CN112430562A

CN112430562A - Genetically engineered bacterium for producing N-acetylglucosamine and application thereof

Info

Publication number: CN112430562A
Application number: CN202011398296.9A
Authority: CN
Inventors: 马倩; 孙全伟; 谭淼; 夏利; 张颖; 杨蒙雅; 谢希贤; 陈宁; 徐庆阳; 张成林; 李燕军; 范晓光
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2021-03-02
Anticipated expiration: 2040-12-04
Also published as: CN112430562B

Abstract

The invention relates to the technical field of genetic engineering, in particular to a genetic engineering bacterium for industrial production of N-acetylglucosamine based on composite carbon source metabolism and application thereof. Through constructing a synthesis way for strengthening N-acetylglucosamine, knocking out a 6-phosphofructokinase gene and directionally mutating a glycerol kinase expression gene, the utilization of a glycerol glucose composite carbon source is subjected to metabolic division, so that glucose is mainly used for producing a target product N-acetylglucosamine, and glycerol is mainly used for cell growth to improve the synthesis efficiency of the N-acetylglucosamine. After the mixed carbon source culture medium is subjected to shake flask fermentation for 48 hours, the yield of the N-acetylglucosamine reaches 107.8g/L to the maximum, the conversion rate of the composite carbon source reaches 49% to the maximum, compared with a glucose utilization strain, the yield is improved by 151.7%, the conversion rate is improved by 60.2%, and the method has strong industrial production potential.

Description

Genetically engineered bacterium for producing N-acetylglucosamine and application thereof

The technical field is as follows:

the invention relates to the technical field of genetic engineering, in particular to a genetic engineering bacterium for industrial production of N-acetylglucosamine based on composite carbon source metabolism and application thereof.

Background art:

n-acetylglucosamine is a functional monosaccharide, and has important effects on repairing and health care of bone joints and relieving osteoarthritis, so that the N-acetylglucosamine has wide market application requirements as a health care product and a medicine. The traditional production method mainly comprises the step of extracting the shrimp shells and the crab shells through acid hydrolysis, and has the problems of environmental pollution, easy sensitization and the like. In recent years, reports of the production of N-acetylglucosamine by a microbial fermentation method have been increasing with the rapid progress of metabolic engineering. At present, fermentation production of microorganisms such as Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum and the like has been realized. The biosynthesis process of N-acetylglucosamine needs fructose-6-phosphate in glycolysis pathway as precursor, so that the synthesis process and cell growth metabolism form a competitive relationship, and the conversion rate of N-acetylglucosamine is limited.

With the vigorous development of the biodiesel industry, a large amount of crude glycerol is remained and is used as a cheap carbon source and has rich sources. According to the invention, through constructing the Escherichia coli of the N-acetylglucosamine production bacterium, knocking out the pfkA gene, blocking the glycolysis process, dividing the carbon metabolism process, and using glycerol as a cheap carbon source for cell growth, the glucose can be more efficiently used for synthesizing the N-acetylglucosamine, and the synthesis efficiency and the conversion rate are obviously improved.

The invention content is as follows:

the invention aims to provide a genetic engineering bacterium for producing N-acetylglucosamine by metabolic division based on a composite carbon source and a method for producing the N-acetylglucosamine by using the genetic engineering bacterium.

One of the technical schemes to be provided by the invention is as follows: the engineering strain takes escherichia coli as a host, expresses glmS gene and Sc-gna1 gene, constructs a synthetic pathway of N-acetylglucosamine, constructs a plasmid-free system, and utilizes a xylose promoter (P)_xylF) RNA polymerase genes from the T7 phage were induced to initiate expression of the gene of interest in combination with a strong promoter system of T7. Meanwhile, site-directed knockout is carried out on 6-phosphofructokinase gene in glycolysis process, and the metabolic division of complex carbon source is realized by introducing the Escherichia coli glycerol kinase gene glpK (G35913) of literature report (Pettigrew: A single amino acid change in Escherichia coli glycerol kinase enzymes glucose control of glycerol fermentation in vivo. J Bacteriol,1996,178(10): 2846-2852).

The genetically engineered bacterium for producing the N-acetylglucosamine comprises glmS from E.coli K-12MG1655 bacteria controlled by a T7 promoter, and a Sc-gna1 gene from saccharomyces cerevisiae; escherichia coli glycerol kinase mutant gene glpK; an RNA polymerase gene (T7RNAP) from a T7 bacteriophage source under the control of a xylose promoter; and nine gene-defects of nagA, nagB, nagC, nagE, manX, manY, manZ, nanE, pfkA.

The nucleotide sequence of the coding gene Sc-gna1 is shown in a sequence table SEQ ID No. 1;

the nucleotide sequence of the coding gene glmS is shown as a sequence table SEQ ID No. 2;

the nucleotide sequence of the coding gene nagA is shown in a sequence table SEQ ID No. 3;

the nucleotide sequence of the coding gene nagB is shown in a sequence table SEQ ID No. 4;

the nucleotide sequence of the coding gene nagC is shown in a sequence table SEQ ID No. 5;

the nucleotide sequence of the coding gene nagE is shown in a sequence table SEQ ID No. 6;

the nucleotide sequence of the coding gene manX is shown in a sequence table SEQ ID No. 7;

the nucleotide sequence of the coding gene manY is shown in a sequence table SEQ ID No. 8;

the nucleotide sequence of the coding gene manZ is shown in a sequence table SEQ ID No. 9;

the nucleotide sequence of the coding gene pfkA is shown in a sequence table SEQ ID No. 10;

the nucleotide sequence of the coding gene glpK is shown in a sequence table SEQ ID No. 11;

the nucleotide sequence of the promoter of the coding gene T7 is shown in a sequence table SEQ ID No. 12;

the coding gene P_xylFThe nucleotide sequence of the promoter is shown as a sequence table SEQ ID No. 13;

the nucleotide sequence of the RNA polymerase coding gene derived from the T7 phage is shown in a sequence table SEQ ID No. 14;

the nucleotide sequence of the coding gene nanE is shown as a sequence table SEQ ID No. 15;

the genetically engineered bacterium for producing N-acetylglucosamine takes E.coli W3110(ATCC 27325) as a host cell.

The second technical scheme provided by the invention is as follows: the construction method of the genetic engineering bacteria for producing the N-acetylglucosamine by the division and metabolism of the escherichia coli composite carbon source comprises the following steps:

(1) integration at the lacIZ Gene site was initiated by xyloseMover P_xylFControlled 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_T7The fructose-6-phosphate transaminase gene glmS under the control of a promoter.

(3) Knocking out expression 6 phosphofructokinase gene pfkA in glycolysis pathway; meanwhile, Escherichia coli glycerol kinase gene mutation glpK is introduced to realize metabolism and division of the composite carbon source.

The third technical scheme provided by the invention is as follows: the method for producing the N-acetylglucosamine by the fermentation of the genetic engineering bacteria comprises the following specific steps:

(1) seed culture: inoculating slant strain into seed culture medium, culturing at 32-37 deg.C and 200-300rpm for 6-12h, and maintaining pH at 6.8-7.2.

(2) Fermentation culture: inoculating the fermentation culture medium according to the inoculation amount of 10-15%, and culturing at the temperature of 32-37 ℃ and the speed of 200-300rpm for 36-72 h; maintaining the pH at 7.0-7.2, maintaining 60% (m/v) of glycerol and glucose composite carbon source for fermentation (taking phenol red as an indicator, continuously keeping the color of fermentation liquor as sugar deficiency, supplementing 60% (m/v) of glycerol and glucose composite carbon source during sugar deficiency), and initially adding xylose solution with the final concentration of 5-20g/L to induce the expression of target genes;

after 36-72h of shake flask fermentation, the yield of N-acetylglucosamine reaches 80-107.8 g/L.

