream and downstream sequences of the gene. The upstream and downstream homology arms were first obtained by PCR, and then the overlapping fragments (iclR-U—iclR-D) were obtained by overlapping PCR. The appropriate gRNA sequence was found using the gRNA search tool (http: / / www.rgenome.net / cas-designer), and the gRNA-iclR-S and gRNA-iclR-A sequences were synthesized. The two single-stranded primers were complementary paired by PCR annealing to obtain double-stranded gRNA-iclR, which was homologously recombined with the pGRB linearized vector to obtain pGRB-iclR. The overlapping fragments and pGRB-iclR were electroporated into the knockout vector containing pREDCas9. ​ 、 ​ In the competent cells, the revived bacteria after electroporation were spread on LB plates containing ampicillin and spectinomycin. After overnight culture at 32°C, positive recombinants were verified by colony PCR, and then pGRB-iclR used for gene editing was eliminated, and finally a successful knockout was achieved. ​ 、 ​ strains ​ Δ ​ Δ ​ Δ ​ -Δ ​ .

[0034] ​ The construction of gene knockout fragments and electrophoresis verification are shown in the attached figure. ​ Among them: M: 1kb DNA marker; 1: upstream homology arm 507 bp; 2: downstream homology arm 549 bp; 3: overlapping fragment 1019 bp; 4: positive bacteria identification fragment 1019 bp; 5: original bacteria genome PCR fragment 1482 bp.

[0035] Detection of starting strains ​ Process strains ​ -Δ ​ Process strains ​ Δ ​ Δ ​ -Δ ​ and strains ​ Δ ​ Δ ​ Δ ​ -Δ ​ To determine the acetic acid accumulation, the experiment was set up as follows: Shake flask fermentation was used to verify the acetic acid accumulation of the above strains.

[0036] (1) Activated slant culture: Use an inoculation loop to inoculate 2 loops of bacteria from a -80°C freezer tube, evenly spread on the slant culture medium, culture at 36°C for 13 h, transfer to the second generation slant culture medium, and culture at 36°C for 13 h; (2) Seed bottle culture: Under strict aseptic conditions, first add 1 mL of 60% sterile glucose solution as a base sugar to a 500 mL Erlenmeyer flask containing 29 mL of seed culture medium. Use a sterile inoculation loop to gently scrape 2 loops of bacteria from the cultured second-generation slant and seal the flask with nine layers of gauze. Shake and culture at 37°C, 220 rpm for 12 h until the OD600 is about 5. The pH is maintained at 7.0 during the culture process. (3) Shake flask fermentation: Inoculate the seed liquid into the fermentation medium at a 10% inoculum volume to a final volume of 30 mL. Seal the flask with nine layers of gauze and shake culture at 37°C and 220 r / min. During the fermentation process, add ammonia water to maintain the pH at 7.0. When the pH does not decrease slowly or even increases as observed by the phenol red indicator, indicating that the bacteria are lacking sugar, add 1 mL of 60% (m / v) glucose solution.

[0037] The slant culture medium consists of 7 g / L yeast powder, 14 g / L peptone, 8 g / L NaCl, 6 g / L beef extract, 2 g / L sucrose, 25 g / L agar powder, and the rest is water, pH 7.0.

[0038] The composition of the seed culture medium is as follows: glucose 20 g / L, yeast powder 4 g / L, (NH4)2SO4 3 g / L, KH2PO4 5 g / L, MgSO4·7H2O 2 g / L, FeSO4·7H2O 3 mg / L, MnSO4·7H2O 4.5 mg / L, V H 3 mg / L, V B1 2 mg / L, trace element mixture 2 mL / L, 2 drops of defoamer, and the rest is water, pH 7.1.

