A method for producing glucosamine by mixed bacteria division of labor fermentation

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to a method for producing glucosamine by fermentation using mixed bacteria with labor division. An enhanced N-acetylglucosamine synthesis pathway and a deacetylation functional module are constructed in two Escherichia coli, respectively, and the problem of feedback inhibition of the key enzyme fructose-6-phosphate aminotransferase in the glucosamine synthesis pathway by the metabolite glucosamine-6-phosphate is solved through the functional division strategy of the two bacteria. After 24 hours of shake flask fermentation, the maximum yield of glucosamine reaches 11.21g / L, and the production intensity reaches 0.47g / (L×h), which has strong potential for industrial production.

Description

Technical field:

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for producing glucosamine by fermenting with mixed bacteria for division of labor. Background technology:

[0002] Glucosamine (2-amino-2deoxy-D-glucose, GlcN) is a compound in which one hydroxyl group of glucose is replaced by an amino group. It is an important functional monosaccharide and the first amino monosaccharide with a confirmed structure. Glucosamine is the main component of glycoproteins and proteoglycans, and is also one of the important nutrients for the formation of chondrocytes. It has a special therapeutic effect on arthritis by stimulating the biosynthesis of cartilage proteoglycans, and is therefore widely used in the prevention and treatment of bone and joint diseases.

[0003] At present, the main production method of glucosamine is chitin hydrolysis. Since a large amount of acid is required for hydrolysis, the environmental pollution caused is the main factor limiting the production of glucosamine. There are also problems such as raw material allergenicity. In recent years, with the rapid development of metabolic engineering, the production of glucosamine by microbial fermentation can solve the above problems to a certain extent. However, the current microbial fermentation method mainly synthesizes N-acetylglucosamine first, and then hydrolyzes it with extracellular acid to generate glucosamine, which makes it difficult to directly and efficiently synthesize glucosamine in microorganisms. This is mainly because the key enzyme for the production of glucosamine in microorganisms, fructose-6-phosphate aminotransferase, is severely feedback inhibited by the metabolite glucosamine-6-phosphate of the synthesis pathway, so glucosamine cannot accumulate in large quantities in normal metabolic synthesis.

[0004] The present invention utilizes the mixed bacteria division of labor to directly produce glucosamine, designs the synthesis of glucosamine into two steps of efficient synthesis and deacetylation of N-acetylglucosamine in two Escherichia coli, avoids the feedback inhibition of fructose-6-phosphate aminotransferase by the accumulation of the intermediate product glucosamine-6-phosphate; further, through the construction and optimization of the mixed bacteria fermentation system, the mixed bacteria direct fermentation method for producing glucosamine is realized. Summary of the invention:

[0005] In order to solve the above technical problems, the present invention provides a method for producing glucosamine by mixed bacteria fermentation.

[0006] One of the technical solutions provided by the present invention is a genetically engineered bacterium that produces N-acetylglucosamine. The genetically engineered strain uses Escherichia coli as a chassis cell, expresses the glmS gene and the Sc-gna1 gene, constructs a synthetic pathway for N-acetylglucosamine, constructs a plasmid-free system, and utilizes a xylose promoter (P xylF) induces the RNA polymerase gene from T7 bacteriophage and deletes the expression of eight genes: nagA, nagB, nagC, nagE, manX, manY, manZ, and nanE;

[0007] Furthermore, the genetically engineered bacteria producing N-acetylglucosamine include glmS and Sc-gna1 genes controlled by a T7 promoter; an RNA polymerase gene (T7RNAP) derived from a T7 bacteriophage controlled by a xylose promoter; and eight gene defective types of nagA, nagB, nagC, nagE, manX, manY, manZ, and nanE;

[0008] Furthermore, the glmS gene is expressed in double copies;

[0009] Preferably, the genetically engineered bacteria for producing N-acetylglucosamine use E. coli K-12MG1655 as chassis cells.

[0010] The second technical solution provided by the present invention is a genetically engineered bacterium for producing glucosamine by deacetylation, which uses Escherichia coli as the base cell, overexpresses the yqaB gene, strengthens intracellular dephosphorylation, and introduces the Pyrococcus pyrolyticus diacetylchitobiose deacetylase gene phD (Dac ph ) to construct a plasmid-free system using the xylose promoter (P xylF ) induces the RNA polymerase gene from T7 bacteriophage and deletes the expression of five genes: nagE, manX, manY, manZ, and nanE;

[0011] Furthermore, the genetically engineered bacteria for producing glucosamine by deacetylation include the yqaB gene controlled by the Trc promoter; the diacetylchitobiose deacetylase gene phD from Pyrococcus controlled by the T7 promoter; the RNA polymerase gene (T7RNAP) derived from T7 bacteriophage controlled by the xylose promoter; and five gene defective types of nagE, manX, manY, manZ, and nanE;

[0012] Furthermore, the phD gene is expressed in double copies;

[0013] Preferably, the genetically engineered bacteria for producing glucosamine by deacetylation use E. coli K-12MG1655 as the chassis cell.

