METHOD FOR PRODUCING N-ACETYL-D-GLUCOSAMINE AND/OR D-GLUCOSAMINE SALT BY MICROBIAL FERMENTATION

Abstract

The present invention discloses a process for producing N-acetyl-D-glucosamine and D-glucosamine salts by microbial fermentation. The invention includes a method to produce N-acetyl-D-glucosamine and/or D-glucosamine salts with higher efficiency and higher yield by increasing the effect of N-acetyl-D-aminomannose-6-phosphate epimerase in microorganisms.

Claims

1. A method for producing N-Acetyl-D-Glucosamine and/or D-Glucosamine salt by microbial fermentation, comprising: A) culturing a microorganism in a fermentation medium, wherein said microorganism comprises at least one genetic modification that increases the activity of N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) in said microorganism; and B) collecting N-Acetyl-D-Glucosamine produced in the culturing step A).

2. The method of claim 1, wherein said genetic modification that increases the activity of N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) is selected from: a) increased enzymatic activity of N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) in said microorganism; and/or b) N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) is overexpressed in said microorganism.

3. The method of claim 2, wherein said microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding N-acetyl-D-aminomannose-6-phosphate epimerase (NanE); said nucleic acid sequence encoding N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) comprises at least one genetic modification that increases the activity of N-acetyl-D-aminomannose-6-phosphate epimerase (NanE).

4. The method of claim 2, said genetic modification comprises one or two of substitutions at positions corresponding to the amino acid sequence of SEQ ID NO.: 17: the 133th cysteine is substituted with arginine and the 187th tyrosine is substituted with histidine.

5. The method of claim 3, said nucleic acid sequence encoding N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) is SEQ ID NO.: 26.

6. The method of claim 3, said N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) has at least about 30% identity to the amino acid sequence of SEQ ID NO.: 17, preferably at least about 50% identity, further preferably at least about 70% identity, further preferably at least about 80% identity, still more preferably at least about 90% identity, most preferably at least about 95% identity, wherein said N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) has enzymatic activity; further preferably, said N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) has the amino acid sequence of SEQ ID NO.: 17.

7. The method of claim 2, wherein said microorganism comprises at least one genetic modification of an endogenous natural promoter of a gene encoding N-acetyl-D-aminomannose-6-phosphate epimerase (NanE); said endogenous natural promoter of said gene encoding N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) is replaced by a promoter having a higher expression level; said promoter having a higher expression level than said endogenous natural promoter is selected from the group consisting of: HCE promoter, gap promoter, trc promoter, and T7 promoter; most preferably, said promoter with a higher expression level is a trc promoter.

8. The method of any one of claims 1 to 7, wherein said microorganism further comprises one or more of the following genetic modifications: (1) at least one genetic modification capable of enhancing the activity of D-glucosamine-6-phosphate deaminase in said microorganism, preferably simultaneously comprising at least one genetic modification capable of reducing the activity of glucosamine-6-phosphate synthase; (2) at least one genetic modification capable of increasing the activity of glucosamine-6-phosphate synthase in said microorganism, and simultaneously comprising at least one genetic modification capable of reducing the activity of D-glucosamine-6-phosphate deaminase; (3) at least one genetic modification capable of enhancing the activity of UDP-N-acetyl-D-glucosamine-2-epimerase (WecB) in said microorganism.

9. The method of claim 8, wherein said genetic modification for enhancing the activity of UDP-N-acetyl-D-glucosamine-2-epimerase in said microorganism is selected from the group consisting of a) increased enzymatic activity of UDP-N-acetyl-D-glucose-2-epimerase; and/or b) overexpression of UDP-N-acetyl-D-glucosamine-2-epimerase in said microorganism.

10. The method of claim 9, wherein said microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding UDP-N-acetyl-D-glucosamine-2-epimerase; said nucleic acid sequence encoding UDP-N-acetyl-D-glucosamine-2-epimerase has a nucleic acid sequence of SEQ ID NO.: 49.

11. The method of any of claims 1-10, wherein said microorganism further comprises one or more of the following genetic modifications: (1) at least one genetic modification capable of reducing the activity of mannose transporter EIIM, P/III.sup.man (ManXYZ) in said microorganism; (2) at least one genetic modification capable of reducing the activity of N-acetylneuraminic acid lyase (NanA) in said microorganism; (3) at least one genetic modification capable of reducing the activity of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in said microorganism; (4) at least one genetic modification capable of reducing the activity of N-acetyl-D-glucosamine specific enzyme II.sup.Nag (NagE) in said microorganism; (5) at least one genetic modification capable of increasing the activity of a phosphoglucosamine mutase (GlmM) in said microorganism; and (6) at least one genetic modification capable of enhancing the activity of bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in said microorganism.

12. The method of any one of claims 1 to 11, wherein said culturing step A) is carried out at a temperature of from about 20 C. to about 45 C.; said culturing step A) is carried out at a pH value of from about pH 4.5 to about pH 8.5; said culturing step A) uses a sugar-retaining liquid fed fermentation process.

13. A microorganism comprising at least one genetic modification that enhances the activity of N-acetyl-D-aminomannose-6-phosphate epimerase in said microorganism.

14. The microorganism of claim 13, wherein said microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding N-acetyl-D-aminomannose-6-phosphate epimerase; said nucleic acid sequence encoding N-acetyl-D-aminomannose-6-phosphate epimerase is SEQ ID NO.: 26.

15. The microorganism of any one of claim 13 or 14, wherein said microorganism further comprises one or more of the following genetic modifications: (1) at least one genetic modification capable of enhancing the activity of D-glucosamine-6-phosphate deaminase in said microorganism, preferably comprising at least one genetic modification capable of reducing the activity of glucosamine-6-phosphate synthase; (2) at least one genetic modification capable of increasing the activity of glucosamine-6-phosphate synthase in said microorganism, and simultaneously comprising at least one genetic modification capable of reducing the activity of D-glucosamine-6-phosphate deaminase; (3) at least one genetic modification capable of enhancing the activity of UDP-N-acetyl-D-glucosamine-2-epimerase (WecB) in said microorganisms.

16. The method of any one of claims 1-12, or the microorganism of any one of claims 13-15, wherein said microbial microorganism is a bacterium, a yeast or a fungus.

17. An N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) having a higher enzymatic activity, said enzyme having the amino acid sequence of SEQ ID NO.: 27.

18. A nucleic acid molecule encoding the N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) of claim 17, which has the nucleic acid sequence of SEQ ID NO.: 26.

19. A vector comprising said nucleic acid molecule of claim 18.

20. A microorganism comprising a nucleic acid molecule of claim 18 in its genome.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0174] FIG. 1 illustrates the scheme of N-acetyl-D-glucosamine biosynthesis pathway and metabolic engineering strategy in Escherichia coli.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0175] The invention will be further described in detail below with reference to specific embodiments. The following examples are merely illustrative of the invention and are not to be construed as limiting the scope of the invention. The technology implemented based on the present invention is intended to be within the scope of the present invention.

[0176] The starting materials and reagents used in the examples are commercially available unless otherwise stated.

[0177] The following is a catalog of various genetically modified microorganisms relating to and/or described in the present invention.

TABLE-US-00001 Strain Number Genotype Description Note AT-001 ATCC 27325, F-IN(rrnD-rrnE)1 lambda-, Parent the primary culture derivative strains of strain of E coli K-12 engineered bacteria, from American Type Culture Collection (ATCC) AT-002-01 AT-001, manXYZ::fKanrf Example 1 AT-002-02 AT-001, manXYZ Example 1 AT-003-01 AT-002-02, nanA::fKanrf Example 1 AT-003-02 AT-002-02, nanA Example 1 AT-004-01 AT-003-02, nagA::fKanrf Example 1 AT-004-02 AT-003-02, nagA Example 1 AT-005-01 AT-004-02, nagE::fKanrf Example 1 AT-005-02 AT-004-02, nagE Example 1 AT-030-01 AT-004-02, nagE::pTrc-nanE-fKanrf Example 2 AT-030-02 AT-004-02, nagE::pTrc-nanE Example 2 AT-031-01 AT-004-02, nagE::pTrc-nanEM-fKanrf Example 3 AT-031-02 AT-004-02, nagE::pTrc-nanEM Example 3 AT-032 AT-031-02, nagB promotor::Trc promoter Example 4 AT-033 AT-032, glmS promotor Example 4 AT-034 AT-031-02, glmS promotor::Trc promoter Example 4 AT-035 AT-034, nagB promotor Example 4 AT-036 AT-031-02, wecB/pTrc99A Example 5 AT-037 AT-031-02, wecB promotor::Trc promoter Example 5 AT-038 AT-033, wecB/pTrc99A Example 6 AT-039 AT-033, wecB promotor::Trc promoter Example 6 AT-040 AT-035, wecB/pTrc99A Example 6 AT-041 AT-035, wecB promotor::Trc promoter Example 6 AT-042-01 AT-004-02, nagE::pTrc-wecB-fKanrf Example 9 AT-042-02 AT-004-02, nagE::pTrc-wecB Example 9 AT-043-01 AT-004-02, nagE::pTrc-wecBM-fKanrf Example 9 AT-043-02 AT-004-02, nagE::pTrc-wecBM Example 9

Example 1

[0178] This example describes the construction of an E. coli mutant that blocks the metabolic pathway associated with the uptake of N-acetyl-D-glucosamine and the degradation of beneficial intermediates.

[0179] The parent strain of the production strain was AT-001 (Escherichia coli ATCC 27325), belonging to the E. coli K-12 derivative, and came from the American Type Culture Collection.

[0180] Blocking the N-acetyl-D-glucosamine uptake and degradation of intermediate metabolites by the strain can reduce the loss in the metabolic process and increase the accumulation of the target product (N-acetyl-D-glucosamine).

[0181] Construction of this mutant host strain can be achieved by completely or partially deleting the manXYZ, nanA, nagA and nagE gene sequences on its chromosomal genome to disable their functions, and thus cause the accumulation of N-acetyl-D-glucosamine.

[0182] The deletion of the gene sequence on the chromosome can be done using Red recombination technology. Red recombination is a DNA homologous recombination technique mediated by the lambda phage Red operon and the Rac phage RecE/RecT system. By this technique, it is possible to easily and rapidly perform various modifications such as insertion, knockout, and mutation in any large DNA molecules. Simply stated, the Red Recombination Technology is: the pKD46 plasmid carrying the recombinase gene is first transferred into the cells, and then the linear DNA segment for targeting is prepared by electroporation, and the positive clones are screened, finally, the resistance genes in the recombinant strain are eliminated.

[0183] The following describes the specific operation procedures:

[0184] 1. Deletion of the manXYZ Gene Sequence

[0185] The mannose transporter EIIM, P/III.sup.man (mannose transporter EIIM, P/III.sup.Man, ManXYZ) can be used as a second transporter protein of N-acetyl-D-glucosamine, which can transport hexoses such as N-acetyl-D-glucosamine into the cells, thus the target product excreted and accumulated outside the cells can be transported back for intracellular degradation. Deletion of the manXYZ gene sequence prevents extracellular N-acetyl-D-glucosamine from being transported back into the cell for degradation.

[0186] (1) Preparation of a Linear DNA Full-Length PCR Fragment for Red Recombination Targeting

[0187] 1) PCR Amplification of fKanrf Segments

[0188] The fKanrf segment, that is, the FRT-Kanr-FRT segment, refers to a FRT site base sequence for specific recognition by FLP recombinase, mounted at both ends of the kanamycin resistance gene (Kanr).

[0189] Design of Primers: forward primer (mfKanf-F) SEQ ID No: 1, and reverse primer (mfKanf-R) SEQ ID No: 2.

[0190] Template: pPic9K.

[0191] PCR reaction conditions: first step: denaturation at 94 C. for 1 min; second step: incubation at 94 C. for 30 s, at 55 C. for 30 s, at 72 C. for 40 s, and carrying out 30 cycles; third step: extension at 72 C. for 10 min.

[0192] fKanrf size: 1.28 kb. Its nucleotide sequence is SEQ ID No: 3.

[0193] The PCR product was separated by 1% agarose gel electrophoresis and purified to recover the segment.

[0194] 2) PCR Amplification of Full-Length Linear DNA Segments for Red Recombination Targeting

[0195] Design of a homology arm primer: according to the manXYZ sequence of SEQ ID No:4, designing a homologous arm forward primer with the manXYZ sequence deleted (manXYZKO-F) and having the sequence of SEQ ID No: 5, and reverse primer (manXYZKO-R) having the sequence of SEQ ID No: 6.

[0196] Template: Amplified FKANRF PCR segment.

[0197] PCR reaction conditions: the first step: denaturation at 94 C. for 1 min; the second step: incubation at 94 C. for 30 s, at 55 C. for 30 s, at 72 C. for 40 s, and carrying out for 30 cycles; the third Step: extension at 72 C. for 10 min.

[0198] Amplification Product: Homologous arm+fkanrf+homologous arm.

[0199] The PCR products were separated by agarose gel electrophoresis and purified and recovered to obtain 100 ng/l linear DNA full-length PCR segment for Red recombinant targeting.

[0200] (2) Red Recombination Operation

[0201] First, the pKD46 vector was introduced into the AT-001 strain of E. coli. Then, a linear DNA segment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance genes were removed.

[0202] 1) Transformation of pKD46 Plasmid

[0203] The pKD46 vector is a plasmid carrying the gene for expression of the Red recombinase, which expresses the three gene segments of Exo, Bet and Gam. The three genes are placed under the arabinose promoter and can be expressed in a large amount by L-arabinose induction. In order to achieve the purpose of modifying the target gene on the chromosome by Red recombination, it is necessary to transform the pKD46 plasmid into E. coli.

