CN115895992A - Method for producing glucosamine by using mixed strain labor and division fermentation - Google Patents
Method for producing glucosamine by using mixed strain labor and division fermentation Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a method for producing glucosamine by division of fermentation by using mixed strain labor. An enhanced N-acetylglucosamine synthesis path and a deacetylation function module are respectively constructed in two types of escherichia coli, and the problem that key enzyme fructose-6-phosphate transaminase in the glucosamine synthesis path is subjected to feedback inhibition of a metabolite glucosamine-6-phosphate is solved through a two-bacterium function division strategy. After 24 hours of shake flask fermentation, the yield of glucosamine can reach 11.21g/L to the maximum extent, the production intensity can reach 0.47 g/(L multiplied by h), and the method has strong industrial production potential.
Description
The technical field is as follows:
the invention belongs to the technical field of genetic engineering, and particularly relates to a method for producing glucosamine by utilizing mixed bacteria labor and performing separated fermentation.
Background art:
glucosamine (GlcN), a compound in which one hydroxyl group of glucose is substituted with an amino group, is an important functional monosaccharide, and is also the first aminosugar having a confirmed structure. Glucosamine, which is a major constituent of glycoproteins and proteoglycans and one of important nutrients for forming chondrocytes, has a special therapeutic effect on arthritis by stimulating the biosynthesis of cartilage proteoglycans, and thus is widely used for the prevention and treatment of osteoarticular diseases.
The main production method of glucosamine at present is a chitin hydrolysis method, and because a large amount of acid is needed for hydrolysis, the environmental pollution caused by hydrolysis is a main factor for limiting the production of glucosamine, and simultaneously, the problems of raw material sensitization and the like also exist. In recent years, with the rapid development of metabolic engineering, the above problems have been solved to some extent by producing glucosamine by microbial fermentation, but currently, it is difficult to directly and efficiently synthesize glucosamine in a microorganism because the microbial fermentation method mainly synthesizes N-acetylglucosamine first and then carries out extracellular acid hydrolysis to produce glucosamine. Mainly because the key enzyme in the microorganism for producing glucosamine, fructose-6-phosphate transaminase, is severely inhibited by the feedback of glucosamine-6-phosphate, a metabolite of the synthetic pathway, and thus glucosamine cannot be accumulated in large quantities in normal metabolic synthesis.
The invention utilizes the labor of mixed bacteria to directly produce the glucosamine by division, designs the synthesis of the glucosamine into two steps of high-efficiency synthesis and deacetylation of the N-acetylglucosamine in two types of escherichia coli, and avoids the feedback inhibition of the accumulation of an intermediate product glucosamine-6-phosphate on fructose-6-phosphate transaminase; further realizes the mixed bacteria direct fermentation method production of the glucosamine by the construction and optimization of a mixed bacteria fermentation system.
The invention content is as follows:
in order to solve the technical problems, the invention provides a method for producing glucosamine by mixed fermentation.
One of the technical schemes to be provided by the invention is as follows: the strain is a genetic engineering strain for producing N-acetylglucosamine, wherein the genetic engineering strain takes escherichia coli as a chassis cell, expresses a glmS gene and a Sc-gna1 gene, constructs a synthetic pathway of the N-acetylglucosamine, constructs a plasmid-free system, and utilizes a xylose promoter (P) xylF ) Inducing RNA polymerase genes from T7 bacteriophage, and deleting the expression of eight genes of nagA, nagB, nagC, nagE, manX, manY, manZ and nanE;
further, the genetically engineered bacterium producing N-acetylglucosamine comprises glmS controlled by a T7 promoter and a Sc-gna1 gene; an RNA polymerase gene (T7 RNAP) derived from a T7 bacteriophage under the control of a xylose promoter; and nagA, nagB, nagC, nagE, manX, manY, manZ, nanE eight gene defects;
further, the glmS gene is expressed as a double copy;
preferably, the genetically engineered bacterium for producing N-acetylglucosamine takes E.coli K-12MG1655 as a basal disc cell.
The second technical scheme provided by the invention is as follows: is a genetically engineered bacterium for producing glucosamine by deacetylation, takes escherichia coli as a chassis cell, overexpresses yqaB gene, strengthens intracellular dephosphorylation, introduces pyrococcus diacetylchitobiose deacetylase gene phD (Dac) ph ) Construction of a plasmid-free System Using the xylose promoter (P) xylF ) Inducing RNA polymerase gene from T7 bacteriophage, and deleting the expression of five genes of nagE, manX, manY, manZ and nanE;
further, the genetically engineered bacterium for producing glucosamine by deacetylation comprises a yqaB gene controlled by a Trc promoter; the T7 promoter controls the diacetylchitobiose deacetylase gene phD from Pyrococcus spp; an RNA polymerase gene (T7 RNAP) derived from a T7 bacteriophage under the control of a xylose promoter; and nagE, manX, manY, manZ, nanE five gene defects;
further, the phD gene is expressed in double copies;
preferably, the genetically engineered bacterium for producing glucosamine by deacetylation takes E.coli K-12MG1655 as a basal disc cell.
