Method for producing N-Acetyl-D-Glucosamine and/or D-Glucosamine salt by microbial fermentation
11466300 · 2022-10-11
Inventors
Cpc classification
C12Y501/03009
CHEMISTRY; METALLURGY
C12N9/1205
CHEMISTRY; METALLURGY
C12N9/80
CHEMISTRY; METALLURGY
C12Y501/03014
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C12Y203/01157
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C12Y206/01016
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C12N9/78
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C12N9/1029
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C12N15/70
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C12P19/26
CHEMISTRY; METALLURGY
C12Y305/01025
CHEMISTRY; METALLURGY
International classification
C12P19/26
CHEMISTRY; METALLURGY
C12N9/12
CHEMISTRY; METALLURGY
C12N9/80
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C12N9/78
CHEMISTRY; METALLURGY
Abstract
This Invention discloses a method for production of N-Acetyl-D-Glucosamine and/or D-Glucosamine Salt by microbial fermentation. The method is intended to manufacture N-Acetyl-D-Glucosamine and/or D-Glucosamine Salt in higher efficiency and higher yield, by increasing the effects of N-Acetyl-D-Mannosamine Kinase.
Claims
1. A method for production of N-Acetyl-D-Glucosamine and/or D-Glucosamine by microbial fermentation, comprising: A) Cultivating of a microorganism in a fermentation medium, where the said microorganism contains at least one genetic modification that may increase the effects of N-Acetyl-D-Mannosamine Kinase in microorganism; and B) Collecting N-Acetyl-D-Glucosamine produced in the step A); wherein the said genetic modification contains substitutions at the following corresponding sites of SEQ ID NO: 17: substitution of glutamine at Site 36 by arginine, substitution of isoleucine at Site 103 by methionine, and substitution of arginine at Site 223 by serine.
2. The method based on claim 1, wherein the genetic modification that may increase the effects of N-Acetyl-D-Mannosamine Kinase in microorganism is selected from: a) increased effects of N-Acetyl-D-Mannosamine Kinase in microorganism; and/or b) overexpression of N-Acetyl-D-Mannosamine Kinase in microorganism.
3. The method based on claim 2, where the microorganism is transformed by molecules of at least one recombinant nucleic acid, containing one nucleotide sequence encoding N-Acetyl-D-Mannosamine Kinase; the nucleic acid sequence encoding N-Acetyl-D-Mannosamine Kinase contains at least one genetic modification increasing the effects of N-Acetyl-D-Mannosamine Kinase.
4. The method based on claim 3, the one nucleic acid sequence encoding N-Acetyl-D-Mannosamine Kinase is SEQ ID NO: 26.
5. The method based on claim 3, at least approximately 30% of the nucleic acid sequence of the said N-Acetyl-D-Mannosamine Kinase is the same as that of SEQ ID NO: 17, wherein the said N-Acetyl-D-Mannosamine Kinase (NanK) is active; and, the said N-Acetyl-D-Mannosamine Kinase has the amino acid sequence of SEQ ID NO: 17.
6. The method based on claim 2, wherein the microorganism contains at least one genetic modification of the natural endogenous promoter to a gene encoding N-Acetyl-D-Mannosamine Kinase; the natural endogenous promoter of the gene encoding N-Acetyl-D-Mannosamine Kinase is replaced by a promoter with higher level; the promoter with higher level is selected from HCE promoter, gap promoter, trc promoter, and T7 promoter; the promoter with higher level is trc promoter.
7. The method based on claim 1, wherein the said microorganism contains one or more of the following genetic modifications: (1) Contain at least one genetic modification that may increase the effects of N-Acetyl-D-Mannosamine-6-Phosphate Epimerase in microorganism; (2) Contain at least one genetic modification that may increase the effects of D-Glucosamine-6-Phosphate Deaminase in microorganism, or contain at least one genetic modification that may decrease the effects of Glucosamine-6-Phosphate Synthase; (3) Contain at least one genetic modification that may increase the effects of D-Glucosamine-6-Phosphate Synthase in microorganism, or contain at least one genetic modification that may decrease the effects of D-Glucosamine-6-Phosphate Deaminase; (4) Contain at least one genetic modification that may increase the effects of UDP-N-Acetyl-D-Glucosamine-2-Epimerase (WecB) in microorganism.
8. The method based on claim 7, wherein the genetic modification increasing the effects of UDP-N-Acetyl-D-Glucosamine-2-Epimerase in microorganism is selected from a) increasing the effects of UDP-N-Acetyl-D-Glucosamine-2-Epimerase in microorganism; and/or b) overexpression of UDP-N-Acetyl-D-Glucosamine-2-Epimerase in microorganism.
9. The method based on claim 8, wherein the microorganism is transformed by molecules of at least one recombinant nucleic acid, containing one nucleotide sequence encoding UDP-N-Acetyl-D-Glucosamine-2-Epimerase; the nucleic acid sequence encoding UDP-N-Acetyl-D-Glucosamine-2-Epimerase (WecB) is SEQ ID NO: 58.
10. The method based on claim 1, wherein the said microorganism contains one or more of the following genetic modifications: (1) Contain at least genetic modification that may decrease the effects of Mannose transporter EIIM, P/III.sup.man(ManXYZ) in microorganism; (2) Contain at least one genetic modification that may decrease the effects of N-Acetylneuraminate Lyase (NanA) in microorganism; (3) Contain at least one genetic modification that may decrease the effects of N-Acetyl-D-Glucosamine-6-Phosphate Deactylase (NagA) in microorganism; (4) Contain at least one genetic modification that may decrease the effects of N-Acetyl-D-Glucosamine Specific Enzyme II.sup.Nag(NagE) in microorganism; (5) Contain at least one genetic modification that may increase the effects of PhosphoGlucosamine Mutase (GlmM) in microorganism; (6) Contain at least one genetic modification that may increase the effects of bifunctional N-acetyl Glucosamine-1-Phosphate Uridyltransferase (GlmU) in microorganism.
11. The method based on claim 1, wherein the said cultivation step A) is carried at approximately 20° C.˜approximately 45° C.; the said cultivation step A) is carried out at approximately pH 4.5˜approximately pH 8.5; the said cultivation step A) uses feed-batch fermentation method.
12. The method based on claim 1, wherein the said microorganism is bacteria, yeasts, or fungi.
Description
DESCRIPTION FOR ATTACHED FIGURES
(1)
ACTUAL IMPLEMENTATION MODES
(2) Hereafter this Invention is further described detailedly in combination with the implementation examples. The following examples are just used as ones to clarify and explain this Invention, and cannot be explained as any limitation to the protection range of this Invention. The technology realized, based on the contents of this Invention, are covered in the protection range of this Invention.
(3) Unless otherwise specified, the raw materials and reagents used in the implementation examples are commercially available goods.
(4) The list of various modified microorganisms involved in and/or described by this Invention is provided below.
(5) TABLE-US-00001 Strain No. Genotype Description Remarks AT-001 ATCC 27325, F-IN(rrnD-rrnE)1 Parent strain of lambda-, a prototrophic derivative engineering strain of Escherichia coli K-12 bacterial, obtained 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-006-01 AT-004-02,
nagE::pTrc-nanK-fKanrf Example 2 AT-006-02 AT-004-02,
nagE::pTrc-nanK Example 2 AT-007-01 AT-004-02,
nagE::pTrc-nanKM-fKanrf Example 3 AT-007-02 AT-004-02,
nagE::pTrc-nanKM Example 3 AT-008 AT-007-02,
nanE/pTrc99A Example 4 AT-009 AT-007-02, nanE promotor::Trc Example 4 promoter AT-010 AT-007-02,
nagB promotor::Trc Example 5 promoter AT-011 AT-010,
glmS promotor Example 5 AT-012 AT-007-02,
glmS promotor::Trc Example 5 promoter AT-013 AT-012,
nagB promotor Example 5 AT-014 AT-011, nanE/pTrc99A Example 6 AT-015 AT-011,
nanE promotor::Trc promoter Example 6 AT-016 AT-013, nanE/pTrc99A Example 6 AT-017 AT-013,
nanE promotor::Trc promoter Example 6 AT-018 AT-007-02, wecB/pTrc99A Example 7 AT-019 AT-007-02,
wecB promotor::Trc Example 7 promoter AT-020 AT-009, wecB/pTrc99A Example 8 AT-021 AT-009,
wecB promotor::Trc promoter Example 8 AT-022 AT-011, wecB/pTrc99A Example 9 AT-023 AT-011,
wecB promotor::Trc promoter Example 9 AT-024 AT-013, wecB/pTrc99A Example 9 AT-025 AT-013,
wecB promotor::Trc promoter Example 9 AT-026 AT-015, wecB/pTrc99A Example 10 AT-027 AT-015,
wecB promotor::Trc promoter Example 10 AT-028 AT-017, wecB/pTrc99A Example 10 AT-029 AT-017,
wecB promotor::Trc promoter Example 10 AT-030-01 AT-004-02,
nagE::pTrc-nanE-fKanrf Example 13 AT-030-02 AT-004-02,
nagE::pTrc-nanE Example 13 AT-031-01 AT-004-02,
nagE:: pTrc-nanEM-fKanrf Example 13 AT-031-02 AT-004-02,
nagE::pTrc-nanEM Example 13 AT-042-01 AT-004-02,
nagE::pTrc-wecB-f Kanrf Example 14 AT-042-02 AT-004-02,
nagE::pTrc-wecB Example 14 AT-043-01 AT-004-02,
nagE::pTrc-wecBM-fKanrf Example 14 AT-043-02 AT-004-02,
nagE::pTrc-wecBM Example 14
EXAMPLE 1
(6) This implementation describes construction of a mutant strain of Escherichia coli, for which the relevant metabolic pathways for intake of N-Acetyl-D-Glucosamine is blocked and degradation of beneficial intermediate products are blocked.