[0204] {circle around (1)} Preparation of Competence:

[0205] First, Escherichia coli ATCC 27325 stock solution stored at 20 C. was inoculated in 10 ml of LB liquid medium in a ration of 1:50-100, and shake-cultured at 37 C. and 225 rpm for 2-3 hours. The culture solution was further added to a 10 ml centrifuge tube, centrifuged at 4000 g5 min, the supernatant was discarded, and the solution was suspended with 5 ml of 0.1M CaCl.sub.2 on an ice bath of for 5 min. Finally, centrifuged at 4000 g5 min, the supernatant was discarded, and the solution was suspended with 5 ml 0.1M CaCl.sub.2 on an ice bath. It was allowed to stand at 4 C. for 12 hours and naturally settled. Preparation of 0.1M CaCl.sub.2: using anhydrous CaCl.sub.2 to make 1M CaCl.sub.2), autoclaved with a vapor pressure of 15 lbf/in.sup.2 for 20 min, and 1.5 ml of the mixture was packaged and stored at 20 C.; for use, allowed to thaw, and then diluted in a ratio of 1:10 to make 0.1M CaCl.sub.2 solution.

[0206] {circle around (2)} Plasmid transformation: 250 l of the naturally-precipitated bacterial solution were taken, and 5 l of pKD46 plasmid was added and cultured at 4 C. for 30 min. Then, it was heated on a 42 C. water bath for 1.5 min, and 0.7 ml of SOC medium was added, and the mixture was shaken at 30 C. for 2 hours. 0.2 ml of bacterial solution was transferred and smeared on a penicillin plate. Incubated overnight (12-16 hours) at 30 C. Monoclones were picked and cultured in 5 ml of LB liquid medium, and plasmid was extracted for identification. Positive strains were preserved for use.

[0207] 2) Electrotransform the Prepared Linear DNA Segment for Targeting, and Screen for Positive Clones

[0208] {circle around (1)} Preparation of Electrotransform Competence:

[0209] AT-001 strain of Escherichia coli ATCC 27325 containing pKD46 was inoculated into a test tube containing ampicillin (Amp) LB medium, shake-cultured at 250 rpm overnight, and inoculated in a ratio of 1% into LB medium containing Amp the next day, then cultured at 30 C. When OD.sub.600 reached about 0.2, added 0.2% L-arabinose and induced at 30 C. for 35 minutes until OD.sub.600 reached about 0.4. The solution was cooled on an ice bath, then washed once with ultrapure water, washed twice with 10% glycerol, and finally resuspended with 10% glycerol. The amount of glycerin used was to produce a final concentration of the bacterial solution concentrated by 500-1000 folds.

[0210] {circle around (2)} Transformation by Electric Shock:

[0211] took out the 2 mm electrotransformation cup from 70% ethanol, washed twice with sterile ultrapure water, and irradiated with ultraviolet light for 30 minutes. It was pre-cooled for 30 minutes at 4 C. 90 l of the final resuspended cells were taken and transferred to a pre-cooled centrifuge tube, and added 5 l (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in step (1), gently suction mixed with a gun, and ice bathed for 30 minutes. Electrotransformation parameters: 2500V, 200, 25 F.

[0212] {circle around (3)} Resuscitation and Screening Positive Clones:

[0213] 1 ml of LB liquid medium was added, and cultured at 37 C. and 100 rpm for 1 hour. Then, one kanamycin (Kan) plate was coated with every 200 l, five plates in total. The coating was even and allowed to dry. The plates were incubated at 30 C. for 24 hours. Clones grown under resistance to kanamycin were picked for PCR identification and positive clones were screened.

[0214] The obtained strain number: AT-002-01 (AT-001, manXYZ:: fKanrf).

[0215] Removal of the Resistance Gene

[0216] To facilitate subsequent work, the resistance gene in the obtained strain (positive clones) may be removed. Removal of the resistance gene may be accomplished by pCP20 plasmid. pCP20 is a plasmid with ampicillin and chloramphenicol resistance genes, and may express FLP recombinase after thermal induction, the FLP recombinase may specifically identify FRT sites. The sequence between FRT sites may be deleted by recombination, and only one FRT site is retained.

[0217] pCP20 was introduced into the above kanamycin-resistant clones, cultured at 30 C. for 8 h, then increased to 42 C. and cultured overnight, and thermally induced to express FLP recombinase; the plasmids were lost gradually. Inoculating loop was dipped with bacterial suspension and streaked the plate of antibiotics-free culture medium, grown monoclones were picked up and dotted on the kanamycin-resistant plate; those that did not grow were clones of which the kanamycin resistance gene had been removed by FLP recombination. Carried out PCR with identification primer to identify clones losing kanamycin resistance.

[0218] The obtained strain number: AT-002-02 (AT-001, A manXYZ).

[0219] 2. Deletion of nanA Gene Sequence

[0220] N-Acetylneuraminate lyase (NanA) can degrade N-Acetyl-D-Mannosamine (ManNAc) in microorganism to produce N-Acetyl-D-Neuraminic Acid (Neu5Ac). Deletion of the nanA gene sequence in nanKETA operon may block degradation of N-Acetyl-D-Mannosamine (ManNAc) into N-Acetyl-D-Neuraminic Acid (Neu5Ac).

[0221] (1) Preparation of Linear DNA Full-length PCR Segment for Red Recombination Targeting

[0222] Design of Homologous Arm Primer: According to the nanA sequence of SEQ ID No:7 in the former segment of nanE-nanK, the homologous arm primers for deletion of nanA sequence were designed: Forward primer (nanAKO-F) SEQ ID No: 8 and reverse primer (nanAKO-R) SEQ ID No: 9.

[0223] Template: Amplification of fKanrfPCR Segment.

[0224] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0225] Amplification Product: Homologous Arm+fKanrf+Homologous Arm.

[0226] The PCR product was separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/L linear DNA full-length PCR segment for Red recombination targeting.

[0227] (2) Red Recombination Operation

[0228] First, pKD46 carrier was introduced into the AT-002-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting was electrotransformed, and positive clones were screened. Finally, the resistance genes were removed.

[0229] 1) Transformation of pKD46 Plasmid

[0230] {circle around (1)} Preparation of Competence:

[0231] First, inoculated the bacterial suspension of Escherichia coli AT-002-02 (AT-001, manXYZ) stored at 20 C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultured at 37 C. and 225 rpm for 2-3 h. then added the culture solution to a 10-mL centrifuge tube, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allowed to stand at 4 C. for 12 h for natural sedimentation.

[0232] {circle around (2)} Plasmid Transformation:

[0233] Transferred 250 L of naturally settled bacterial solution, added 5 L of pKD46 plasmid, and cultured at 4 C. for 30 min. Then heated on a water bath at 42 C. for 1.5 min, added 0.7 mL of SOC medium, and shook at 30 C. for 2 h. Transferred 0.2 mL of the bacterial suspension, and smeared on a penicillin plate. Cultured overnight (for 12-16 h) at 30 C. Monoclone was picked up, added 5 mL of LB broth medium and cultured, and plasmid was extracted for identification. Stored the positive strain for use.

[0234] 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone

[0235] {circle around (1)} Preparation of Electrotransform Competence:

[0236] Inoculated the AT-002-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultured at 250 rpm. On the next day, inoculated in a ratio of 1% into LB medium containing Amp, and cultured at 30 C.; when OD.sub.600 reached about 0.2, added 0.2% L-Arabinose, and induced at 30 C. for 35 min until OD.sub.600 reached about 0.4. Cooled on an ice bath. Washed once with ultrapure water, washed twice with 10% glycerin, and finally resuspended with 10% glycerin; the amount of glyerin used was to produce a final concentration of the bacterial soluion concentrated by 500-1000 folds.

[0237] {circle around (2)} Transformation by Electric Shock:

[0238] Took out a 2-mm electrotransformation cup from 70% ethanol, washed twice with sterilized ultrapure water, and irradiated by UV lamp for 30 min. Precooled at 4 C. for 30 min. Transferred 90 L of finally resuspended cells to a precooled centrifuge tube, added 5 L (more than 100 ng) of the full-length PCR segement (linear DNA) obtained in Step (1), gently suction mixed with a gun, and maintained on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 200, 25 F.

[0239] {circle around (3)} Resuscitate and Screen Positive Clones:

[0240] added 1 mL of LB broth medium, and cultured at 37 C. and 100 rpm for 1 h. Then smeared one kanamycin (Kan) plate with every 200 L, 5 plates in total. Smeared evenly and allowed to dry. Cultured at 30 C. for 24 h. Picked up clones grown under kanamycin resistance, and carried out PCR identification to screen positive clones.

[0241] The obtained strain number: AT-003-01 (AT-002-02, nanA::fKanrf).

[0242] (3) Removal of the Resistance Gene

[0243] Introduced pCP20 into the above kanamycin-resistant clones, cultured at 30 C. for 8 h, then increases to 42 C. and cultured overnight, and thermally induced to express FLP recombinase; the plasmids were lost gradually. Streaked the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, picked up grown monoclones and dotted on the kanamycin-resistant plate; those that did not grow were clones of which the kanamycin resistance gene had been removed by FLP recombination. Carried out PCR with identification primer to identify clones losing kanamycin resistance.

[0244] The obtained strain number: AT-003-02 (AT-002-02, nanA).

[0245] 3. Deletion of nagA Gene Sequence

[0246] N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) can transform N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P) in microorganism into D-glucosamine-6-phosphate (GlcN-6-P). Deletion of nagA gene sequence in nag operon (nagE-nagBACD) may block transformation of N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P) into D-Glucosamine-6-phosphate (GlcN-6-P).

[0247] (1) Preparation of Linear DNA Full-length PCR Segment for Red Recombination Targeting

[0248] Design of Homologous Arm Primers: From NCBI, looked up NC_000913, nagA sequence SEQ ID No: 10 for N-acetyl-D-glucosamine-6-phosphate deacetylase, Escherichia coli str.K-12, designed the homologous arm primers for deletion of nagA sequence: Forward primer (nagAKO-F) SEQ ID No: 11 and reverse primer (nagAKO-R) SEQ ID No: 12.

[0249] Template: Amplification of fKanrfPCR Segment.

[0250] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0251] Amplification Product: Homologous Arm+fKanf+Homologous Arm.

[0252] The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/L linear DNA full-length PCR segment for targeting of Red recombination.

[0253] (2) Red Recombination Operation

[0254] First, pKD46 carrier was introduced into the AT-003-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting was electrotransformed, and positive clones were screened. Finally, the resistance genes were removed.

[0255] 1) Transformation of pKD46 Plasmid

[0256] {circle around (1)} Preparation of Competence:

[0257] First, inoculateed the bacterial suspension of Escherichia coli AT-003-02 (AT-002-02, nanA) stored at 20 C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultured at 37 C. and 225 rpm for 2-3 h. then added the culture solution to a 10-mL centrifuge tube, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL 0.1M CaC.sub.2 on an ice bath for 5 min. Finally, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allowed to stand at 4 C. for 12 h for natural edimentation.

[0258] {circle around (2)} Plasmid Transformation:

[0259] Transferred 250 L of naturally settled bacterial solution, add 5 L of pKD46 plasmid, and cultured at 4 C. for 30 min. Then heated on a water bath at 42 C. for 1.5 min, added 0.7 mL of SOC medium, and shook at 30 C. for 2 h. Transferred 0.2 mL of the bacterial suspension, and smeared on a penicillin plate. Cultured overnight (for 12-16 h) at 30 C. Picked up monoclone, added 5 mL of LB broth medium and cultured, and plasmid was extracted for identification. Storeed the positive strain for use.

[0260] 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone

[0261] {circle around (1)} Preparation of Electrotransform Competence:

[0262] Inoculated the AT-003-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultured at 250 rpm, on the next day, inoculated in a ratio of 1% into LB medium containing Amp, and cultured at 30 C.; when OD.sub.600 reached about 0.2, added 0.2% L-Arabinose, and induced at 30 C. for 35 min until OD.sub.600 reached about 0.4. Cooled on an ice bath. Washed once with ultrapure water, washed twice with 10% glycerin, and finally resuspended with 10% glycerin; the amount of glycerin used is to produce a final concentration of the bacterial solution concentrated by 500-1000 folds.

[0263] {circle around (2)} Transformation by Electric Shock:

[0264] Took out a 2-mm electrotransformation cup from 70% ethanol, washed twice with sterilized ultrapure water, and irradiated by UV lamp for 30 min. Precooled at 4 C. for 30 min. Transferred 90 L of finally resuspended cells to a precooled centrifuge tube, added 5 L (more than 100 ng) of the full-length PCR segement (linear DNA) obtained in Step (1), gently suction mixed with a gun, and maintained on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 20011, 25 F.

[0265] {circle around (3)} Resuscitate and Screen Positive Clones:

[0266] added 1 mL of LB broth medium, and cultured at 37 C. and 100 rpm for 1 h. Then smeared one kanamycin (Kan) plate with every 200 L, 5 plates in total. Smeared evenly and allowed to dry. Cultured at 30 C. for 24 h. Picked up clones grown under kanamycin resistance, and carried out PCR identification to screen positive clones.

[0267] The obtained strain number: AT-004-01 (AT-003-02, nagA::fKanrf).

[0268] (3) Removal of the Resistance Gene

[0269] Introduced pCP20 into the above kanamycin-resistant clones, cultured at 30 C. for 8 h, then increased to 42 C. and cultured overnight, and thermally induced to express FLP recombinase; the plasmids were lost gradually. Streaked the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, picked up grown monoclones and dotted on the kanamycin-resistant plate; those that did not grow were clones of which the kanamycin resistance gene had been removed by FLP recombination. Carried out PCR with identification primer to identify clones losing kanamycin resistance.

[0270] The obtained strain number: AT-004-02 (AT-003-02, nagA).

[0271] 4. Deletion of nagE Gene Sequence

[0272] Deletion of the gene sequence nagE for N-acetyl-D-glucosamine specific enzyme II.sup.Nag (NagE) may block transport of extracellular GlcNAc into cells for degradation.