[0014] The nucleotide sequence of Sc-gna1 is shown in the sequence table SEQ ID No.1;

[0015] The nucleotide sequence of glmS is shown in the sequence listing as SEQ ID No. 2;

[0016] The nucleotide sequence of the nagA is shown in the sequence table SEQ ID No.3;

[0017] The nucleotide sequence of nagB is shown in the sequence table as SEQ ID No.4;

[0018] The nucleotide sequence of nagC is shown in the sequence table as SEQ ID No.5;

[0019] The nucleotide sequence of the nagE is shown in the sequence listing as SEQ ID No.6;

[0020] The nucleotide sequence of manX is shown in the sequence listing as SEQ ID No.7;

[0021] The nucleotide sequence of manY is shown in the sequence listing as SEQ ID No.8;

[0022] The nucleotide sequence of manZ is shown in the sequence listing SEQ ID No.9;

[0023] The nucleotide sequence of phD is shown in the sequence table SEQ ID No. 10

[0024] The nucleotide sequence of the T7 promoter is shown in the sequence listing as SEQ ID No.11;

[0025] The P xylF The nucleotide sequence of the promoter is shown in the sequence listing as SEQ ID No.12;

[0026] The nucleotide sequence of the RNA polymerase encoding gene derived from T7 bacteriophage is shown in the sequence table as SEQ ID No.13;

[0027] The nucleotide sequence of the nanE is shown in the sequence listing as SEQ ID No.14;

[0028] The nucleotide sequence of the Trc promoter is shown in the sequence listing as SEQ ID No.15;

[0029] The nucleotide sequence of yqaB is shown in the sequence listing as SEQ ID No.16;

[0030] Preferably, the genetically engineered bacteria for producing N-acetylglucosamine uses E. coli K-12MG1655 as a host cell.

[0031] The third technical solution provided by the present invention: the construction method of the genetically engineered bacteria for producing N-acetylglucosamine (GlcNAc):

[0032] (1) Integrate the xylose promoter P at the lacIZ gene locus xylFControlled by T7 RNA polymerase.

[0033] (2) Constructing the GlcNAc synthesis pathway. First, we knocked out the GlcNAc decomposition pathway nagA, nagB, nagC, nagE, manX, manY, manZ, and nanE. At the same time, we integrated the glucosamine-6-phosphate N-acetyltransferase gene Sc-gna1 into the nagE gene locus. We also integrated the P-terminal mitochondria into the pseudogene loci yjiV and ycjV. T7 Promoter-controlled fructose-6-phosphate aminotransferase gene glmS.

[0034] The fourth technical solution provided by the present invention is a method for constructing the above-mentioned genetically engineered bacteria for producing glucosamine (GlcN) by deacetylation:

[0035] (1) Integrate the xylose promoter P at the lacIZ gene locus xylF Controlled by T7 RNA polymerase.

[0036] (2) Constructing the GlcN synthesis pathway. First, we knocked out the phosphorylation pathways for GlcNAc to enter the cell, nagE, manX, manY, manZ, and nanE, and integrated P T7 The diacetylchitobiose deacetylase gene phD controlled by the promoter; the fructose-1-phosphate phosphatase gene yqaB controlled by the Trc promoter was integrated into the pseudogene site yeeP.

[0037] The fifth technical solution provided by the present invention is a method for producing glucosamine by mixed fermentation of genetically engineered bacteria as described in the above technical solution one and the technical solution two, which is specifically as follows:

[0038] Fermentation culture: The seed liquid of the two strains was inoculated at OD 600 The ratio of the two bacteria is 1:1-1:4 (N-acetylglucosamine-producing bacteria: glucosamine-producing bacteria), and the two bacteria are mixed or inoculated into the fermentation medium respectively, with the total inoculation amount being 10%-15% of the fermentation medium, and cultured under shaking conditions of 35-37°C and 180-240r / min. During the fermentation process, ammonia water is added to maintain the pH at 6.8-7.2. When the fermentation system lacks sugar, 60% (m / v) glucose solution is added to maintain the glucose concentration in the fermentation broth not exceeding 5g / L. The total fermentation period is 24-36h;

[0039] Furthermore, when the pH did not decrease slowly or even increased as observed by the phenol red indicator, it indicated that the bacteria lacked sugar;

[0040] Furthermore, a xylose solution with a final concentration of 5-20 g / L was added at the beginning of fermentation to induce the expression of the target gene;

[0041] After 24-36h of shake flask fermentation, the glucosamine production reached 5.1-11.21g / L;

[0042] Furthermore, the fermentation broth components are: glucose 10-30 g / L, yeast powder 1-6 g / L, (NH 4 ) 2 SO 4 1-5g / L, KH 2 PO 4 3-8g / L, MgSO 4 7H 2 O 1-5g / L, citric acid 1-5g / L, FeSO 4 7H 2 O 10-30mg / L, MnSO 4 7H 2 O 1-5mg / L, NaCl 0.5-2g / L, CaCl 2 ·2H 2 O 10-30 mg / L, V H 0.05-2mg / L, V B1 0.1-1mg / L, trace element mixture 1-3mL / L, defoaming agent 1-2 drops, the rest is water, pH 6.8-7.2, 115℃ high pressure steam sterilization for 15min.

[0043] The trace element mixture is composed of: Na 2 MoO 4 ·2H 2 O 1-3g / L, NiCl 2 6H 2 O 0.5-1.5 g / L, CaCl 2 ·2H 2 O 2-8g / L, CuSO 4 ·5H 2 O 0.1-0.5g / L, Al 2 (SO 4 ) 3 18H 2 O 0.1-0.3 g / L, CoCl 2 6H 2 O 0.5-1.5g / L, ZnSO 4 ·2H 2 O 0.1-0.5 g / L, H 3 BO 3 0.05-0.2g / L, the rest is water.

[0044] Beneficial effects:

[0045] The invention transfers the glucosamine-6-phosphate acetyltransferase gene (Sc-gna1) from yeast (Saccharomyces cerevisiae) into Escherichia coli, and expresses the fructose-6-phosphate aminotransferase gene (glmS) in multiple copies, thereby reconstructing and strengthening the synthesis pathway of N-acetylglucosamine in Escherichia coli. The recombinant strain can catalyze the synthesis of N-acetylglucosamine using glucose as a raw material.