The nucleotide sequence of the Sc-gna1 is shown as a sequence table SEQ ID No. 1;
the nucleotide sequence of the glmS is shown as a sequence table SEQ ID No. 2;
the nucleotide sequence of nagA is shown in a sequence table SEQ ID No. 3;
the nucleotide sequence of nagB is shown in a sequence table SEQ ID No. 4;
the nucleotide sequence of nagC is shown in a sequence table SEQ ID No. 5;
the nucleotide sequence of nagE is shown in a sequence table SEQ ID No. 6;
the nucleotide sequence of manX is shown in a sequence table SEQ ID No. 7;
the nucleotide sequence of manY is shown in a sequence table SEQ ID No. 8;
the nucleotide sequence of manZ is shown in a sequence table SEQ ID No. 9;
the nucleotide sequence of the phD is shown in a sequence table SEQ ID No.10
The nucleotide sequence of the T7 promoter is shown in a sequence table SEQ ID No. 11;
said P is xylF The nucleotide sequence of the promoter is shown in a sequence table SEQ ID No. 12;
the nucleotide sequence of the RNA polymerase coding gene derived from the T7 bacteriophage is shown in a sequence table SEQ ID No. 13;
the nucleotide sequence of nanE is shown in a sequence table SEQ ID No. 14;
the nucleotide sequence of the Trc promoter is shown in a sequence table SEQ ID No. 15;
the nucleotide sequence of the yqaB is shown as a sequence table SEQ ID No. 16;
preferably, the genetically engineered bacterium for producing N-acetylglucosamine uses E.coli K-12MG1655 as a host cell.
The third technical scheme provided by the invention is as follows: the construction method of the genetic engineering bacteria for producing N-acetylglucosamine (GlcNAc) comprises the following steps:
(1) Integration of xylose promoter P at the lacIZ Gene site xylF Controlled T7RNA polymerase.
(2) Constructing a GlcNAc synthesis pathway. Firstly, knocking out catabolic pathways nagA, nagB, nagC, nagE, manX, manY, manZ and nanE of GlcNAc, and simultaneously integrating a glucosamine-6-phosphate N-acetyltransferase gene Sc-gna1 on a nagE gene locus; integration of yjiV and ycjV from P at the pseudogene locus T7 The fructose-6-phosphate transaminase gene glmS under the control of a promoter.
The fourth technical scheme provided by the invention is as follows: the construction method of the genetically engineered bacterium for producing glucosamine (GlcN) by deacetylation comprises the following steps:
(1) Integration of xylose promoter P at lacIZ Gene site xylF Controlled T7RNA polymerase.
(2) Constructing a GlcN synthetic pathway. Firstly, the phosphorylation pathways nagE, manX, manY, manZ and nanE of GlcNAc entering cells are knocked out, and ygaY and yiQ are integrated at pseudogene sites and are integrated by P T7 A diacetylchitobiose deacetylase gene phD under the control of a promoter; the fructose-1-phosphate phosphatase gene yqaB controlled by the Trc promoter was integrated at the pseudogene site yeeP.
The fifth technical scheme provided by the invention is as follows: the method for producing glucosamine by mixed fermentation of genetically engineered bacteria according to the first technical scheme and the second technical scheme comprises the following specific steps:
fermentation culture: inoculating seed solutions of the two strains according to the OD 600 The ratio range of 1-1;
further, when no slow decrease or even increase in pH was observed by phenol red indicator, indicating that the cells were sugar deficient;
further, xylose solution with the final concentration of 5-20g/L is added at the beginning of fermentation to induce the expression of target genes;
after 24-36h of shake flask fermentation, the glucosamine yield reaches 5.1-11.21g/L;
further, the fermentation culture solution comprises the following components: 10-30g/L glucose, 1-6g/L yeast powder, (NH) 4 ) 2 SO 4 1-5g/L，KH 2 PO 4 3-8g/L，MgSO 4 ·7H 2 O1-5 g/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 10-30mg/L，V H 0.05-2mg/L，V B1 0.1-1mg/L, 1-3mL/L of mixed solution of trace elements, 1-2 drops of defoaming agent and the balance of water, the pH value is 6.8-7.2, and the sterilization is carried out for 15min by high-pressure steam at 115 ℃.
The microelement mixed solution comprises the following components: na (Na) 2 MoO 4 ·2H 2 O 1-3g/L，NiCl 2 ·6H 2 O 0.5-1.5g/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 0.1-0.3g/L，CoCl 2 ·6H 2 O 0.5-1.5g/L，ZnSO 4 ·2H 2 O 0.1-0.5g/L，H 3 BO 3 0.05-0.2g/L, and the balance of water.
Has the advantages that:
the invention transfers glucosamine-6-phosphate acetyl transferase gene (Sc-gna 1) derived from saccharomycetes (Saccharomyces cerevisiae) into Escherichia coli, and expresses fructose-6-phosphate acetyl transferase gene (glmS) in multiple copies, thereby reconstructing and strengthening the synthetic pathway of N-acetylglucosamine in the Escherichia coli. The recombinant strain can catalyze and synthesize the N-acetylglucosamine by taking glucose as a raw material.
The diacetyl chitobiose deacetylase gene (phD) derived from Pyrococcus horikoshii is transferred into escherichia coli and is subjected to multi-copy expression, the deacetylation way of non-phosphorylated substrate N-acetylglucosamine is enhanced, the recombinant strain can deacetylate the N-acetylglucosamine to generate glucosamine, and the mass accumulation of glucosamine-6-phosphate is avoided, so that the problem of feedback inhibition of key enzyme (fructose-6-phosphate aminotransferase) is solved.