[0039] The fermentation medium composition is as follows: glucose 15 g / L, xylose 20 g / L, yeast powder 4 g / L, (NH4)2SO4 8 g / L, KH2PO4 4 g / L, MgSO4·7H2O 6 g / L, NaCl 2 g / L, FeSO4·7H2O 25 mg / L, MnSO4·7H2O 4 mg / L, CaCl2·2H2O 20 mg / L, V H 2mg / L, V B11mg / L, trace element mixture 2mL / L, phenol red indicator 1%, 2 drops of defoaming agent, and the rest is water, pH 6.8-7.2.

[0040] The components of the trace element mixture are: Na2MoO4·2H2O 2g / L, NiCl2·6H2O 1g / L, CaCl2·2H2O5g / L, CuSO4·5H2O 0.1g / L, Al2(SO4)3·18H2O 1g / L, CoCl2·6H2O 1.5g / L, ZnSO4·2H2O0.5g / L, H3BO30.2g / L, and the rest is water.

[0041] The fermentation period was 30 h, and the acetic acid accumulation of each strain was shown in the following table.

[0042]

[0043] Table 1 Primers involved in strain construction

[0044] Example 2: ​ Construction of Genetically Engineered Bacteria Producing Neu5Ac in MG1655 Chassis In this embodiment, by ​ The ptrc-99A plasmid (containing the glucosamine-6-phosphate N-acetyltransferase gene) was introduced into ​ , fructose-6-phosphate aminotransferase gene ​ , phosphotransferase gene ​ , N-acetylglucosamine 2-epimerase gene ​ , N-acetylneuraminic acid synthetase gene ​ Fragment), constructing a genetically engineered bacterium producing Neu5Ac ​ -ptrc- ​ .

[0045] Further adopt the operating method in Example 1, ​ -ptrc- ​ strains ​ 、 ​ 、 ​ 、 ​ Knockout was performed to obtain strains ​ ptrc-NEA ​ ​ ptrc-NEA ​ -ptrc-NEA-Δ ​ -Δ ​ -Δ ​ -Δ ​ .

[0046] The strains were subjected to Neu5Ac shake flask fermentation and acetic acid accumulation verification using the same method as in Example 1.

[0047] The fermentation period was 30 h and the accumulation of acetic acid was shown in the following table.

[0048]

[0049] It can be seen that by knocking out ​ 、 ​ The effect of reducing acetic acid accumulation can be achieved in different strains.

[0050] Example 3: Method for producing Neu5Ac using the strains constructed in Examples 1 and 2 above as production strains (1) Activated slant culture: Use an inoculation loop to inoculate 2 loops of bacteria from a -80°C freezer tube, evenly spread on the slant culture medium, culture at 36°C for 13 h, transfer to the second generation slant culture medium, and culture at 36°C for 13 h; (2) Seed bottle culture: Under strict aseptic conditions, first add 1 mL of 60% sterilized glucose solution as the base sugar to a 500 mL triangular flask containing 29 mL of seed culture medium. Use a sterile inoculation loop to gently scrape 2 loops of bacteria from the cultured second-generation slant and seal the flask with nine layers of gauze. Shake and culture at 37°C and 220 rpm for 12 h until the OD reaches 0. 600 5, and the pH was maintained at 7.0 during the culture process; (3) Shake flask fermentation: Inoculate the seed liquid into the fermentation medium at a 10% inoculum volume to a final volume of 30 mL. Seal the flask with nine layers of gauze and shake culture at 37°C and 220 r / min. During the fermentation process, add ammonia water to maintain the pH at 7.0. When the pH does not decrease slowly or even increases as observed by the phenol red indicator, indicating that the bacteria are lacking sugar, add 1 mL of 60% (m / v) glucose solution.

[0051] The slant culture medium consists of 7 g / L yeast powder, 14 g / L peptone, 8 g / L NaCl, 6 g / L beef extract, 2 g / L sucrose, 25 g / L agar powder, and the rest is water, pH 7.0.

[0052] The composition of the seed culture medium is as follows: glucose 20 g / L, yeast powder 4 g / L, (NH4)2SO4 3 g / L, KH2PO4 5 g / L, MgSO4·7H2O 2 g / L, FeSO4·7H2O 3 mg / L, MnSO4·7H2O 4.5 mg / L, V H 3 mg / L, V B12 mg / L, trace element mixture 2 mL / L, 2 drops of defoamer, and the rest is water, pH 7.1.