[0046] The invention transfers the diacetylchitobiose deacetylase gene (phD) from Pyrococcus horikoshii into Escherichia coli and performs multi-copy expression, thereby strengthening the deacetylation pathway of non-phosphorylated substrate N-acetylglucosamine. The recombinant strain can deacetylate N-acetylglucosamine to generate glucosamine, thereby avoiding the large accumulation of glucosamine-6-phosphate, thereby solving the feedback inhibition problem of the key enzyme (fructose-6-phosphate aminotransferase).

[0047] The present invention uses E. coli K-12MG1655 as the starting strain and utilizes the xylose promoter (P xylF ) induced the RNA polymerase gene from T7 bacteriophage, combined with the T7 strong promoter system to strengthen the key genes (Sc-gna1, glmS, phD) in the N-acetylglucosamine and glucosamine synthesis pathways, and blocked the N-acetylglucosamine phosphorylation pathway, thus realizing the efficient synthesis of N-acetylglucosamine and the deacetylation to produce glucosamine in the mixed bacteria fermentation system. In the mixed bacteria labor division system, by combining and optimizing the copy numbers of different key enzymes of the two bacteria and balancing the metabolic intensity of the target pathways of the two bacteria, the efficient synthesis of the target product was obtained, and the optimal combination of key enzyme expression intensity was established as double copies of glms, single copy of Sc-gna1, and double copies of phD. After 24-36h of shake flask fermentation, the glucosamine production of the constructed mixed bacteria system reached 5.1-11.21g / L, which has strong potential for industrial production. Description of the drawings:

[0048] Figure 1 Fermentation Validation

[0049] Among them, GLA is a strain that produces N-acetylglucosamine, DA-1 is a strain with two copies of the phD gene, DA-2 is a strain with three copies of the phD gene (with the phD gene integrated at the ilvG site), and DA-3 is a strain with the yqaB gene integrated on the basis of DA-1.

[0050] Figure 2Verification of phD gene expression. Wherein: M: 1kb DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: target gene; 4: overlapping fragment; 5: original bacterial genome PCR fragment; 6: positive bacteria identification fragment. Specific implementation method:

[0051] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence 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 composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0052] Some gene sequences involved in the present invention and embodiments are shown in the sequence listing SEQ ID NO.1-16.

[0053] Example 1 Construction of N-acetylglucosamine producing strain:

[0054] (1) Integration of T7 RNA polymerase gene (T7-RNAP)

[0055] The T7 RNA polymerase gene (T7-RNAP) was integrated using Red recombination technology.

[0056] ① Using PCR technology and E. coli K12MG1655 genome as template, according to the xylose promoter P xylF Gene sequence design a pair of primers (PxylF-F / R) to amplify the xylose promoter P xylF ;

[0057] ②Using PCR technology with the E. coli BL21 genome as a template, a pair of primers (T7-F / R) were designed according to the T7 RNA polymerase gene sequence to amplify the T7 RNA polymerase gene;

[0058] ③Use PCR technology to design primers (Cm r -F / R) amplification of chloramphenicol resistance gene fragment;

[0059] ④Using PCR technology with the E.coli K12 MG1655 genome as a template, according to the LacIZ gene sequence, upstream homology arm primers (LacIZ-UF / UR) and downstream homology arm primers (LacIZ-DF / DR) were designed at both ends of the gene, and PCR amplification was performed to obtain the upstream and downstream homology arms of the gene to be integrated;

[0060] ⑤ Using the amplified fragment obtained in steps ①②③④ as a template, obtain the T7 RNA polymerase gene integration fragment by overlapping PCR technology, wherein the gene integration fragment consists of the upstream and downstream homology arm gene fragments of the LacIZ gene with the integration site, the chloramphenicol resistance gene fragment, and the T7 RNA polymerase gene fragment with the xylose promoter added;

[0061] ⑥ The above gene fragment was introduced into the starting bacteria E. coli K12 MG1655 competent cells containing the pKD46 plasmid to obtain positive transformants, and the pCP20 plasmid was further used to eliminate the chloramphenicol resistance in the transformants to obtain the LacIZ gene replaced by P xylF Positive transformants of the T7 RNAP gene controlled by the promoter.

[0062] (2) Knockout of nagBAC gene cluster, manXYZ gene cluster, and nanE gene

[0063] Using CRISPR / Cas9 gene editing technology to knock out the nagBAC gene cluster:

[0064] ① Using PCR technology and the E. coli K12 MG1655 genome as a template, according to the nagBAC gene sequence, upstream homology arm primers (nagBAC-UF / UR) and downstream homology arm primers (nagBAC-DF / DR) were designed at both ends of the gene, and PCR amplification was performed to obtain the upstream and downstream homology arms of the nagBAC gene;

[0065] ②Using overlapping PCR technology, taking the upstream and downstream homology arms of the nagBAC gene as templates, PCR amplification was performed to obtain overlapping fragments of the upstream and downstream homology arms of the nagBAC gene;

[0066] ③Construct a gRNA plasmid containing the Cas9 cleavage recognition sequence. The DNA fragment containing the target sequence is prepared by annealing the primers gRNA-nagBAC-F' and gRNA-nagBAC-R'. The constructed gRNA plasmid is transformed into DH5α competent cells to screen positive transformants.

[0067] ④ Extract the recombinant gRNA plasmid of the positive transformant in step ③, and electroporate it into the competent cells of the positive transformant obtained in step (1) containing the pRedCas9 plasmid at the same time as the nagBAC gene knockout fragment constructed in step ②, screen the positive transformant with successful knockout of the nagBAC gene, eliminate the pRedCas9 plasmid and gRNA plasmid in the positive transformant to obtain the engineered strain with knockout of the nagBAC gene. The knockout method of the manXYZ gene cluster and the nanE gene is consistent with the above steps.