Coli K-12MG1655 is used as starting strain, xylose promoter (P) is utilized xylF ) Inducing RNA polymerase genes from T7 bacteriophage, combining with a T7 strong promoter system to strengthen key genes (Sc-gna 1, glmS and phD) in the synthesis pathway of N-acetylglucosamine and glucosamine, and blocking the phosphorylation pathway of N-acetylglucosamine, thereby realizing the labor division of efficient synthesis and deacetylation of N-acetylglucosamine to generate glucosamine in a mixed fermentation system. In a mixed bacteria labor division system, the copy numbers of different key enzymes of two bacteria are optimized in a combined manner, the metabolic intensity of target pathways of the two bacteria is balanced, the efficient synthesis of a target product is obtained, the optimal combination of the expression intensity of the key enzymes is determined to be glms double copies, sc-gna1 single copies and phD double copies, the yield of glucosamine reaches 5.1-11.21g/L after the constructed mixed bacteria system is fermented for 24-36h in a shake flask, and the method has strong industrial production potential.
Description of the drawings:
FIG. 1 fermentation validation
Wherein GLA is a strain for producing N-acetylglucosamine, DA-1 is a strain with two phD gene copies, DA-2 is a strain with three phD gene copies (increasing the integration of phD genes on ilvG sites), and DA-3 is a strain integrating yqaB genes on the basis of DA-1.
FIG. 2phD Gene expression validation. Wherein: m:1kb DNA marker;1: an upstream homology arm; 2: downstream homology arm 3: a gene of interest; 4: overlapping segments; 5: PCR fragment of original bacterium genome; 6: and (4) identifying fragments of positive bacteria.
The specific implementation mode is as follows:
the invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
The partial gene sequence related to the invention and the embodiment is shown in a sequence table SEQ ID NO. 1-16.
Example 1 construction of N-acetylglucosamine-producing strains:
(1) Integration of the T7RNA polymerase gene (T7-RNAP)
Integration of the T7RNA polymerase gene (T7-RNAP) was performed using Red recombination technology.
(1) Adopting PCR technology, taking E.coli K12MG1655 genome as template, according to xylose promoter P xylF Gene sequence design a pair of primers (PxylF-F/R) for amplifying xylose promoter P xylF ；
(2) Designing a pair of primers (T7-F/R) according to a T7RNA polymerase gene sequence by using E.coli BL21 genome as a template by adopting a PCR technology to amplify the T7RNA polymerase gene;
(3) the PCR technology is adopted, pKD3 plasmid is taken as a template, and a primer (Cm) is designed r -F/R) amplification of a chloramphenicol resistance gene fragment;
(4) adopting a PCR technology to design an upstream homology arm primer (LacIZ-UF/UR) and a downstream homology arm primer (LacIZ-DF/DR) at two ends of a gene by taking an E.coli K12MG1655 genome as a template according to a LacIZ gene sequence, and carrying out PCR amplification to obtain upstream and downstream homology arms of a to-be-integrated site gene;
(5) taking the amplified fragments obtained in the steps (1), (2), (3) and (4) as templates, and obtaining a T7RNA polymerase gene integration fragment by an overlapping PCR technology, wherein the gene integration fragment consists of gene fragments of upper and lower homologous arms of a LacIZ gene with an integration site, a chloramphenicol resistance gene fragment and a T7RNA polymerase gene fragment added with a xylose promoter;
(6) introducing the gene fragment into a starting bacterium E.coli K12MG1655 competent cell containing pKD46 plasmid to obtain a positive transformant, and further eliminating the transformant by utilizing pCP20 plasmidThe obtained LacIZ gene is replaced by P xylF Positive transformants of the T7RNAP gene under the control of the promoter.
(2) Knock-out of nagBAC gene cluster, manXYZ gene cluster and nanE gene
Knocking out nagBAC gene cluster by CRISPR/Cas9 gene editing technology:
(1) designing an upstream homology arm primer (nagBAC-UF/UR) and a downstream homology arm primer (nagBAC-DF/DR) at two ends of a gene by using an E.coli K12MG1655 genome as a template according to a nagBAC gene sequence by adopting a PCR technology, and carrying out PCR amplification to obtain upstream and downstream homology arms of a nagBAC gene;
(2) adopting an overlapping PCR technology to obtain overlapping fragments of the upstream and downstream homologous arms of the nagBAC gene by PCR amplification by taking the upstream and downstream homologous arms of the nagBAC gene as templates;
(3) constructing a gRNA plasmid containing a Cas9 cutting recognition sequence, annealing a DNA fragment containing a target sequence by primers gRNA-nagBAC-F 'and gRNA-nagBAC-R', transferring the constructed gRNA plasmid into DH5 alpha transformation competent cells, and screening positive transformants;
(4) and (3) extracting recombinant gRNA plasmids of the positive transformants in the step (3), simultaneously electrically transferring the recombinant gRNA plasmids and the nagBAC gene knockout fragment constructed in the step (2) into positive transformant competent cells obtained in the step (1) containing pRedda9 plasmids, screening the positive transformants successfully knocking out the nagBAC genes, and eliminating the pRedda9 plasmids and the gRNA plasmids in the positive transformants to obtain the nagBAC gene knockout engineering strains. The manXYZ gene cluster and the nanE gene knockout method are the same as the above steps.