(7) The said parent strain of the production strain is AT-001 (Escherichia coli ATCC 27325), belonging to K-12 derivative strain of Escherichia coli, and is obtained from American Type Culture Collection.
(8) Blockage of intake of N-Acetyl-D-Glucosamine into the strain, and of degradation of intermediate products may reduce consumption during metabolism, and increase accumulation of the target product (N-Acetyl-D-Glucosamine).
(9) To construct such mutant host strain, manXYZ, nanA, nagA, and nagE gene sequences in its chromosomal genomes may be deleted completely or partially, to deactivate its function, and thus result in accumulation of N-Acetyl-D-Glucosamine.
(10) Such deletion of gene sequence in the chromosome may be accomplished by Red recombinant technology. Red recombination is an homologous recombination technology, based on Red operon in λ phage, and mediated by RecE/RecT system in Rac phage. By the technology, it may carry out simply and rapidly multiple modifications such as insertion, knockout, and mutation to any large DNA molecules. Simply speaking, for Red recombination technology, pKD46 plasmids with the recombinase-expressing gene are introduced into thalli, then the prepared linear DNA segment for targeting is electrotransformed, positive clones are screened, and finally the resistance gene in the strain is removed.
(11) The specific operational process is described hereafter:
(12) 1. Deletion of manXYZ Gene Sequence
(13) Mannose transporter EIIM, P/III.sup.man (mannose transporter EIIM, P/III.sup.man, ManXYZ) may be used as a second transporter of N-Acetyl-D-Glucosamine; it may transport hexose such as N-Acetyl-D-Glucosamine into cells, and thus the target product excreted out of cells and accumulated is transported into cells for degradation. Deletion of manXYZ gene sequence may block transportation of extracellular N-Acetyl-D-Glucosamine into cells for degradation.
(14) (1) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(15) 1) fKanrf Segment Amplified by PCR
(16) fKanrf, i.e. FRT-Kanr-FRT segment means a FRT site base sequence for specific recognition of FLP recombinase, mounted at both ends of kalamycin resistance gene (Kanr).
(17) Primer Design: Forward primer (mfKanf-F) SEQ ID No.1, and reverse primer (mfKanf-R) SEQ ID No.2.
(18) Template: pPic9K.
(19) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(20) fKanrf size: 1.28 kb. Its nucleotide sequence is SEQ ID No.3.
(21) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(22) 2) PCR Amplification of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(23) Design of Homologous Arm Primer: According to the manXYZ sequence SEQ ID No.4, it is designed to delete the homologous arm forward primer (manXYZKO-F) SEQ ID No.5 and reverse primer (manXYZKO-R) SEQ ID No.6 of the manXYZ sequence.
(24) Template: Amplification of fKanrf PCR Segment.
(25) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(26) Amplification Product: Homologous Arm+fKanrf+Homologous Arm.
(27) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(28) (2) Red Recombination Operation
(29) First, pKD46 carrier is introduced into the AT-001 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(30) 1) Transformation of pKD46 Plasmid
(31) pKD46 carrier is a plasmid with Red recombinase-expressing gene, which is 3-gene segment expressing Exo, Bet, and Gam; the 3 genes are placed below the arabinose promoter, and may express in a large amount by induction of L-arabinose. It is necessary to transform pKD46 plasmid into Escherichia coli, to achieve purpose to modify the target gene in the chromosome by Red recombination.
(32) {circle around (1)} Preparation of Competence: First, inoculate the bacterial suspension of Escherichia coli ATCC 27325 stored at −20° C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultivate at 37° C. and 225 rpm for 2-3 h. then add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation. Here, preparation of 0.1M CaCl.sub.2: Prepare a 1M CaCl.sub.2 solution with anhydrous CaCl.sub.2, autoclave at a steam pressure of 15 lbf/in2 for 20 min, and dispense 1.5 mL and store at −20° C.; for use, allow to thaw, and then dilute in a ratio of 1:10 to produce a 0.1M CaCl.sub.2 solution.
(33) {circle around (2)} Plasmid Transformation: Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of pKD46 plasmid, and cultivate at −4° C. for 30 min. Then heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h. Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate. Cultivate overnight (for 12-16 h) at 30° C. Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification. Store the positive strain for use.
(34) 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone
(35) {circle around (1)} Preparation of electrotransformed competence: Inoculate the AT-001 strain of Escherichia coli ATCC 27325, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultivate overnight at 250 rpm; On the next day, inoculate in a ratio of 1% into LB medium containing Amp, and cultivate at 30° C.; when OD.sub.600 reaches approximately 0.2, add 0.2% L-Arabinose, and induce at 30° C. for 35 min until OD.sub.600 reaches approximately 0.4. Cool on an ice bath. Wash once with ultrapure water, wash twice with 10% glycerin, and finally resuspend with 10% glycerin; the amount of glycerin used is to produce a final concentration of the thalli concentrated by 500-1000 folds.
(36) {circle around (2)} Transformation by electric shock: Take out a 2-mm electrotransformation cup from 70% ethanol, wash with sterilized ultrapure water, and irradiate by UV lamp for 30 min. Precool at 4° C. for 30 min. Transfer 90 μL of finally resuspended cells to a precooled centrifuge tube, add 5 μL (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (1), suction gently by a gun, and maintain on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 200Ω, 25 μF.
(37) {circle around (3)} Resuscitate and screen positive clones: add 1 mL of LB broth medium, and cultivate at 37° C. and 100 rpm for 1 h. Then smear one kanamycin (Kan) plate with every 200 μL, 5 plates in total. Smear evenly and allow to air-dry. Cultivate at 30° C. for 24 h. Pick up clones grown under kanamycin resistance, and carry out PCR identification to screen positive clones.
(38) No. of the obtained strain: AT-002-01 (AT-001, manXYZ::fKanrf).
(39) (3) Removal of the Resistance Gene
(40) 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, which may identify specifically FRT sites. The sequence between FRT sites may be deleted by recombination, to retain only one FRT site.
(41) Introduce pCP20 into the above kanamycin-resistant clones, cultivate at 30° C. for 8 h, then increase to 42° C. and cultivate overnight, and thermally induce to express FLP recombinase; the plasmids are lost gradually. Streak the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, pick up grown monoclones and dot on the kanamycin-resistant plate; those that cannot grown are clones of which the kanamycin resistance gene has been removed by FLP recombination. Carry out PCR with identification primer to identify clones losing kanamycin resistance.
(42) No. of the obtained strain: AT-002-02 (AT-001, manXYZ).
(43) 2. Deletion of nanA Gene Sequence
(44) N-Acetylneuraminate Lyase (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).
(45) (1) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(46) Design of Homologous Arm Primer: According to the nanA sequence SEQ ID No.7 in the former segment of nanE-nanK, the homologous arm primers for deletion of nanA sequence are designed: Forward primer (nanAKO-F) SEQ ID No.8 and reverse primer (nanAKO-R) SEQ ID No.9.
(47) Template: Amplification of fKanrf PCR Segment.
(48) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(49) Amplification Product: Homologous Arm+fKanrf+Homologous Arm.