[0068] (3) Expression of Sc-gna1 and glmS genes

[0069] The Sc-gna1 gene was integrated into the genome of the positive transformant strain obtained in step (2) using CRISPR / Cas9 gene editing technology:

[0070] ① Using PCR technology and the Saccharomyces cerevisiae genome as a template, a pair of primers (Sc-gna1-F, Sc-gnal-R) were designed according to the Sc-gna1 gene sequence to amplify the Sc-gna1 gene, and the T7 promoter and T7 terminator sequences were added to the 5' and 3' ends of the gna1 fragment amplification primers to amplify P T7 -Sc-gna1 fragment (can also be synthesized according to the gene sequence);

[0071] ②Using PCR technology and the E. coli K12MG1655 genome as a template, according to the nagE gene sequence, upstream homology arm primers (nagE-UF, nagE-UR) and downstream homology arm primers (nagE-DF, nagE-DR) were designed at both ends of the gene, and PCR amplification was performed to obtain the upstream and downstream homology arms of the gene to be integrated;

[0072] ③ Using the amplified fragments obtained in step ① and step ② as templates, the Sc-gna1 gene integration fragment was obtained by overlapping PCR technology. The gene integration fragment consisted of the upstream and downstream homologous arm gene fragments of the nagE gene to be integrated and the P T7 -Sc-gna1 gene fragment composition;

[0073] ④ The DNA fragment containing the target sequence used to construct the gRNA plasmid was prepared by annealing the primers gRNA-nagE-F' and gRNA-nagE-R'; the constructed gRNA plasmid was transformed into DH5α competent cells to screen positive transformants;

[0074] ⑤ Extract the recombinant gRNA plasmid of the positive transformant of step ④, and electroporate it into the positive transformant obtained in step (2) containing the pRedCas9 plasmid at the same time as the gna1 gene integration fragment constructed in step ③, screen the positive transformant with successful integration of the gna1 gene, eliminate the pRedCas9 plasmid and gRNA plasmid in the positive transformant to obtain the genetically engineered bacteria with integrated gna1 gene. The method of integrating the glmS gene from E. coli K-12 MG1655 into the pseudogenes yjiV and ycjV is consistent with the above steps.

[0075] Thus, a genetically engineered bacterium with E. coli K-12 MG1655 as the chassis cell was obtained, which contained glmS (double copy expression) and Sc-gna1 gene controlled by T7 promoter; RNA polymerase gene (T7RNAP) derived from T7 bacteriophage controlled by xylose promoter; and eight gene-deficient N-acetylglucosamine-producing genetically engineered bacteria, namely nagA, nagB, nagC, nagE, manX, manY, manZ, and nanE.

[0076] Example 2 Construction of glucosamine-producing strains

[0077] (1) Integration of T7 RNA polymerase gene (T7-RNAP)

[0078] The T7 RNA polymerase gene (T7-RNAP) was integrated using Red recombination technology.

[0079] ① Using PCR technology and E. coli K12MG1655 genome as template, according to the xylose promoter P xylF Gene sequence design a pair of primers (PxylF-F / R) to amplify the xylose promoter P xylF ;

[0080] ②Using PCR technology with the E. coli BL21 genome as a template, a pair of primers (T7-F / R) were designed according to the T7 RNA polymerase gene sequence to amplify the T7 RNA polymerase gene;

[0081] ③Use PCR technology to design primers (Cm r -F / R) amplification of chloramphenicol resistance gene fragment;

[0082] ④Using PCR technology with the E. coli K12MG1655 genome as a template, according to the LacIZ gene sequence, upstream homology arm primers (LacIZ-UF / UR) and downstream homology arm primers (LacIZ-DF / DR) were designed at both ends of the gene, and PCR amplification was performed to obtain the upstream and downstream homology arms of the gene to be integrated;

[0083] ⑤ Using the amplified fragment obtained in steps ①②③④ as a template, obtain the T7 RNA polymerase gene integration fragment by overlapping PCR technology, wherein the gene integration fragment consists of the upstream and downstream homology arm gene fragments of the LacIZ gene to be integrated, the chloramphenicol resistance gene fragment, and the T7 RNA polymerase gene fragment with the xylose promoter added;

[0084] ⑥ The above gene fragment was introduced into the starting bacteria E. coli K12 MG1655 competent cells containing the pKD46 plasmid to obtain positive transformants, and the pCP20 plasmid was further used to eliminate the chloramphenicol resistance in the transformants to obtain the LacIZ gene replaced by P xylF Positive transformants of the T7 RNAP gene controlled by the promoter.

[0085] (2) Knockout of manXYZ gene cluster, nagE, and nanE genes

[0086] Using CRISPR / Cas9 gene editing technology to knock out the manXYZ gene cluster:

[0087] ① Using PCR technology and the E. coli K12 MG1655 genome as a template, according to the manXYZ gene sequence, upstream homology arm primers (manXYZ-UF / UR) and downstream homology arm primers (manXYZ-DF / DR) were designed at both ends of the gene, and PCR amplification was performed to obtain the upstream and downstream homology arms of the manXYZ gene;

[0088] ② Using overlapping PCR technology with the upstream and downstream homology arms of the manXYZ gene as templates, PCR amplification was performed to obtain overlapping fragments of the upstream and downstream homology arms of the manXYZ gene;

[0089] ③Construct a gRNA plasmid containing the Cas9 cutting recognition sequence. The DNA fragment containing the target sequence is prepared by annealing the primers gRNA-manXYZ-F' and gRNA-manXYZ-R'. The constructed gRNA plasmid is transformed into DH5α transfected competent cells to screen positive transformants.

[0090] ④ Extract the recombinant gRNA plasmid of the positive transformant in step ③, and electroporate it into the competent cells of the positive transformant obtained in step (1) containing the pRedCas9 plasmid at the same time as the manXYZ gene knockout fragment constructed in step ②, screen the positive transformant with successful knockout of the manXYZ gene, eliminate the pRedCas9 plasmid and gRNA plasmid in the positive transformant to obtain the engineered strain with knockout of the manXYZ gene. The knockout method of nagE and nanE genes is the same as the above steps.