(3) Expression of Sc-gna1, glmS Gene
Integrating the Sc-gna1 gene into the positive transformant strain genome obtained in the step (2) by using a CRISPR/Cas9 gene editing technology:
(1) adopting PCR technology to take Saccharomyces cerevisiae genome as a template, designing a pair of primers (Sc-gna 1-F and Sc-gnal-R) according to Sc-gna1 gene sequence to amplify Sc-gna1 gene, adding T7 promoter and T7 terminator sequence to 5 'and 3' ends of amplification primer of gna1 fragment, and amplifying P T7 Sc-gna1 fragment (which can also be carried out on the basis of the gene sequence)Synthesis);
(2) designing upstream homology arm primers (nagE-UF and nagE-UR) and downstream homology arm primers (nagE-DF and nagE-DR) at two ends of a gene by using an E.coli K12MG1655 genome as a template according to a nagE gene sequence by adopting a PCR technology, and carrying out PCR amplification to obtain upstream and downstream homology arms of a to-be-integrated site gene;
(3) taking the amplified fragments obtained in the step (1) and the step (2) as templates, and obtaining Sc-gna1 gene integration fragments by an overlapping PCR technology, wherein the gene integration fragments comprise gene fragments of upstream and downstream homology arms of nagE gene of a site to be integrated and P T7 -Sc-gna1 gene fragment;
(4) a DNA fragment containing a target sequence used for constructing a gRNA plasmid is prepared by annealing primers gRNA-nagE-F 'and gRNA-nagE-R'; transforming the constructed gRNA plasmid into DH5 alpha transformation competent cells, and screening positive transformants;
(5) and (3) extracting recombinant gRNA plasmids of the positive transformants in the step (4), simultaneously electrotransferring the recombinant gRNA plasmids and the integration fragment of the gna1 gene constructed in the step (3) into the positive transformants obtained in the step (2) containing pReddas 9 plasmids, screening the positive transformants successfully integrating the gna1 gene, and eliminating the pReddas 9 plasmids and the gRNA plasmids in the positive transformants to obtain the genetic engineering bacteria integrating the gna1 gene. The method for integrating the glmS gene from E.coli K-12MG1655 source on the pseudogene yjiV, ycjV is identical to the above procedure.
Coli K-12MG1655 as a basal disc cell containing glmS (double copy expression) under the control of a T7 promoter, and a Sc-gna1 gene were obtained; an RNA polymerase gene (T7 RNAP) derived from a T7 bacteriophage under the control of a xylose promoter; and nagA, nagB, nagC, nagE, manX, manY, manZ and nanE eight gene-deficient N-acetylglucosamine-producing genetic engineering bacteria.
EXAMPLE 2 construction of glucosamine-producing strains
(1) Integration of the T7RNA polymerase gene (T7-RNAP)
Integration of the T7RNA polymerase gene (T7-RNAP) was performed using Red recombination technology.
(1) Adopting PCR technology, taking E.coli K12MG1655 genome as template, according to xylose promoter P xylF Design of a pair of primers (PxylF-F/R) for amplifying xylose promoter P by gene sequence xylF ；
(2) Using E.coli BL21 genome as a template by adopting PCR technology, designing a pair of primers (T7-F/R) according to the T7RNA polymerase gene sequence, and amplifying the T7RNA polymerase gene;
(3) the PCR technology is adopted, pKD3 plasmid is taken as a template, and a primer (Cm) is designed r -F/R) amplification of a chloramphenicol resistance gene fragment;
(4) adopting a PCR technology to design an upstream homology arm primer (LacIZ-UF/UR) and a downstream homology arm primer (LacIZ-DF/DR) at two ends of a gene by taking an E.coli K12MG1655 genome as a template according to a LacIZ gene sequence, and carrying out PCR amplification to obtain upstream and downstream homology arms of a to-be-integrated site gene;
(5) taking the amplified fragments obtained in the steps (1), (2), (3) and (4) as templates, and obtaining a T7RNA polymerase gene integration fragment by an overlapping PCR technology, wherein the gene integration fragment consists of gene fragments of upper and lower homologous arms of a LacIZ gene of a site to be integrated, a chloramphenicol resistance gene fragment and a T7RNA polymerase gene fragment added with a xylose promoter;
(6) introducing the gene fragment into a starting bacterium E.coli K12MG1655 competent cell containing pKD46 plasmid to obtain a positive transformant, further utilizing pCP20 plasmid to eliminate chloramphenicol resistance in the transformant, and replacing the LacIZ gene with P xylF Positive transformants of the T7RNAP gene under the control of the promoter.
(2) Knock-out of manXYZ gene cluster, nagE and nanE genes
Knocking out manXYZ gene cluster by using CRISPR/Cas9 gene editing technology:
(1) adopting a PCR technology, taking an E.coli K12MG1655 genome as a template, designing an upstream homology arm primer (manXYZ-UF/UR) and a downstream homology arm primer (manXYZ-DF/DR) at two ends of a gene according to a manXYZ gene sequence, and carrying out PCR amplification to obtain upstream and downstream homology arms of a manXYZ gene;
(2) adopting an overlapped PCR technology to obtain overlapped fragments of the upstream and downstream homology arms of manXYZ gene by taking the upstream and downstream homology arms of the manXYZ gene as templates through PCR amplification;
(3) constructing a gRNA plasmid containing a Cas9 cutting recognition sequence, annealing a DNA fragment containing a target sequence by primers gRNA-manXYZ-F 'and gRNA-manXYZ-R', transforming the constructed gRNA plasmid into a DH5 alpha transforming competent cell, and screening a positive transformant;
(4) and (3) extracting recombinant gRNA plasmids of the positive transformants in the step (3), simultaneously transferring the recombinant gRNA plasmids and the manXYZ gene knockout fragment constructed in the step (2) into positive transformant competent cells obtained in the step (1) containing pReddaS 9 plasmids, screening the positive transformants successfully knocking out the manXYZ genes, and eliminating the pReddaS 9 plasmids and gRNA plasmids in the positive transformants to obtain engineering strains for knocking out the manXYZ genes. The method for knocking out nagE and nanE genes is consistent with the steps.