The seed culture solution comprises the following components: 10-30g/L of glucose or glycerol glucose composite carbon source, 2-10g/L of yeast powder, (NH)₄)₂SO₄ 1-5g/L，KH₂PO₄ 1-5g/L，MgSO₄·7H₂O1-5 g/L, citric acid 1-5g/L, FeSO₄·7H₂O 1-5mg/L，MnSO₄·7H₂O 1-5mg/L，V_H 0.05-2mg/L，V_B10.1-2mg/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 mixture is sterilized by high-pressure steam at 115 ℃ for 15 min;

preferably, the carbon source adopted by the seed culture solution is a glycerol glucose complex carbon source;

the fermentation culture solution comprises the following components: 10-30g/L of glycerol glucose composite carbon source, 1-6g/L of yeast powder, (NH)₄)₂SO₄ 1-5g/L，KH₂PO₄ 3-8g/L，MgSO₄·7H₂O1-5 g/L, citric acid 1-5g/L, FeSO₄·7H₂O 30-90mg/L，MnSO₄·7H₂O 1-5mg/L，NaCl 0.5-2g/L，CaCl₂·2H₂O 10-30mg/L，V_H 0.05-2mg/L，V_B10.1-1mg/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 glycerol glucose composite carbon source consists of glycerol and glucose in a mass ratio of 1-4: 1-8;

preferably, the glycerol glucose complex carbon source is prepared by mixing glycerol and glucose in a mass ratio of 1: 8, preparing a mixture;

the trace element mixed liquid comprises the following components: na (Na)₂MoO₄·2H₂O 1-3g/L，NiCl₂·6H₂O 0.5-1.5g/L，CaCl₂·2H₂O 2-8g/L，CuSO₄·5H₂O 0.1-0.5g/L，Al₂(SO₄)₃·18H₂O 0.1-0.3g/L，CoCl₂·6H₂O 0.5-1.5g/L，ZnSO₄·2H₂O 0.1-0.5g/L，H₃BO₃0.05-0.2g/L, and the balance of water.

Has the advantages that:

the invention transfers the N-acetylglucosamine phosphotransferase gene (Sc-gna1) derived from microzyme (Saccharomyces cerevisiae) into Escherichia coli, and expresses fructose-6-phosphotransferase 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.

According to the invention, 6-phosphofructokinase gene pfkA is knocked out, and the site-directed mutation glycerol kinase gene is introduced, so that the metabolic division of a composite carbon source is realized, glycerol is more used for thallus growth, glucose is mainly used for the generation of N-acetylglucosamine products, and the synthesis efficiency of N-acetylglucosamine is improved.

The constructed strain for producing the N-acetylglucosamine enhances the metabolic flux from glucose to the N-acetylglucosamine through a series of transformation, so that the engineering bacteria can efficiently produce the N-acetylglucosamine by using glycerol and glucose, the yield of the N-acetylglucosamine is up to 107.8g/L after the shake flask fermentation of a composite carbon source culture medium is carried out for 48 hours, the conversion rate of a composite carbon source is up to 49%, compared with a glucose utilization strain, the yield is increased by 151.7%, the conversion rate is increased by 60.2%, and the industrial production potential is stronger.

Description of the drawings:

FIG. 1 validation of nagBAC gene cluster knock-out. Wherein: m: 1kb DNA marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping segments; 4: PCR fragment of original bacterium genome; 5: and (4) identifying fragments of positive bacteria.

FIG. 2Sc-gna1 gene expression validation. Wherein: m: 1kb DNA marker; 1: an upstream homology arm; 2: target gene 3: a downstream homology arm; 4: overlapping segments; 5: PCR fragment of original bacterium genome; 6: and (4) identifying fragments of positive bacteria.

FIG. 3 fermentation validation

Wherein Glucose is Glucose utilization strain (constructed in example 1) and Glucose is used as carbon source to produce N-acetylglucosamine by fermentation;

mix carbon sources were a complex carbon source-utilizing strain (constructed in example 2) that utilized glycerol glucose complex carbon source to produce N-acetylglucosamine.

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.

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 technique.

Coli W3110 genome as template by PCR technique, based on xylose promoter P_xylFGene sequence design a pair of primers (PxylF-F/R) for amplifying xylose promoter P_xylF；

Secondly, a PCR technology is adopted, E.coli BL21 genome is used as a template, a pair of primers (T7-F/R) is designed according to a T7RNA polymerase gene sequence, and a T7RNA polymerase gene is amplified;

thirdly, designing a primer (Cm) by adopting PCR technology and taking pKD3 plasmid as a template^r-F/R) amplification of a chloramphenicol resistance gene fragment;

fourthly, designing upstream homology arm primers (T7-UF/UR) and downstream homology arm primers (T7-DF/DR) at two ends of a gene by adopting a PCR technology and taking an E.coli W3110 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;

fifthly, taking the amplified fragment obtained in the third step as a template, and obtaining a T7RNA polymerase gene integration fragment by an overlapping PCR technology, wherein the gene integration fragment consists of gene fragments with the upper and lower homologous arms of an integration site gene, chloramphenicol resistance gene fragments and a T7RNA polymerase gene fragment added with a xylose promoter;

sixthly, the gene fragment is led into a starting bacterium E.coli W3110 competent cell containing pKD46 plasmid to obtain a positive transformant, and chloramphenicol resistance in the transformant is eliminated to obtain a LacIZ gene which is replaced by P_xylFPositive transformants of the T7RNAP gene under the control of the promoter.

(2) Knock-out of nagBAC gene cluster, manXYZ gene cluster, nagE gene and nanE gene

Knocking out nagBAC gene cluster by using CRISPR/Cas9 gene editing technology:

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 adopting a PCR technology and taking an E.coli W3110 genome as a template according to a nagBAC gene sequence, and carrying out PCR amplification to obtain upstream and downstream homology arms of a nagBAC gene;

secondly, using the upstream and downstream homologous arms of the nagBAC gene as a template by adopting an overlapping PCR technology, and carrying out PCR amplification to obtain overlapping fragments of the upstream and downstream homologous arms of the nagBAC gene;

thirdly, constructing a gRNA plasmid containing a Cas9 cutting recognition sequence, annealing a DNA fragment containing a target sequence by primers nagBAC-F 'and nagBAC-R', transforming the constructed gRNA plasmid into DH5 alpha transformation competent cells, and screening positive transformants;

and fourthly, extracting recombinant gRNA plasmids of the positive transformants in the third step, simultaneously electrically transferring the recombinant gRNA plasmids of the positive transformants into the positive transformant competent cells obtained in the step (1) containing pRedda 9 plasmid together with the nagBAC gene knockout fragment constructed in the second step, screening the positive transformants successfully knocking out the nagBAC gene, and obtaining the nagBAC gene knockout engineering strains after eliminating the pRedda 9 plasmid and the gRNA plasmid in the positive transformants.

The electrophoretogram for construction of overlapping fragments and PCR validation of positive strains is shown in FIG. 1. Wherein: m: 1kb DNA marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping segments; 4: PCR fragment of original bacterium genome; 5: and (4) identifying fragments of positive bacteria.