[0091] (3) Expression of phD gene

[0092] The phD gene was integrated into the genome of the positive transformant strain obtained in step (2) using CRISPR / Cas9 gene editing technology:

[0093] ① Using synthetic gene technology, the diacetylchitobiose deacetylase gene phD (Dac ph) was synthesized into pUC-GW plasmid, and a pair of primers (phD-F, phD-R) were designed based on the phD gene sequence to amplify the phD gene. T7 promoter and T7 terminator sequences were added to the 5' and 3' ends of the phD fragment amplification primers to amplify P T7 -phD fragment;

[0094] ②Using PCR technology with the E. coli K12 MG1655 genome as a template, according to the pseudogene site ygaY gene sequence, upstream homology arm primers (ygaY-UF, ygaY-UR) and downstream homology arm primers (ygaY-DF, ygaY-DR) were designed at both ends of the gene, and PCR amplification was performed to obtain the upstream and downstream homology arms of the gene to be integrated;

[0095] ③ Using the amplified fragments obtained in step ① and step ② as templates, the phD gene integration fragment was obtained by overlapping PCR technology, such as Figure 2 As shown, the gene integration fragment is composed of upstream and downstream homologous arm gene fragments of the gene to be integrated and the phD gene fragment;

[0096] ④ The DNA fragment containing the target sequence used to construct the gRNA plasmid was prepared by annealing the primers gRNA-ygaY-F' and gRNA-ygaY-R'; the constructed gRNA plasmid was transformed into DH5α competent cells to screen positive transformants;

[0097] ⑤ Extract the recombinant gRNA plasmid of the positive transformant of step ④, and electroporate it into the positive transformant obtained in step (2) containing the pRedCas9 plasmid at the same time as the phD gene integration fragment constructed in step ③, screen the positive transformants that have successfully integrated the phD gene, eliminate the pRedCas9 plasmid and gRNA plasmid in the positive transformants to obtain the genetically engineered bacteria with integrated phD gene. The method of integrating the phD gene on the pseudogene yciQ is consistent with the above steps. (For comparison, when constructing a strain with three copies of the phD gene, another phD gene is integrated on ilvG).

[0098] (4) Expression of yqaB gene

[0099] The yqaB gene was integrated into the genome of the positive transformant strain obtained in step (3) using CRISPR / Cas9 gene editing technology:

[0100] ① Using PCR technology and E. coli K12 MG1655 genome as template, a pair of primers (yqaB-F, yqaB-R) were designed according to the yqaB gene sequence to amplify the yqaB gene, and the Trc promoter and Trc terminator sequences were added to the 5' and 3' ends of the yqaB fragment amplification primers to amplify P Trc-yqaB fragment;

[0101] ②Using PCR technology with the E. coli K12 MG1655 genome as a template, according to the pseudogene site yeeP gene sequence, upstream homology arm primers (yeeP-UF, yeeP-UR) and downstream homology arm primers (yeeP-DF, yeeP-DR) were designed at both ends of the gene, and PCR amplification was performed to obtain the upstream and downstream homology arms of the gene to be integrated;

[0102] ③ Using the amplified fragments obtained in step ① and step ② as templates, the yqaB gene integration fragment is obtained by overlapping PCR technology, wherein the gene integration fragment consists of the upstream and downstream homologous arm gene fragments of the yeeP gene to be integrated and the yqaB gene fragment;

[0103] ④ The DNA fragment containing the target sequence used to construct the gRNA plasmid was prepared by annealing the primers gRNA-yeeP-F' and gRNA-yeeP-R'; the constructed gRNA plasmid was transformed into DH5α competent cells to screen positive transformants;

[0104] ⑤ Extract the recombinant gRNA plasmid of the positive transformant of step ④, and simultaneously electroporate the yqaB gene integration fragment constructed in step ③ into the positive transformant obtained in step (3) containing the pRedCas9 plasmid, screen the positive transformants with successful integration of the yqaB gene, eliminate the pRedCas9 plasmid and gRNA plasmid in the positive transformants, and obtain the genetically engineered bacteria with integrated yqaB gene.

[0105] Thus, a genetically engineered bacterium with E. coli K-12 MG1655 as the chassis cell was obtained, which contained the yqaB gene controlled by the Trc promoter; the diacetylchitobiose deacetylase gene phD from Pyrococcus controlled by the T7 promoter (double copy expression); the RNA polymerase gene (T7RNAP) derived from T7 phage controlled by the xylose promoter; and the deacetylation-producing glucosamine-producing genetically engineered bacteria with five gene defects, namely nagE, manX, manY, manZ, and nanE.

[0106] Example 3 Shake flask fermentation experiment

[0107] The strains constructed in the above-mentioned Examples 1 and 2 were used as production strains to produce glucosamine by mixed bacteria:

[0108] (1) Activated slant culture: Use an inoculation loop to inoculate 1-2 loops of bacteria from a -80°C freezer tube, evenly spread on the slant culture medium, culture at 37°C for 12 h, transfer to the second generation slant culture medium, and culture at 37°C for 12 h;

[0109] (2) Seed bottle culture: Use an inoculation loop to inoculate the bacteria on the slant into a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium to prepare the seed solution. Seal the flask with twelve layers of gauze and culture at 37°C and 220 rpm with shaking for 12 h.