[0053] The fermentation medium composition was as follows: glucose 20 g / L, xylose 20 g / L, yeast powder 4 g / L, (NH4)2SO4 10 g / L, KH2PO4 8 g / L, MgSO4·7H2O 5 g / L, NaCl 0.5 g / L, FeSO4·7H2O 20 mg / L, MnSO4·7H2O 4 mg / L, CaCl2·2H2O 20 mg / L, V H 1 mg / L, V B1 0.5 mg / L, trace element mixture 2 mL / L, phenol red indicator 1%, 2 drops of defoaming agent, and the rest is water, pH 7.0.

[0054] The components of the trace element mixture are: Na2MoO4·2H2O 1 g / L, NiCl2·6H2O 1.5 g / L, CaCl2·2H2O 8 g / L, CuSO4·5H2O 0.5 g / L, Al2(SO4)3·18H2O 1.5 g / L, CoCl2·6H2O 1 g / L, ZnSO4·2H2O 0.5 g / L, H3BO3 0.2 g / L, and the rest is water.

[0055] The shake flask fermentation cycle was 30 h, and the Neu5Ac yield and production intensity indicators are shown in the following table.

[0056]

[0057] As can be seen from the above table, E.coli W3110 NEA-1- Δ iclR- Δ poxB- Δ ackA -Δ pta After 30h of shake flask fermentation, the yield of Neu5Ac reached 17.5 g / L and the production intensity reached 0.58 g / (L×h). E.coli W3110 NEA-1 was compared to the strain in this application E.coli W3110 NEA-1- ΔiclR-ΔpoxB-Δ ackA-Δpta Neu5Ac production increased by 54.8% and production intensity increased by 52.6%. MG1655 The latter also has similar performance.

[0058] It can be seen that the genetic modification performed in the present invention is beneficial to the improvement of Neu5Ac production.

[0059] Example 4: Method for producing Neu5Ac using the strains constructed in Examples 1 and 2 above as production strains (1) Slant activation culture: Under strict aseptic conditions, use a sterilized inoculation loop to take out the microbial strains stored in glycerol at -80°C, and evenly and densely streak the bacterial liquid on the solid culture medium slant. Incubate at 37°C for about 12 hours to obtain the primary colony slant. Then, use the same method to inoculate the new eggplant-shaped flask solid culture medium slant to obtain the second-generation colony slant. (2) Seed tank culture: First, in a sterile clean bench, add 100 mL of sterile deionized water to the eggplant-shaped flask to elute the bacteria. Then, using flame inoculation, quickly transfer the seed substrate and bacterial liquid into the pre-prepared seed fermentation tank. The final volume of the fermentation tank is 3 L. The culture temperature is 37 ° C, the pH is 7.0, and the rotation speed is 200 r / min. To ensure normal growth of the bacteria, adjust the stirring speed or ventilation volume in time to ensure that the dissolved oxygen level is maintained at 40%; (3) Fermentation culture in fermenter: OD of seed culture medium 600 When the value reached 13, the fermentation medium was transferred to a 5-L fermentor containing 3 L of fermentation medium at a 12% volume ratio for subsequent fermentation experiments. During fermentation, the pH was maintained at 7.0, the temperature was maintained at 37°C, and the dissolved oxygen level was controlled at 30%. Once the glucose in the fermentor was completely consumed, an 80% glucose solution was added at a constant rate, maintaining the glucose concentration in the tank at 0.5 g / L.

[0060] The fermentation cycle was 42 h, and the yield and production intensity indicators of Neu5Ac were shown in the following table.

[0061]

[0062] Under the same fermentation conditions, the E.coli W3110 Compared with NEA-1, in this application E.coli W3110 NEA-1 - Δ iclR- Δ poxB- Δ ackA -Δ pta The Neu5Ac yield increased by 39.3% and the production intensity increased by 38.8%. MG1655 The latter also has similar performance.