The manXYZ gene cluster and the nagE and nanE gene knockout method are consistent with the 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:

firstly, adopting PCR technology to use Saccharomyces cerevisiae genome as a template, designing a pair of primers (Sc-gna1-F, 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-a Sc-gna1 fragment;

secondly, designing upstream homology arm primers (Sc-gna1-UF and Sc-gna1-UR) and downstream homology arm primers (Sc-gna1-DF and Sc-gna1-DR) at two ends of a gene by using an E.coli W3110 genome as a template according to an integration site gene sequence by adopting a PCR technology, and performing PCR amplification to obtain upstream and downstream homology arms of a to-be-integrated site gene;

thirdly, taking the amplified fragment obtained in the first step and the second step as a template, and obtaining a Sc-gna1 gene integration fragment by an overlapping PCR technology, wherein the gene integration fragment consists of gene fragments with integration site genes, upstream and downstream homologous arms and a Sc-gna1 gene fragment;

fourthly, DNA fragments containing target sequences used for constructing the gRNA plasmids are prepared by annealing primers gna1-F 'and gna 1-R'; the constructed gRNA is transformed into DH5 alpha transformation competent cells through plasmid transformation, and positive transformants are screened;

fifthly, extracting recombinant gRNA plasmids of the positive transformants in the step (iv), simultaneously electrically transferring the recombinant gRNA plasmids and the integration fragments of the gna1 gene constructed in the step (iii) into the positive transformants obtained in the step (2) containing the pRedCas9 plasmid, screening the positive transformants successfully integrating the gna1 gene, and eliminating the pRedCas9 plasmid and the gRNA plasmid in the positive transformants to obtain the genetically engineered bacteria integrating the gna1 gene.

The electrophorograms of the construction of overlapping fragments and PCR validation of positive strains are shown in FIG. 2. Wherein: m: 1kb DNA marker; 1: an upstream homology arm; 2: target gene 3: a downstream homology arm; 4: overlapping segments; 5: PCR fragment of original bacterium genome; 6: and (4) identifying fragments of positive bacteria.

The procedure for integration of glmS gene from E.coli K-12MG1655 source on pseudogenes yjiv, ycjV was identical to the procedure described above.

Example 2 construction of Complex carbon Source metabolizing and Industrial production of N-acetylglucosamine strains

(1) pfkA gene knockout by CRISPR/Cas9 gene editing technology

Designing an upstream homology arm primer (pfkA-UF/UR) and a downstream homology arm primer (pfkA-DF/DR) at two ends of a gene according to a pfkA gene sequence by adopting a PCR technology and taking an E.coli W3110 genome as a template, and carrying out PCR amplification to obtain upstream and downstream homology arms of the pfkA gene;

secondly, an overlapping PCR technology is adopted, and the upstream and downstream homologous arms of the pfkA gene are taken as templates, and PCR amplification is carried out to obtain overlapping fragments of the upstream and downstream homologous arms of the pfkA gene;

thirdly, constructing a gRNA plasmid containing a Cas9 cutting recognition sequence, annealing a DNA fragment containing a target sequence by primers pfkA-F 'and pfkA-R', transforming the constructed gRNA plasmid into DH5 alpha transformation competent cells, and screening positive transformants;

and fourthly, extracting recombinant gRNA plasmids of the positive transformants in the third step, simultaneously transferring the recombinant gRNA plasmids and the pfkA gene knockout fragment constructed in the second step into positive transformant competent cells constructed in the example 1 and containing pRedda 9 plasmids, screening the positive transformants successfully knocking out the pfkA genes, and obtaining the pfkA gene knockout engineering strains after eliminating the pRedda 9 plasmids and gRNA plasmids in the positive transformants.

(2) The site-directed mutagenesis of glpK gene is carried out by using CRISPR/Cas9 gene editing technology:

designing an upstream homology arm primer (glpK-UF/UR) and a downstream homology arm primer (glpK-DF/DR) at two ends of a gene according to a glpK gene sequence by adopting a PCR (polymerase chain reaction) technology and taking an E.coli W3110 genome as a template, and carrying out PCR amplification to obtain upstream and downstream homology arms of the glpK gene;

secondly, an overlapping PCR technology is adopted, and the upstream and downstream homologous arms of the glpK gene are taken as templates, and PCR amplification is carried out to obtain overlapping fragments of the upstream and downstream homologous arms of the glpK gene;

thirdly, constructing a gRNA plasmid containing a Cas9 cutting recognition sequence, annealing a DNA fragment containing a target sequence by primers glpK-F 'and glpK-R', transforming the constructed gRNA plasmid into a DH5 alpha transformation competent cell, and screening a positive transformant;

fourthly, extracting recombinant gRNA plasmids of the positive transformants in the third step, simultaneously electrically transferring the recombinant gRNA plasmids and the glpK gene knockout fragment constructed in the second step into positive transformant competent cells obtained in the step (1) containing pReddAS 9 plasmids, screening the positive transformants successfully knocking out the glpK gene, and obtaining strains knocking out the glpK gene after eliminating the pReddAS 9 plasmids and the gRNA plasmids in the positive transformants;

using E.coli W3110 genome as template by PCR technique, designing a pair of primers (glpK-U, glpK-D) to amplify mutant gene glpK according to the E.coli glpK gene mutation information reported in literature (Pettigrew: A single amino acid change in Escherichia coli glycerol kinase enzymes glucose control of glycerol inactivation in vivo. J Bacteriol,1996,178(10):2846 and 2852);

integrating glpK at the position of the knocked-out glpK by adopting a PCR technology and taking an E.coli W3110 genome as a template, designing upstream homology arm primers (glpK-UF and glpK-UR) and downstream homology arm primers (glpK-DF and glpK-DR) at two ends of the gene according to an integration site gene sequence, and carrying out PCR amplification to obtain upstream and downstream homology arms of the gene of the site to be integrated;

seventhly, obtaining a glpK gene integration fragment by using the amplification fragments obtained in the fifth step and the sixth step as templates through an overlapped PCR technology, wherein the gene integration fragment consists of gene fragments with homologous arms at the upstream and the downstream of the integration site gene and the glpK gene fragment;

the DNA fragment containing the target sequence used for constructing the gRNA plasmid is prepared by annealing primers glpK-F 'and glpK-R'; the constructed gRNA is transformed into DH5 alpha transformation competent cells through plasmid transformation, and positive transformants are screened;

ninthly, extracting recombinant gRNA plasmids of positive transformants in the step ((c)), and simultaneously electrically transferring the recombinant gRNA plasmids and the glpK gene integration fragments constructed in the step ((c)) into positive transformant competent cells of the step ((2)) containing pReddCas 9 plasmid, screening the positive transformants successfully integrating the glpK gene, and eliminating the pReddCas 9 plasmid and the gRNA plasmid in the positive transformants to obtain genetically engineered bacteria integrating the glpK gene.

Example 3 Shake flask fermentation experiment

The strain constructed in the above example 2 was used as a production strain to produce N-acetylglucosamine:

(1) seed culture: a loopful of the strain was scraped using an inoculating loop into a 500mL shaking flask of 30mL volume and cultured at 37 ℃ and 220rpm for 12 hours.

(2) And (3) shaking flask fermentation: inoculating a fermentation culture medium according to the inoculation amount of 10%, and culturing at 37 ℃ and 220rpm for 48 h; maintaining the pH at 7.0-7.2, maintaining 60% (m/v) of glycerol and glucose composite carbon source for fermentation (taking phenol red as an indicator, regarding the color of fermentation liquor as sugar deficiency, supplementing 60% (m/v) of glycerol and glucose composite carbon source during sugar deficiency), and initially adding xylose solution with the final concentration of 10g/L to induce the expression of target genes.

(3) Collecting fermentation liquor, centrifuging at 13000rpm, collecting supernatant to determine N-acetylglucosamine content, and allowing the content in the fermentation liquor to reach 107.8g/L in 48h (FIG. 3).

(4) The detection method of the N-acetylglucosamine comprises the following steps: the chromatographic column is

HPX-87H column (7.8 mm. times.300 mm); the detector is an RID-20A refractive index detector; the mobile phase is 5mmol/L sulfuric acid solution; the column temperature is set to be 30-40 ℃, and the flow rate is set to be 0.5 mL/min. The peak time of N-acetylglucosamine was about 14.2 minutes.