[0110] (3) Shake flask fermentation: The above two bacteria were inoculated according to the OD 600 The value ratio is 1:1, 1:2, and 1:3, the inoculation volume of the two bacteria is adjusted, the total inoculation volume is maintained at 10%, and it is inoculated into the fermentation medium, and cultured at 37°C, 220rpm for 24h; the pH is maintained at 7.0-7.2 (phenol red is used as an indicator, and the color of the fermentation liquid remains unchanged, which is considered to be sugar deficiency. When sugar is deficient, 60% (m / v) glucose solution is added), the concentration of glucose in the fermentation liquid is maintained at no more than 5g / L, and a xylose solution with a final concentration of 10g / L is added at the beginning of fermentation to induce the expression of the target gene.

[0111] (4) After fermentation, the fermentation broth was collected and centrifuged at 13,000 rpm. The supernatant was collected and the glucosamine content was determined.

[0112] (5) Detection method of glucosamine: The chromatographic column used was a ZORBAX Eclipse Plus C18 chromatographic column (4.6 mm × 250 mm); the detector was a RID-20A refractive index detector; the mobile phase was methanol: buffer 2:8 (v / v), and the buffer was 0.005 M sodium octane sulfonate solution (pH = 5.1); the column temperature was set at 30-37°C, and the flow rate was set at 0.7 mL / min. The elution time of glucosamine was about 15.8 minutes.

[0113] Seed culture medium composition: glucose 20g / L, yeast powder 3g / L, (NH 4 ) 2 SO 4 2 g / L, KH 2 PO 4 2 g / L, MgSO 4 7H 2 O 1g / L, citric acid 2g / L, FeSO 4 7H 2 O 2.8mg / L, MnSO 4 7H 2 O 1.2mg / L, V H 0.1mg / L,V B1 0.5mg / L, trace element mixture 1mL / L, 1 drop of defoamer, the rest is water, pH 6.8-7.2, 115℃ high pressure steam sterilization for 15min;

[0114] Fermentation medium composition: glucose 20g / L, yeast powder 3g / L, (NH4 ) 2 SO 4 4g / L, KH 2 PO 4 6.67 g / L, MgSO 4 7H 2 O 2.5g / L, citric acid 3.55g / L, FeSO 4 7H 2 O 20mg / L, MnSO 4 7H 2 O 1.2mg / L, NaCl 1g / L, CaCl 2 ·2H 2 O 25mg / L,V H 0.1mg / L,V B1 0.5mg / L, trace element mixture 1mL / L, 1 drop of defoamer, the rest is water, pH 6.8-7.2, 115℃ high pressure steam sterilization for 15min;

[0115] The trace element mixture is composed of: Na 2 MoO 4 ·2H 2 O 1.25g / L, NiCl 2 6H 2 O 0.8g / L, CaCl 2 ·2H 2 O 6g / L, CuSO 4 ·5H 2 O 0.2g / L, Al 2 (SO 4 ) 3 18H 2 O 0.25g / L, CoCl 2 6H 2 O 1.25g / L, ZnSO 4 ·2H 2 O 0.35g / L, H 3 BO 3 0.07g / L, the rest is water.

[0116] The fermentation inoculation ratio of the present embodiment is: 600 The inoculation values ​​are 1:1, 1:2, and 1:3.

[0117] from Figure 1 It can be known that Figure 1In the figure, GLA represents the GlcNAC production strain constructed in Example 1; DA-3 is a strain into which the yqaB gene is integrated on the basis of DA-1, i.e., the GlcN production strain constructed in Example 2; DA-1 is a strain having two copies of the phD gene; and DA-2 is a strain into which one copy of the phD gene is integrated on the basis of DA-1. ), the two final engineered bacteria can realize the division of labor in mixed fermentation to produce glucosamine. After 24 hours of shake flask fermentation, when the inoculation ratio is 1:1, 1:2, and 1:3, the glucosamine production in the fermentation broth reaches 11.21 g / L, 8.13 g / L, and 5.1 g / L, respectively. When the inoculation ratio is 1:1, the production intensity reaches a maximum of 0.47 g / (L×h), which has strong industrial production potential.

[0118] Example 4 Shake flask fermentation experiment

[0119] The strains constructed in the above-mentioned Examples 1 and 2 were used as production strains to produce glucosamine by mixed bacteria:

[0120] (1) Activated slant culture: Use an inoculation loop to inoculate 1-2 loops of bacteria from a -80°C freezer tube, evenly spread on the slant culture medium, culture at 37°C for 12 h, transfer to the second generation slant culture medium, and culture at 37°C for 12 h;

[0121] (2) Seed bottle culture: Use an inoculation loop to inoculate the bacteria on the slant into a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium to prepare the seed solution. Seal the flask with twelve layers of gauze and culture at 37°C and 220 rpm with shaking for 12 h.

[0122] (3) Shake flask fermentation: The above two bacteria were inoculated according to OD 600 The ratio of the value is 1:1 (the fermentation inoculation ratio is N-acetylglucosamine producing bacteria and glucosamine producing bacteria according to OD 600 The inoculation volume of the two bacteria was adjusted to keep the total inoculum volume at 12%, and the culture was carried out at 36°C, 210rpm for 24h; the pH was maintained at 7.0-7.2 (phenol red was used as an indicator, and the color of the fermentation liquid remained unchanged, which was considered to be sugar deficiency. When sugar was deficient, 60% (m / v) glucose solution was added), the glucose concentration in the fermentation liquid was kept below 5g / L, and xylose solution with a final concentration of 10g / L was added at the beginning of fermentation to induce the expression of the target gene.

[0123] (4) After fermentation, the fermentation broth was collected and centrifuged at 13,000 rpm. The supernatant was collected and the glucosamine content was determined. The content in the fermentation broth after 24 hours reached 9.06 g / L.