(50) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(51) (2) Red Recombination Operation
(52) First, pKD46 carrier is introduced into the AT-002-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(53) 1) Transformation of pKD46 Plasmid
(54) {circle around (1)} Preparation of Competence: First, inoculate 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-cultivate at 37° C. and 225 rpm for 2-3 h. then add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation.
(55) {circle around (2)} Plasmid Transformation: Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of pKD46 plasmid, and cultivate at −4° C. for 30 min. Then heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h. Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate. Cultivate overnight (for 12-16 h) at 30° C. Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification. Store the positive strain for use.
(56) 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone
(57) {circle around (1)} Preparation of Electrotransformed Competence: Inoculate the AT-002-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultivate at 250 rpm. On the next day, inoculate in a ratio of 1% into LB medium containing Amp, and cultivate at 30° C.; when OD.sub.600 reaches approximately 0.2, add 0.2% L-Arabinose, and induce at 30° C. for 35 min until OD.sub.600 reaches approximately 0.4. Cool on an ice bath. Wash once with ultrapure water, wash twice with 10% glycerin, and finally resuspend with 10% glycerin; the amount of glycerin used is to produce a final concentration of the thalli concentrated by 500-1000 folds.
(58) {circle around (2)} Transformation by electric shock: Take out a 2-mm electrotransformation cup from 70% ethanol, wash with sterilized ultrapure water, and irradiate by UV lamp for 30 min. Precool at 4° C. for 30 min. Transfer 90 μL of finally resuspended cells to a precooled centrifuge tube, add 5 μL (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (1), suction gently by a gun, and maintain on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 200Ω, 25 μF.
(59) {circle around (3)} Resuscitate and screen positive clones: add 1 mL of LB broth medium, and cultivate at 37° C. and 100 rpm for 1 h. Then smear one kanamycin (Kan) plate with every 200 μL, 5 plates in total. Smear evenly and allow to air-dry. Cultivate at 30° C. for 24 h. Pick up clones grown under kanamycin resistance, and carry out PCR identification to screen positive clones.
(60) No. of the obtained strain: AT-003-01 (AT-002-02, nanA::fKanrf).
(61) (3) Removal of the Resistance Gene
(62) Introduce pCP20 into the above kanamycin-resistant clones, cultivate at 30° C. for 8 h, then increase to 42° C. and cultivate overnight, and thermally induce to express FLP recombinase; the plasmids are lost gradually. Streak the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, pick up grown monoclones and dot on the kanamycin-resistant plate; those that cannot grown are clones of which the kanamycin resistance gene has been removed by FLP recombination. Carry out PCR with identification primer to identify clones losing kanamycin resistance.
(63) No. of the obtained strain: AT-003-02 (AT-002-02, nanA).
(64) 3. Deletion of nagA Gene Sequence
(65) 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).
(66) (1) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(67) Design of Homologous Arm Primers: From NCBI, look up NC_000913, nagA sequence SEQ ID No.10 for N-Acetyl-D-Glucosamine-6-Phosphate Deacetylase, Escherichia coli str.K-12, design 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.
(68) Template: Amplification of fKanrf PCR Segment.
(69) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(70) Amplification Product: Homologous Arm+fKanf+Homologous Arm.
(71) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(72) (2) Red Recombination Operation
(73) First, pKD46 carrier is introduced into the AT-003-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(74) 1) Transformation of pKD46 Plasmid
(75) {circle around (1)} Preparation of Competence: First, inoculate 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-cultivate at 37° C. and 225 rpm for 2-3 h. then add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation.
(76) {circle around (2)} Plasmid Transformation: Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of pKD46 plasmid, and cultivate at −4° C. for 30 min. Then heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h. Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate. Cultivate overnight (for 12-16 h) at 30° C. Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification. Store the positive strain for use.
(77) 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone
(78) {circle around (1)} Preparation of Electrotransformed Competence: Inoculate the AT-003-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultivate at 250 rpm, on the next day, inoculate in a ratio of 1% into LB medium containing Amp, and cultivate at 30° C.; when OD.sub.600 reaches approximately 0.2, add 0.2% L-Arabinose, and induce at 30° C. for 35 min until OD.sub.600 reaches approximately 0.4. Cool on an ice bath. Wash once with ultrapure water, wash twice with 10% glycerin, and finally resuspend with 10% glycerin; the amount of glycerin used is to produce a final concentration of the thalli concentrated by 500-1000 folds.
(79) {circle around (2)} Transformation by electric shock: Take out a 2-mm electrotransformation cup from 70% ethanol, wash with sterilized ultrapure water, and irradiate by UV lamp for 30 min. Precool at 4° C. for 30 min. Transfer 90 μL of finally resuspended cells to a precooled centrifuge tube, add 5 μL (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (1), suction gently by a gun, and maintain on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 200Ω, 25 μL.
(80) {circle around (3)} Resuscitate and screen positive clones: add 1 mL of LB broth medium, and cultivate at 37° C. and 100 rpm for 1 h. Then smear one kanamycin (Kan) plate with every 200 μL, 5 plates in total. Smear evenly and allow to air-dry. Cultivate at 30° C. for 24 h. Pick up clones grown under kanamycin resistance, and carry out PCR identification to screen positive clones.
(81) No. of the obtained strain: AT-004-01 (AT-003-02, nagA::fKanrf).
(82) (3) Removal of the Resistance Gene
(83) Introduce pCP20 into the above kanamycin-resistant clones, cultivate at 30° C. for 8 h, then increase to 42° C. and cultivate overnight, and thermally induce to express FLP recombinase; the plasmids are lost gradually. Streak the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, pick up grown monoclones and dot on the kanamycin-resistant plate; those that cannot grown are clones of which the kanamycin resistance gene has been removed by FLP recombination. Carry out PCR with identification primer to identify clones losing kanamycin resistance.
(84) No. of the obtained strain: AT-004-02 (AT-003-02, nagA).
(85) 4. Deletion of nagE Gene Sequence
(86) 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.
(87) (1) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(88) Design of Homologous Arm Primers: From NCBI, look up NC_000913, NagB promoter and nagA gene sequence SEQ ID No.13, Escherichia coli str.K-12, design 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.
(89) Template: Amplification of fKanrf PCR Segment.
(90) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(91) Amplification Product: Homologous Arm+fKanrf+Homologous Arm.
(92) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(93) (2) Red Recombination Operation
(94) First, pKD46 carrier is introduced into the AT-004-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(95) 1) Transformation of pKD46 Plasmid
(96) {circle around (1)} Preparation of Competence: First, inoculate 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-cultivate at 37° C. and 225 rpm for 2-3 h. then add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation.
(97) {circle around (2)} Plasmid Transformation: Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of pKD46 plasmid, and cultivate at −4° C. for 30 min. Then heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h. Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate. Cultivate overnight (for 12-16 h) at 30° C. Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification. Store the positive strain for use.
(98) 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone
(99) {circle around (1)} Preparation of Electrotransformed Competence: Inoculate the AT-004-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultivate at 250 rpm. On the next day, inoculate in a ratio of 1% into LB medium containing Amp, and cultivate at 30° C.; when OD.sub.600 reaches approximately 0.2, add 0.2% L-Arabinose, and induce at 30° C. for 35 min until OD.sub.600 reaches approximately 0.4. Cool on an ice bath. Wash once with ultrapure water, wash twice with 10% glycerin, and finally resuspend with 10% glycerin; the amount of glycerin used is to produce a final concentration of the thalli concentrated by 500-1000 folds.
(100) {circle around (2)} Transformation by electric shock: Take out a 2-mm electrotransformation cup from 70% ethanol, wash with sterilized ultrapure water, and irradiate by UV lamp for 30 min. Precool at 4° C. for 30 min. Transfer 90 μL of finally resuspended cells to a precooled centrifuge tube, add 5 μL (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (1), suction gently by a gun, and maintain on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 200Ω, 25 μF.
(101) {circle around (3)} Resuscitate and screen positive clones: add 1 mL of LB broth medium, and cultivate at 37° C. and 100 rpm for 1 h. Then smear one kanamycin (Kan) plate with every 200 μL, 5 plates in total. Smear evenly and allow to air-dry. Cultivate at 30° C. for 24 h. Pick up clones grown under kanamycin resistance, and carry out PCR identification to screen positive clones.
(102) No. of the obtained strain: AT-005-01 (AT-004-02, nagE::fKanrf).
(103) (3) Removal of the Resistance Gene
(104) Introduce pCP20 into the above kanamycin-resistant clones, cultivate at 30° C. for 8 h, then increase to 42° C. and cultivate overnight, and thermally induce to express FLP recombinase; the plasmids are lost gradually. Streak the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, pick up grown monoclones and dot on the kanamycin-resistant plate; those that cannot grown are clones of which the kanamycin resistance gene has been removed by FLP recombination. Carry out PCR with identification primer to identify clones losing kanamycin resistance.