Seed culture medium components: 20g/L of glycerol glucose composite carbon source, 3g/L of yeast powder, (NH)₄)₂SO₄ 2g/L，KH₂PO₄2g/L，MgSO₄·7H₂O1 g/L, citric acid 2g/L, FeSO₄·7H₂O 2.8mg/L，MnSO₄·7H₂O 1.2mg/L，V_H0.1mg/L，V_B10.5mg/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 culture components: 20g/L of glycerol glucose composite carbon source, 3g/L of yeast powder, (NH)₄)₂SO₄ 4g/L，KH₂PO₄6.67g/L，MgSO₄·7H₂O2.5 g/L, citric acid 3.55g/L, FeSO₄·7H₂O 30mg/L，MnSO₄·7H₂O 1.2mg/L，NaCl 2g/L，CaCl₂·2H₂O 25mg/L,V_H 0.1mg/L，V_B10.5mg/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 ℃;

the glycerol-glucose composite carbon source related in the embodiment is prepared by mixing glycerol and glucose in a mass ratio of 1: 8 (comparative example, the complex carbon sources were replaced with equal amounts of glucose carbon sources, the strains were replaced with the glucose utilization strains constructed in example 1, and other conditions were unchanged);

trace element mixed liquor componentComprises the following steps: na (Na)₂MoO₄·2H₂O 1.25g/L，NiCl₂·6H₂O 0.8g/L，CaCl₂·2H₂O 5g/L，CuSO₄·5H₂O 0.2g/L，Al₂(SO₄)₃·18H₂O 0.25g/L，CoCl₂·6H₂O 1.25g/L，ZnSO₄·2H₂O 0.25g/L，H₃BO₃0.07g/L, and the balance of water.

As can be seen from FIG. 3, the final engineering bacteria can efficiently produce N-acetylglucosamine by using glycerol and glucose, the yield of the N-acetylglucosamine after 48h shake flask fermentation of the composite carbon source culture medium reaches 107.8g/L, the conversion rate of the composite carbon source reaches 49% at most, compared with the glucose utilization strain (the strain constructed in example 1), the yield is increased by 151.7%, the conversion rate is increased by 60.2%, and the industrial production potential is stronger.

Example 4 Shake flask fermentation experiment

(1) seed culture: inoculating slant strain into seed culture medium, culturing at 32 deg.C and 220rpm for 12 hr, and maintaining pH at 6.8-7.2.

(2) Fermentation culture: inoculating a fermentation culture medium according to the inoculation amount of 12%, and culturing at 32 ℃ and 220rpm for 60 hours; maintaining the pH at 7.0-7.2, maintaining 60% (m/v) of glycerol and glucose composite carbon source for fermentation (taking phenol red as an indicator, continuously keeping the color of fermentation liquor as sugar deficiency, supplementing 60% (m/v) of glycerol and glucose composite carbon source during sugar deficiency), and initially adding xylose solution with the final concentration of 10g/L to induce the expression of target genes;

after the fermentation is carried out for 60 hours in a shake flask, the yield of the N-acetylglucosamine reaches 87.15 g/L;

the seed culture solution comprises the following components: 20g/L of glycerol glucose composite carbon source, 4g/L of yeast powder, (NH)₄)₂SO₄ 3g/L，KH₂PO₄ 3g/L，MgSO₄·7H₂O1 g/L, citric acid 2g/L, FeSO₄·7H₂O 2mg/L，MnSO₄·7H₂O 1.2mg/L，V_H0.1mg/L，V_B10.5mg/L, trace1mL/L of element mixed liquor, 1 drop of defoaming agent and the balance of water, the pH value is 6.8-7.2, and the high-pressure steam sterilization is carried out for 15min at the temperature of 115 ℃;

the fermentation culture solution comprises the following components: 20g/L of glycerol glucose composite carbon source, 4g/L of yeast powder, (NH)₄)₂SO₄ 5g/L，KH₂PO₄ 7g/L，MgSO₄·7H₂O3 g/L, citric acid 4g/L, FeSO₄·7H₂O 40mg/L，MnSO₄·7H₂O 1.2mg/L，NaCl 2g/L，CaCl₂·2H₂O 25mg/L,V_H 0.1mg/L，V_B10.5mg/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 ℃;

the glycerol-glucose composite carbon source is prepared by mixing glycerol and glucose in a mass ratio of 1: 4, preparing a composition;

the trace element mixed liquid comprises the following components: na (Na)₂MoO₄·2H₂O 1.25g/L，NiCl₂·6H₂O 0.8g/L，CaCl₂·2H₂O 5g/L，CuSO₄·5H₂O 0.2g/L，Al₂(SO₄)₃·18H₂O 0.25g/L，CoCl₂·6H₂O 1.25g/L，ZnSO₄·2H₂O 0.25g/L，H₃BO₃0.07g/L, and the balance of water.

Example 5 Shake flask fermentation experiment

(1) seed culture: inoculating slant strain into seed culture medium, culturing at 35 deg.C and 200rpm for 10 hr, and maintaining pH at 6.8-7.2.

(2) Fermentation culture: inoculating a fermentation culture medium according to the inoculation amount of 10%, and culturing at 35 ℃ and 300rpm for 72 h; maintaining the pH at 7.0-7.2, maintaining 60% (m/v) of glycerol and glucose composite carbon source for fermentation (taking phenol red as an indicator, continuously keeping the color of fermentation liquor as sugar deficiency, supplementing 60% (m/v) of glycerol and glucose composite carbon source during sugar deficiency), and initially adding xylose solution with the final concentration of 20g/L to induce the expression of target genes;

after the fermentation is carried out for 72 hours in a shake flask, the yield of the N-acetylglucosamine reaches 93.2 g/L;

the seed culture solution comprises the following components: 30g/L of glycerol glucose composite carbon source, 3g/L of yeast powder, (NH)₄)₂SO₄ 2g/L，KH₂PO₄ 4g/L，MgSO₄·7H₂O3 g/L, citric acid 4g/L, FeSO₄·7H₂O 3mg/L，MnSO₄·7H₂O 1.2mg/L，V_H0.1mg/L，V_B10.5mg/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 ℃;

the fermentation culture solution comprises the following components: 30g/L of glycerol glucose composite carbon source, 6g/L of yeast powder, (NH)₄)₂SO₄ 5g/L，KH₂PO₄ 7g/L，MgSO₄·7H₂O3 g/L, citric acid 4g/L, FeSO₄·7H₂O 35mg/L，MnSO₄·7H₂O 1.2mg/L，NaCl 2g/L，CaCl₂·2H₂O 25mg/L,V_H 0.1mg/L，V_B10.5mg/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 ℃;

the glycerol-glucose composite carbon source is prepared by mixing glycerol and glucose in a mass ratio of 1: 5, preparing a composition;

In conclusion, the genetically engineered bacterium realizes the efficient production of the N-acetylglucosamine by the metabolic division of the escherichia coli composite carbon source through directionally modifying the metabolic pathways of the glycerol and the glucose. The reinforcement of the N-acetylglucosamine synthesis pathway is constructed by the CRISPR/Cas9 technology according to the following method: eight genes of nagA, nagB, nagC, nagE, manX, manY, manZ and nanE are knocked out in starting escherichia coli W3110, a xylose strong promoter is adopted to induce RNA polymerase synthesis from T7 bacteriophage so as to realize induction enhancement of an N-acetylglucosamine synthesis pathway regulated and controlled by a T7 promoter, and the induction enhancement comprises introducing a gna1 gene (Sc-gna1) derived from saccharomyces cerevisiae and carrying out multi-copy enhancement on a glmS gene in escherichia coli K-12MG 1655. On the basis, 6-phosphofructokinase gene pfkA is knocked out, and mutant gene glpK is introduced, so that the division metabolism of the composite carbon source is realized. And finally, after the compound carbon source culture medium is subjected to shake flask fermentation for 48 hours, the yield of the N-acetylglucosamine is up to 107.8g/L, the conversion rate of the compound carbon source is up to 49%, compared with the yield of a glucose utilization strain, the yield is increased by 151.7%, the conversion rate is increased by 60.2%, and the compound carbon source culture medium has strong industrial production potential.