[0273] (1) Preparation of Linear DNA Full-length PCR Segment for Red Recombination Targeting

[0274] Design of Homologous Arm Primers: From NCBI, looked up NC_000913, nagB promoter and nagA gene sequence SEQ ID No: 13, Escherichia coli str.K-12, designed the homologous arm primers for deletion of nagA sequence: Forward primer (nagEKO-F1) SEQ ID No:14 and reverse primer (nagEKO-R1) SEQ ID No:15.

[0275] Template: Amplification of fKanrfPCR Segment.

[0276] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0277] Amplification Product: Homologous Arm+fKanrf+Homologous Arm.

[0278] The PCR product was separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/L linear DNA full-length PCR segment for Red recombination targeting.

[0279] (2) Red Recombination Operation

[0280] First, pKD46 carrier was introduced into the AT-004-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting was electrotransformed, and positive clones were screened. Finally, the resistance genes were removed.

[0281] 1) Transformation of pKD46 Plasmid

[0282] {circle around (1)} Preparation of Competence:

[0283] First, inoculated the bacterial suspension of Escherichia coli AT-004-02 (AT-003-02, nagA) stored at 20 C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultured at 37 C. and 225 rpm for 2-3 h. then added the culture solution to a 10-mL centrifuge tube, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allowed to stand at 4 C. for 12 h for natural sedimentation.

[0284] {circle around (2)} Plasmid Transformation: Transferred 250 L of naturally settled bacterial solution, add 5ed L of pKD46 plasmid, and cultured at 4 C. for 30 min. Then heated on a water bath at 42 C. for 1.5 min, added 0.7 mL of SOC medium, and shook at 30 C. for 2 h. Transferred 0.2 mL of the bacterial suspension, and smeared on a penicillin plate. Cultured overnight (for 12-16 h) at 30 C. Picked up monoclones, added 5 mL of LB broth medium and cultured, and plasmid was extracted for identification. Stored the positive strain for use.

[0285] 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone

[0286] {circle around (1)} Preparation of Electrotransform Competence: Inoculated the AT-004-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultured at 250 rpm. On the next day, inoculated in a ratio of 1% into LB medium containing Amp, and cultured at 30 C.; when OD.sub.600 reached about 0.2, added 0.2% L-Arabinose, and induced at 30 C. for 35 min until OD.sub.600 reached about 0.4. Cooled on an ice bath. Washed once with ultrapure water, washed twice with 10% glycerin, and finally resuspended with 10% glycerin; the amount of glycerin used was to produce a final concentration of the bacterial solution concentrated by 500-1000 folds.

[0287] {circle around (2)} Transformation by Electric Shock:

[0288] Took out a 2-mm electrotransformation cup from 70% ethanol, washed twice with sterilized ultrapure water, and irradiated by UV lamp for 30 min. Precooled at 4 C. for 30 min. Transferred 90 L of finally resuspended cells to a precooled centrifuge tube, added 5 L (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (1), gently suction mixed with a gun, and maintained on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 200, 25 F.

[0289] {circle around (3)} Resuscitate and screen positive clones: added 1 mL of LB broth medium, and cultured at 37 C. and 100 rpm for 1 h. Then smeared one kanamycin (Kan) plate with every 200 L, 5 plates in total. Smeared evenly and allowed to dry. Cultured at 30 C. for 24 h. Picked up clones grown under kanamycin resistance, and carried out PCR identification to screen positive clones.

[0290] The obtained strain number: AT-005-01 (AT-004-02, nagE::fKanrf).

[0291] (3) Removal of the Resistance Gene

[0292] Introduced pCP20 into the above kanamycin-resistant clones, cultured at 30 C. for 8 h, then increased to 42 C. and cultured overnight, and thermally induced to express FLP recombinase; the plasmids were lost gradually. Streaked the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, picked up grown monoclones and dotted on the kanamycin-resistant plate; those that did not grow were clones of which the kanamycin resistance gene had been removed by FLP recombination. Carried out PCR with identification primer to identify clones losing kanamycin resistance.

[0293] The obtained strain number: AT-005-02 (AT-004-02, nagE).

Example 2

[0294] This example describes the gene NanE cloning of N-acetyl-D-aminomannose-6-phosphate epimerase (NanE), and transformed nanE/pTrc99A plasmids in Escherichia coli, as well as integration of ptrc-nanE gene cassette into the chromosome of Escherchia coli.

[0295] 1. nanE Gene Cloning, Transformation of nanE/pTrc99A plasmid in Escherichia coli, and its Influence on Output of N-acetyl-D-glucosamine

[0296] Amplification of the gene nanE of Escherichia coli NanE (N-acetyl-D-aminomannose-6-phosphate epimerase), that is controlled by Trc promoter to transform the strain for overexpression of the enzyme, may strengthen the conversion of N-acetyl-D-mannosamine-6-phosphate (ManNAc-6-P) into N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P).

[0297] 1) Cloning of Escherichia coli nanE Gene

[0298] From NCBI, looked up U00096, to obtain the nucleotide sequence SEQ ID No: 16 of the nanE gene of Escherichia coli, whose amino acid sequence is SEQ ID No: 17.

[0299] Primer Design: Forward primer (nanE-F) SEQ ID No: 18 and reverse primer (nanE-R) SEQ ID No:19.

[0300] Template: AT-001 (Escherichia coli ATCC 27325) genome

[0301] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0302] Amplification Product Size: 690 bp.

[0303] The PCR product was separated by 1% agarose gel electrophoresis, and purified to recover the segment.

[0304] Connected the obtained PCR amplification segment with pUC57-T carrier, and carried out sequencing for identification, to obtain nanE/pUC57.

[0305] ) Construction and Transformation of Plasmid in which nanE Gene is Controlled by Trc Promoter.

[0306] {circle around (1)} Plasmid Construction:

[0307] Amplified plasmid nanE/pUC57, digested enzymatically nanE/pUC57 and carrier pTrc99A with Nco I and Hind III, respectively, separated by agarose gel electrophoresis, purified to recover nanE segment and pTrc99A segment, connected overnight with T4 DNA ligase at 16 C., and carried out identification to obtain nanE/pTrc99A plasmid.

[0308] {circle around (2)} Preparation of Competence:

[0309] First, inoculated the bacterial suspension of AT-005-02 stored at 20 C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultured at 37 C. and 225 rpm for 2-3 h. then added the culture solution to a 10-mL centrifuge tube, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allowed to stand at 4 C. for 12 h for natural sedimentation.

[0310] {circle around (3)} Plasmid Transformation:

[0311] Transferred 250 L of naturally settled bacterial solution, added 5 L of nanE/pTrc99A plasmid, and cultured at 4 C. for 30 min. Then heated on a water bath at 42 C. for 1.5 min, added 0.7 mL of SOC medium, and shook at 30 C. for 2 h. Transferred 0.2 mL of the bacterial suspension, and smeared on a penicillin plate. Cultured overnight (for 12-16 h) at 30 C. Picked up monoclone, added 5 mL of LB broth medium and cultured, and plasmids were extracted for identification. Storeed the positive strain for use. Obtained Recombinant Strain nanE/pTrc99A (AT-005-02)

[0312] 3) Influence of nanE/pTrc99A Plasmid Transformation on the Output of N-Acetyl-D-Glucosamine

[0313] Comparing the recombinant nanE/pTrc99A (AT-005-02) strain with reference strain using shake-flask fermentation trial. Transferred the monoclonal strain freshly cultured in culture medium of the LB plate, inoculated into a test tube (13150 mm) containing 3 mL of the LB broth culture medium, and shake-cultured at 30 C. for 8 h. Components of LB Broth Medium: 5 g/L yeast powder, 10 g/L peptone, and 10 g/L NaCl. Then transferred the seed culture solution, inoculated 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD.sub.600 was about 0.5; shake-cultured at 37 C. and 225 rpm. The fermentation cycle was 72 h. At 24 h and 48 h, adjusted the pH value of the fermentation solution to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, added 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transferred 1 mL of the fermentation broth and centrifuged. Measured the content of N-acetyl-D-glucosamine by HPLC method.

[0314] {circle around (1)} HPLC Method to Measure the Content of N-acetyl-D-glucosamine

[0315] Buffer: Added 3.5 g of dipotassium hydrogen phosphate to 1-L volumetric flask, added water to dissolve, added 0.25 mL of 0.25 mL of ammonia water, then diluted with water and mixed well, adjusted to pH 7.5, and added water to volume.

[0316] 1 Mobile Phase: Acetonitrile: Buffer (75:25).

[0317] Diluent: Acetonitrile and water (50:50).

[0318] Standard Solution: Dissolved 1.0 mg/mL USP N-acetyl-D-glucosamine

[0319] Reference Substance (RS) in the diluent.

[0320] Sample Solution: Dissolved 1.0 mg/mL N-acetyl-D-glucosamine sample in the diluent.

[0321] Liquid Phase Conditions:

[0322] Model: LC

[0323] Detector: UV 195 nm

[0324] Chromatographic Column: 4.6-mm15-cm; 3-m packing L8

[0325] Flow Rate: 1.5 mL/min

[0326] Column Temperature: 35 C.

[0327] Injection Volume: 10 L

[0328] Preparation of M9 Culture Solution

[0329] First prepared 5M9 culture medium: added into approximately 800 mL of double distilled water (ddH.sub.2O) 64 g of Na.sub.2HPO.sub.4.7H.sub.2O, 15 g of KH.sub.2PO.sub.4, 2.5 g of NaCl, and 5.0 g of NH.sub.4Cl, and after dissolution, added water to 1000 mL. Sterilized at 121 C. for 30 min. Then prepared 1M MgSO.sub.4, 1M CaCl.sub.2, and 20% glucose, respectively, and sterilized them separately. Then prepared M9 culture solution according to Table 1, while 1000 microelement solution was prepared according to Table 2.

TABLE-US-00002 TABLE 1 2) Components of M9 Culture Solution Ingredients Amount (mL/L) 5 M9 200 1M MgSO.sub.4 2 1M CaCl.sub.2 0.1 20% Glucose 20 1000 Microelement 1 Solution ddH.sub.2O to 1000 pH 6.9

TABLE-US-00003 TABLE 2 3) Components of 1000 Microelement Solution Ingredients Amount (g/L) CoCl.sub.26H.sub.2O 0.01 CuSO.sub.45H.sub.2O 0.01 MnSO.sub.4H.sub.2O 0.033 FeSO.sub.47H.sub.2O 0.50 ZnSO.sub.47H.sub.2O 0.38 H.sub.3BO.sub.3 0.01 NaMoO.sub.42H.sub.2O 0.01 pH 3

[0330] {circle around (3)} Influence of nanE/pTrc99A Plasmid Transformation on the Output of N-Acetyl-D-Glucosamine from Shake-Flask Fermentation

[0331] See Table 3 for the output from shake-flask fermentation. The results show that: The output by the reference strain AT-005-02 was very low and was not detected, while the output from overexpressed recombinant nanE/pTrc99A (AT-005-02), by nanE gene controlled by Trc promoter, increased significantly.

TABLE-US-00004 TABLE 3 Output from Shake-flask Fermentation by the Recombinant nanE/pTrc99A (AT-005-02) Output of N-acetyl-D-glucosamine Strains (g/L) AT-005-02 (AT-004-02, nagE) Not detected (Reference) NanE/pTrc99A (AT-005-02) 1.3 0.3

[0332] 2. Integration of pTrc-nanE Gene Cassette into the Chromosome of Escherichia Coli

[0333] The nagE gene site was used as integration site of pTrc-nanE gene cassette into the chromosome. To achieve integration of pTrc-nanE gene cassette into the chromosome of Escherichia coli, first the nanE segment containing Trc promoter pTrc-nanE was amplified, as well as the kanamycin resistance gene segment with FLP recognition site (FRT site) at its both ends: FRT-Kanr-FRT (fXanrf), and they were spliced. Then homologous arm primers for deletion of the nagE gene sequence was designed, and using the spliced segment of pTrc-nanK and fKanrf as template, amplified the linear DNA full-length segment for Red recombination targeting.

[0334] The specific operation process is provided below:

[0335] (1) PCR Amplification of pTrc-nanE segment

[0336] Template: nanE/pTrc99A.

[0337] Primer Design: Forward primer (Treff-F) SEQ ID No:20, and reverse primer (Treff-R) SEQ ID No:21.

[0338] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0339] Produce Size: 0.86 kb.

[0340] The PCR product was separated by 1% agarose gel electrophoresis, and purified to recover the segment.

[0341] (2) fKanrf Segment Amplified by PCR

[0342] Primer Design: Forward primer (mfKanf-F) SEQ ID No:1, and reverse primer (mfKanf-R) SEQ ID No:2.

[0343] Template: pPic9K.

[0344] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0345] fKanrf size: 1.28 kb. Its nucleotide sequence is SEQ ID No:3.

[0346] The PCR product was separated by 1% agarose gel electrophoresis, and purified to recover the segment.

[0347] (3) Amplification of fKanrf Spliced with pTrc-nanE

[0348] Primer Design: Forward primer (fKanf-F) SEQ ID No: 22, and reverse primer (fKanf-R) SEQ ID No: 23.

[0349] Template: fKanrf.

[0350] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0351] fKanrf size from secondary amplification: 1.3 kb.

[0352] The PCR product was separated by 1% agarose gel electrophoresis, and purified to recover the segment.

[0353] (4) Preparation of Linear DNA Full-Length PCR Segment for Red Recombination Targeting

[0354] Design of Homologous Arm Primers: Design again homologous arm primers for deletion of the nagE gene sequence: Forward primer (nagEKO-F2) SEQ ID No: 24, and reverse primer (nagEKO-R2) SEQ ID No: 25.

[0355] Template: PTrc-nanE PCR segment and fKanrfPCR segment from secondary amplification mixed in the ratio of 1:1.

[0356] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0357] Amplification Product: Homologous Arm+pTrc-nanE-fKanrf+Homologous Arm

[0358] The PCR product was separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/L linear DNA full-length PCR segment for Red recombination targeting.

[0359] (5) Red Recombination Operation

[0360] First, pKD46 carrier was introduced into the AT-004-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting was electrotransformed, and positive clones were screened. Finally, the resistance genes were removed.