(105) No. of the obtained strain: AT-005-02 (AT-004-02, nagE).
EXAMPLE 2
(106) This example describes gene nanK cloning of N-Acetyl-D-Mannosamine Kinase (NanK), and transformed nanK/pTrc99A plasmids in Escherichia coli, as well as integration of ptrc-nanK gene cassette into the chromosome of Escherichia coli.
(107) 1. nanK Gene Cloning, Transformation of nanK/pTrc99A Plasmid in Escherichia coli, and its Influence on Output of N-Acetyl-D-Glucosamine
(108) Amplification of the gene nanK of Escherichia coli NanK (N-acetylmannosamine kinase, N-Acetyl-D-Mannosamine Kinase), that is controlled by Trc promoter to transform the strain for overexpression of the enzyme, may strengthen phosphorylation of ManNAc (N-Acetyl-D-mannosamine, N-Acetyl-D-Mannosamine or N-Acetyl-D-Mannosamine) into ManNAc-6-P (N-Acetyl-D-mannosamine-6-P, N-Acetyl-D-Mannosamine-6-Phosphate).
(109) 1) nanK Gene Cloning of Escherichia coli
(110) From NCBI, look up U00096, to obtain the nucleotide sequence SEQ ID No.16 of the nanK gene of Escherichia coli, which amino acid sequence is SEQ ID No.17.
(111) Primer Design: Forward primer (nanK-F) SEQ ID No.18 and reverse primer (nanK-R) SEQ ID No.19.
(112) Template: Escherichia coli AT-001.
(113) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(114) Amplification Product Size: 0.9 kb.
(115) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(116) Connect the obtained PCR amplification segment with pUC57-T carrier, and carry out sequencing for identification, to obtain nanK/pUC57.
(117) 2) Construction and Transformation of Plasmid in which nanK Gene is Controlled by Trc Promoter
(118) {circle around (1)} Plasmid Construction: Amplify plasmid nanK/pUC57, digest enzymatically nanK/pUC57 and carrier pTrc99A with Nco I and Hind III, separate by agarose gel electrophoresis, purify to recover nanK segment and pTrc99A segment, connect overnight with T4 DNA ligase at 16° C., and carry out identification to obtain nanK/pTrc99A plasmid.
(119) {circle around (2)} Preparation of Competence: First, inoculate 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-cultivate at 37° C. and 225 rpm for 2-3 h. then add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation.
(120) {circle around (3)} Plasmid Transformation: Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of nanK/pTrc99A plasmid, and cultivate at −4° C. for 30 min. Then heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h. Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate. Cultivate overnight (for 12-16 h) at 30° C. Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification. Store the positive strain for use. Obtain Recombinant Strain nanK/pTrc99A (AT-005-02)
(121) 3) Influence of nanK/pTrc99A Plasmid Transformation on the Output of N-Acetyl-D-Glucosamine
(122) Carry out shake-flask fermentation trial with the recombinant strain and nanK/pTrc99A (AT-005-02) reference strain. Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate 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 transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD.sub.600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(123) {circle around (1)} HPLC Method to Measure the Content of N-Acetyl-D-Glucosamine
(124) Buffer: Add 3.5 g of dipotassium hydrogen phosphate to 1-L volumetric flask, add water to dissolve, add 0.25 mL of 0.25 mL of ammonia water, then dilute with water and mix well, adjust to pH 7.5, and add water to volume.
(125) Mobile Phase: Acetonitrile:Buffer (75:25).
(126) Diluent: Acetonitrile and water (50:50).
(127) Standard Solution: Dissolve 1.0 mg/mL USP N-Acetyl-D-Glucosamine Reference Substance (RS) in the diluent.
(128) Sample Solution: Dissolve 1.0 mg/mL N-Acetyl-D-Glucosamine sample in the diluent.
(129) Liquid Phase Conditions:
(130) Model: LC
(131) Detector: UV 195 nm
(132) Chromatographic Column: 4.6-mm×15-cm; 3-μm packing L8
(133) Flow Rate: 1.5 mL/min
(134) Column Temperature: 35° C.
(135) Injection Volume: 10 μL
(136) {circle around (2)} Preparation of M9 Culture Solution
(137) First prepare 5×M9 culture medium: To approximately 800 mL of double distilled water (ddH.sub.2O), add 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, add water to 1000 mL. Sterilize at 121° C. for 30 min. Then prepare 1M MgSO.sub.4, 1M CaCl.sub.2, and 20% glucose, respectively, and sterilize them separately. Then prepare M9 culture solution according to Table 1, while 1000× microelement solution is prepared according to Table 2.
(138) TABLE-US-00002 TABLE 1 Components of M9 Culture Solution Ingredients Amount used (mL/L) 5xM9 200 1M MgSO.sub.4 2 1M CaCl.sub.2 0.1 20% Glucose 20 1000x 1 Microelement Solution ddH.sub.2O to 1000 pH 6.9
(139) TABLE-US-00003 TABLE 2 Components of 1000x Microelement Solution Ingredients Amount used (g/L) CoCl.sub.2•6H.sub.2O 0.01 CuSO.sub.4•5H.sub.2O 0.01 MnSO.sub.4•H.sub.2O 0.033 FeSO.sub.4•7H.sub.2O 0.50 ZnSO.sub.4•7H.sub.2O 0.38 H.sub.3BO.sub.3 0.01 NaMoO.sub.4•2H.sub.2O 0.01 pH 3
(140) {circle around (3)} Influence of nanK/pTrc99A Plasmid Transformation on the Output of N-Acetyl-D-Glucosamine from Shake-Flask Fermentation
(141) See Table 3 for the output from shake-flask fermentation. The results show that: The output by the reference strain AT-005-02 is very low and is not detected, while the output from overexpression by nanK gene of nanK/pTrc99A (AT-005-02), controlled by Trc promoter, is increased obviously.
(142) TABLE-US-00004 TABLE 3 Output from Shake-flask Fermentation by the Recombinant nanK/pTrc99A (AT-005-02) Output of species N-Acetyl-D-Glucosamine (g/L) AT-005-02 (AT-004-02, nagE) Not detected (Reference) NanK/pTrc99A (AT-005-02) 2.9 ± 0.4
(143) 2. Integration of pTrc-nanK Gene Cassette into the Chromosome of Escherichia coli
(144) Using the nagE gene site as integration site of pTrc-nanK gene cassette into the chromosome. To achieve integration of pTrc-nanK gene cassette into the chromosome of Escherichia coli, first amplify the nanK segment pTrc-nanK of Trc promoter, as well as the kanamycin resistance gene segment with FLP recognition site (FRT site) at its both ends: FRT-Kanr-FRT (fKanrf), and splice them. Then design homologous primers for deletion of the nagE gene sequence, and using the spliced segment of pTrc-nanK and fKanrf as template, amplify the linear DNA full-length segment for targeting of Red recombination.
(145) The specific operating process is provided below:
(146) (1) PCR Amplification of pTrc-nanK Segment
(147) Template: nanK/pTrc99A.
(148) Primer Design: Forward primer (Trcff-F) SEQ ID No.20, and reverse primer (Trcff-R) SEQ ID No.21.
(149) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(150) Produce Size: 1.05 kb.
(151) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(152) (2) fKanrf Segment Amplified by PCR
(153) Primer Design: Forward primer (mfKanf-F) SEQ ID No.1, and reverse primer (mfKanf-R) SEQ ID No.2.
(154) Template: pPic9K.
(155) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(156) fKanrf size: 1.28kb. Its nucleotide sequence is SEQ ID No.3.
(157) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(158) (3) Amplification of fKanrf Spliced with pTrc-nanK
(159) Primer Design: Forward primer (fKanf-F) SEQ ID No.22, and reverse primer (fKanf-R) SEQ ID No.23.
(160) Template: fKanrf.
(161) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(162) fKanrf size from secondary amplification: 1.3 kb.
(163) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(164) (4) Preparation of Linear DNA Full-length PCR Segment for Targeting of Red Recombination
(165) 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.
(166) Template: Mix pTrc-nanK PCR segment, and fKanrf PCR segment from secondary amplification in the ratio of 1:1.