(3) Expression of the phD Gene
Integrating phD gene into positive transformant strain genome obtained in step (2) by CRISPR/Cas9 gene editing technology:
(1) the diacetyl chitobiose deacetylase gene phD (Dac) shown in SEQ ID NO.10 is synthesized by using a gene synthesis technology ph ) Synthesizing the fragment to pUC-GW plasmid, designing a pair of primers (phD-F and phD-R) according to the phD gene sequence by using the plasmid as a template to amplify phD gene, adding the T7 promoter and T7 terminator sequences to 5 'and 3' ends of phD fragment amplification primers, and amplifying P T7 -a phD fragment;
(2) designing upstream homology arm primers (ygaY-UF and ygaY-UR) and downstream homology arm primers (ygaY-DF and ygaY-DR) at two ends of a gene by using an E.coli K12MG1655 genome as a template according to a pseudogene locus ygaY gene sequence by adopting a PCR technology, and performing PCR amplification to obtain upstream and downstream homology arms of a locus gene to be integrated;
(3) taking the amplified fragments obtained in the step (1) and the step (2) as templates, and obtaining a phD gene integration fragment by an overlapping PCR technology, wherein the gene integration fragment is composed of gene fragments of upstream and downstream homology arms of a gene of a locus to be integrated and the phD gene fragment as shown in figure 2;
(4) a DNA fragment containing a target sequence used for constructing a gRNA plasmid is prepared by annealing primers gRNA-ygaY-F 'and gRNA-ygaY-R'; transforming the constructed gRNA into DH5 alpha transformation competent cells through plasmid transformation, and screening positive transformants;
(5) and (3) extracting recombinant gRNA plasmids of the positive transformants in the step (4), simultaneously electrotransfering the recombinant gRNA plasmids and the phD gene integration fragments constructed in the step (3) into the positive transformants obtained in the step (2) containing pRedCas9 plasmids, screening the positive transformants successfully integrating the phD genes, and eliminating the pRedCas9 plasmids and the gRNA plasmids in the positive transformants to obtain the genetic engineering bacteria integrating the phD genes. The method for integration of the phD gene into the pseudogene yciQ was identical to the procedure described above. (for comparison, when a strain with three copies of the phD gene was constructed, one phD gene was integrated into ilvG).
(4) Expression of the yqaB Gene
Integrating yqaB gene into the positive transformant strain genome obtained in the step (3) by using CRISPR/Cas9 gene editing technology:
(1) adopting PCR technology to take E.coli K12MG1655 genome as template, designing a pair of primers (yqaB-F, yqaB-R) according to yqaB gene sequence to amplify yqaB gene, adding Trc promoter and Trc terminator sequence to yqaB fragment amplification primer 5 'and 3' ends, amplifying P Trc -a yqaB fragment;
(2) adopting a PCR technology to design upstream homology arm primers (yeeP-UF and yeeP-UR) and downstream homology arm primers (yeeP-DF and yeeP-DR) at two ends of a gene by taking an E.coli K12MG1655 genome as a template and according to a yeeP gene sequence of a pseudogene locus, and carrying out PCR amplification to obtain upstream and downstream homology arms of the gene of the locus to be integrated;
(3) taking the amplified fragments obtained in the step (1) and the step (2) as templates, and obtaining yqaB gene integration fragments through an overlapping PCR technology, wherein the gene integration fragments consist of gene fragments of upstream and downstream homologous arms of yeeP gene of a site to be integrated and yqaB gene fragments;
(4) a DNA fragment containing a target sequence and used for constructing a gRNA plasmid is prepared by annealing primers gRNA-yeeP-F 'and gRNA-yeeP-R'; transforming the constructed gRNA plasmid into DH5 alpha transformation competent cells, and screening positive transformants;
(5) and (5) extracting recombinant gRNA plasmids of the positive transformants in the step (4), simultaneously electrotransferring the recombinant gRNA plasmids and the yqaB gene integration fragments constructed in the step (3) into the positive transformants obtained in the step (3) containing pRedCas9 plasmids, screening the positive transformants successfully integrating the yqaB genes, and eliminating the pRedCas9 plasmids and gRNA plasmids in the positive transformants to obtain the genetic engineering bacteria integrating the yqaB genes.
Coli K-12MG1655 as a chassis cell, containing yqaB gene controlled by Trc promoter; diacetylchitobiose deacetylase gene phD from Pyrococcus (double copy expression) controlled by the T7 promoter; an RNA polymerase gene (T7 RNAP) derived from a T7 bacteriophage under the control of a xylose promoter; and the deacetylation of five gene defects of nagE, manX, manY, manZ and nanE to produce the gene engineering bacteria of the glucosamine.