TABLE 1 primers involved in the construction of the strains

The above detailed description of the genetically engineered bacterium for the metabolic production of N-acetylglucosamine by using complex carbon source and its application are illustrative and not restrictive, and several examples can be cited within the scope of the present invention, therefore, variations and modifications thereof without departing from the general concept of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

<110> Tianjin science and technology university

<120> genetic engineering bacterium for producing N-acetylglucosamine and application thereof

<130> 1

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 480

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 1

atgagcttac ccgatggatt ttatataagg cgaatggaag agggggattt ggaacaggtc 60

actgagacgc taaaggtttt gaccaccgtg ggcactatta cccccgaatc cttcagcaaa 120

ctcataaaat actggaatga agccacagta tggaatgata acgaagataa aaaaataatg 180

caatataacc ccatggtgat tgtggacaag cgcaccgaga cggttgccgc tacggggaat 240

atcatcatcg aaagaaagat cattcatgaa ctggggctat gtggccacat cgaggacatt 300

gcagtaaact ccaagtatca gggccaaggt ttgggcaagc tcttgattga tcaattggta 360

actatcggct ttgactacgg ttgttataag attattttag attgcgatga gaaaaatgtc 420

aaattctatg aaaaatgtgg gtttagcaac gcaggcgtgg aaatgcaaat tagaaaatag 480

<210> 2

<211> 1830

<212> DNA

<213> E.coli K-12 MG1655

<400> 2

atgtgtggaa ttgttggcgc gatcgcgcaa cgtgatgtag cagaaatcct tcttgaaggt 60

ttacgtcgtc tggaataccg cggatatgac tctgccggtc tggccgttgt tgatgcagaa 120

ggtcatatga cccgcctgcg tcgcctcggt aaagtccaga tgctggcaca ggcagcggaa 180

gaacatcctc tgcatggcgg cactggtatt gctcacactc gctgggcgac ccacggtgaa 240

ccttcagaag tgaatgcgca tccgcatgtt tctgaacaca ttgtggtggt gcataacggc 300

atcatcgaaa accatgaacc gctgcgtgaa gagctaaaag cgcgtggcta taccttcgtt 360

tctgaaaccg acaccgaagt gattgcccat ctggtgaact gggagctgaa acaaggcggg 420

actctgcgtg aggccgttct gcgtgctatc ccgcagctgc gtggtgcgta cggtacagtg 480

atcatggact cccgtcaccc ggataccctg ctggcggcac gttctggtag tccgctggtg 540

attggcctgg ggatgggcga aaactttatc gcttctgacc agctggcgct gttgccggtg 600

acccgtcgct ttatcttcct tgaagagggc gatattgcgg aaatcactcg ccgttcggta 660

aacatcttcg ataaaactgg cgcggaagta aaacgtcagg atatcgaatc caatctgcaa 720

tatgacgcgg gcgataaagg catttaccgt cactacatgc agaaagagat ctacgaacag 780

ccgaacgcga tcaaaaacac ccttaccgga cgcatcagcc acggtcaggt tgatttaagc 840

gagctgggac cgaacgccga cgaactgctg tcgaaggttg agcatattca gatcctcgcc 900

tgtggtactt cttataactc cggtatggtt tcccgctact ggtttgaatc gctagcaggt 960

attccgtgcg acgtcgaaat cgcctctgaa ttccgctatc gcaaatctgc cgtgcgtcgt 1020

aacagcctga tgatcacctt gtcacagtct ggcgaaaccg cggataccct ggctggcctg 1080

cgtctgtcga aagagctggg ttaccttggt tcactggcaa tctgtaacgt tccgggttct 1140

tctctggtgc gcgaatccga tctggcgcta atgaccaacg cgggtacaga aatcggcgtg 1200

gcatccacta aagcattcac cactcagtta actgtgctgt tgatgctggt ggcgaagctg 1260

tctcgcctga aaggtctgga tgcctccatt gaacatgaca tcgtgcatgg tctgcaggcg 1320

ctgccgagcc gtattgagca gatgctgtct caggacaaac gcattgaagc gctggcagaa 1380

gatttctctg acaaacatca cgcgctgttc ctgggccgtg gcgatcagta cccaatcgcg 1440

ctggaaggcg cattgaagtt gaaagagatc tcttacattc acgctgaagc ctacgctgct 1500

ggcgaactga aacacggtcc gctggcgcta attgatgccg atatgccggt tattgttgtt 1560

gcaccgaaca acgaattgct ggaaaaactg aaatccaaca ttgaagaagt tcgcgcgcgt 1620

ggcggtcagt tgtatgtctt cgccgatcag gatgcgggtt ttgtaagtag cgataacatg 1680

cacatcatcg agatgccgca tgtggaagag gtgattgcac cgatcttcta caccgttccg 1740

ctgcagctgc tggcttacca tgtcgcgctg atcaaaggca ccgacgttga ccagccgcgt 1800

aacctggcaa aatcggttac ggttgagtaa 1830

<210> 3

<211> 1149

<212> DNA

<213> E.coli W3110

<400> 3

atgtatgcat taacccaggg ccggatcttt accggccacg aatttcttga tgaccacgcg 60

gttgttatcg ctgatggcct gattaaaagc gtctgtccgg tagcggaact gccgccagag 120

atcgaacaac gttcactgaa cggggccatt ctctcccccg gttttatcga tgtgcagtta 180

aacggctgcg gcggcgtaca gtttaacgac accgctgaag cggtcagcgt ggaaacgctg 240

gaaatcatgc agaaagccaa tgagaaatca ggctgtacta actatctgcc gacgcttatc 300

accaccagcg atgagctgat gaaacagggc gtgcgcgtta tgcgcgagta cctggcaaaa 360

catccgaatc aggcgttagg tctgcatctg gaaggtccgt ggctgaatct ggtaaaaaaa 420

ggcacccata atccgaattt tgtgcgtaag cctgatgccg cgctggtcga tttcctgtgt 480

gaaaacgccg acgtcattac caaagtgacc ctggcaccgg aaatggttcc tgcggaagtc 540

atcagcaaac tggcaaatgc cgggattgtg gtttctgccg gtcactccaa cgcgacgttg 600

aaagaagcaa aagccggttt ccgcgcgggg attacctttg ccacccatct gtacaacgcg 660

atgccgtata ttaccggtcg tgaacctggc ctggcgggcg cgatcctcga cgaagctgac 720

atttattgcg gtattattgc tgatggcctg catgttgatt acgccaacat tcgcaacgct 780

aaacgtctga aaggcgacaa actgtgtctg gttactgacg ccaccgcgcc agcaggtgcc 840

aacattgaac agttcatttt tgcgggtaaa acaatatact accgtaacgg actttgtgtg 900

gatgagaacg gtacgttaag cggttcatcc ttaaccatga ttgaaggcgt gcgtaatctg 960

gtcgaacatt gcggtatcgc actggatgaa gtgctacgta tggcgacgct ctatccggcg 1020

cgtgcgattg gcgttgagaa acgtctcggc acactcgccg caggtaaagt agccaacctg 1080

actgcattca cacctgattt taaaatcacc aagaccatcg ttaacggtaa cgaggtcgta 1140

actcaataa 1149

<210> 4

<211> 801

<212> DNA

<213> E.coli W3110

<400> 4

atgagactga tccccctgac taccgctgaa caggtcggca aatgggctgc tcgccatatc 60

gtcaatcgta tcaatgcgtt caaaccgact gccgatcgtc cgtttgtact gggcctgccg 120

actggcggca cgccgatgac cacctataaa gcgttagtcg aaatgcataa agcaggccag 180

gtcagcttta agcacgttgt caccttcaac atggacgaat atgtcggtct gccgaaagag 240

catccggaaa gctactacag ctttatgcac cgtaatttct tcgatcacgt tgatattcca 300

gcagaaaaca tcaaccttct caacggcaac gccccggata tcgacgccga gtgccgccag 360

tatgaagaaa aaatccgttc ttacggaaaa attcatctgt ttatgggcgg tgtaggtaac 420

gacggtcata ttgcatttaa cgaaccggcg tcttctctgg cttctcgtac tcgtatcaaa 480

accctgactc atgacactcg cgtcgcaaac tctcgtttct ttgataacga tgttaatcag 540

gtgccaaaat atgccctgac tgtcggtgtt ggtacactgc tggatgccga agaagtgatg 600

attctggtgc tgggtagcca gaaagcactg gcgctgcagg ccgccgttga aggttgcgtg 660