[0063] It can be seen that the genetic modification performed in the present invention is beneficial to the improvement of Neu5Ac production.

[0064] The components of the slant culture medium are: peptone 12 g / L, yeast powder 3 g / L, NaCl 8 g / L, beef extract 15 g / L, sucrose 0.8 g / L, agar powder 20 g / L, and the rest is water, pH 6.8-7.2.

[0065] The composition of the seed culture medium is as follows: glucose 20 g / L, yeast powder 5 g / L, (NH4)2SO4 3 g / L, KH2PO4 3 g / L, MgSO4·7H2O 2.5 g / L, FeSO4·7H2O 3 mg / L, MnSO4·7H2O 4 mg / L, V H 0.05mg / L, V B1 0.5mg / L, trace element mixture 2mL / L, 2 drops of defoaming agent, the rest is water, pH 6.8-7.2.

[0066] The fermentation medium composition is as follows: glucose 25 g / L, xylose 15 g / L, yeast powder 5 g / L, (NH4)2SO4 7 g / L, KH2PO4 10 g / L, MgSO4·7H2O 5 g / L, NaCl 3 g / L, FeSO4·7H2O 30 mg / L, MnSO4·7H2O 3 mg / L, CaCl2·2H2O 30 mg / L, V H 0.5mg / L, V B1 1mg / L, trace element mixture 2mL / L, phenol red indicator 1%, 2 drops of defoaming agent, the rest is water, pH 6.8-7.2.

[0067] The components of the trace element mixture are: Na2MoO4·2H2O 3g / L, NiCl2·6H2O 1.5g / L, CaCl2·2H2O8g / L, CuSO4·5H2O 0.5g / L, Al2(SO4)3·18H2O 1.5g / L, CoCl2·6H2O 1.5g / L, ZnSO4·2H2O0.5g / L, H3BO30.05g / L, and the rest is water.

[0068] Example 5: Method for producing Neu5Ac using the strains constructed in Examples 1 and 2 above as production strains The fermentation temperature of the Neu5Ac-producing strain was adjusted in a 2 L quadruple fermenter, and the effects of fermentation temperatures of 28°C, 30°C, 32°C, 35°C, and 37°C on Neu5Ac production were tested respectively.

[0069] (1) Slant activation culture: Under strict aseptic conditions, use a sterilized inoculation loop to take out the microbial strains stored in glycerol at -80°C, and evenly and densely streak the bacterial liquid on the solid culture medium slant. Incubate at 37°C for about 12 hours to obtain the primary colony slant. Then, use the same method to inoculate the new eggplant-shaped flask solid culture medium slant to obtain the second-generation colony slant. (2) Seed tank culture: First, in a sterile clean bench, add 100 mL of sterile deionized water to the eggplant-shaped bottle to elute the bacteria. Then, use flame inoculation to quickly transfer the seed substrate and bacterial liquid into the pre-prepared seed fermentation tank. The culture temperature is 37 ° C, pH 7.0, and the rotation speed is 200 r / min. To ensure normal growth of the bacteria, adjust the stirring speed or ventilation volume in time to ensure that the dissolved oxygen level is maintained at 40%; (2) Fermentation culture in fermenter: OD of seed culture medium 600 When the value increased to 10, the fermented liquid was transferred to a 2-L fermentor at a volume ratio of 15%, with a final volume of 600 mL per fermentor. The fermentation temperatures were set at 28°C, 30°C, 32°C, 35°C, and 37°C, respectively. The pH was maintained at 7.0, the dissolved oxygen was controlled at 40%, and the initial rotational speed was 200 r / min. After the glucose in the fermentor was completely consumed, an 80% glucose solution was added at a constant rate, maintaining the glucose concentration at 1 g / L. The fermentation cycle lasted 52 hours. The Neu5Ac yield, acetic acid accumulation, conversion rate, and production intensity indicators are shown in the table below.