(167) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(168) Amplification Product: Homologous Arm+pTrc-nanK-fKanrf+Homologous Arm
(169) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(170) (5) Red Recombination Operation
(171) First, pKD46 carrier is introduced into the AT-004-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(172) 1) Transformation of pKD46 Plasmid
(173) {circle around (1)} Preparation of Competence: First, inoculate 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-cultivate at 37° C. and 225 rpm for 2-3 h. then add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation.
(174) {circle around (2)} Plasmid Transformation: Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of pKD46 plasmid, and cultivate at −4° C. for 30 min. Then heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h. Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate. Cultivate overnight (for 12-16 h) at 30° C. Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification. Store the positive strain for use.
(175) 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone
(176) {circle around (1)} Cool on an ice bath. Wash once with ultrapure water, wash twice with 10% glycerin, and finally resuspend with 10% glycerin; the amount of glycerin used is to produce a final concentration of the thalli concentrated by 500-1000 folds.
(177) {circle around (2)} Transformation by electric shock: Take out a 2-mm electrotransformation cup from 70% ethanol, wash with sterilized ultrapure water, and irradiate by UV lamp for 30 min. Precool at 4° C. for 30 min. Transfer 90 μL of finally resuspended cells to a precooled centrifuge tube, add 5 μL (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (4), suction gently by a gun, and maintain on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 200Ω, 25 μF.
(178) {circle around (3)} Resuscitate and screen positive clones: add 1 mL of LB broth medium, and cultivate at 37° C. and 100 rpm for 1 h. Then smear one kanamycin (Kan) plate with every 200 μL, 5 plates in total. Smear evenly and allow to air-dry. Cultivate at 30° C. for 24 h. Pick up clones grown under kanamycin resistance, and carry out PCR identification to screen positive clones.
(179) No. of the obtained strain: AT-006-01 (AT-004-02, nagE::pTrc-nanK-fKanrf).
(180) (6) Removal of the Resistance Gene
(181) Introduce pCP20 into the above kanamycin-resistant clones, cultivate at 30° C. for 8 h, then increase to 42° C. and cultivate overnight, and thermally induce to express FLP recombinase; the plasmids are lost gradually. Streak the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, pick up grown monoclones and dot on the kanamycin-resistant plate; those that cannot grown are clones of which the kanamycin resistance gene has been removed by FLP recombination. Carry out PCR with identification primer to identify clones losing kanamycin resistance.
(182) No. of the obtained strain: AT-006-02 (AT-004-02, nagE::pTrc-nanK).
(183) 3. Influence of pTrc-nanK Gene Cassette Integration on the Output of N-Acetyl-D-Glucosamine
(184) Carry out a shake-flask fermentation trial with the recombinant strain AT-006-02 of which the nagE gene site in the chromosome is integrated with pTrc-nanK gene cassette, and the reference strain. Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD.sub.600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(185) 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 are very low and are not detected, while the output by the recombinant strain integrated with pTrc-nanK gene cassette is increased obviously, and is also increased significantly than that by the recombinant strain nanK/pTrc99A (AT-005-02) not integrated.
(186) TABLE-US-00005 TABLE 4 Output from Shake-flask Fermentation by the Recombinant Strain integrated with pTrc-nanK Gene Cassette Output of N-Acetyl-D-Glucosamine species (g/L) AT-001 Not detected (Reference) AT-005-02 (AT-004-02, nagE) Not detected (Reference) nanK/pTrc99A(AT-005-02) 2.8 ± 0.5 AT-006-02 (AT-004-02,
nagE::pTrc-nanK) 4.2 ± 0.5
EXAMPLE 3
(187) This implementation example describes screening for a gene mutant of N-Acetyl-D-Mannosamine Kinase (NanK); the said gene encodes N-Acetyl-D-Mannosamine Kinase (NanK) with increased activities.
(188) To further increase synthetic quantity of N-Acetyl-D-Glucosamine by the production strain, screen a gene mutant encoding N-Acetyl-D-Mannosamine Kinase (NanK) with increased activities. To achieve the purpose, amplify the cloned gene by error-prone PCR technology; used for amplified DNA polymerase, amplify the said gene under conditions leading to high-frequency mismatch, so as to obtain a high-frequency mutation in PCR products.
(189) The specific operating process is provided below:
(190) 1. Amplification of the Gene nanK of N-Acetyl-D-Mannosamine Kinase in Escherichia coli by Error-Prone PCR
(191) By means of Taq DNA polymerase without the property of 3′-5′ proofreading function, control the frequency of random mutation under high magnesium ion concentration (8 mmol/L) and different dNTP concentrations (where, the concentration of dATP and dGTP is 1.5 mmol/L; and the concentration of dTTP and dCTP is 3.0 mmol/L), introduce random mutations into the target gene, and construct a mutant library; The template concentration A260 value is 1000 ng/mL, the enzyme concentration is 5 U/μL, and the primer concentration is 100 μM.
(192) Error-prone PCR reaction system (50 μL): 10×PCR reaction buffer 5 μl, dNTP (22.5 mM) 5 μL, MgCl.sub.2 (2.5 mM) 5 μL, Forward primer (nanK-F, SEQ ID No.18) 1 μL, reverse primer (nanK-R, SEQ ID No.19) 1 μL, DNA template (nanK/pUC57) 0.1 μL, Taq DNA polymerase 0.5 μL, and ddH.sub.2O 32.4 μL.
(193) PCR procedure: Predegenerate at 96° C. for 4 min; degenerate at 94° C. for 1 min, anneal at 56° C. for 1 min, extend at 75° C. for 2 min, and repeat for 45 cycles; finally extend at 75° C. for 15 min, recover PCR product (product size: 0.9 kb) by gel recovery method; transfer 5 μL of the product and carry out 1% agarose gel electrophoresis test; the product is store at −20° C. for use.
(194) 2. Construction of the Gene Mutant Library of N-Acetyl-D-Mannosamine Kinase
(195) Digest the above PCR product by two enzymes of restriction endonuclease Nco I and Hind III, carry out a ligation reaction with pTrc99A digested by Nco I and Hind III, then transform Escherichia coli AT-005-02 with the mixture of the ligated products to obtain a large amount of cloned transformants, and construct a mutant library of transformed thalli.
(196) 3. Screening for Mutants with High Enzyme Activities
(197) Using the wild type NanK/pTrc99A (AT-005-02) as reference, pick up randomly 300 mutant clones from the mutant library of transformed thalli, inoculate into 5 mL of LB medium containing 50 μg/mL Ampicillin (Amp), shake-cultivate at 37° C. and 150 rpm for 18 h, and then centrifugate at 10000 rpm for 5 mim and collect thalli. Discard the supernatant, then resuspend at 4° C. in 1 mL of PBS solution (pH 7.5, 10 mmol/L), carry out ultrasonicate at a voltage of 300 V for 10 min (ultrasonicate for 3 s and pause for 6 s), centrifugate, transfer the supernatant as crude extract of enzyme, and carry out a method for determination of enzyme activity.
(198) Determination of N-Acetyl-D-Mannosamine Kinase (NanK) Activity: Based on the phosphorylated amount of N-Acetyl-D-Mannosamine (ManNAc); that is to say, using the reduced amount of N-Acetyl-D-Mannosamine as test marker. Definition of Enzyme Activity Unit: Under the enzymatic reaction conditions, the enzyme amount reduced by the reducing sugar, equivalent to 1 μmol N-Acetyl-D-Mannosamine per minute, is defined as one enzyme activity unit (IU). The specific procedure is provided as follows: Transfer 5 mL of the reaction system as the system for determination of enzyme activity, which contains 500 mmol/L N-Acetyl-D-Mannosamine, 5 mmol/L glucose, 100 mmol/L Tris-HCl (pH8.0), and 100 μL of crude enzyme solution. Carry out the reaction for determination of enzyme activity on a water bath at 37° C., maintain for 4 h, and then place the enzymatic hydrolysate at 70° C. for 10 min to stop the reaction. Centrifugate at 3000 rpm for 10 min, and transfer the supernatant. Measure the content of N-Acetyl-D-Mannosamine by HPLC method.
(199) The results show that: The enzyme activity of the mutant strain with maximum activity is 77.5 IU/mL, and the enzyme activity of the reference is 16.3 IU/mL. Transform NanK by error-prone PCR, to obtain a mutant strain with enzyme activity increased by 5 folds. Pick up the mutant strain with maximum enzyme activity and extract plasmids for sequencing. The results show that: The gene sequence of the mutant of N-Acetyl-D-Mannosamine Kinase 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 the type N-Acetyl-D-Mannosamine Kinase, 4 base point mutations occur in total: 107A/G, 309T/G, 669G/C, and 783A/G; There occur 3 missense mutations of amino acids, of which the mutation points are: Q36R (glutamine at Site 36 is replaced by arginine), I103M (isoleucine at Site 103 is replaced by methionine), and R223S (arginine at Site 223 is replaced by serine) The mutant gene is named as nanKM.