aaccatatgt ggaccatcag ctgtctgcaa ctgcatccga aagcgatcat ggtgtgcgat 720

gaaccttcca ccatggagct gaaagttaag actttaagat atttcaatga attagaagca 780

gaaaatatca aaggtctgta a 801

<210> 5

<211> 1221

<212> DNA

<213> E.coli W3110

<400> 5

atgacaccag gcggacaagc tcagataggt aatgttgatc tcgtaaaaca gcttaacagc 60

gcggcggttt atcgcctgat tgaccagtac gggccaatct cgcggattca gattgccgag 120

caaagccagc ttgcccccgc cagcgtaacc aaaattacgc gtcagcttat cgaacgcggg 180

ctgatcaaag aagttgatca gcaggcctcc accgggggcc gccgcgctat ctccatcgtc 240

accgaaaccc gcaatttcca cgcaatcggc gtacggcttg gtcgtcatga cgccaccatc 300

actctgtttg atctcagcag caaagtgctg gcagaagaac attacccgct gccggaacgt 360

acccagcaaa cgctggaaca tgccctgttg aatgccattg ctcagtttat tgatagctac 420

cagcgcaaac tgcgcgagct gatcgcgatt tcggtgatcc tgccagggct tgttgacccg 480

gacagcggca aaattcatta catgccgcat attcaggtag aaaactgggg gctggtagaa 540

gctctggaag aacgttttaa agtgacctgt ttcgttggtc acgatatccg tagtctggcg 600

ctggcggagc actacttcgg tgcaagtcag gattgcgaag actccattct ggtgcgtgtc 660

catcgcggaa ccggggccgg gattatctct aacgggcgca tttttattgg ccgcaacggc 720

aacgtcggtg aaattggcca tattcaggtc gaaccgctgg gtgaacgctg ccactgcggc 780

aactttggct gcctggaaac tatcgctgcc aacgctgcca ttgaacaacg ggtgttgaat 840

ctgttaaagc agggctacca gagccgcgtg ccgctggacg actgcaccat caaaactatc 900

tgcaaagccg cgaacaaagg cgatagtctg gcgtcggaag taattgagta tgtcggtcgt 960

catctgggta aaaccatcgc cattgctatc aacttattta atccgcaaaa aattgttatt 1020

gccggtgaaa tcaccgaagc cgataaagtg ctgctccctg ctattgaaag ctgcattaat 1080

acccaggcgc tgaaggcgtt tcgcactaat ctgccggtgg tacgttctga gctggatcac 1140

cgctcggcaa tcggcgcttt tgcgctggta aaacgcgcca tgctcaacgg tattttgctc 1200

cagcatttgc tggaaaatta a 1221

<210> 6

<211> 1947

<212> DNA

<213> E.coli W3110

<400> 6

atgaatattt taggtttttt ccagcgactc ggtagggcgt tacagctccc tatcgcggtg 60

ctgccggtgg cggcactgtt gctgcgattc ggtcagccag atttacttaa cgttgcgttt 120

attgcccagg cgggcggtgc gatttttgat aacctcgcat taatcttcgc catcggtgtg 180

gcatccagct ggtcgaaaga cagcgctggt gcggcggcgc tggcgggtgc ggtaggttac 240

tttgtgttaa ccaaagcgat ggtgaccatc aacccagaaa ttaacatggg tgtactggcg 300

ggtatcatta ccggtctggt tggtggcgca gcctataacc gttggtccga tattaaactg 360

ccggacttcc tgagcttctt cggcggcaaa cgctttgtgc cgattgccac cggattcttc 420

tgcctggtgc tggcggccat ttttggttac gtctggccgc cggtacagca cgctatccat 480

gcaggcggcg agtggatcgt ttctgcgggc gcgctgggtt ccggtatctt tggtttcatc 540

aaccgtctgc tgatcccaac cggtctgcat caggtactga acaccatcgc ctggttccag 600

attggtgaat tcaccaacgc ggcgggtacg gttttccacg gtgacattaa ccgcttctat 660

gccggtgacg gcaccgcggg gatgttcatg tccggcttct tcccgatcat gatgttcggt 720

ctgccgggtg cggcgctggc gatgtacttc gcagcaccga aagagcgtcg tccgatggtt 780

ggcggtatgc tgctttctgt tgctgttact gcgttcctga ccggtgtgac tgagccgctg 840

gaattcctgt tcatgttcct tgctccgctg ctgtacctcc tgcacgcact gctgaccggt 900

atcagcctgt ttgtggcaac gctgctgggt atccacgcgg gcttctcttt ctctgcgggg 960

gctatcgact acgcgttgat gtataacctg ccggccgcca gccagaacgt ctggatgctg 1020

ctggtgatgg gcgttatctt cttcgctatc tacttcgtgg tgttcagttt ggttatccgc 1080

atgttcaacc tgaaaacgcc gggtcgtgaa gataaagaag acgagatcgt tactgaagaa 1140

gccaacagca acactgaaga aggtctgact caactggcaa ccaactatat tgctgcggtt 1200

ggcggcactg acaacctgaa agcgattgac gcctgtatca cccgtctgcg ccttacagtg 1260

gctgactctg cccgcgttaa cgatacgatg tgtaaacgtc tgggtgcttc tggggtagtg 1320

aaactgaaca aacagactat tcaggtgatt gttggcgcga aagcagaatc catcggcgat 1380

gcgatgaaga aagtcgttgc ccgtggtccg gtagccgctg cgtcagctga agcaactccg 1440

gcaactgccg cgcctgtagc aaaaccgcag gctgtaccaa acgcggtatc tatcgcggag 1500

ctggtatcgc cgattaccgg tgatgtcgtg gcactggatc aggttcctga cgaagcattc 1560

gccagcaaag cggtgggtga cggtgtggcg gtgaaaccga cagataaaat cgtcgtatca 1620

ccagccgcag ggacaatcgt gaaaatcttc aacaccaacc acgcgttctg cctggaaacc 1680

gaaaaaggcg cggagatcgt cgtccatatg ggtatcgaca ccgtagcgct ggaaggtaaa 1740

ggctttaaac gtctggtgga agagggtgcg caggtaagcg cagggcaacc gattctggaa 1800

atggatctgg attacctgaa cgctaacgcc cgctcgatga ttagcccggt ggtttgcagc 1860

aatatcgacg atttcagtgg cttgatcatt aaagctcagg gccatattgt ggcgggtcaa 1920

acaccgctgt atgaaatcaa aaagtaa 1947

<210> 7

<211> 972

<212> DNA

<213> E.coli W3110

<400> 7

gtgaccattg ctattgttat aggcacacat ggttgggctg cagagcagtt gcttaaaacg 60

gcagaaatgc tgttaggcga gcaggaaaac gtcggctgga tcgatttcgt tccaggtgaa 120

aatgccgaaa cgctgattga aaagtacaac gctcagttgg caaaactcga caccactaaa 180

ggcgtgctgt ttctcgttga tacatgggga ggcagcccgt tcaatgctgc cagccgcatt 240

gtcgtcgaca aagagcatta tgaagtcatt gcaggcgtta acattccaat gctcgtggaa 300

acgttaatgg cccgtgatga tgacccaagc tttgatgaac tggtggcact ggcagtagaa 360

acaggccgtg aaggcgtgaa agcactgaaa gccaaaccgg ttgaaaaagc cgcgccagca 420

cccgctgccg cagcaccaaa agcggctcca actccggcaa aaccaatggg gccaaacgac 480

tacatggtta ttggccttgc gcgtatcgac gaccgtctga ttcacggtca ggtcgccacc 540

cgctggacca aagaaaccaa tgtctcccgt attattgttg ttagtgatga agtggctgcg 600

gataccgttc gtaagacact gctcacccag gttgcacctc cgggcgtaac agcacacgta 660

gttgatgttg ccaaaatgat tcgcgtctac aacaacccga aatatgctgg cgaacgcgta 720

atgctgttat ttaccaaccc aacagatgta gagcgtctcg ttgaaggcgg cgtgaaaatc 780

acctctgtta acgtcggtgg tatggcattc cgtcagggta aaacccaggt gaataacgcg 840

gtttcggttg atgaaaaaga tatcgaggcg ttcaagaaac tgaatgcgcg cggtattgag 900

ctggaagtcc gtaaggtttc caccgatccg aaactgaaaa tgatggatct gatcagcaaa 960

atcgataagt aa 972

<210> 8

<211> 801

<212> DNA

<213> E.coli W3110

<400> 8

atggagatta ccactcttca aattgtgctg gtatttatcg tagcctgtat cgcaggtatg 60

ggatcaatcc tcgatgaatt tcagtttcac cgtccgctaa tcgcgtgtac cctggtgggt 120

atcgttcttg gggatatgaa aaccggtatt attatcggtg gtacgctgga aatgatcgcg 180

ctgggctgga tgaacatcgg tgctgcagtt gcgcctgacg ccgctctggc ttctatcatt 240

tctaccattc tggttatcgc aggtcatcag agcattggtg caggtatcgc actggcaatc 300

cctctggccg ctgcgggcca ggtactgacc atcatcgttc gtactattac cgttgctttc 360

cagcacgctg cggataaggc tgctgataac ggcaacctga cagcgatttc ctggatccac 420