[0070]

[0071] Fermentation in a 2-L quadruple fermenter revealed that temperature further affected Neu5Ac production, with significant differences in yield at different temperatures. Neu5Ac yield exhibited nonlinear changes at 28°C, 30°C, 32°C, 35°C, and 37°C, with the highest yield not occurring at its optimal growth temperature of 37°C.

[0072] It is known in the art that during the fermentation process using E. coli, E. coli When BL 21 strain is used as the chassis, the target gene is usually placed in a plasmid vector for overexpression. Excessive fermentation temperature often leads to overexpression of protein, which in turn causes the protein to form insoluble inclusion bodies, affecting the catalytic efficiency. Therefore, a temperature of about 30°C is usually used for fermentation. E. coli BL 21 strain fermentation; E.coli W3110 and E.coliMG1655 chassis, when the target gene is expressed in the genome, the copy number is generally less than the copy number in the plasmid vector, and the optimal growth temperature of 37°C is usually used as the fermentation temperature. E.coli W3110 commonly used fermentation temperature is different, when the fermentation temperature is 30 ℃, E.coli W3110 WHAT- 1- Δ iclR- Δ poxB- Δ ackA -Δ pta The fermentation effect of Neu5Ac in the 2000-0100 flavonoids was the best, with a yield of 40 g / L, a conversion rate of 20.3%, and a production intensity of 0.77 g / (L×h). E.coli The engineered strain MG1655 also reached its highest Neu5Ac production at 30°C. These results indicate that fermentation temperature significantly influences Neu5Ac production and acetic acid accumulation. At 30°C, production significantly increased compared to other temperatures, while acetic acid accumulation significantly decreased. Conversely, conversion and production intensity indicators also significantly improved.

[0073] The above results show that the expression of key enzymes in the engineered strain constructed in the present invention is most suitable at 30°C, the intracellular metabolism is most coordinated, and the fermentation effect of high yield and low acetic acid is presented.

[0074] The components of the slant culture medium are: peptone 12 g / L, yeast powder 3 g / L, NaCl 8 g / L, beef extract 15 g / L, sucrose 0.8 g / L, agar powder 20 g / L, and the rest is water, pH 6.8-7.2.

[0075] The composition of the seed culture medium is as follows: glucose 20 g / L, yeast powder 5 g / L, (NH4)2SO4 3 g / L, KH2PO4 3 g / L, MgSO4·7H2O 2.5 g / L, FeSO4·7H2O 3 mg / L, MnSO4·7H2O 4 mg / L, V H 0.05mg / L, V B1 0.5mg / L, trace element mixture 2mL / L, 2 drops of defoaming agent, the rest is water, pH 6.8-7.2.

[0076] The fermentation medium composition is as follows: glucose 22 g / L, xylose 14 g / L, yeast powder 4 g / L, (NH4)2SO4 6 g / L, KH2PO4 7 g / L, MgSO4·7H2O 6 g / L, NaCl 2.5 g / L, FeSO4·7H2O 22 mg / L, MnSO4·7H2O 3 mg / L, CaCl2·2H2O 20 mg / L, V H 1.5mg / L, V B11.5 mg / L, trace element mixture 2 mL / L, phenol red indicator 1%, 2 drops of defoaming agent, and the rest is water, pH 6.8-7.2.

[0077] The components of the trace element mixture are: Na2MoO4·2H2O 3g / L, NiCl2·6H2O 1.5g / L, CaCl2·2H2O8g / L, CuSO4·5H2O 0.5g / L, Al2(SO4)3·18H2O 1.5g / L, CoCl2·6H2O 1.5g / L, ZnSO4·2H2O0.5g / L, H3BO30.05g / L, and the rest is water.