(200) 4. Integration of pTrc-nanK Gene Cassette into the nagE Gene Site in the Chromosome of Escherichia coli
(201) Using the nagE gene site as integration site of pTrc-nanKM gene cassette into the chromosome. To achieve integration of pTrc-nanKM gene cassette into the chromosome of Escherichia coli, first amplify the nanK segment pTrc-nanKM of Trc promoter, as well as the kanamycin resistance gene segment with FLP recognition site (FRT site) at its both ends: FRT-Kanr-FRT (fKanrf), and splice them. Then design homologous primers for deletion of the nagE gene sequence, and using the spliced segment of pTrc-nanKM and fKanrf as template, amplify the linear DNA full-length segment for targeting of Red recombination.
(202) The specific operating process is provided below:
(203) (1) PCR Amplification of pTrc-nanKM Segment
(204) Template: nanKM/pTrc99A.
(205) Primer Design: Forward primer (Trcff-F) SEQ ID No.20, and reverse primer (Trcff-R) SEQ ID No.21.
(206) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(207) Produce Size: 1.05kb.
(208) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(209) (2) fKanrf Segment Amplified by PCR
(210) Primer Design: Forward primer (mfKanf-F) SEQ ID No.1, and reverse primer (mfKanf-R) SEQ ID No.2.
(211) Template: pPic9K.
(212) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(213) fKanrf size: 1.28kb. Its nucleotide sequence is SEQ ID No.3.
(214) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(215) (3) Amplification of fKanrf Spliced with pTrc-nanKM
(216) Primer Design: Forward primer (fKanf-F) SEQ ID No.22, and reverse primer (fKanf-R) SEQ ID No.23.
(217) Template: fKanrf.
(218) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(219) fKanrf size from secondary amplification: 1.3 kb.
(220) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(221) (4) Preparation of Linear DNA Full-length PCR Segment for Targeting of Red Recombination
(222) 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.
(223) Template: Mix pTrc-nanKM PCR segment, and fKanrf PCR segment from secondary amplification in the ratio of 1:1.
(224) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(225) Amplification Product: Homologous Arm+pTrc-nanKM-fKanrf+Homologous Arm
(226) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(227) (5) Red Recombination Operation
(228) First, pKD46 carrier is introduced into the AT-004-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(229) 1. Transformation of pKD46 Plasmid
(230) {circle around (1)} Preparation of Competence: First, inoculate 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-cultivate at 37° C. and 225 rpm for 2-3 h. Then add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation.
(231) {circle around (2)} Plasmid Transformation: Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of pKD46 plasmid, and cultivate at −4° C. for 30 min. Then heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h. Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate. Cultivate overnight (for 12-16 h) at 30° C. Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification. Store the positive strain for use.
(232) 2. Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone
(233) {circle around (1)} Preparation of Electrotransformed Competence: Inoculate the AT-004-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultivate at 250 rpm, on the next day, inoculate in a ratio of 1% into LB medium containing Amp, and cultivate at 30° C.; when OD.sub.600 reaches approximately 0.2, add 0.2% L-Arabinose, and induce at 30° C. for 35 min until OD.sub.600 reaches approximately 0.4. Cool on an ice bath. Wash once with ultrapure water, wash twice with 10% glycerin, and finally resuspend with 10% glycerin; the amount of glycerin used is to produce a final concentration of the thalli concentrated by 500-1000 folds.
(234) {circle around (2)} Transformation by electric shock: Take out a 2-mm electrotransformation cup from 70% ethanol, wash with sterilized ultrapure water, and irradiate by UV lamp for 30 min. Precool at 4° C. for 30 min. Transfer 90 μL of finally resuspended cells to a precooled centrifuge tube, add 5 μL (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (4), suction gently by a gun, and maintain on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 2000, 25 μF.
(235) Resuscitate and screen positive clones: add 1 mL of LB broth medium, and cultivate at 37° C. and 100 rpm for 1 h. Then smear one kanamycin (Kan) plate with every 200 μL, 5 plates in total. Smear evenly and allow to air-dry. Cultivate at 30° C. for 24 h. Pick up clones grown under kanamycin resistance, and carry out PCR identification to screen positive clones.
(236) No. of the obtained strain: AT-007-01 (AT-004-02, ΔnagE::pTrc-nanKM-fKanrf).
(237) (6) Removal of the Resistance Gene
(238) Introduce pCP20 into the above kanamycin-resistant clones, cultivate at 30° C. for 8 h, then increase to 42° C. and cultivate overnight, and thermally induce to express FLP recombinase; the plasmids are lost gradually. Streak the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, pick up grown monoclones and dot on the kanamycin-resistant plate; those that cannot grown are clones of which the kanamycin resistance gene has been removed by FLP recombination. Carry out PCR with identification primer to identify clones losing kanamycin resistance.
(239) No. of the obtained strain: AT-007-02 (AT-004-02, ΔnagE::pTrc-nanKM).
(240) 5. Influence of pTrc-nanKM Gene Cassette Integration on the Output of N-Acetyl-D-Glucosamine
(241) Carry out a shake-flask fermentation trial with the recombinant strain AT-007-02 of which the nagE gene site in the chromosome is integrated with pTrc-nanKM gene cassette, and the reference strain. Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD.sub.600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(242) See Table 5 for the output from shake-flask fermentation. The results show that: The output by the reference strain AT-005-02 is very low and is not detected, while the output by the recombinant strain AT-007-02 integrated with pTrc-nanKM gene cassette is increased obviously, and is also increased significantly than that by the unmutant reference strain (AT-006-02).
(243) TABLE-US-00006 TABLE 5 Output from Shake-flask Fermentation by the Recombinant Strain integrated with pTrc-nanKM Gene Cassette Output of N-Acetyl-D-Glucosamine Species (g/L) AT-005-02 (AT-004-02, nagE) Not detected (Reference) AT-006-02 (AT-004-02,
nagE::pTrc-nanK) 4.5 ± 0.4 AT-007-02 (AT-004-02,
nagE::pTrc-nanKM) 11.2 ± 1.2
(244) The above results show that: The output of N-Acetyl-D-Glucosamine may be increased obviously by overexpression of N-Acetyl-D-Mannosamine Kinase; moreover, the output of N-Acetyl-D-Glucosamine may also be greatly by the mutant screened by error-prone PCR technology, due to increased activities of the obtained mutant of the enzyme.
EXAMPLE 4
(245) This implementation example describes Escherichia coli strains integrated with pTrc-nanKM gene cassette, and the gene (NanE) thereof for overexpression of N-Acetyl-D-Mannosamine-6-P Epimerase as well as its influence on the output of N-Acetyl-D-Glucosamine.
(246) Amplify the gene nanE of Escherichia coli NanE (N-acetylmannosamine-6-phosphate epimerase, N-Acetyl-D-Mannosamine-6-P Epimerase), insert pTrc99A so that nanE is controlled by Trc promoter yp transform the strains, or the natural endogenous promoter with nanE gene is replaced by Trc promoter for overexpression of the enzyme, to strengthen transformation of N-Acetyl-D-Mannosamine-6-Phosphate (ManNAc-6-P) into N-Acetyl-D-Glucosamine-6-Phosphate (GlcNAc-6-P).
(247) 1. Amplify the nanE Gene and Insert λ pTrc99A
(248) From NCBI, look up U00096, to obtain the nucleotide sequence SEQ ID No.28 of the nanE gene of Escherichia coli, which amino acid sequence is SEQ ID No.29.
(249) Primer Design: Forward primer (nanE-F) SEQ ID No.30, and reverse primer (nanE-R) SEQ ID No.31.
(250) Template: AT-001 (Escherichia coli ATCC 27325) Genome.
(251) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(252) Amplification Product Size: 690 kb.
(253) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(254) Digest enzymatically the obtained PCR amplification segment and carrier pTrc99A with Nco I and Hind III, separate by agarose gel electrophoresis, purify and recover nanE segment and pTrc99A segment, connect overnight with T4 DNA ligase at 16° C., and carry out identification to obtain nanE/pTrc99A plasmid.
(255) 2. Transformation and Integration of Escherichia coli Strains with pTrc-NanKM Gene Cassette by nanE/pTrc99A
(256) (1) Preparation of Competence
(257) {circle around (1)} Inoculate the bacterial suspension of AT-007-02 stored at −20° C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultivate at 37° C. and 225 rpm for 2-3 h.