gtttcttctc tgttcctgca agcaatgcgt gtggctattc cggccgtcat cgttgcgctg 480

tctgttggta ccagcgaagt acagaacatg ctgaatgcga ttccggaagt ggtgaccaat 540

ggtctgaata tcgccggtgg catgatcgtg gtggttggtt atgcgatggt tatcaacatg 600

atgcgtgctg gctacctgat gccgttcttc tacctcggct tcgtaaccgc agcattcacc 660

aactttaacc tggttgctct gggtgtgatt ggtactgtta tggcagtgct ctacatccaa 720

cttagcccga aatacaaccg cgtagccggt gcgcctgctc aggcagctgg taacaacgat 780

ctcgataacg aactggacta a 801

<210> 9

<211> 861

<212> DNA

<213> E.coli W3110

<400> 9

gtgagcgaaa tggttgatac aactcaaact accaccgaga aaaaactcac tcaaagtgat 60

attcgtggcg tcttcctgcg ttctaacctc ttccagggtt catggaactt cgaacgtatg 120

caggcactgg gtttctgctt ctctatggta ccggcaattc gtcgcctcta ccctgagaac 180

aacgaagctc gtaaacaagc tattcgccgt cacctggagt tctttaacac ccagccgttc 240

gtggctgcgc cgattctcgg cgtaaccctg gcgctggaag aacagcgtgc taatggcgca 300

gagatcgacg acggtgctat caacggtatc aaagtcggtt tgatggggcc actggctggt 360

gtaggcgacc cgatcttctg gggaaccgta cgtccggtat ttgcagcact gggtgccggt 420

atcgcgatga gcggcagcct gttaggtccg ctgctgttct tcatcctgtt taacctggtg 480

cgtctggcaa cccgttacta cggcgtagcg tatggttact ccaaaggtat cgatatcgtt 540

aaagatatgg gtggtggctt cctgcaaaaa ctgacggaag gggcgtctat cctcggcctg 600

tttgtcatgg gggcattggt taacaagtgg acacatgtca acatcccgct ggttgtctct 660

cgcattactg accagacggg caaagaacac gttactactg tccagactat tctggaccag 720

ttaatgccag gcctggtacc actgctgctg acctttgctt gtatgtggct actgcgcaaa 780

aaagttaacc cgctgtggat catcgttggc ttcttcgtca tcggtatcgc tggttacgct 840

tgcggcctgc tgggactgta a 861

<210> 10

<211> 963

<212> DNA

<213> E.coli W3110

<400> 10

atgattaaga aaatcggtgt gttgacaagc ggcggtgatg cgccaggcat gaacgccgca 60

attcgcgggg ttgttcgttc tgcgctgaca gaaggtctgg aagtaatggg tatttatgac 120

ggctatctgg gtctgtatga agaccgtatg gtacagctag accgttacag cgtgtctgac 180

atgatcaacc gtggcggtac gttcctcggt tctgcgcgtt tcccggaatt ccgcgacgag 240

aacatccgcg ccgtggctat cgaaaacctg aaaaaacgtg gtatcgacgc gctggtggtt 300

atcggcggtg acggttccta catgggtgca atgcgtctga ccgaaatggg cttcccgtgc 360

atcggtctgc cgggcactat cgacaacgac atcaaaggca ctgactacac tatcggtttc 420

ttcactgcgc tgagcaccgt tgtagaagcg atcgaccgtc tgcgtgacac ctcttcttct 480

caccagcgta tttccgtggt ggaagtgatg ggccgttatt gtggagatct gacgttggct 540

gcggccattg ccggtggctg tgaattcgtt gtggttccgg aagttgaatt cagccgtgaa 600

gacctggtaa acgaaatcaa agcgggtatc gcgaaaggta aaaaacacgc gatcgtggcg 660

attaccgaac atatgtgtga tgttgacgaa ctggcgcatt tcatcgagaa agaaaccggt 720

cgtgaaaccc gcgcaactgt gctgggccac atccagcgcg gtggttctcc ggtgccttac 780

gaccgtattc tggcttcccg tatgggcgct tacgctatcg atctgctgct ggcaggttac 840

ggcggtcgtt gtgtaggtat ccagaacgaa cagctggttc accacgacat catcgacgct 900

atcgaaaaca tgaagcgtcc gttcaaaggt gactggctgg actgcgcgaa aaaactgtat 960

taa 963

<210> 11

<211> 1509

<212> DNA

<213> Artificial sequence

<400> 11

atgactgaaa aaaaatatat cgttgcgctc gaccagggca ccaccagctc ccgcgcggtc 60

gtaatggatc acgatgccaa tatcattagc gtgtcgcagc gcgaatttga gcaaatctac 120

ccaaaaccag gttgggtaga acacgaccca atggaaatct gggccaccca aagctccacg 180

ctggtagaag tgctggcgaa agccgatatc agttccgatc aaattgcagc tatcggtatt 240

acgaaccagc gtgaaaccac tattgtctgg gaaaaagaaa ccggcaagcc tatctataac 300

gccattgtct ggcagtgccg tcgtaccgca gaaatctgcg agcatttaaa acgtgacggt 360

ttagaagatt atatccgcag caataccggt ctggtgattg acccgtactt ttctggcacc 420

aaagtgaagt ggatcctcga ccatgtggaa ggctctcgcg agcgtgcacg tcgtggtgaa 480

ttgctgtttg gtacggttga tacgtggctt atctggaaaa tgactcaggg ccgtgtccat 540

gtgaccgatt acaccaacgc ctctcgtacc atgttgttca acatccatac cctggactgg 600

gacgacaaaa tgctggaagt gctggatatt ccgcgcgaga tgctgccaga agtgcgtcgt 660

tcttccgaag tatacggtca gactaacatt ggcggcaaag gcggcacgcg tattccaatc 720

tccgggatcg ccggtgacca gcaggccgcg ctgtttggtc agttgtgcgt gaaagaaggg 780

atggcgaaga acacctatgg cactggctgc tttatgctga tgaacactgg cgagaaagcg 840

gtgaaatcag aaaacggcct gctgaccacc atcgcctgcg gcccgactgg cgaagtgaac 900

tatgcgttgg aaagtgcggt gtttatggca ggcgcatcca ttcagtggct gcgcgatgaa 960

atgaagttga ttaacgacgc ctacgattcc gaatatttcg ccaccaaagt gcaaaacacc 1020

aatggtgtgt atgtggttcc ggcatttacc gggctgggtg cgccgtactg ggacccgtat 1080

gcgcgcgggg cgattttcgg tctgactcgt ggggtgaacg ctaaccacat tatacgcgcg 1140

acgctggagt ctattgctta tcagacgcgt gacgtgctgg aagcgatgca ggccgactct 1200

ggtatccgtc tgcacgccct gcgcgtggat ggtggcgcag tagcaaacaa tttcctgatg 1260

cagttccagt ccgatattct cggcacccgc gttgagcgcc cggaagtgcg cgaagtcacc 1320

gcattgggtg cggcctatct cgcaggcctg gcggttggct tctggcagaa cctcgacgag 1380

ctgcaagaga aagcggtgat tgagcgcgag ttccgtccag gcatcgaaac cactgagcgt 1440

aattaccgtt acgcaggctg gaaaaaagcg gttaaacgcg cgatggcgtg ggaagaacac 1500

gacgaataa 1509

<210> 12

<211> 61

<212> DNA

<213> E.coli BL21

<400> 12

taatacgact cactataggg tctagaaata attttgttta actttaagaa ggagatatac 60

c 61

<210> 13

<211> 48

<212> DNA

<213> E.coli W3110

<400> 13

ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttg 48

<210> 14

<211> 2652

<212> DNA

<213> E.coli BL21

<400> 14

atgaacacga ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg 60

ttcaacactc tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag 120

catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa 180

gctggtgagg ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag 240

atgattgcac gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg 300

acagccttcc agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag 360

accactctgg cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca 420

atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480

cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa 540

gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg 600

tggtcttcgt ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc 660

attgagtcaa ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac 720

tctgagacta tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg 780

ctggctggca tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc 840