[0361] 1) Transformation of pKD46 Plasmid

[0362] {circle around (1)} Preparation of Competence:

[0363] First, inoculated the bacterial suspension of Escherichia coli AT-004-02 stored at 20 C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultured at 37 C. and 225 rpm for 2-3 h. then added the culture solution to a 10-mL centrifuge tube, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL 0.1M CaC.sub.2 on an ice bath for 5 min. Finally, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allowed to stand at 4 C. for 12 h for natural sedimentation.

[0364] {circle around (2)} Plasmid Transformation:

[0365] Transferred 250 L of naturally settled bacterial solution, added 5 L of pKD46 plasmid, and cultured at 4 C. for 30 min. Then heated on a water bath at 42 C. for 1.5 min, added 0.7 mL of SOC medium, and shook at 30 C. for 2 h. Transferred 0.2 mL of the bacterial suspension, and smeared on a penicillin plate. Cultured overnight (for 12-16 h) at 30 C. Picked up monoclone, added 5 mL of LB broth medium and cultured, and plasmids were extracted for identification. Storeed the positive strain for use.

[0366] 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone

[0367] {circle around (1)} Preparation of Electrotransform Competence: Inoculated the AT-004-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultured at 250 rpm. On the next day, inoculated in a ratio of 1% into LB medium containing Amp, and cultured at 30 C.; when OD.sub.600 reached about 0.2, added 0.2% L-Arabinose, and induced at 30 C. for 35 min until OD.sub.600 reached about 0.4. Cooled on an ice bath. Washed once with ultrapure water, washed twice with 10% glycerin, and finally resuspended with 10% glycerin; the amount of glycerin used was to produce a final concentration of the bacterial solution concentrated by 500-1000 folds.

[0368] {circle around (2)} Transformation by Electric Shock:

[0369] Took out a 2-mm electrotransformation cup from 70% ethanol, washed twice with sterilized ultrapure water, and irradiated by UV lamp for 30 min. Precooled at 4 C. for 30 min. Transferred 90 L of finally resuspended cells to a precooled centrifuge tube, added 5 L (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (4), gently suction mixed with a gun, and maintained on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 20011, 25 F.

[0370] {circle around (3)} Resuscitate and Screen Positive Clones:

[0371] added 1 mL of LB broth medium, and cultured at 37 C. and 100 rpm for 1 h. Then smeared one kanamycin (Kan) plate with every 200 L, 5 plates in total. Smeared evenly and allowed to dry. Cultured at 30 C. for 24 h. Picked up clones grown under kanamycin resistance, and carried out PCR identification to screen positive clones.

[0372] The obtained strain number: AT-030-01 (AT-004-02, nagE::pTrc-nanE-fKanrf).

[0373] (6) Removal of the Resistance Gene

[0374] Introduced pCP20 into the above kanamycin-resistant clones, cultured at 30 C. for 8 h, then increased to 42 C. and cultured overnight, and thermally induced to express FLP recombinase; the plasmids were lost gradually. Streaked the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, picked up grown monoclones and dotted on the kanamycin-resistant plate; those that did not grow were clones of which the kanamycin resistance gene had been removed by FLP recombination. Carried out PCR with identification primer to identify clones losing kanamycin resistance.

[0375] The obtained strain number: AT-030-02 (AT-004-02, nagE::pTrc-nanE).

[0376] 3) Influence of pTrc-nanE Gene Cassette Integration on the Output of N-Acetyl-D-Glucosamine

[0377] Carried out a shake-flask fermentation trial with the recombinant strain AT-030-02 of which the nagE gene site in the chromosome was integrated with pTrc-nanE gene cassette, and the reference strain. Transferred the monoclonal strain freshly cultured in culture medium of the LB plate, inoculated into a test tube (13150 mm) containing 3 mL of the LB broth culture medium, and shake-cultured at 30 C. for 8 h. Then transferred the seed culture solution, inoculated 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD.sub.600 was about 0.5; shake-cultured at 37 C. and 225 rpm. The fermentation cycle was 72 h. At 24 h and 48 h, adjusted the pH of the fermentation broth to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, added 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transferred 1 mL of the fermentation broth and centrifuged. Measured the content of N-cetyl-D-glucosamine by HPLC method.

[0378] See Table 4 for the output from shake-flask fermentation. The results show that: The outputs by the reference strains AT-001 and AT-005-02 were very low and were not detected, while the output by the recombinant strain integrated with pTrc-nanE gene cassette increased significantly, and also increased significantly than that by the not integrated recombinant strain nanE/pTrc99A (AT-005-02).

TABLE-US-00005 TABLE 4 Output from Shake-flask Fermentation by the Recombinant Strain integrated with pTrc-nanE Gene Cassette Output of N-acetyl-D- Strains glucosamine (g/L) AT-001 (Reference) Not detected AT-005-02 (AT-004-02, nagE) (Reference) Not detected nanE/pTrc99A(AT-005-02) 1.2 0.3 AT-030-02 (AT-004-02, nagE::pTrc-nanE) 2.5 0.5

Example 3

[0379] This example describes screening for a gene mutant of N-acetyl-D-aminomannose-6-phosphate epimerase (NanE); said gene encodes N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) with increased enzyme activities.

[0380] To further increase synthetic quantity of N-acetyl-D-glucosamine by the production strain, screening a gene mutant encoding N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) with increased enzyme activities. To achieve the purpose, error-prone PCR technology was used to amplify the cloned gene; through DNA polymerase used for amplification, amplified said gene under conditions leading to high-frequency mismatch, so as to obtain a high-frequency mutation in PCR products.

[0381] The specific operating process is provided below:

[0382] 1. Amplification of the Gene nanE of N-Acetyl-D-Aminomannose-6-Phosphate Epimerasein Escherichia coli by Error-Prone PCR

[0383] By means of Taq DNA polymerase without the property of 3-5 proofreading function, controlled the frequency of random mutation under high magnesium ion concentration (8 mmol/L) and different dNTP concentrations (where, the concentration of dATP and dGTP was 1.5 mmol/L; and the concentration of dTTP and dCTP was 3.0 mmol/L), introduced random mutations into the target gene, and constructed a mutation library; the template concentration A260 value was 1000 ng/mL, the enzyme concentration was 5 U/L, and the primer concentration was 100 M.

[0384] Error-prone PCR reaction system (50 L): 10PCR reaction buffer 5 l, dNTP (22.5 mM) 5 L, MgCl.sub.2 (2.5 mM) 5 L, Forward primer (nanE-F, SEQ ID No:18) 1 L, reverse primer (nanE-R, SEQ ID No: 19) 1 L, DNA template (nanE/pUC57) 0.1 L, Taq DNA polymerase 0.5 L, and ddH.sub.2O 32.4 L.

[0385] PCR procedure: Pre-degeneration at 96 C. for 4 min; degeneration at 94 C. for 1 min, annealing at 56 C. for 1 min, extension at 75 C. for 2 min, and repeated for 45 cycles; finally extended at 75 C. for 15 min, recovered PCR product (product size: 0.7 kb) by gel recovery method; transferred 5 L of the product and tested with 1% agarose gel electrophoresis; the product was store at 20 C. for use.

[0386] 2. Construction of the Gene Mutation Library of N-Acetyl-D-Aminomannose-6-Phosphate Epimerase

[0387] Digested the above PCR product by two enzymes of restriction endonuclease, Nco I and Hind III, carried out a ligation reaction with pTrc99A digested by Nco I and HindIII, then transformed Escherichia coli AT-005-02 with the mixture of the ligated products to obtain a large amount of cloned transformants, and constructed a mutation library of transformed strains.

[0388] 3. Screening for Mutants with High Enzyme Activities

[0389] Using the wild type NanE/pTrc99A (AT-005-02) as reference, randomly picked up 350 mutant clones from the mutation library of transformed strains, inoculated into 5 mL of LB medium containing 50 g/mL Ampicillin (Amp), shake-cultured at 37 C. and 150 rpm for 18 h, and then centrifuged at 10000 rpm for 5 mim and collected bacterial solution. Discarded the supernatant, then resuspended at 4 C. in 1 mL of PBS solution (pH 7.5, 10 mmol/L), carried out ultrasonicate at a voltage of 300 V for 10 min (ultrasonicated for 3 s and paused for 6 s), centrifuged, transferred the supernatant as crude extract of enzyme for determination of enzyme activity.

[0390] Determination of N-acetyl-D-aminomannose-6-phosphate epimerase (NanE) Activity: Based on the amount of N-Acetyl-D-Mannosamine-6-phosphate (ManNAc-6-p) converted to N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P), that is, using the reduced amount of N-Acetyl-D-Mannosamine-6-phosphate as test marker. Definition of Enzyme Activity Unit: Under the enzymatic reaction conditions, the enzyme amount needed to reduce equivalent to 1 mol of N-Acetyl-D-Mannosamine-6-phosphate per minute is defined as one enzyme activity unit (IU). The specific procedure is provided as follows: isotope-labeled ManNAc-6-P was prepared as a substrate. Prepared a total volume of 225 ul of reaction solution containing ManNAc kinase (NanK) crude enzyme solution (containing 1-5 mg protein), 20 mM ATP disodium salt, 60 mM Tris-HCl, pH 8.1, 20 mM MgCl2 and 5 mM ManNAc, 50 nCi.sup.[14C]ManNAc. Incubated at 37 C. for 30 min. The reaction was terminated by the addition of 350 ul of ethanol. The product was eluted with water and lyophilized. Next, a total volume of 26.5 ul of the reaction solution was prepared as an enzyme activity assay system containing 1 mM isotope-labeled ManNAc-6-P, 37 mM Tris-HCl, pH 8.0 and 19 mM MgCl2. After incubation at 37 C. for 30 min, the reaction was boiled for 3 min, then 0.1 volume of alkaline phosphatase buffer was added to adjust the pH and 20 units of alkaline phosphatase. After incubation at 37 C. for 1 hour, samples were taken onto dry chromatography paper and pre-soaked with 1% sodium tetraborate. The solvent system used was ethyl acetate:isopropanol:pyridine:water (50:22:14:14). The radioactive compound was separated by paper chromatography. The radioactivity was measured by a liquid scintillation counter, and the activity unit of N-acetyl-D-aminomannose-6-P epimerase was calculated based the amount of ManNAc-6-P converted to GlcNAc-6-P.

[0391] The results show that: The enzyme activity of the mutant strain with maximum activity was 72 IU/mL, and the enzyme activity of the reference control was 9.5 IU/mL. Through transforming NanE by error-prone PCR, a mutant strain with significantly increased enzyme activity was obtained. The mutant strain with maximum enzyme activity was picked up and plasmids were extracted for sequencing. The results showed that: The gene sequence of the mutant of N-acetyl-D-aminomannose-6-P epimerase is shown as SEQ ID No: 26, and the corresponding amino acid is shown as SEQ ID No: 27. By sequence alignment with the gene sequence of wild type N-acetyl-D-aminomannose-6-P epimerase, 3 base point mutations occurred in total: 198C/T, 397T/C, and 559T/C; and caused two amino acid missense mutation, the mutation points are: C133R (the 133th cysteine to arginine), Y187H (the 187th tyrosine to histidine). The mutant gene was named nanEM.

[0392] 4. Integration of pTrc-nanE Gene Cassette into the nagE Gene Site in the Chromosome of Escherichia Coli

[0393] The nagE gene site was used as integration site of pTrc-nanEM gene cassette into the chromosome. To achieve integration of pTrc-nanEM gene cassette into the chromosome of Escherichia coli, first the nanEM segment containing Trc promoter pTrc-nanEM was amplified, as well as the kanamycin resistance gene segment with FLP recognition site (FRT site) at its both ends: FRT-Kanr-FRT (fXanrf), and they were spliced. Then homologous arm primers were designed for deletion of the nagE gene sequence, and using the spliced segment of pTrc-nanEM and fKanrf as template, amplified the linear DNA full-length segment for Red recombination targeting.

[0394] The specific operating process is provided below:

[0395] (1) PCR Amplification of pTrc-nanEM Segment

[0396] Template: nanEM/pTrc99A.

[0397] Primer Design: Forward primer (Treff-F) SEQ ID No: 20, and reverse primer (Treff-R) SEQ ID No: 21.

[0398] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0399] Produce Size: 0.86 kb.

[0400] The PCR product was separated by 1% agarose gel electrophoresis, and purified to recover the segment.

[0401] (2) fKanrf Segment Amplified by PCR

[0402] Primer Design: Forward primer (mfKanf-F) SEQ ID No:1, and reverse primer (mfKanf-R) SEQ ID No: 2.

[0403] Template: pPic9K.

[0404] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0405] fKanrf size: 1.28 kb. Its nucleotide sequence is SEQ ID No: 3.

[0406] The PCR product was separated by 1% agarose gel electrophoresis, and purified to recover the segment.

[0407] (3) Amplification of fKanrf Spliced with pTrc-nanEM

[0408] Primer Design: Forward primer (fKanf-F) SEQ ID No: 22, and reverse primer (fKanf-R) SEQ ID No: 23.

[0409] Template: fKanrf.

[0410] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0411] fKanrf size from secondary amplification: 1.3 kb.

[0412] The PCR product was separated by 1% agarose gel electrophoresis, and purified to recover the segment.

[0413] (4) Preparation of Linear DNA Full-Length PCR Segment for Red Recombination Targeting

[0414] Design of Homologous Arm Primers: Designed again homologous arm primers for deletion of the nagE gene sequence: Forward primer (nagEKO-F2) SEQ ID No: 24, and reverse primer (nagEKO-R2) SEQ ID No: 25.

[0415] Template: pTrc-nanEM PCR segment and fKanrf PCR segment from secondary amplification were mixed in the ratio of 1:1.

[0416] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0417] Amplification Product: Homologous Arm+pTrc-nanEM-fKanrf+Homologous Arm

[0418] The PCR product was separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/L linear DNA full-length PCR segment for Red recombination targeting.