[0124] Seed culture medium composition: glucose 20g / L, yeast powder 3g / L, (NH 4 )2 SO 4 2 g / L, KH 2 PO 4 2 g / L, MgSO 4 7H 2 O 1g / L, citric acid 2g / L, FeSO 4 7H 2 O 2.8mg / L, MnSO 4 7H 2 O 1.2mg / L, V H 0.1mg / L,V B1 0.5mg / L, trace element mixture 1mL / L, 1 drop of defoamer, the rest is water, pH 6.8-7.2, 115℃ high pressure steam sterilization for 15min;

[0125] Fermentation medium composition: glucose 30g / L, yeast powder 4g / L, (NH 4 ) 2 SO 4 3g / L, KH 2 PO 4 6.5g / L, MgSO 4 7H 2 O 3.5g / L, citric acid 3.5g / L, FeSO 4 7H 2 O 30mg / L, MnSO 4 7H 2 O 3.5mg / L, NaCl 1.5g / L, CaCl 2 ·2H 2 O 20mg / L,V H 0.5mg / L,V B1 0.5mg / L, trace element mixture 1.5mL / L, 1 drop of defoamer, the rest is water, pH 6.8-7.2, 115℃ high pressure steam sterilization for 15min;

[0126] The trace element mixture is composed of: Na 2 MoO 4 ·2H 2 O 1.25g / L, NiCl 2 6H 2 O 0.8g / L, CaCl 2 ·2H 2 O 5g / L, CuSO 4 ·5H 2 O 0.2g / L, Al 2 (SO 4 ) 3 18H2 O 0.25g / L, CoCl 2 6H 2 O 1.25g / L, ZnSO 4 ·2H 2 O 0.25g / L, H 3 BO 3 0.07g / L, the rest is water.

[0127] Example 5 Shake flask fermentation experiment

[0128] The strains constructed in the above-mentioned Examples 1 and 2 were used as production strains to produce glucosamine by mixed bacteria:

[0129] (1) Activated slant culture: Use an inoculation loop to inoculate 1-2 loops of bacteria from a -80°C freezer tube, evenly spread on the slant culture medium, culture at 37°C for 12 h, transfer to the second generation slant culture medium, and culture at 37°C for 12 h;

[0130] (2) Seed bottle culture: Use an inoculation loop to inoculate the bacteria on the slant into a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium to prepare the seed solution. Seal the flask with twelve layers of gauze and culture at 37°C and 220 rpm with shaking for 12 h.

[0131] (3) Shake flask fermentation: The above two bacteria were inoculated according to OD 600 The ratio of the value is 1:1 (the fermentation inoculation ratio is N-acetylglucosamine producing bacteria and glucosamine producing bacteria according to OD 600 The inoculation volume of the two bacteria was adjusted to keep the total inoculum at 15%, and the culture was carried out at 37°C, 230rpm for 36h; the pH was maintained at 7.0-7.2 (phenol red was used as an indicator, and the color of the fermentation liquid remained unchanged, which was considered to be sugar deficiency. When sugar was deficient, 60% (m / v) glucose solution was added), the glucose concentration in the fermentation liquid was kept below 5g / L, and xylose solution with a final concentration of 10g / L was added at the beginning of fermentation to induce the expression of the target gene.

[0132] (4) The fermentation broth was collected and centrifuged at 13,000 rpm. The supernatant was collected and the glucosamine content was determined. The content in the fermentation broth after 36 hours reached 9.8 g / L.

[0133] Seed culture medium composition: glucose 20g / L, yeast powder 3g / L, (NH 4 ) 2 SO 4 2g / L, KH 2 PO 4 2g / L, MgSO 4 7H 2O 1g / L, citric acid 2g / L, FeSO 4 7H 2 O 2.8mg / L, MnSO 4 7H 2 O 1.2mg / L, V H 0.1mg / L,V B1 0.5mg / L, trace element mixture 1mL / L, 1 drop of defoamer, the rest is water, pH 6.8-7.2, 115℃ high pressure steam sterilization for 15min;

[0134] Fermentation medium composition: glucose 25g / L, yeast powder 4g / L, (NH 4 ) 2 SO 4 4g / L, KH 2 PO 4 8g / L, MgSO 4 7H 2 O 4g / L, citric acid 5g / L, FeSO 4 7H 2 O 15mg / L, MnSO 4 7H 2 O 3mg / L, NaCl 1.5g / L, CaCl 2 ·2H 2 O 20mg / L,V H 1.5mg / L,V B1 1mg / L, trace element mixture 1mL / L, 1 drop of defoamer, the rest is water, pH 6.8-7.2, 115℃ high pressure steam sterilization for 15min;

[0135] The trace element mixture is composed of: Na 2 MoO 4 ·2H 2 O 1.25g / L, NiCl 2 6H 2 O 0.8g / L, CaCl 2 ·2H 2 O 5g / L, CuSO 4 ·5H 2 O 0.3g / L, Al 2 (SO 4 ) 3 18H 2 O 0.25g / L, CoCl 2 6H 2 O 1g / L, ZnSO 4 ·2H 2 O 0.25g / L, H 3BO 3 0.07g / L, the rest is water.

[0136] In summary, the genetically engineered bacteria of the present invention achieve the efficient synthesis of N-acetylglucosamine and the division of labor of deacetylation to generate glucosamine in a mixed fermentation system by strengthening the synthesis pathway of N-acetylglucosamine and introducing a heterologous deacetylase. The strengthening of the synthesis pathway of N-acetylglucosamine is constructed using CRISPR / Cas9 technology according to the following method: eight genes, nagA, nagB, nagC, nagE, manX, manY, manZ, and nanE, are knocked out in the starting strain E.coli K12 MG1655, and a strong xylose promoter is used to induce the synthesis of RNA polymerase derived from T7 phage to achieve the induction and strengthening of the synthesis pathway of N-acetylglucosamine regulated by the T7 promoter, including the introduction of the gna1 gene (Sc-gna1) from Saccharomyces cerevisiae, and the multi-copy strengthening of the glmS gene in Escherichia coli K-12MG1655. The deacetylation of N-acetylglucosamine was constructed using CRISPR / Cas9 technology according to the following method: the five genes nagE, manX, manY, manZ, and nanE were knocked out in the starting strain E. coli K12 MG1655, the xylose strong promoter was used to induce the synthesis of RNA polymerase from T7 phage, and the diacetylchitobiose deacetylase gene phD (Dac ph ), combined with the T7 strong promoter system to start the expression of the target gene and perform multi-copy enhancement, the yqaB gene in E. coli K-12 MG1655 was enhanced. Finally, after 24 hours of shake flask fermentation, the maximum yield of glucosamine reached 11.21g / L, and the production intensity reached 0.47g / (L×h), which has strong industrial production potential.