attactggtg gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac 900

agtaagaaag cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt 960

aacattgcgc aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta 1020

atcaccaagt ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc 1080

ccgatgaaac cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct 1140

gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc 1200

atgcttgagc aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg 1260

gactggcgcg gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc 1320

aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg 1380

aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag 1440

ttcattgagg aaaaccacga gaacatcatg gcttgcgcta agtctccact ggagaacact 1500

tggtgggctg agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg 1560

gtacagcacc acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc 1620

tctggcatcc agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac 1680

ttgcttccta gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag 1740

attctacaag cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag 1800

aacactggtg aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg 1860

ctggcttacg gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggcttacggg 1920

tccaaagagt tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat 1980

tccggcaagg gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg 2040

atttgggaat ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag 2100

tctgctgcta agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc 2160

aagcgttgcg ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag 2220

aagcctattc agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc 2280

attaacacca acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct 2340

aactttgtac acagccaaga cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag 2400

aagtacggaa tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac 2460

gctgcgaacc tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat 2520

gtactggctg atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa 2580

atgccagcac ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc 2640

gcgttcgcgt aa 2652

<210> 15

<211> 690

<212> DNA

<213> E.coli W3110

<400> 15

atgtcgttac ttgcacaact ggatcaaaaa atcgctgcta acggtggcct gattgtctcc 60

tgccagccgg ttccggacag cccgctcgat aaacccgaaa tcgtcgccgc catggcatta 120

gcggcagaac aggcgggcgc ggttgccatt cgcattgaag gtgtggcaaa tctgcaagcc 180

acgcgtgcgg tggtgagcgt gccgattatt ggaattgtga aacgcgatct ggaggattct 240

ccggtacgca tcacggccta tattgaagat gttgatgcgc tggcgcaggc gggcgcggac 300

attatcgcca ttgacggcac cgaccgcccg cgtccggtgc ctgttgaaac gctgctggca 360

cgtattcacc atcacggttt actggcgatg accgactgct caacgccgga agacggcctg 420

gcatgccaaa agctgggagc cgaaattatt ggcactacgc tttctggcta taccacgcct 480

gaaacgccag aagagccgga tctggcgctg gtgaaaacgt tgagcgacgc cggatgtcgg 540

gtgattgccg aagggcgtta caacacgcct gctcaggcgg cggatgcgat gcgccacggc 600

gcgtgggcgg tgacggtcgg ttctgcaatc acgcgtcttg agcacatttg tcagtggtac 660

aacacagcga tgaaaaaggc ggtgctatga 690

Claims

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 controlled by a T7 promoter and a Sc-gna1 gene; escherichia coli glycerol kinase mutant gene glpK; an RNA polymerase gene from a T7 bacteriophage source under the control of a xylose promoter; and nine gene-defects of nagA, nagB, nagC, nagE, manX, manY, manZ, nanE, pfkA.

2. The genetically engineered bacterium of claim 1, wherein the host cell is E.coli W3110.

3. The genetically engineered Escherichia coli for producing N-acetylglucosamine of claim 1, wherein the nucleotide sequence of the coding gene Sc-gna1 is represented by SEQ ID No.1 of the sequence table;

the nucleotide sequence of the coding gene nanE is shown in a sequence table SEQ ID No. 15.

4. The method for constructing engineering bacteria of any one of claims 1 to 3, characterized by comprising the steps of:

(1) integration of xylose promoter P at the lacIZ Gene site_xylFControlled 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_T7The fructose-6-phosphate transaminase gene glmS under the control of a promoter;

5. Use of the engineered bacterium of any one of claims 1-3 for the production of N-acetylglucosamine.

6. Use according to claim 5, characterized in that the process for the fermentative production of N-acetylglucosamine comprises the following steps:

inoculating the strain to a fermentation medium according to the inoculation amount of 10-15%, and culturing at the temperature of 32-37 ℃ and the speed of 200-300rpm for 36-72 h; maintaining the pH value at 7.0-7.2, maintaining the fermentation of 60% glycerol glucose composite carbon source, and initially adding xylose solution with final concentration of 5-20g/L to induce the expression of target genes.

7. The use according to claim 6, wherein the fermentation broth comprises the following components: 10-30g/L of glycerol glucose composite carbon source, 1-6g/L of yeast powder, (NH)₄)₂SO₄ 1-5g/L，KH₂PO₄ 3-8g/L，MgSO₄·7H₂O1-5 g/L, citric acid 1-5g/L, FeSO₄·7H₂O 30-90mg/L，MnSO₄·7H₂O 1-5mg/L，NaCl 0.5-2g/L，CaCl₂·2H₂O 10-30mg/L，V_H 0.05-2mg/L，V_B10.1-1mg/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 mixture is sterilized by high-pressure steam at 115 ℃ for 15 min;

the glycerol-glucose composite carbon source is composed of glycerol and glucose according to the mass ratio of 1-4: 1-8.

8. The use of claim 7, wherein the glycerol glucose complex carbon source is glycerol and glucose in a mass ratio of 1: 8.

9. The use of claim 6, wherein the mixed solution of trace elements comprises: na (Na)₂MoO₄·2H₂O 1-3g/L，NiCl₂·6H₂O 0.5-1.5g/L，CaCl₂·2H₂O 2-8g/L，CuSO₄·5H₂O 0.1-0.5g/L，Al₂(SO₄)₃·18H₂O 0.1-0.3g/L，CoCl₂·6H₂O 0.5-1.5g/L，ZnSO₄·2H₂O 0.1-0.5g/L，H₃BO₃0.05-0.2g/L, and the balance of water.