[0419] (5) Red Recombination Operation

[0420] First, pKD46 carrier was introduced into the AT-004-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting was electrotransformed, and positive clones were screened. Finally, the resistance genes were removed.

[0421] 1) Transformation of pKD46 Plasmid

[0422] {circle around (1)} Preparation of Competence:

[0423] First, inoculated the bacterial suspension of Escherichia coli AT-004-02 stored at 20 C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultured at 37 C. and 225 rpm for 2-3 h. Then added the culture solution to a 10 mL centrifuge tube, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL of 0.1M CaC.sub.2 on an ice bath. Allowed to stand at 4 C. for 12 h for natural sedimentation.

[0424] {circle around (2)} Plasmid Transformation:

[0425] Transferred 250 L of naturally settled bacterial solution, added 5 L of pKD46 plasmid, and cultured at 4 C. for 30 min. Then heated on a water bath at 42 C. for 1.5 min, added 0.7 mL of SOC medium, and shook at 30 C. for 2 h. Transferred 0.2 mL of the bacterial suspension, and smeared on a penicillin plate. Cultured overnight (for 12-16 h) at 30 C. Picked up monoclone, added 5 mL of LB broth medium and cultured, and plasmids were extracted for identification. Stored the positive strain for use.

[0426] 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone

[0427] {circle around (1)} Preparation of Electrotransform Competence:

[0428] Inoculated the AT-004-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultured at 250 rpm, on the next day, inoculated in a ratio of 1% into LB medium containing Amp, and cultured at 30 C.; when OD.sub.600 reached about 0.2, added 0.2% L-Arabinose, and induced at 30 C. for 35 min until OD.sub.600 reached about 0.4. Cooled on an ice bath. Washed once with ultrapure water, washed twice with 10% glycerin, and finally resuspended with 10% glycerin; the amount of glycerin used was to produce a final concentration of the bacterial solution concentrated by 500-1000 folds.

[0429] {circle around (2)} Transformation by Electric Shock:

[0430] Took out a 2-mm electrotransformation cup from 70% ethanol, washed twice with sterilized ultrapure water, and irradiated by UV lamp for 30 min. Precooled at 4 C. for 30 min. Transferred 90 L of finally resuspended cells to a precooled centrifuge tube, added 5 L (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (4), gently suction mixed with a gun, and maintained on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 20011, 25 F.

[0431] {circle around (3)} Resuscitate and screen positive clones: added 1 mL of LB broth medium, and cultured at 37 C. and 100 rpm for 1 h. Then smeared one kanamycin (Kan) plate with every 200 L, 5 plates in total. Smeared evenly and allowed to dry. Cultured at 30 C. for 24 h. Picked up clones grown under kanamycin resistance, and carried out PCR identification to screen positive clones.

[0432] The obtained strain number: AT-031-01 (AT-004-02, nagE::pTrc-nanEM-fKanrf).

[0433] (6) Removal of the Resistance Gene

[0434] Introduced pCP20 into the above kanamycin-resistant clones, cultured at 30 C. for 8 h, then increased to 42 C. and cultured overnight, and thermally induced to express FLP recombinase; the plasmids were lost gradually. Streaked the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, picked up grown monoclones and dotted on the kanamycin-resistant plate; those that did not grow were clones of which the kanamycin resistance gene had been removed by FLP recombination. Carried out PCR with identification primer to identify clones that had lost kanamycin resistance.

[0435] The obtained strain number: AT-031-02 (AT-004-02, nagE::pTrc-nanEM).

[0436] 5. Influence of pTrc-nanEM Gene Cassette Integration on the Output of N-Acetyl-D-Glucosamine

[0437] Carrying out a shake-flask fermentation trial comparing the recombinant strain AT-031-02 of which the nagE gene site in the chromosome was integrated with pTrc-nanEM gene cassette, with the reference strain. Transferred the monoclonal strain freshly cultured in culture medium of the LB plate, inoculated into a test tube (13150 mm) containing 3 mL of the LB broth culture medium, and shake-cultured at 30 C. for 8 h. Then transferred the seed culture solution, inoculated 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD.sub.600 was about 0.5; shake-cultured at 37 C. and 225 rpm. The fermentation cycle was 72 h. At 24 h and 48 h, adjusted the pH value to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, added 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transferred 1 mL of the fermentation broth and centrifuged. Measured the content of N-acetyl-D-glucosamine by HPLC method.

[0438] See Table 5 for the output from shake-flask fermentation. The results show that: The output by the reference strain AT-005-02 was very low and was not detected, while the output by the recombinant strain AT-031-02 integrated with pTrc-nanEM gene cassette increased significantly, and also increased significantly than that by the reference strain (AT-030-02) without mutation.

TABLE-US-00006 TABLE 5 Output from Shake-flask Fermentation by the Recombinant Strain integrated with pTrc-nanEM Gene Cassette Output of N-acetyl-D- Strains glucosamine (g/L) AT-005-02 (AT-004-02, nagE) (Reference) Not detected AT-030-02 (AT-004-02, nagE::pTrc-nanE) 2.6 0.5 AT-031-02 (AT-004-02, nagE::pTrc-nanEM) 5.9 0.8

[0439] The above results show that: The output of N-acetyl-D-glucosamine may be increased significantly by overexpression of N-acetyl-mannosamine-6-phosphate epimerase; moreover, the output of N-acetyl-D-glucosamine may also be greatly increased by the mutant screened by error-prone PCR technology, due to the increased enzyme activities of the obtained mutant of the enzyme.

Example 4

[0440] This example describes Escherichia coli strains integrated with pTrc-nanEM cassette, and wherein the effect on N-acetyl-D-glucosamine production yield of replacing and/or removing the endogenous natural promoter of the glucosamine-6-phosphate synthase (GlmS) gene glmS and/or D-Glucosamine-6-phosphate deaminase (NagB) gene nagB

[0441] 1. Replacing the endogenous natural promoter of nagB gene with the Trc promoter, and further deleting the endogenous natural promoter of glmS gene, and the effect on N-acetyl-D-glucosamine production yield by Escherichia coli strains integrated with pTrc-NanEM gene cassette.

[0442] (1) The natural endogenous promoter of the nagB gene was replaced by Trc promoter

[0443] The gene promoter of D-Glucosamine-6-Phosphate Deaminase (NagB) is nag regulon (nagE-nagBACD) was replaced by Trc promoter. The reaction catalyzed by D-Glucosamine-6-Phosphate Deaminase (NagB) is reversible; overexpression of nagB may accelerate the forward catalytic reaction by NagB, to achieve the purpose of increasing D-Glucosamine-6-Phosphate (GlcN-6-P).

[0444] First, amplified Trc promoter segment and fKanrf segment, and spliced them together. Then designed homologous arm primers, and amplified linear DNA full-length segment for Red recombination targeting.

[0445] 1. Amplifying the Trc Promoter Sequence

[0446] Based on published information, obtained Trc promoter sequence: SEQ ID No: 28.

[0447] Primers design: forward primer (KanTrcRed-F) SEQ ID No: 29, reverse primer (KanTrcRed-R) SEQ ID No: 30.

[0448] Template: pTrc99A

[0449] PCR reaction conditions: first step: denaturation at 94 C. for 1 min; second step: incubation at 94 C. for 30 s, at 55 C. for 30 s, at 72 C. for 40 s, carrying out 30 cycles; third step: extension at 72 C. for 10 min.

[0450] Product size: 166 bp.

[0451] The PCR product was separated by 1% agarose gel electrophoresis and purified to recover segment.

[0452] 2. Amplifying the Kanamycin Resistance Gene with FLP Recombinase Recognition Site (FRT Site) on Both Sides: fKanrf

[0453] Primers design: forward primer (mfKanf-F) SEQ ID No: 1, reverse primer (mfKanf-R) SEQ ID No: 2.

[0454] Template: pPic9K.

[0455] PCR reaction conditions: first step: denaturation at 94 C. for 1 min; second step: incubation at 94 C. for 30 s, at 55 C. for 30 s, at 72 C. for 40 s, carrying out 30 cycles; third step: extension at 72 C. for 10 min.

[0456] fKanrf size: 1.28 kb. Its nucleotide sequence is SEQ ID No: 3.

[0457] The PCR product was separated by 1% agarose gel electrophoresis and purified to recover segment.

[0458] 3. Amplifying fKanrf Docked with the Trc Promoter

[0459] Primers design: forward primer (fKanfRed-F1) SEQ ID No: 31, reverse primer (fKanfRed-R1) SEQ ID No: 32.

[0460] Template: fKanrf.

[0461] PCR reaction conditions: first step: denaturation at 94 C. for 1 min; second step: incubation at 94 C. for 30 s, at 55 C. for 30 s, at 72 C. for 40 s, carrying out 30 cycles; third step: extension at 72 C. for 10 min.

[0462] The size of the second amplified fKanrf: 1.3 kb.

[0463] The PCR product was separated by 1% agarose gel electrophoresis and purified to recover segment.

[0464] 4. Preparation of a Linear DNA Full-Length PCR Fragment for Red Recombinant Targeting

[0465] Homology arm primers design: According to NCBI, found NC_000913, Escherichia coli str. K-12 nagB promoter sequence and nagE gene sequence SEQ ID No: 13, designed homology arm primer for deletion of nagB promoter gene sequence: forward primer (nagBKO-F1) SEQ ID No: 33, reverse primer (nagBKO-R1) SEQ ID No: 34.

[0466] Template: mixed Trc promoter PCR fragment and secondary amplified fKanrfPCR fragment at a ratio of 1:1.

[0467] PCR reaction conditions: first step: denaturation at 94 C. for 1 min; second step: incubation at 94 C. for 30 s, at 55 C. for 30 s, at 72 C. for 40 s, carrying out 30 cycles; third step: extension at 72 C. for 10 min.

[0468] Amplification product: homology arm+fKanrf+Trc promoter+homology arm.

[0469] The PCR product was separated by agarose gel electrophoresis, purified and recovered to obtain a 100 ng/l linear DNA full-length PCR fragment for Red recombinant targeting.

[0470] 5, Red Recombination Operation

[0471] First, the pKD46 vector was transferred into the E. coli AT-031-02 strain. Then, a linear DNA fragment for targeting was prepared by electrotransformation, and positive clones were selected. Finally, the resistance genes were eliminated.

[0472] The obtained strain number: AT-032 (AT-031-02, AnagB promotor::Trc promoter).

[0473] (2) Deletion of the glmS Gene Endogenous Natural Promoter

[0474] The Glucosamine-6-phosphate synthase (glmS) gene promoter sequence was deleted. Glucosamine-6-phosphate synthase (GlmS), also known as L-glutamine-6-phosphate aminotransferase (L-glutamine; D-fructose-6-phosphate aminotransferase), catalyzes the amination of glucose-6-phosphate (Glc-6)-P) to D-glucosamine-6-phosphate (GlcN-6-P), but it has serious product inhibition problems. With the deletion of its promoter sequence, expression of the enzyme is lost, and GlcN-6-P product inhibition is eliminated.

[0475] First, the fKanrf fragment was amplified, and then the homology arm primer was designed to amplify the full-length linear DNA fragment of Red recombination targeting.

[0476] 1) Amplification of the kanamycin resistance gene with FLP recombinase recognition site (FRT site) on both sides: fKanrf

[0477] Primers design: forward primer (mfKanf-F) SEQ ID No:1, reverse primer (mfKanf-R) SEQ ID No:2.

[0478] Template: pPic9K.

[0479] PCR reaction conditions: first step: denaturation at 94 C. for 1 min; second step: incubation at 94 C. for 30 s, 55 C. for 30 s, 72 C. for 40 s, carrying out 30 cycles; third step: extension at 72 C. for 10 min.

[0480] fKanrf size: 1.28 kb. Its nucleotide sequence is SEQ ID No: 3.

[0481] The PCR product was separated by 1% agarose gel electrophoresis and purified to recover segment.

[0482] 2) Preparation of a linear DNA full-length PCR fragment for Red recombination targeting

[0483] Design of the homology arm primer: According to NCBI, found the NC_000913, Escherichia coli str. K-12 L-glutamine-6-phosphate fructose aminotransferase (GlmS) gene promoter sequence SEQ ID No: 35, designed homology arm primer for deletion of gimS gene promoter sequence: forward primer (Proglms KO-F) SEQ ID No: 36, reverse primer (Proglms KO-R) SEQ ID No: 37.

[0484] Template: fKanrfPCR fragment.

[0485] PCR reaction conditions: first step: denaturation at 94 C. for 1 min; second step: incubation at 94 C. for 30 s, at 55 C. for 30 s, at 72 C. for 40 s, carrying out 30 cycles; third step: extension at 72 C. for 10 min.

[0486] Amplification product: homology arm+fKanf+homology arm.

[0487] The PCR product was separated by agarose gel electrophoresis, purified and recovered to obtain 100 ng/l linear DNA full-length PCR fragment for Red recombination targeting.

[0488] 3) Red Recombination Operation

[0489] First, the pKD46 vector was transferred into E. coli AT-032 strain. Then, a linear DNA fragment for targeting was prepared by electrotransformation, and positive clones were selected. Finally, the resistance gene was eliminated.

[0490] The obtained strain number: AT-033 (AT-032, glmS promotor).

[0491] (3) Effect of Replacing nagB Promoter with a Higher Expression Level Promoter and Further Deleting of glmS Promoter on N-Acetyl-D-Glucosamine Production.

[0492] For the strains in which the pTrc-nanEM cassette was integrated, the nagB promoter was replaced with a promoter of a higher expression level, and the glmS promoter was further deleted in the recombinant strain. The strains were subjected to a shake flask fermentation test. The monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13150 mm), and shake-cultured at 30 C. at 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium). The initial OD.sub.600 was about 0.5, shake-cultured at 225 rpm at 37 C., and the fermentation cycle was 72 hours. At 24 hours and 48 hours, the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH. According to the sugar consumption of the fermentation broth, 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L. At the end of the fermentation, 1 ml of the fermentation broth was taken and centrifuged. The N-acetyl-D-glucosamine content was determined by HPLC.