[0137] Table 1 Primers involved in the construction of the strain of the present invention

[0138]

[0139]

[0140] The above-mentioned reference examples describe in detail the construction of a genetically engineered bacterium for producing N-acetylglucosamine and a genetically engineered bacterium for producing glucosamine by deacetylation, and the method of fermentation using mixed bacteria with division of labor. These are illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for producing glucosamine by fermentation, characterized in that: The details are as follows: The mixed fermentation is carried out using genetically engineered Escherichia coli bacteria that produce N-acetylglucosamine and genetically engineered Escherichia coli bacteria that produce glucosamine; The genetically engineered Escherichia coli bacteria producing N-acetylglucosamine is based on Escherichia coli as a host and is controlled by the introduction of a T7 promoter. glmS Genes and Sc-gna1 gene; RNA polymerase gene from T7 phage controlled by xylose promoter; and deletion of nagA, nagB, nagC, nagE, manX, manY, manZ, nanE The expression of eight genes; Sc- gna1 The nucleotide sequence is shown in the sequence table SEQ ID No.1; glmS The nucleotide sequence of the gene encoding RNA polymerase from T7 bacteriophage is shown in SEQ ID No. 2 in the sequence list; the nucleotide sequence of the gene encoding RNA polymerase from T7 bacteriophage is shown in SEQ ID No. 13 in the sequence list; glmS The gene is expressed in two copies; The genetically engineered Escherichia coli bacteria producing glucosamine is based on Escherichia coli as a host and introduces a Trc promoter to control Yj Y Gene, Pyrococcus diacetylchitobiose deacetylase gene controlled by T7 promoter phD , using the xylose promoter P xylF Induce the RNA polymerase gene from T7 bacteriophage and delete the nagE, manX, manY, manZ, nanE The expression of five genes; phD The nucleotide sequence is shown in the sequence table SEQ ID No.10; Yj Y The nucleotide sequence is shown in the sequence table SEQ ID No.16; phD The gene is expressed in two copies; The genetically engineered Escherichia coli bacteria producing N-acetylglucosamine or the genetically engineered Escherichia coli bacteria producing glucosamine, the host cell is E. coli K-12 MG1655; The seed liquid of Escherichia coli genetically engineered bacteria producing N-acetylglucosamine and Escherichia coli genetically engineered bacteria producing glucosamine was inoculated according to the OD 600 The value is 1:1-1:4, and 10%-15% of the total inoculum is inoculated into the fermentation medium. The culture is carried out at 32-37°C and 200-300rpm for 24-36h. The pH is maintained at 6.8-7.2, and the fermentation is maintained with 60% glucose solution. At the beginning of the fermentation, a xylose solution with a final concentration of 5-20g / L is added to induce the expression of the target gene.

2. The method for producing glucosamine by fermentation according to claim 1, characterized in that: Said nagA The nucleotide sequence is shown in the sequence listing SEQ ID No.3; Said nagB The nucleotide sequence is shown in the sequence listing SEQ ID No.4; Said nagC The nucleotide sequence is shown in the sequence listing SEQ ID No.5; Said nagE The nucleotide sequence is shown in the sequence listing as SEQ ID No.6; Said manX The nucleotide sequence is shown in the sequence listing SEQ ID No.7; Said manY The nucleotide sequence is shown in the sequence listing as SEQ ID No.8; Said manZ The nucleotide sequence is shown in the sequence listing SEQ ID No.9; The nucleotide sequence of the T7 promoter is shown in the sequence listing as SEQ ID No.11; Said P xylF The nucleotide sequence of the promoter is shown in the sequence listing as SEQ ID No.12; Said E The nucleotide sequence is shown in the sequence listing as SEQ ID No.14; The nucleotide sequence of the Trc promoter is shown in the sequence table as SEQ ID No.

15.

3. The method for producing glucosamine by fermentation according to claim 1, characterized in that: The fermentation medium components are: glucose 10-30g / L, yeast powder 1-6g / L, (NH4)2SO4 1-5g / L, KH2PO4 3-8g / L, MgSO4·7H2O 1-5g / L, citric acid 1-5g / L, FeSO4·7 H2O 10-30mg / L, MnSO4·7H2O 1-5mg / L, NaCl 0.5-2g / L, CaCl2·2H2O 10-30mg / L, V H 0.05-2mg / L, V B1 0.1-1mg / L, trace element mixture 1-3mL / L, defoaming agent 1-2 drops, the rest is water, pH 6.8-7.2, 115℃ high pressure steam sterilization for 15-20 min.

4. The method for producing glucosamine by fermentation as claimed in claim 3, characterized in that: The components of the trace element mixed solution are: Na2MoO4·2H2O 1-3g / L, NiCl2·6H2O 0.5-1.5g / L, CaCl2·2H2O 2-8g / L, CuSO4·5H2O0.1-0.5g / L, Al2(SO4)3·18H2O 0.1-0.3g / L, CoCl2·6H2O 0.5-1.5g / L, ZnSO4·2H2O 0.1-0.5g / L, H3BO3 0.05-0.2g / L, and the rest is water.

Images