[0078] Example 6: Utilization E.coli W3110 NEA-1- Δ iclR- Δ poxB- Δ ackA -Δ pta Method for producing Neu5Ac as a production strain (1) Slant activation culture: Under strict aseptic conditions, use a sterilized inoculation loop to take out the microbial strains stored in glycerol at -80°C, and evenly and densely streak the bacterial liquid on the solid culture medium slant. Incubate at 37°C for about 12 hours to obtain the primary colony slant. Then, use the same method to inoculate the new eggplant-shaped flask solid culture medium slant to obtain the second-generation colony slant. (2) Seed tank culture: First, in a sterile clean bench, add 100 mL of sterile deionized water to the eggplant-shaped bottle to elute the bacteria. Then, use flame inoculation to quickly transfer the seed base sugar and bacterial liquid into the pre-prepared seed tank. The final volume of the seed tank is 3 L. The initial seed culture temperature is 35 ° C, pH 7.0, and the rotation speed is 200 r / min. To ensure normal growth of the bacteria, adjust the stirring speed or ventilation volume in time to ensure that the dissolved oxygen level remains within 40%; (3) Fermentation culture in fermenter: OD of seed culture medium 600 When the value increased to 15, the fermentation medium was transferred at a 10% volume ratio to a 5-L fermentor containing 3 L of fermentation medium for subsequent fermentation experiments. During fermentation, the pH was maintained at 7.0, the temperature was maintained at 30°C, and the dissolved oxygen content was controlled at approximately 35%. Once the glucose in the fermentor was completely consumed, an 80% glucose solution was added at a constant rate, maintaining the glucose concentration at approximately 2 g / L. After a 58-hour fermentation period, the Neu5Ac fermentation broth yield reached 70 g / L, with a production rate of 1.2 g / (L×h).

[0079] The components of the slant culture medium are: peptone 12 g / L, yeast powder 3 g / L, NaCl 8 g / L, beef extract 15 g / L, sucrose 0.8 g / L, agar powder 20 g / L, and the rest is water, pH 6.8-7.2.

[0080] The composition of the seed culture medium is as follows: glucose 20 g / L, yeast powder 5 g / L, (NH4)2SO4 3 g / L, KH2PO4 3 g / L, MgSO4·7H2O 2.5 g / L, FeSO4·7H2O 3 mg / L, MnSO4·7H2O 4 mg / L, V H 0.05mg / L, V B1 0.5mg / L, trace element mixture 2mL / L, 2 drops of defoaming agent, the rest is water, pH 6.8-7.2.

[0081] The fermentation medium composition is as follows: glucose 30 g / L, xylose 20 g / L, yeast powder 5 g / L, (NH4)2SO4 8 g / L, KH2PO4 8 g / L, MgSO4·7H2O 8 g / L, NaCl 2 g / L, FeSO4·7H2O 25 mg / L, MnSO4·7H2O 4 mg / L, CaCl2·2H2O 25 mg / L, V H 1mg / L, V B1 0.5 mg / L, trace element mixture 3 mL / L, phenol red indicator 1%, 2 drops of defoaming agent, and the rest is water, pH 6.8-7.2.

[0082] The components of the trace element mixture are: Na2MoO4·2H2O 3g / L, NiCl2·6H2O 1.5g / L, CaCl2·2H2O8g / L, CuSO4·5H2O 0.5g / L, Al2(SO4)3·18H2O 1.5g / L, CoCl2·6H2O 1.5g / L, ZnSO4·2H2O0.5g / L, H3BO30.05g / L, and the rest is water.

[0083] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that, without departing from the concept of this patent, a person skilled in the art would be able to make various variations, combinations, and improvements to the above-described embodiments, all of which fall within the scope of protection of this patent. Therefore, the scope of protection of this patent shall be determined by the claims.

Claims

1. A method for improving the ability of Escherichia coli to produce Neu5Ac, characterized in that: The method is to attenuate the acetic acid synthesis-related genes in the Neu5Ac production strain poxB , ackA , pta and glyoxylate cycle transcriptional regulator genes iclR expression to achieve this.