[0493] The yield of shake flask fermentation is shown in Table 6. The results showed that N-acetyl-D-glucosamine yield of the recombinant strain that replaced the nagB promoter with the Trc promoter increased significantly, and after further deleting glmS promoter, the yield of N-acetyl-D-glucosamine greatly improved.

TABLE-US-00007 TABLE 6 Output from Shake-flask Fermentation by the Recombinant Strain with replacement of nagB promoter and further removal of glmS promoter Output of N-acetyl-D- Strains glucosamine (g/L) AT-031-02 (reference) 6.0 0.9 AT-032 (AT-031-02, nagB promotor::Trc 9.3 0.9 promoter) AT-033 (AT-032, glmS promotor) 12.0 1.1

[0494] Replacing the endogenous natural promoter of glmS gene with the Trc promoter, and further deleting the endogenous natural promoter of nagB gene, and the effect on N-acetyl-D-glucosamine production yield by Escherichia coli strains integrated with pTrc-NanEM gene cassette.

[0495] (1) Replacing the glmS Gene Endogenous Natural Promoter with the Trc Promoter

[0496] The L-glutamine-6-phosphate aminotransferase (L-glutamine: D-fructose-6-phosphate aminotransferase) gene promoter sequence was replaced with a Trc promoter sequence. L-Glutamine-6-phosphate fructose aminotransferase, also known as Glucosamine-6-phosphate synthase (GlmS); its promoter sequence was replaced with the Trc promoter sequence and led to overexpression of gimS, and accelerated GlmS catalyzing function, and achieved the goal of increasing D-glucosamine-6-phosphate (GlcN-6-P).

[0497] First, the Trc promoter sequence fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombination targeting.

[0498] 1) Amplification of Full-Length PCR Fragments of Linear DNA for Red Recombination Targeting

[0499] Homology arm primer design: according to glmS gene promoter sequence of SEQ ID No: 35, designed Trc promoter homologous arm primer: forward primer (ProglmspTrc-F) SEQ ID No: 38, reverse primer (ProglmspTrc-R), SEQ ID No: 39.

[0500] Template: Trc promoter PCR fragment and secondary amplified fKanrfPCR fragment were mixed at a ratio of 1:1.

[0501] PCR reaction conditions: first step: denaturation at 94 C. for 1 min; second step: incubation at 94 C. for 30 s, at 55 C. for 30 s, at 72 C. for 40 s, carrying out 30 cycles; third step: extension at 72 C. for 10 min.

[0502] Amplification product: homology arm+fKanrf+Trc promoter+homology arm.

[0503] The PCR product was separated by agarose gel electrophoresis, purified and recovered to obtain a 100 ng/l linear DNA full-length PCR fragment for Red recombination targeting.

[0504] 2) Red Recombination Operation

[0505] First, the pKD46 vector was transferred into the E. coli AT-031-02 strain. Then, a linear DNA fragment for targeting was prepared by electrotransformation, and positive clones were selected. Finally, the resistance gene was eliminated.

[0506] The obtained strain number: AT-034 (AT-031-02, glmS promotor::Trc promoter).

[0507] (2) Deletion of the NagB Gene Endogenous Natural Promoter

[0508] Deletion of the D-glucosamine-6-phosphate deaminase (NagB) gene promoter sequence in nag regulon (nagE-nagBACD) caused the loss of function of nagB, and eliminated NagB reverse catalysis, and reduced the production of GlcN-6-P to Glc-6-P.

[0509] First, the fKanrf fragment was amplified, and then the homology arm primer was designed to prepare a full-length linear DNA fragment for Red recombination targeting.

[0510] 1) Preparation of a Linear DNA Full-Length PCR Fragment for Red Recombination Targeting

[0511] Homology arm primer design: according to the nagB promoter and nagE gene sequence SEQ ID NO: 13, desgiend the homology arm primer for deletion of nagB promoter gene sequence: forward primer (NagBKO-F2) SEQ ID No: 40, reverse primer (NagBKO-R2) SEQ ID No: 41.

[0512] Template: fKanrfPCR Fragment

[0513] PCR reaction conditions: first step: denaturation at 94 C. for 1 min; second step: incubation at 94 C. for 30 s, at 55 C. for 30 s, at 72 C. for 40 s, carrying out 30 cycles; third step: extension at 72 C. for 10 min.

[0514] Amplification product: homology arm+fKanrf+homology arm.

[0515] The PCR product was separated by agarose gel electrophoresis, purified and recovered to obtain a 100 ng/l linear DNA full-length PCR fragment for Red recombination targeting.

[0516] 2) Red Recombination Operation

[0517] First, the pKD46 vector was transferred into E. coli AT-034 strain. Then, a linear DNA fragment for targeting was prepared by electrotransformation, and positive clones were selected. Finally, the resistance gene was eliminated.

[0518] The obtained strain number: AT-035 (AT-034, nagB promotor).

[0519] (3) The Effect of Replacing the glmS Promoter with a Promoter with a Higher Expression Level and Further Deleting the nagB Promoter on the Production of N-Acetyl-Glucosamine

[0520] For the strains in which the pTrc-nanEM cassette was integrated, the glmS promoter was replaced with a promoter of a higher expression level, and the recombinant strains in which the nagB promoter was further deleted were subjected to a shake flask fermentation test. The monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13150 mm), and shake-cultured at 30 C. at 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium). The initial OD.sub.600 was about 0.5, shake-cultured at 225 rpm at 37 C., and the fermentation cycle was 72 hours. At 24 hours and 48 hours, the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH. According to the sugar consumption of the fermentation broth, 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L. At the end of the fermentation, 1 ml of the fermentation broth was taken and centrifuged. The N-acetyl-D-glucosamine content was determined by HPLC method.

[0521] The yield of shake flask fermentation is shown in Table 7. The results showed that the recombinant strain that replaced the glmS promoter with the Trc promoter had no obvious effect on the increase of N-acetyl-D-glucosamine production, but there is a significant improvement in N-acetyl-D-glucosamine production when simultaneously deleting the nagB promoter.

TABLE-US-00008 TABLE 7 Output from Shake-flask Fermentation by the Recombinant Strain with replacement of glmS promoter and further removal of nagB promoter Output of N-acetyl-D- Strains glucosamine (g/L) AT-031-02 (Reference) 5.9 1.0 AT-034 (AT-031-02, glmS promotor::Trc 6.2 1.1 promoter) AT-035 (AT-034, nagB promotor) 9.9 1.0

Example 5

[0522] This example describes Escherichia coli strains integrated with pTrc-nanEM gene cassette, and overexpression the gene wecB of UDPN-Acetyl-D-Glucosamine-2-Epimerase, as well as its influence on the output of N-Acetyl-D-Glucosamine.

[0523] The gene wecB of UDPN-AcetylGlucosamine-2-Epimerase (WecB) was controlled by Trc promoter to transform strains, or the natural endogenous promoter of the wecB gene was replaced by Trc promoter for overexpression of the enzyme, to strengthen production of ManNAc (N-Acetyl-D-mannosamine) from UDP-GlcNAc (UDPN-Acetyl Glucosamine).

[0524] 1. Transformation of Escherichia Coli Strains Integrated with pTrc-NanEM Gene Cassette by wecB/pTrc99A

[0525] (1) Amplifying the wecB Gene of Escherichia coli and Inserting pTrc99A

[0526] According to NCBI, looked up the nucleotide sequence SEQ ID No: 42 of the wecB gene of Escherichia coli, and its amino acid sequence is SEQ ID No43.

[0527] Primer Design: Forward primer (TrcwecB-F) SEQ ID No: 44, and reverse primer (TrcwecB-R) SEQ ID No:45.

[0528] Template: AT-001 (Escherichia coli ATCC 27325) Genome.

[0529] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0530] Amplification Product Size: 1.13 kb.

[0531] The PCR product was separated by 1% agarose gel electrophoresis, and purified to recover the segment.

[0532] Digested enzymatically the obtained PCR amplification segment and carrier pTrc99A with Nco I and Hind III, separated by agarose gel electrophoresis, purified to recover wecB segment and pTrc99A segment, connected with T4 DNA ligase at 16 C. overnight, and identified to obtain wecB/pTrc99A plasmid.

[0533] (2) Transformation of Escherichia Coli Strains Integrated with pTrc-NanEM Gene Cassette by wecB/pTrc99A

[0534] (1) Preparation of Competence:

[0535] First, inoculated the bacterial suspension of AT-031-02 stored at 20 C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultured at 37 C. and 225 rpm for 2-3 h. Then added the culture solution to a 10-mL centrifuge tube, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allowed to stand at 4 C. for 12 h for natural sedimentation.

[0536] (2) Plasmid Transformation:

[0537] Took 250 L of naturally settled bacterial solution, added 5 L of wecB/pTrc99A plasmid, and cultured at 4 C. for 30 min. Then heated on a water bath at 42 C. for 1.5 min, added 0.7 mL of SOC medium, and shook at 30 C. for 2 h. Took 0.2 mL of the bacterial suspension, and smeared on a penicillin plate. Cultured overnight (for 12-16 h) at 30 C. Picked up monoclone, added 5 mL of LB broth medium and cultured, and plasmids were extracted for identification. Positive clones were stored for use.

[0538] No of the obtained strain: AT-036 (AT-031-02, wecB/pTrc99A).

[0539] 2. The Natural Endogenous Promoter of the wecB Gene of Escherichia coli Strains Integrated with pTrc-nanEM Cassette was Replaced with Trc Promoter

[0540] First, amplified Trc promoter sequence segment and fKanrf segment, and spliced them together. Then designed homologous primers, and amplified linear DNA full-length segment for Red recombination targeting.

[0541] (1) Preparation of Linear DNA Full-Length PCR Segment for Red Recombination Targeting

[0542] Design of Homologous Arm Primers: According to NCBI, looked up NC_000913 to obtain the nucleotide sequence SEQ ID No: 46 of the gene promoter of Escherichia coli UDPN-AcetylGlucosamine-2-Epimerase (wecB), designed homologous arm primers with the promoter replaced by Trc promoter: Forward primer (ProwecBpTrc-F) SEQ ID No: 47, and reverse primer (Pro wecBpTrc-R) SEQ ID No:48.

[0543] Template: Mixed Trc promoter PCR segment and fKanrf PCR segment from secondary amplification at a ratio of 1:1.

[0544] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0545] Amplification Product: Homologous Arm+fKanrf+Trc Promoter+Homologous Arm.

[0546] The PCR product was separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/L linear DNA full-length PCR segment for Red recombination targeting.

[0547] (2) Red Recombination Operation

[0548] First, pKD46 carrier was introduced into the AT-007-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting was electrotransformed, and positive clones were screened. Finally, the resistance gene was removed.

[0549] No of the obtained strain: AT-037 (AT-031-02, wecB promotor::Trc promoter).

[0550] 3. The Effect of Recombinant Strain Integrated with pTrc-nanEM Gene Cassette, Strains Transformed with wecB/pTrc99A Plasmid, and Strains Having wecB Promoter Replaced by Trc Promoter on the Output of N-Acetyl-D-Glucosamine.

[0551] Carrying out a shake-flask fermentation trial with the strain integrated with pTrc-nanEM gene cassette, and the recombinant strain produced for overexpression of wecB (including the strain with transformed wecB/pTrc99A and the strain with wecB promoter replaced by Trc promoter). Transferred the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculated into a test tube (13150 mm) containing 3 mL of the LB broth culture medium, and shake-cultured at 30 C. for 8 h. Then transferred the seed culture solution, inoculated 3% into a 250-mL shake-flask containing 50 mL of M9 culture solution. The initial OD.sub.600 was about 0.5; shake-cultured at 37 C. and 225 rpm. The fermentation cycle was 72 h. At 24 h and 48 h, adjusted the pH value to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, added 65% glucose solution in portions to maintain a glucose concentration of 20 g/L. After completion of fermentation, transferred 1 mL of the fermentation broth and centrifuged. Measured the content of N-Acetyl-D-Glucosamine by HPLC method.

[0552] See Table 8 for the output from shake-flask fermentation. The results show that: Compared with the reference strain AT-031-02, the output of N-Acetyl-D-Glucosamine increased significantly in recombinant strain with transformed wecB/pTrc99A, and increased more greatly in recombinant strain with wecB promoter replaced by Trc promoter.

TABLE-US-00009 TABLE 8 Output from Shake-flask Fermentation by Recombinant Strains with Transformed wecB/pTrc99A Plasmid or with wecB promoter replaced by Trc Promoter Output of N-Acetyl-D- Strains Glucosamine (g/L) AT-031-02 (Reference) 6.0 1.0 AT-036 (AT-031-02, wecB/pTrc99A) 10.3 1.1 AT-037 (AT-031-02, wecB promotor::Trc 13.2 1.1 promoter)

Example 6

[0553] This example describes influences of the Escherichia coli strains integrated with pTrc-nanEM gene cassette on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the glmS gene and nagB gene was replaced and/or deleted, transformed wecB/pTrc99A plasmid or replaced the natural endogenous promoter of the wecB gene by Trc promoter

[0554] 1. Escherichia coli strains integrated with pTrc-nanKM cassette were used to transform wecB/pTrc99A plasmid, of which the natural endogenous promoter of the nagB gene was replaced by Trc promoter, and meanwhile the natural endogenous promoter of the glmS gene was deleted

[0555] Preparation of competence: Transformed wecB/pTrc99A plasmid with CaCl.sub.2 into Escherichia coli strain AT-033 integrated with pTrc-NanEM gene cassette, of which the natural endogenous promoter of the nagB gene was replaced by Trc promoter and meanwhile the natural endogenous promoter of the glmS gene was deleted; monoclones were picked up and cultured, and plasmids were extracted to identify positive clones.

[0556] No of the obtained strain: AT-038 (AT-033, wecB/pTrc99A).