Example 3 Shake flask fermentation experiment
Glucosamine is produced by mixing the strains constructed in the above examples 1 and 2 as production strains:
(1) Activated slant culture: inoculating 1-2 ring strains from a refrigerator bacteria-protecting tube at-80 deg.C with an inoculating ring, uniformly coating on a slant culture medium, culturing at 37 deg.C for 12h, transferring to the second generation slant culture medium, and culturing at 37 deg.C for 12h;
(2) Seed bottle culture: inoculating the thalli on the inclined plane into a 500mL triangular flask filled with 30mL seed culture medium by using an inoculating loop for preparing seed liquid, sealing the triangular flask by using twelve layers of gauze, and carrying out shake culture for 12h at 37 ℃ and 220 r/min;
(3) And (3) shake flask fermentation: respectively inoculating the two bacteria according to the inoculated OD 600 Adjusting the volume of two bacterium inocula according to the value proportion of 1; maintaining the pH at 7.0-7.2 (taking phenol red as an indicator, continuously changing the color of the fermentation liquor as sugar deficiency, supplementing 60% (m/v) of glucose solution during sugar deficiency), keeping the concentration of glucose in the fermentation liquor not more than 5g/L, and initially adding xylose solution with the final concentration of 10g/L to induce the expression of the target gene.
(4) And (4) collecting fermentation liquor after the fermentation is finished, centrifuging at 13000rpm, collecting supernate and determining the content of glucosamine.
(5) The detection method of glucosamine comprises the following steps: the column used was a ZORBAX Eclipse Plus C18 column (4.6 mm. Times.250 mm); the detector is an RID-20A refractive index detector; the mobile phase is methanol: buffer 2 (v/v), buffer 0.005M sodium octane sulfonate solution (pH = 5.1); the column temperature is set to 30-37 ℃ and the flow rate is set to 0.7mL/min. The time to peak of glucosamine was about 15.8 minutes.
Seed culture medium components: 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 O1 g/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, 1mL/L of mixed solution of trace elements, 1 drop of defoamer and the balance of water, the pH value is 6.8-7.2, and the mixture is sterilized by high-pressure steam at 115 ℃ for 15min;
fermentation medium components: glucose 20g/L, yeast powder 3g/L, (NH) 4 ) 2 SO 4 4g/L，KH 2 PO 4 6.67g/L，MgSO 4 ·7H 2 O2.5 g/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, 1mL/L of mixed solution of trace elements, 1 drop of defoamer and the balance of water, the pH value is 6.8-7.2, and the mixture is sterilized by high-pressure steam at 115 ℃ for 15min;
the trace element mixed liquid comprises the following components: na (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, and the balance of water.
The fermentation inoculation ratio related in this example is that the yield of N-acetyl glucosamine bacteria and the yield of glucosamine bacteria is OD after inoculation 600 Value 1, 1.
As can be seen from FIG. 1 (in FIG. 1, GLA represents a GlcNAC production strain constructed in example 1; DA-3 is a strain integrating yqaB gene on the basis of DA-1, namely a GlcN production strain constructed in example 2; DA-1 is a strain integrating two phD gene copies; DA-2 is a strain integrating one more phD gene copy on the basis of DA-1.), the final two engineering bacteria can realize labor division production of glucosamine in mixed fermentation, and after 24h of shake flask fermentation, when the inoculation ratios are 1.
Example 4 Shake flask fermentation experiment
Glucosamine is produced by mixing the strains constructed in the above examples 1 and 2 as production strains:
(1) Activated slant culture: inoculating 1-2 ring strains from a refrigerator bacteria-protecting tube at-80 ℃ by using an inoculating loop, uniformly coating the strains in a slant culture medium, culturing for 12 hours at 37 ℃, transferring the strains to a second generation slant culture medium, and culturing for 12 hours at 37 ℃;
(2) Seed bottle culture: inoculating the thallus on the inclined plane into a 500mL triangular flask filled with 30mL seed culture medium by using an inoculating loop for preparing seed liquid, sealing the triangular flask by using twelve layers of gauze, and performing shake culture for 12 hours at 37 ℃ and 220 r/min;
(3) And (3) shake flask fermentation: inoculating the two kinds of bacteria to OD 600 The value ratio is 1 600 Value 1 for inoculation), adjusting the volume of two-strain inoculation, keeping the total inoculation amount to be 12%, inoculating to a fermentation medium, and culturing at 36 ℃ and 210rpm for 24h; maintaining the pH at 7.0-7.2 (taking phenol red as an indicator, continuously changing the color of the fermentation liquor as sugar deficiency, supplementing 60% (m/v) of glucose solution during sugar deficiency), keeping the concentration of glucose in the fermentation liquor not more than 5g/L, and initially adding xylose solution with the final concentration of 10g/L to induce the expression of the target gene.
(4) And (4) after the fermentation is finished, collecting fermentation liquor, centrifuging at 13000rpm, collecting supernate and determining the content of glucosamine, wherein the content in the fermentation liquor reaches 9.06g/L in 24 hours.
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 O1 g/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, 1mL/L of mixed solution of trace elements, 1 drop of defoaming agent and the balance of water, the pH value is 6.8-7.2, and the sterilization is carried out for 15min by high-pressure steam at 115 ℃;
fermentation medium components: 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 O3.5 g/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, 1.5mL/L of mixed solution of trace elements, 1 drop of antifoaming agent and the balance of water, the pH value is 6.8-7.2, and the mixture is sterilized by high-pressure steam at 115 ℃ for 15min;
the microelement mixed solution comprises the following components: na (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 ·18H 2 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, and the balance of water.