(258) {circle around (2)} Add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min.
(259) {circle around (3)} Centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation.
(260) (2) Plasmid Transformation
(261) {circle around (1)} Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of nanE/pTrc99A plasmid, and cultivate at −4° C. for 30 min. {circle around (2)} Heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h.
(262) {circle around (3)} Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate.
(263) {circle around (4)} Cultivate overnight (for 12-16 h) at 30° C.
(264) {circle around (5)} Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification.
(265) {circle around (6)} Positive clones are stored for use.
(266) No. of the obtained strain: AT-008 (AT-007-02, nanE/pTrc99A).
(267) 3. The Natural Endogenous Promoter with nanE gene of Escherichia coli Strains Integrated with pTrc-nanKM Cassette is Replaced with Trc Promoter
(268) First, amplify Trc promoter sequence segment and fKanrf segment, and splice together. Then design homologous primers, and amplify linear DNA full-length segment for targeting of Red recombination.
(269) (1) Amplification of Trc Promoter Sequence
(270) According to public information, look up Trc promoter sequence: SEQ ID No.32.
(271) Primer Design: Forward primer (KanTrcRed-F) SEQ ID No.33, and reverse primer (KanTrcRed-R) SEQ ID No.34.
(272) Template: pTrc99A
(273) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(274) Product Size: 166 bp.
(275) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(276) (2) Amplify the Kanamycin Resistance Gene with Recognition Sites (FRT Sites) for FLP Recombinase at its Both Ends: fKanrf
(277) Primer Design: Forward primer (mfKanf-F) SEQ ID No.1, and reverse primer (mfKanf-R) SEQ ID No.2.
(278) Template: pPic9K.
(279) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(280) fKanrf size: 1.28 kb. Its nucleotide sequence is SEQ ID No.3.
(281) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(282) (3) Amplification of fKanrf Spliced with Trc Promotor
(283) Primer Design: Forward primer (fKanfRed-F1) SEQ ID No.35, and reverse primer (fKanfRed-R1) SEQ ID No.36.
(284) Template: fKanrf.
(285) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(286) fKanrf size from secondary amplification: 1.3 kb.
(287) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(288) (4) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(289) Design homologous primers: Based on promoter sequence SEQ ID No.37 of the nanE gene. Design primers to be replaced with Trc promoter: Forward primer (ProNanEpTrc-F) SEQ ID No.38, and reverse primer (ProNanEpTrc-R) SEQ ID No.39.
(290) Template: Mix Trc promoter PCR segment and fKanrf PCR segment from secondary amplification in the ratio of 1:1.
(291) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(292) Amplification Product: Homologous Arm+fKanrf+Trc Promoter+Homologous Arm.
(293) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(294) (5) Red Recombination Operation
(295) First, pKD46 carrier is introduced into the AT-007-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(296) No. of the obtained strain: AT-009 (AT-007-02, nanE promotor::Trc promoter).
(297) 4. Recombinant Strain Integrated with pTrc-nanKM Gene Cassette, and Influences of the Strain Thereof with nanE/pTrc99A by Transformation, and of the Strain Thereof with nanE Promoter Replaced by Trc Promoter on the Output of N-Acetyl-D-Glucosamine.
(298) Carry out a shake-flask fermentation trial with the strain integrated with pTrc-nanKM gene cassette, and the strain thereof for overexpression of NanE (including the strain thereof with nanE/pTrc99A by transformation and the strain thereof with NanE promoter replaced by Trc promoter). Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of M9 culture solution. The initial OD.sub.600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(299) See Table 6 for the output from shake-flask fermentation by recombinant strains. The results show that: The output may be increased obviously from overexpression of NanE from whether NanE promoter is replaced by Trc promoter, or nanE/pTrc99A plasmid is transformed; moreover, the output is increased more significant from the recombinant strain with NanE promoter replaced by Trc promoter than that with transformed nanE/pTrc99A plasmid.
(300) TABLE-US-00007 TABLE 6 Output from Shake-flask Fermentation by the Recombinant Strain integrated with pTrc-nanKM Gene Cassette, for Overexpression of NanE Output of N-Acetyl-D-Glucosamine species (g/L) AT-007-02 11.5 ± 1.2 (Reference) AT-008 (AT-007-02, nanE/pTrc99A) 16.3 ± 1.1 AT-009 19.7 ± 1.4 (AT-007-02, nanE promotor::Trc promoter)
EXAMPLE 5
(301) This implementation example describes Escherichia coli strains integrated with pTrc-nanKM, and influences of those with the natural endogenous promoter is replaced and/or deleted in the gene glmS of Glucosamine-6-Phosphate Synthase (GlmS) and/or the nagB gene of D-Glucosamine-6-Phosphate Deaminase (NagB) on the output of N-Acetyl-D-Glucosamine.
(302) 1. Influences of Escherichia coli strains integrated with pTrc-NanKM gene cassette on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the nagB gene is replaced by Trc promoter, or the natural endogenous promoter of the glmS gene is further deleted.
(303) (1) The Natural Endogenous Promoter of the nagB Gene is Replaced by Trc Promoter
(304) The gene promoter of D-Glucosamine-6-Phosphate Deaminase (NagB) in nag regulon (nagE-nagBACD) is 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).
(305) First, amplify Trc promoter segment and fKanrf segment, and splice together. Then design homologous primers, and amplify linear DNA full-length segment for targeting of Red recombination.
(306) 1) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(307) Design of Homologous Arm Primers: From NCBI, look up NC_000913, nagB promoter sequence and nagA gene sequence SEQ ID No.13 of Escherichia coli str.K-12, design the homologous arm primers for deletion of nagB promoter: Forward primer (nagBKO-F1) SEQ ID No.40 and reverse primer (nagBKO-R1) SEQ ID No.41.
(308) Template: Mix Trc promoter PCR segment and fKanrf PCR segment from secondary amplification in the ratio of 1:1.
(309) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(310) Amplification Product: Homologous Arm+fKanrf+Trc Promoter+Homologous Arm.
(311) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(312) 2) Red Recombination Operation
(313) First, pKD46 carrier is introduced into the AT-007-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(314) No. of the obtained strain: AT-010 (AT-007-02, nagB promotor::Trc promoter).
(315) (2) Deletion of the Natural Endogenous Promoter of the glmS Gene
(316) Deletion of the gene promoter of Glucosamine-6-Phosphate Synthase (glmS). Glucosamine-6-Phosphate Synthase (GlmS), also called as L-Glutamine-D-fructose-6-phosphate aminotransferase, can catalyze amination of Glucose-6-Phosphate (Glc-6-P) into D-Glucosamine-6-Phosphate (GlcN-6-P), but has a severe problem for product inhibition. When the promoter sequence is deleted so that the enzyme cannot be expressed, it may solve product inhibition of GlcN-6-P.
(317) First, amplify fKanrf segment; then design homologous primers, and amplify linear DNA full-length segment for targeting of Red recombination.
(318) 1) Amplify the Kanamycin Resistance Gene with Recognition Sites (FRT Sites) for FLP Recombinase at its Both Ends: fKanrf
(319) Primer Design: Forward primer (mfKanf-F) SEQ ID No.1, and reverse primer (mfKanf-R) SEQ ID No.2.
(320) Template: pPic9K.
(321) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(322) fKanrf size: 1.28 kb. Its nucleotide sequence is SEQ ID No.3.
(323) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(324) 2) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(325) Design of Homologous Primers: From NCBI, look up NC_000913, the gene promoter sequence SEQ ID No.42 of Glucosamine-6-Phosphate Synthase (GlmS) of Escherichia coli str.K-12, design the homologous arm primers for deletion of glmS gene promoter: Forward primer (ProglmsKO-F) SEQ ID No.43 and reverse primer (ProglmsKO-R) SEQ ID No.44.
(326) Template: fKanrf PCR Segment.
(327) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(328) Amplification Product: Homologous Arm+fKanf+Homologous Arm.
(329) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(330) 3) Red Recombination Operation
(331) First, pKD46 carrier is introduced into the AT-010 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(332) No. of the obtained strain: AT-011 (AT-010, glmS promotor).
(333) (3) Influence of the Strains of which nagB Promoter Replaced by a Promoter with Higher Expression Level, or glmS Promoter is Further Deleted, on the Output of N-Acetyl-D-Glucosamine
(334) Carry out a shake-flask fermentation trial with the recombinant strains integrated with pTrc-nanKM gene cassette, of which nagB promoter is replaced by a promoter with higher expression level, or glmS promoter is further deleted. Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(335) See Table 7 for the output from shake-flask fermentation. The results show that: The output of N-Acetyl-D-Glucosamine is obviously increased by the recombinant strains of which nagB promoter is replaced by Trc promoter, or glmS promoter is further deleted.