2. The method for improving the Neu5Ac production ability of Escherichia coli according to claim 1, wherein: described poxB Gene encoding pyruvate oxidase; pta Gene encoding phosphotransacetylase; ackA Gene encoding acetate kinase; iclR Gene encoding a glyoxylate cycle transcriptional regulator.

3. The method for improving the Neu5Ac production ability of Escherichia coli according to claim 1, wherein: Ways to reduce the expression of the gene include, but are not limited to, knocking out, inhibiting or inactivating the gene.

4. An engineered Escherichia coli strain producing Neu5Ac, characterized in that: The engineering bacteria is a Neu5Ac production strain poxB , ackA , pta as well as iclR The gene was deleted and expressed.

5. The engineered Escherichia coli strain producing Neu5Ac according to claim 4, wherein: The Neu5Ac-producing strains include but are not limited to: E. coli W3110 、E.coli W3110 NEA-1, E. coli MG1655; described E. coli The W3110 NEA-1 strain and its construction method have been disclosed in Example 1 of Chinese Patent ZL202110973426.

5.

6. The engineered Escherichia coli strain producing Neu5Ac according to claim 4, wherein: Pyruvate oxidase encoding gene poxB , the nucleotide sequence is shown in SEQ ID NO.1; Phosphotransacetylase encoding gene pta , the nucleotide sequence is shown in SEQ ID NO.2; Acetate kinase encoding gene ackA , the nucleotide sequence is shown in SEQ ID NO.3; Gene encoding glyoxylate cycle transcriptional regulator iclR , the nucleotide sequence is shown in SEQ ID NO.

4.

7. Use of the strain according to any one of claims 4 to 6 in producing Neu5Ac.

8. The use according to claim 7, wherein The shake flask fermentation culture method is as follows: the seed liquid is inoculated into the fermentation medium at an inoculum rate of 8-15%, and the culture is shaken at 28-37°C and 180-240 rpm. During the fermentation process, ammonia water is added to maintain the pH at 6.8-7.2, and glucose solution is added to maintain the fermentation process. The fermentation cycle is 24-36 hours. The fermentation method of the fermentation tank is as follows: at the OD of the seed culture solution 600 When the value increases to between 10 and 18, it is transferred to the fermentation medium at a volume ratio of 10 to 20% for subsequent fermentation experiments; during the fermentation process, the pH is maintained at 7.0 to 7.2, the temperature is maintained at 28°C to 37°C, and the dissolved oxygen is controlled between 25% and 45%; when the glucose in the fermentation tank is completely consumed, glucose solution is added, during which the glucose concentration in the tank is controlled between 0.1 and 5 g / L, and the fermentation cycle is approximately 36 h to 58 h.

9. The use according to claim 8, wherein The temperature for shake flask fermentation was 30°C; the temperature for fermentation in fermenter was 30°C.

10. The use according to claim 8, wherein The fermentation medium comprises: 15-30 g / L glucose, 5-20 g / L xylose, 2-5 g / L yeast extract, 2-10 g / L (NH4)2SO4, 4-10 g / L KH2PO4, 2-8 g / L MgSO4·7H2O, 0.5-3 g / L NaCl, 5-30 mg / L FeSO4·7H2O, 1-5 mg / L MnSO4·7H2O, 15-30 mg / L CaCl2·2H2O, V H 0.05-2 mg / L, V B1 0.1-2 mg / L, trace element mixture 1-3 mL / L, phenol red indicator 1-3%, defoamer 1-2 drops, the remainder is water, pH 6.8-7.2; autoclave at 121°C for 20 min; The composition of the trace element mixture is: Na2MoO4·2H2O 1-3 g / L, NiCl2·6H2O 0.5-1.5 g / L, CaCl2·2H2O 2-8 g / L, CuSO4·5H2O 0.1-0.5 g / L, Al2(SO4)3·18H2O 1-1.5 g / L, CoCl2·6H2O 0.5-1.5 g / L, ZnSO4·2H2O 0.1-0.5 g / L, H3BO30.05-0.2 g / L, and the rest is water.

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