Example 5 Shake flask fermentation experiments
Glucosamine is produced by mixing the strains constructed in the above examples 1 and 2 as production strains:
(1) Activated slant culture: inoculating 1-2 ring strains from a refrigerator bacteria-protecting tube at-80 ℃ by using an inoculating loop, uniformly coating the strains in a slant culture medium, culturing for 12 hours at 37 ℃, transferring the strains to a second generation slant culture medium, and culturing for 12 hours at 37 ℃;
(2) Seed bottle culture: inoculating the thalli on the inclined plane into a 500mL triangular flask filled with 30mL seed culture medium by using an inoculating loop for preparing seed liquid, sealing the triangular flask by using twelve layers of gauze, and carrying out shake culture for 12h at 37 ℃ and 220 r/min;
(3) And (3) shaking flask fermentation: inoculating the two kinds of bacteria to OD 600 The value ratio is 1 600 Value 1 inoculation), adjusting the volume of two-strain inoculation, keeping the total inoculation amount at 15%, inoculating to a fermentation medium, and culturing at 37 ℃ and 230rpm for 36h; and (2) maintaining the pH at 7.0-7.2 (taking phenol red as an indicator, regarding the color of the fermentation liquor as sugar deficiency continuously unchanged, supplementing 60% (m/v) of glucose solution when the sugar deficiency occurs), keeping the concentration of the glucose in the fermentation liquor not more than 5g/L, and initially adding xylose solution with the final concentration of 10g/L to induce the expression of the target gene.
(4) Collecting fermentation liquor, centrifuging at 13000rpm, collecting supernatant to determine glucosamine content, wherein the content in the fermentation liquor reaches 9.8g/L after 36 h.
Seed culture medium components: glucose 20g/L, yeast powder 3g/L, (NH) 4 ) 2 SO 4 2g/L，KH 2 PO 4 2g/L，MgSO 4 ·7H 2 O1 g/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, 1mL/L of mixed solution of trace elements, 1 drop of defoaming agent and the balance of water, the pH value is 6.8-7.2, and the sterilization is carried out for 15min by high-pressure steam at 115 ℃;
fermentation medium components: 25g/L glucose, 4g/L yeast powder, (NH) 4 ) 2 SO 4 4g/L，KH 2 PO 4 8g/L，MgSO 4 ·7H 2 O4 g/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, 1mL/L of mixed solution of trace elements, 1 drop of defoamer and the balance of water, the pH value is 6.8-7.2, and the mixture is sterilized by high-pressure steam at 115 ℃ for 15min;
the microelement mixed solution comprises the following components: na (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 3 BO 3 0.07g/L, and the balance of water.
In conclusion, the genetically engineered bacterium provided by the invention realizes the high-efficiency synthesis and deacetylation of N-acetylglucosamine to generate glucosamine in a mixed bacterium fermentation system by strengthening the synthesis way of the N-acetylglucosamine and introducing heterologous deacetylase. The reinforcement of the N-acetylglucosamine synthesis pathway is constructed by using a CRISPR/Cas9 technology according to the following method: eight genes of nagA, nagB, nagC, nagE, manX, manY, manZ and nanE are knocked out from an original strain E.coli K12MG1655, RNA polymerase synthesis from a T7 bacteriophage is induced by adopting a xylose strong promoter so as to realize induction enhancement of an N-acetylglucosamine synthesis path regulated and controlled by the T7 promoter, and the induction enhancement comprises introducing a gna1 gene (Sc-gna 1) from saccharomyces cerevisiae and carrying out multi-copy enhancement on a glmS gene in Escherichia coli K-12MG 1655. The deacetylation of N-acetylglucosamine is constructed by the CRISPR/Cas9 technology according to the following method: knocking out nagE, manX, manY, manZ and nanE five genes in an original strain E.coli K12MG1655, inducing RNA polymerase synthesis from T7 bacteriophage by adopting xylose strong promoter, introducing gene phD (Dac) derived from pyrococcus ph ) And combining a T7 strong promoter system to start the expression of a target gene and carrying out multi-copy enhancement, and enhancing the yqaB gene in the E.coli K-12 MG1655. Finally, after 24 hours of shake flask fermentation, the yield of glucosamine can reach 11.21g/L to the maximum extent, the production intensity can reach 0.47 g/(L multiplied by h), and the method has strong industrial production potential.
TABLE 1 primers involved in the construction of the strains of the invention
The above detailed description of the construction of the genetically engineered bacterium for producing N-acetylglucosamine and a genetically engineered bacterium for producing glucosamine by deacetylation and the process of fractional fermentation using mixed fermentation, which are given by way of illustration and not of limitation, can be regarded as a number of examples within the scope of the invention, and thus variations and modifications thereof without departing from the general concept of the invention are intended to be within the scope of the invention.
Claims (10)
1. The Escherichia coli genetic engineering strain for producing N-acetylglucosamine is characterized in that the genetic engineering strain takes Escherichia coli as a host and comprises a glmS gene and a Sc-gna1 gene which are controlled by a T7 promoter; RNA polymerase gene from T7 phage under the control of xylose promoter; and deletion of expression of eight genes, nagA, nagB, nagC, nagE, manX, manY, manZ, nanE.