[0557] 2. In Escherichia coli strains integrated with pTrc-nanEM cassette, of which the natural endogenous promoter of the nagB gene was replaced by Trc promoter and meanwhile the natural endogenous promoter of the glmS gene was deleted, the natural endogenous promoter of the wecB gene was replaced by Trc promoter

[0558] First, pKD46 carrier was introduced into the AT-033 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting was electrotransformed, and positive clones were screened. Finally, the resistance gene was removed.

[0559] No of the obtained strain: AT-039(AT-033, A wecB promotor::Trc promoter).

[0560] 3. Escherichia coli strains integrated with pTrc-nanEM cassette were used to transform wecB/pTrc99A plasmid, of which the natural endogenous promoter of the glmS gene was replaced by Trc promoter, and meanwhile the natural endogenous promoter of the nagB gene was deleted

[0561] Preparation of competence: Transformed wecB/pTrc99A plasmid with CaCl.sub.2 into Escherichia coli strain AT-035 integrated with pTrc-nanEM gene cassette, of which the natural endogenous promoter of the glmS gene was replaced by Trc promoter and meanwhile the natural endogenous promoter of the nagB gene was deleted, monoclones were picked up and cultured, and plasmids were extracted to identify positive clones.

[0562] No of the obtained strain: AT-040 (AT-035, wecB/pTrc99A).

[0563] 4. In Escherichia coli strains integrated with pTrc-nanEM cassette, of which the natural endogenous promoter of the glmS gene was replaced by Trc promoter and meanwhile the natural endogenous promoter of the nagB gene was deleted, the natural endogenous promoter of the wecB gene was replaced by Trc promoter

[0564] First, pKD46 carrier was introduced into the AT-035 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting was electrotransformed, and positive clones were screened. Finally, the resistance gene was removed.

[0565] No of the obtained strain: AT-041 (AT-035, AwecB promotor::Trc promoter).

[0566] 5. Influences of the Escherichia coli strains integrated with pTrc-nanEM gene cassette on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the nagB gene and glmS gene was replaced and/or deleted to transform wecB/pTrc99A plasmid, and the natural endogenous promoter of the wecB gene was replaced by Trc promoter

[0567] Carrying out a shake-flask fermentation trial with the recombinant strains with different genotypes, obtained from the strains integrated with pTrc-nanEM gene cassette, of which the natural endogenous promoter of the glmS and nagB genes were replaced and/or deleted to transform wecB/pTrc99A plasmid, or the natural endogenous promoter of the wecB gene was replaced by Trc promoter. Transferred the monoclonal strain freshly cultiured in culture medium of the LB plate, inoculated into a test tube (13150 mm) containing 3 mL of the LB broth culture medium, and shake-cultured at 30 C. for 8 h. Then transferred the seed culture solution, inoculate 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD.sub.600 was about 0.5; shake-cultured at 37 C. and 225 rpm. The fermentation cycle was 72 h. At 24 h and 48 h, adjusted the pH value to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, added 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, took 1 mL of the fermentation broth and centrifuged. Measured the content of N-Acetyl-D-Glucosamine by HPLC method.

[0568] See Table 9 for the output from shake-flask fermentation. The results show that: Compared with the reference strains AT-033 or AT-035, the output of N-Acetyl-D-Glucosamine increased significantly by recombinant strain with transformed wecB/pTrc99A, and increased more greatly by recombinant strain with wecB promoter replaced by Trc promoter.

TABLE-US-00010 TABLE 9 Output from Shake-flask Fermentation by Recombinant Strains with Transformed wecB/pTrc99A Plasmid or with wecB promoter replaced by Trc Promoter Output of N-Acetyl-D- Strains Glucosamine (g/L) AT-033 (Reference) 12.1 1.1 AT-038 (AT-033, wecB/pTrc99A) 17.3 1.4 AT-039(AT-033, wecB promotor::Trc 21.6 1.8 promoter) AT-035 (Reference) 10.1 1.1 AT-040(AT-035, wecB/pTrc99A) 16.8 1.3 AT-041(AT-035, wecB promotor::Trc 20.6 1.5 promoter)

Example 7

[0569] This example describes a fermentation experiment for production of N-acetyl-D-glucosamine by a 10-L fermentation tank

[0570] A fermentation experiment was carried out for production of N-Acetyl-D-Glucosamine by a 10-L fermentation tank, using recombinant engineering strain AT-039 as production strain.

1. Seed Cultivation

[0571] (1) Cultivation of Primary Seed: Picked monoclonal strain freshly cultured in the LB plate medium, inoculated into 8 mL of LB broth medium, and shake-cultured at 37 C. and 225 rpm for 8 h. [0572] (2) Cultivation of Secondary Seed: Took 6 mL of the primary seed culture solution, inoculated into 1000-mL shake flask containing 200 mL of M9 culture solution, and shake-cultured at 37 C. and 225 rpm for 16 h, until OD.sub.600 value was 6.0-10, approximately the medium stage of log growth. [0573] (3) Preparation of the fermentation medium according to Table 10, where the microelement solution was prepared according to Table 11, and the complex vitamins solution was prepared according to Table 12.

TABLE-US-00011 TABLE 10 Fermentation Medium Ingredients Amount (/L) K.sub.2HPO.sub.4 1.30 g KH.sub.2PO.sub.4 1.00 g MgSO.sub.47H.sub.2O 0.10 g NH.sub.4Cl 0.02 g (NH.sub.4).sub.2SO.sub.4 0.20 g NaH.sub.2PO.sub.4 0.60 g Polyether Defoamer 10 mL Microelement Solution 4 ml Complex Vitamins 4 ml Solution Glucose 6.00 g

[0574] Note:

[0575] The microelement solution was sterilized separately and then added, and the vitamins solution was filtered and then added;

[0576] Glucose: Concentration of 65% (w/v); it was sterilized separately and added prior to inoculation. Amount to be added: 6.0 g/L;

[0577] The above solutions were combined, and then adjusted to pH 7.0 with 10M NH.sub.4OH;

[0578] The fermentation medium was a basal medium prior to addition of glucose; initial loading amount of the basal medium (initial volume accounting for the total capacity of the fermentation tank): 50%.

TABLE-US-00012 TABLE 11 Microelement Solution Amount used Ingredients (g/L) CaCl.sub.22H.sub.2O 10 FeCl.sub.36H.sub.2O 10 MnSO.sub.45H.sub.2O 2.5 AlCl.sub.36H.sub.2O 2.5 CoCl.sub.26H.sub.2O 1.75 ZnSO.sub.42H.sub.2O 0.5 NaMoO.sub.42H.sub.2O 0.5 CuSO.sub.45H.sub.2O 0.25 H.sub.3BO.sub.3 0.125 pH 3 to 4

TABLE-US-00013 TABLE 12 Complex Vitamins Solution Amount Ingredients (mg/L) Folic Acid 2 Vitamin B.sub.2 Riboflavin 100 Vitamin B.sub.1 Thiamine HCl 1500 Nicotinic Acid 500 Vitamin B.sub.6 Pyridoxine HCl 500 Calcium Pantothenate, Ca- 500 panthothenate Biotin 1 Vitamin B.sub.12 10

2. Inoculation

[0579] Inoculated the secondary seed solution to the fermentation tank at a ratio of 40 mL/L; inoculation size: 2.5-5% (v/v); the initial OD.sub.600 was 0.3-0.5.

3. Process Parameters

[0580] High-density fermentation was carried out by a 10-L self-controlled fermentation tank, data were collected by software equipped in the machine to realize online control by computer. The control parameters were: The air flow was 0.5-1 vvm.; dissolved oxygen is >=20%, to increase regulation of rotational speed and ventilation; temperature 37 C.; pH 7.0, the automatic flow was maintained stable by addition of saturated ammonia water. Supplemented glucose when glucose in the basal medium was consumed up, i.e. dissolved oxygen had risen again. Glucose was supplemented in a speed to control residual glucose concentration of no more than 0.45 g/L. The glucose-supplementing solution contained 65% (w/v) of glucose, was added with 2.5% Sodium Gluconate or 6% Ribose. The fermentation was stopped after 60-72 h. Total loading amount: 75%-80%.

4. Example (10-L Fermentation Tank)

[0581] (1) Strain No: AT-039. Batch No: 00123. [0582] (2) Concentration of Seed Solution: OD.sub.600 was 2.8. [0583] (3) Base Stock: 4 L. [0584] (4) Inoculation Size 200 mL. [0585] (5) Glucose supplementation speed: The residual glucose concentration was controlled of no more than 0.45 g/L. [0586] (6) Glucose-supplementing solution: The solution contains glucose in a concentration of 65% (w/v) and was added with 2.5% sodium gluconate. [0587] (7) Track Indicators: Measured OD.sub.600 and residual glucose content (residual glucose in the fermentation solution). [0588] (8) Product: N-Acetyl-D-Glucosamine. Potency: 72 h, 127.0 g/L.

Example 8

[0589] This example describes the treatment process after separation and purification of N-Acetyl-D-Glucosamine and D-Glucosamine Hydrochloride

[0590] 1. Refinement of N-Acetyl-D-Glucosamine [0591] (1) Deactivation: The fermentation solution was placed at 80 C. for 30 min. [0592] (2) Solid-liquid separation: Centrifuged at 4000-8000 rpm, discarded the bacterial residue and proteins, and transferred the fermentation solution. It may also be filtered via ceramic membrane. [0593] (3) Decoloration: Product: Water: Activated Charcoal=1: (1.5-3): (0.01-0.1); stirred for 0.5-5 h. [0594] (4) Desalt: Desalted by electroosmosis. Initial salt concentration of the fermentation loaded into the concentrated chamber tank: 0.01-0.05 mol/L. Flow rate of the dilute-chamber fermentation solution: 40-80 L/h; flow rate of the concentrated-chamber fermentation solution: 40-80 L/h; the voltage of single membrane pair was 0.5-1.4 V. It may also be desalted by anion or cation ion exchange resin. [0595] (5) Concentration: The fermentation solution desalted was heated at 50-80 C. under vacuum conditions (0.095 MPa) for 8-15 h until oversaturation, by approximately 4-6 folds. [0596] (6) Crystallization: The concentrated fermentation solution was cooled to 25-35 C. in water at 25 C., then cooled for 1-3 h to 0-10 C. in water at 0 C. Anhydrous alcohol was added (in an amount of approximately 5-20 times the product weight), and stirred at 700-1500 rpm for 15 min-1 h. [0597] (7) Washing: Added anhydrous alcohol (same amount as that of the product) and stirred for 10-100 rpm, 0.5-2 h. [0598] (8) Drying: 50-100 C., 3-10 h. Purity: 99.96%. The total yield was 91.5%.

[0599] 2. Refinement of D-Glucosamine Hydrochloride [0600] (1) Deactivation: The fermentation solution was placed at 80 C. for 30 min. [0601] (2) Solid-liquid separation: Centrifuged at 4000-8000 rpm, discarded the bacterial residue and proteins, and transferred the fermentation solution. It may also be filtered via ceramic membrane. [0602] (3) Decoloration: Product: Water: Activated Charcoal=1:(1.5-3): (0.01-0.1); stirred for 0.5-5 h. [0603] (4) Desalt: Desalt by electroosmosis. Initial salt concentration of the fermentation loaded into the concentrated chamber tank: 0.01-0.05 mol/L. Flow rate of the dilute-chamber fermentation solution: 40-80 L/h; flow rate of the concentrated-chamber fermentation solution: 40-80 L/h; the voltage of single membrane pair was 0.5-1.4 V. It may also be desalted by anion or cation ion exchange resin. [0604] (5) Concentration: The fermentation solution desalted was heated at 50-80 C. under vacuum conditions (0.095 MPa) for 8-15 h until oversaturation, by approximately 4-6 folds. [0605] (6) Hydrolysis: Introduced the concentrated fermentation solution to an enamel or glass container, added concentrated hydrochloric acid (37%) to a final concentration of 12%-16%, stirred thoroughly, and maintained at 70 C. for 90 min. Hydrochloric Acid may be used in a recycling way. [0606] (7) Crystallization: First cooled to 25-35 C. in water at 25 C., and then cooled to 4 C. in water at 0 C. for 1-3 h. [0607] (8) Washing: Added anhydrous alcohol (same amount as that of the product) and stirred for 10-100 rpm, 0.5-2 h. Centrifuged at 700-1500 rpm for 15-60 min to obtain Glucosamine Hydrochloride; the conversion rate is 89.7%. [0608] (9) Dissolution: Dissolve the washed product in water in a similar volume to that of the original fermentation solution. [0609] (10) Decoloration: Added activated charcoal (in an amount of 1%). Mixed for 30 min. Then centrifuged at 700-1500 rpm for 15-60 min. Or filtered to obtain a colorless filtrate. [0610] (11) Recrystallization: Evaporate at 50 C. and 55 cmHg vacuum until oversaturation. Added anhydrous alcohol (in an amount of approximately 5-20 times the product weight), and stirred at 700-1500 rpm for 15 min-1 h. [0611] (12) Washing: Add anhydrous alcohol (same amount as that of the product) and stir for 10-100 rpm, 0.5-2 h. Then centrifuged at 700-1500 rpm for 15-60 min. [0612] (13) Drying: 50-100 C., 3-10 h. Purity: 99.92%. The total yield is 84.3%.

Example 9

[0613] This example describes screening for a gene mutant of UDP-N-acetyl-D-glucosamine-2-epimerase (WecB), said gene UDP-N-acetyl-D-glucosamine-2-epimerase, WecB) with increased enzyme activities.

[0614] To further increase synthetic quantity of N-Acetyl-D-Glucosamine by the production strain, screen a gene mutant encoding the enzyme with increased enzyme activities. To achieve the purpose, amplifying the cloned gene by error-prone PCR technology, through DNA polymerase used for amplification, amplify said gene under conditions leading to high-frequency mismatch, so as to obtain a high-frequency mutation in PCR products.

[0615] The specific operating process is provided below:

[0616] 1. Amplification of the Gene wecB of UDP-N-Acetyl-D-Glucosamine-2-Epimerase in Escherichia coli by Error-Prone PCR

[0617] By means of Taq DNA polymerase without the property of 3-5 proofreading function, controlled the frequency of random mutation under high magnesium ion concentration (8 mmol/L) and different dNTP concentrations (the concentration of dATP and dGTP was 1.5 mmol/L; and the concentration of dTTP and dCTP was 3.0 mmol/L), introduced random mutations into the target gene, and constructed a mutation library; the template concentration A260 value was 1000 ng/mL, the enzyme concentration was 5 U/L, and the primer concentration was 100 M.

[0618] Error-prone PCR reaction system (50 L): 10PCR reaction buffer 5 l, dNTP (2.5 mM) 5 L, MgCl.sub.2 (2.5 mM) 5 L, Forward primer (TrcwecB-F, SEQ ID No44) 1 L, reverse primer (TrcwecB-R, SEQ ID No45) 1 L, DNA template (wecB/pUC57) 0.1 L, Taq DNA polymerase 0.5 L, and ddH.sub.2O 32.4 L.

[0619] PCR procedure: Pre-degeneration at 96 C. for 4 min; degeneration at 94 C. for 1 min, annealing at 56 C. for 1 min, extension at 75 C. for 2 min, and repeated for 45 cycles; finally extension at 75 C. for 15 min, recovered PCR product (product size: 1.13 kb) by gel recovery method; took 5 L of the product and carried out 1% agarose gel electrophoresis test; the product was store at 20 C. for use.

[0620] 2. Construction of the Gene Mutation Library of UDP-N-Acetyl-D-Glucosamine-2-Epimerase

[0621] Digested the above PCR product by two enzymes of restriction endonuclease Nco I and Hind III, carried out a ligation reaction with pTrc99A digested by Nco I and Hind III, then transformed Escherichia coli AT-005-02 with the mixture of the ligated products to obtain a large amount of cloned transformants, and constructed a mutation library of transformed strains.

[0622] 3. Screening for Mutants with High Enzyme Activities

[0623] Using the wild type WecB/pTrc99A (AT-005-02) as reference, picked up randomly 640 mutant clones from the mutation library of transformed strains, inoculated into 5 mL of LB medium containing 50 g/mL Ampicillin (Amp), shake-cultured at 37 C. and 150 rpm for 18 h, and then centrifuged at 10000 rpm for 5 mim and collect bacterial solution. Discarded the supernatant, then resuspended at 4 C. in 1 mL of PBS solution (pH 7.5, 10 mmol/L), carried out ultrasonicate at a voltage of 300 V for 10 min (ultrasonicate for 3 s and pause for 6 s), centrifuged, transferred the supernatant as crude extract of enzyme, and carried out a method for determination of enzyme activity.

[0624] 3. Determination of UDP-N-acetyl-D-glucosamine-2-epimerase activity: Based on the amount of UDP-N-acetyl-D-glucosamine transformed into N-acetyl-D-aminomannose; that is, using the reduced amount of UDP-N-acetyl-D-glucosamine as test marker. Definition of Enzyme Activity Unit: Under the enzymatic reaction conditions, the enzyme amount reduced, equivalent to 1 mol UDP-N-acetyl-D-glucosamine per minute, is defined as one enzyme activity unit (IU). The specific procedure is provided as follows: First, a 20 ml reaction system was used as an enzyme activity assay system containing 45 mmol/L phosphate buffer (pH 7.5), 10 mM MgCl.sub.2 and 100 nCi of UDPGlcNAc and 5 mg of crude enzyme solution. The enzyme was incubated for 30 min in a 37 C. water bath. The reaction was terminated by the addition of ethanol. The radioactive compound was separated by paper chromatography. The radioactivity was measured by a liquid scintillation counter. The solvent system used was n-propanol: 1 M sodium acetate, pH 5.0: water (7:1:2). The activity unit of UDP-N-acetylglucosamine-2-epimerase was calculated based on how much UDPGlcNAc was converted to ManNAc.

[0625] The results show that: The enzyme activity of the mutant strain with maximum activity was 653 IU/mL, and the enzyme activity of the reference control was 21 IU/mL. Transforming WecB by error-prone PCR can obtain a mutant strain with increased greatly enzyme activity. Picked up the mutant strain with maximum enzyme activity and extracted plasmids for sequencing. The results show that: The mutant gene sequence of UDP-N-acetylglucosamine-2-epimerase is shown as SEQ ID No49, and the corresponding amino acid sequence is shown as SEQ ID No50. Through gene sequence alignment with the wild type of UDP-N-acetylglucosamine-2-epimerase, 5 base point mutations occurred in total: 101G/C, 433C/G, 677G/T, 734T/G, and 1038T/C. There occurred 4 missense mutations of amino acids, of which the mutation points are: C34S (cysteine at position 34 is changed to serine), H145D (histidine at position 145 is changed to aspartic acid), C226F (cysteine at position 226 is changed to phenylalanine), and V245G (valine at position 245 becomes glycine). The mutant gene is named as wecBM.

[0626] 4. Integration of pTrc-wecBM Gene Cassette into the nagE Gene Site in the Chromosome of Escherichia Coli

[0627] The nagE gene site was used as integration site of pTrc-wecBM gene cassette into the chromosome. To achieve integration of pTrc-wecBM gene cassette into the chromosome of Escherichia coli, first amplified the wecBM segment containing Trc promoter, i.e. pTrc-wecBM, as well as the kanamycin resistance gene segment with FLP recognition site (FRT site) at its both ends: FRT-Kanr-FRT (fKanrf), and spliced them. Then designed homology arm primers for deletion of the nagE gene sequence, and using the spliced segment of pTrc-wecBM and fKanrf as template, amplified the linear DNA full-length segment for Red recombination targeting.

[0628] The specific operating process is provided below:

[0629] (1) PCR Amplification of pTrc-wecBM Segment

[0630] Template: wecBM/pTrc99A.

[0631] Primer Design: Forward primer (Treff-F) SEQ ID No20, and reverse primer (Treff-R) SEQ ID No21.

[0632] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0633] Product Size: 1.3 kb.

[0634] The PCR product was separated by 1% agarose gel electrophoresis, and purified to recover the segment.

[0635] (2) PCR Amplification of fKanrf Segment

[0636] Primer Design: Forward primer (mfKanf-F) SEQ ID No1, and reverse primer (mfKanf-R) SEQ ID No2.

[0637] Template: pPic9K.

[0638] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0639] fKanrf size: 1.28 kb. Its nucleotide sequence is SEQ ID No3.

[0640] The PCR product was separated by 1% agarose gel electrophoresis, and purified to recover the segment.

[0641] (3) Amplification of fKanrf Spliced with pTrc-wecBM

[0642] Primer Design: Forward primer (fKanf-F) SEQ ID No22, and reverse primer (fKanf-R) SEQ ID No23.

[0643] Template: fKanrf.

[0644] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0645] fKanrf size from secondary amplification: 1.3 kb.

[0646] The PCR product was separated by 1% agarose gel electrophoresis, and purified to recover the segment.

[0647] (4) Preparation of Linear DNA Full-Length PCR Segment for Red Recombination Targeting

[0648] Design of Homologous Arm Primers: Designed again homologous arm primers for deletion of the nagE gene sequence: Forward primer (nagEKO-F2) SEQ ID No24, and reverse primer (nagEKO-R2) SEQ ID No25.

[0649] Template: Mixed pTrc-wecBM PCR segment, and fKanrfPCR segment from secondary amplification at a ratio of 1:1.

[0650] PCR reaction conditions: Step 1: Degeneration at 94 C. for 1 min; Step 2: Incubation at 94 C. for 30 s, at 55 C. for 30 s, and at 72 C. for 40 s, and carrying out 30 cycles; Step 3: Extension at 72 C. for 10 min.

[0651] Amplification Product: Homologous Arm+pTrc-wecBM-fKanrf+Homologous Arm

[0652] The PCR product was separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/L linear DNA full-length PCR segment for Red recombination targeting.

[0653] (5) Red Recombination Operation

[0654] First, pKD46 carrier was introduced into the AT-004-02 strain of Escherichia coli Then, the prepared linear DNA segment for targeting was electrotransformed, and positive clones were screened. Finally, the resistance gene was removed.

[0655] 1) Transformation of pKD46 Plasmid

[0656] {circle around (1)} Preparation of Competence:

[0657] First, inoculated the bacterial suspension of Escherichia coli AT-004-02 stored at 20 C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultured at 37 C. and 225 rpm for 2-3 h. Then added the culture solution to a 10-mL centrifuge tube, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL 0.1M CaC.sub.2 on an ice bath for 5 min. Finally, centrifuged at 4000 g5 min, discarded the supernatant, and suspended with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allowed to stand at 4 C. for 12 h for natural sedimentation.

[0658] Plasmid Transformation: Took 250 L of naturally settled bacterial solution, added 5 L of pKD46 plasmid, and cultured at 4 C. for 30 min. Then heated on a water bath at 42 C. for 1.5 min, added 0.7 mL of SOC medium, and shook at 30 C. for 2 h. Took 0.2 mL of the bacterial suspension, and smeared on a penicillin plate. Cultured overnight (for 12-16 h) at 30 C. Picked up monoclone, added 5 mL of LB broth medium and cultured, and plasmid was extracted for identification. Stored the positive strain for use.

[0659] 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone

[0660] {circle around (1)} Preparation of Electrotransform Competence:

[0661] Inoculated the AT-004-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultured at 250 rpm, on the next day, inoculated at a ratio of 1% into LB medium containing Amp, and cultured at 30 C.; when OD.sub.600 reached about 0.2, added 0.2% L-Arabinose, and induced at 30 C. for 35 min until OD.sub.600 reached about 0.4. Cooled on an ice bath. Washed once with ultrapure water, washed twice with 10% glycerin, and finally resuspended with 10% glycerin; the amount of glycerin used was to produce a final concentration of the bacterial solution concentrated by 500-1000 folds.

[0662] Transformation by electric shock: Took out a 2-mm electrotransformation cup from 70% ethanol, washed twice with sterilized ultrapure water, and irradiated by UV lamp for 30 min. Precooled at 4 C. for 30 min. Took 90 L of finally resuspended cells to a precooled centrifuge tube, added 5 L (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (4), gently suction mixed with a gun, and maintained on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 20011, 25 F.

[0663] {circle around (3)} Resuscitate and Screen Positive Clones:

[0664] added 1 mL of LB broth medium, and cultured at 37 C. and 100 rpm for 1 h. Then smeared one kanamycin (Kan) plate with every 200 L, 5 plates in total. Smeared evenly and allowed to dry. Cultured at 30 C. for 24 h. Picked up clones grown under kanamycin resistance, and carried out PCR identification to screen positive clones.

[0665] No of the obtained strain: AT-043-01 (AT-004-02, nagE::pTrc-wecBM-fKanrf).

[0666] As described above, prepared the strain AT-042-01 (AT-004-02, nagE::pTrc-wecB-fKanrf).

[0667] (6) Removal of the Resistance Gene

[0668] Introduced pCP20 into the above kanamycin-resistant clones, cultured at 30 C. for 8 h, then increased to 42 C. and cultured overnight, and thermally induced to express FLP recombinase; the plasmids were lost gradually. Streaked the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, picked up grown monoclones and dotted on the kanamycin-resistant plate; those that did not grow were clones of which the kanamycin resistance gene had been removed by FLP recombination. Carried out PCR with identification primer to identify clones that had lost kanamycin resistance.

[0669] No of the obtained strain: AT-043-02 (AT-004-02, AnagE::pTrc-wecEM).

[0670] As described above, prepared the strain AT-042-02 (AT-004-02, nagE::pTrc-wecB).

[0671] 5. Influence of the Integration of pTrc-wecB and pTrc-wecBM Gene Cassettes on the Output of N-Acetyl-D-Glucosamine

[0672] Carrying out a shake-flask fermentation experiment with the recombinant strains AT-042-02 and AT-043-02, of which the nagE gene site in the chromosome was integrated with pTrc-wecB and pTrc-wecBM gene cassettes, and the reference strain. Transferred the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculated into a test tube (13150 mm) containing 3 mL of the LB broth culture medium, and shake-cultured at 30 C. for 8 h. Then transferred the seed culture solution, inoculated 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD.sub.600 was about 0.5; shake-cultured at 37 C. and 225 rpm. The fermentation cycle was 72 h. At 24 h and 48 h, adjusted the pH value to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, added 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transferred 1 mL of the fermentation broth and centrifuged. Measured the content of N-Acetyl-D-Glucosamine by HPLC method.

[0673] See Table 13 for the output from shake-flask fermentation. The results show that: The output by the reference strain AT-005-02 was very low and was not detected, while the output by the recombinant strain AT-043-02 integrated with pTrc-wecBM gene cassette increased significantly, and also increased significantly than that by the unmutant reference strain AT-042-02.

TABLE-US-00014 TABLE 13 Output from Shake-flask Fermentation by the Recombinant Strain integrated with pTrc-wecBM Gene Cassette Output of N-Acetyl-D- Strains Glucosamine (g/L) AT-005-02 (AT-004-02, nagE) (Reference) Not detected AT-042-02 (AT-004-02, nagE::pTrc-nanE) 7.1 0.8 AT-043-02 (AT-004-02, nagE::pTrc-nanEM) 10.9 0.9

[0674] The above results show that: The output of N-Acetyl-D-Glucosamine may be increased significantly by overexpression of UDP-N-acetyl-D-glucosamine-2-epimerase; moreover, the output of N-Acetyl-D-Glucosamine may also be greatly increased by the mutant screened by error-prone PCR technology, due to increased activities of the obtained mutant of the epimerase.

[0675] Although this Invention is described in detail by common explanations and specific implementation schemes, it may be revised or improved on top of this Invention; this will be apparent to those skilled in the art. Therefore, such revisions or improvements that are not deviated from the spirits of this Invention still fall within the scope of the claimed invention.