2. The Escherichia coli genetic engineering strain for producing glucosamine is characterized in that the genetic engineering strain takes Escherichia coli as a host, overexpresses yqaB gene, introduces a Pyrococcus diacetylchitobiose deacetylase gene phD, and utilizes a xylose promoter (P) xylF ) The RNA polymerase gene from T7 phage is induced, and the expression of five genes of nagE, manX, manY, manZ and nanE is deleted.
3. The genetically engineered bacterium of escherichia coli for producing N-acetylglucosamine according to claim 1 or the genetically engineered bacterium of escherichia coli for producing glucosamine according to claim 2, wherein the host cell is e.
4. The genetically engineered bacterium of Escherichia coli for producing N-acetylglucosamine according to claim 1 or the genetically engineered bacterium of Escherichia coli for producing glucosamine according to claim 2,
the nucleotide sequence of the Sc-gna1 is shown in a sequence table SEQ ID No. 1;
the nucleotide sequence of the glmS is shown as a sequence table SEQ ID No. 2;
the nucleotide sequence of nagA is shown in a sequence table SEQ ID No. 3;
the nucleotide sequence of nagB is shown in a sequence table SEQ ID No. 4;
the nucleotide sequence of nagC is shown in a sequence table SEQ ID No. 5;
the nucleotide sequence of nagE is shown in a sequence table SEQ ID No. 6;
the nucleotide sequence of manX is shown in a sequence table SEQ ID No. 7;
the nucleotide sequence of manY is shown in a sequence table SEQ ID No. 8;
the nucleotide sequence of manZ is shown in a sequence table SEQ ID No. 9;
the nucleotide sequence of the phD is shown in a sequence table SEQ ID No.10
The nucleotide sequence of the T7 promoter is shown as a sequence table SEQ ID No. 11;
the P is xylF The nucleotide sequence of the promoter is shown as a sequence table SEQ ID No. 12;
the nucleotide sequence of the RNA polymerase coding gene derived from the T7 bacteriophage is shown in a sequence table SEQ ID No. 13;
the nucleotide sequence of nanE is shown in a sequence table SEQ ID No. 14;
the nucleotide sequence of the Trc promoter is shown in a sequence table SEQ ID No. 15;
the nucleotide sequence of the yqaB is shown as a sequence table SEQ ID No. 16.
5. The genetically engineered bacterium of claim 1, wherein the glmS gene is expressed in two copies.
6. The glucosamine production escherichia coli genetically engineered bacterium of claim 2, wherein the phD gene is expressed in double copies.
7. Use of the engineered bacterium of any one of claims 1-6 in the production of glucosamine.
8. The use according to claim 7, characterized in that the method for the fermentative production of glucosamine comprises the following steps:
inoculating the seed liquid of Escherichia coli gene engineering bacteria for producing N-acetylglucosamine and Escherichia coli gene engineering bacteria for producing glucosamine according to the OD 600 1-1, and 4, inoculating 10-15% of total inoculum size to a fermentation culture medium, and culturing at 32-37 ℃ and 200-300rpm for 24-36h; maintaining the pH at 6.8-7.2, maintaining the fermentation with 60% glucose solution, and adding xylose solution with final concentration of 5-20g/L to induce the expression of target gene.
9. The use according to claim 7, wherein the fermentation broth comprises the following components: 10-30g/L of glucose, 1-6g/L of yeast powder, (NH) 4 ) 2 SO 4 1-5g/L，KH 2 PO 4 3-8g/L，MgSO 4 ·7H 2 O1-5 g/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 10-30mg/L，V H 0.05-2mg/L，V B1 0.1-1mg/L, 1-3mL/L of mixed solution of trace elements, 1-2 drops of defoaming agent and the balance of water, the pH value is 6.8-7.2, and the sterilization is carried out for 15-20min by high-pressure steam at 115 ℃.
10. The use of claim 9, wherein the mixed solution of trace elements comprises: na (Na) 2 MoO 4 ·2H 2 O 1-3g/L，NiCl 2 ·6H 2 O 0.5-1.5g/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 0.1-0.3g/L，CoCl 2 ·6H 2 O 0.5-1.5g/L，ZnSO 4 ·2H 2 O 0.1-0.5g/L，H 3 BO 3 0.05-0.2g/L, and the balance of water.
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| CN104498517A (en) * | 2014-11-29 | 2015-04-08 | 滨州市金朗生物科技有限公司 | Construction and application methods of escherichia coli with high yield of producing N-acetylglucosamine |
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| CN112430562A (en) * | 2020-12-04 | 2021-03-02 | 天津科技大学 | Genetically engineered bacterium for producing N-acetylglucosamine and application thereof |
| CN112877272A (en) * | 2021-04-28 | 2021-06-01 | 中国农业科学院北京畜牧兽医研究所 | Escherichia coli engineering bacteria of N-acetylglucosamine and fermentation production method |
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| CN104498517A (en) * | 2014-11-29 | 2015-04-08 | 滨州市金朗生物科技有限公司 | Construction and application methods of escherichia coli with high yield of producing N-acetylglucosamine |
| CN108070604A (en) * | 2017-10-10 | 2018-05-25 | 江南大学 | A kind of method of recombination bacillus coli resting cell production Glucosamine |
| CN112430562A (en) * | 2020-12-04 | 2021-03-02 | 天津科技大学 | Genetically engineered bacterium for producing N-acetylglucosamine and application thereof |
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