(336) TABLE-US-00008 TABLE 7 Output from Shake-flask Fermentation by Recombinant Strains of which nagB promoter is replaced or glmS promoter is further deleted Output of N-Acetyl-D-Glucosamine species (g/L) AT-007-02 11.6 ± 1.2 (Reference) AT-010 17.2 ± 1.3 (AT-007-02, nagB promotor::Trc promoter) AT-011 (AT-010,
glmS promotor) 21.4 ± 1.5
(337) 2. Influences of Escherichia coli strains integrated with pTrc-NanKM gene cassette on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the glmS gene is replaced by Trc promoter, or the natural endogenous promoter of the glmS gene is further deleted.
(338) (1) The Natural Endogenous Promoter of the glmS Gene is Replaced by Trc Promoter
(339) The gene promoter sequence of L-Glutamine-D-Fructose-6-Phosphate Aminotransferase is replaced by Trc promoter sequence. L-Glutamine-6-Phosphate Aminotransferase, also called as Glucosamine-6-Phosphate Synthase (GlmS); replacement of its promoter sequence is replaced by Trc promoter sequence may overexpress glmS, and accelerate GlmS catalytic function, to achieve the purpose of increasing D-Glucosamine-6-Phosphate (GlcN-6-P).
(340) First, amplify Trc promoter sequence segment and fKanrf segment, and splice together. Then design homologous primers, and amplify linear DNA full-length segment for targeting of Red recombination.
(341) 1) Amplification of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(342) Design of homologous arm primers: According to the glmS gene promoter sequence SEQ ID No.42, design homologous arm primers with the promoter replaced by Trc promoter: Forward primer (ProglmspTrc-F) SEQ ID No.45, and reverse primer (ProglmspTrc-R) SEQ ID No.46.
(343) Template: Mix Trc promoter PCR segment and fKanrf PCR segment from secondary amplification in the ratio of 1:1.
(344) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(345) Amplification Product: Homologous Arm+fKanrf+Trc Promoter+Homologous Arm.
(346) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(347) 2) Red Recombination Operation
(348) First, pKD46 carrier is introduced into the AT-007-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(349) No. of the obtained strain: AT-012 (AT-007-02, glmS promotor::Trc promoter).
(350) (2) Deletion of the Natural Endogenous Promotor of NagB Gene
(351) Deletion of the gene promoter sequence of D-Glucosamine-6-Phosphate Deaminase (NagB) in the nag regulon (nagE-nagBACD), so that nagB loses its function, may eliminate the reverse catalytic function of NagB and reduce production of Glc-6-P from GlcN-6-P.
(352) First, amplify fKanrf segment; then design homologous primers, and prepare linear DNA full-length segment for targeting of Red recombination.
(353) 1) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(354) Design of Homologous Arm Primers: According to nagB promoter sequence and nagA gene sequence SEQ ID No.13, design the homologous arm primers for deletion of nagB promoter sequence: Forward primer (nagBKO-F2) SEQ ID No.47 and reverse primer (nagBKO-R2) SEQ ID No.48.
(355) Template: fKanrf PCR Segment
(356) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(357) Amplification Product: Homologous Arm+fKanrf+Homologous Arm.
(358) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(359) 2) Red Recombination Operation
(360) First, pKD46 carrier is introduced into the AT-012 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(361) No. of the obtained strain: AT-013 (AT-012, nagB promotor).
(362) (3) Influence of the Strains of which glmS Promoter Replaced by a Promoter with Higher Expression Level, or is Further Deleted, on the Output of N-Acetyl-D-Glucosamine
(363) Carry out a shake-flask fermentation trial with the recombinant strains integrated with pTrc-nanKM gene cassette, of which glmS promoter is replaced by a promoter with higher expression level, or nagB promoter is further deleted. Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(364) See Table 8 for the output from shake-flask fermentation. The results show that: The output of N-Acetyl-D-Glucosamine is not obviously increased by the recombinant strains of which glmS promoter is replaced by Trc promoter; however, the output of N-Acetyl-D-Glucosamine is increased when nagB promoter is meanwhile deleted, than that by the reference strain.
(365) TABLE-US-00009 TABLE 8 Output from Shake-flask Fermentation by Recombinant Strains of which glmS promoter is replaced or nagB promoter is further deleted Output of N-Acetyl-D-Glucosamine species (g/L) AT-007-02 11.6 ± 1.5 (Reference) AT-012 11.9 ± 1.4 (AT-007-02, glmS promotor::Trc promoter) AT-013 (AT-012,
nagB promotor) 21.4 ± 1.6
EXAMPLE 6
(366) This implementation example describes Escherichia coli strains integrated with pTrc-nanKM gene cassette, and influences of those on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the gene nagB of D-Glucosamine-6-Phosphate Deaminase (NagB), and of the gene GlmS of Glucosamine-6-Phosphate Synthase (GlmS) is replaced and/or deleted to transform nanE/pTrc99A plasmid, or the natural endogenous promoter of the nanE gene is replaced by Trc promoter.
(367) 1. Escherichia coli strains integrated with pTrc-nanKM cassette are used to transform nanE/pTrc99A plasmid, of which the natural endogenous promoter of the nagB gene is replaced by Trc promoter, and meanwhile the natural endogenous promoter of the glmS gene is deleted
(368) (1) Preparation of Competence: First, inoculate the bacterial suspension of AT-011 stored at −20° C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultivate at 37° C. and 225 rpm for 2-3 h. Then add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation.
(369) (2) Plasmid Transformation: Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of nanE/pTrc99A plasmid, and cultivate at −4° C. for 30 min. Then heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h. Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate. Cultivate overnight (for 12-16 h) at 30° C. Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification. Positive clones are stored for use.
(370) No. of the obtained strain: AT-014 (AT-011, nanE/pTrc99A).
(371) 2. In Escherichia coli strains integrated with pTrc-nanKM cassette, of which the natural endogenous promoter of the nagB gene is replaced by Trc promoter and meanwhile the natural endogenous promoter of the glmS gene is deleted, the natural endogenous promoter of the NanE gene is replaced by Trc promoter
(372) First, amplify Trc promoter sequence segment and fKanrf segment, and splice together. Then design homologous primers, and amplify linear DNA full-length segment for targeting of Red recombination.
(373) (1) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(374) Design homologous primers: Based on promoter sequence SEQ ID No.37 of the nanE gene. Design primers to be replaced with Trc promoter: Forward primer (ProNanEpTrc-F) SEQ ID No.38, and reverse primer (ProNanEpTrc-R) SEQ ID No.39.
(375) Template: Mix Trc promoter PCR segment and fKanrf PCR segment from secondary amplification in the ratio of 1:1.
(376) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(377) Amplification Product: Homologous Arm+fKanrf+Trc Promoter+Homologous Arm.
(378) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(379) (2) Red Recombination Operation
(380) First, pKD46 carrier is introduced into the AT-011 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(381) No. of the obtained strain: AT-015 (AT-011, nanE promotor::Trc promoter).
(382) 3. Escherichia coli strains integrated with pTrc-nanKM cassette are used to transform nanE/pTrc99A plasmid, of which the natural endogenous promoter of the glmS gene is replaced by Trc promoter, and meanwhile the natural endogenous promoter of the nagB gene is deleted
(383) First, prepare competence of recombinant Escherichia coli strain AT-013; then, NanE/pTrc99A plasmid is transformed into AT-013 by CaCl.sub.2 transformation method, pick up monoclones and cultivate, and pipet plasmid to identify positive clones.
(384) No. of the obtained strain: AT-016 (AT-013, nanE/pTrc99A).
(385) 4. In Escherichia coli strains integrated with pTrc-nanKM cassette, of which the natural endogenous promoter of the glmS gene is replaced by Trc promoter and meanwhile the natural endogenous promoter of the nagB gene is deleted, the natural endogenous promoter of the NanE gene is replaced by Trc promoter
(386) (1) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(387) First, amplify Trc promoter sequence segment and fKanrf segment, and splice together. Then design homologous primers, and amplify linear DNA full-length segment for targeting of Red recombination.
(388) (2) Red Recombination Operation
(389) First, pKD46 carrier is introduced into the AT-013 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(390) No. of the obtained strain: AT-017 (AT-013, nanE promotor::Trc promoter).
(391) 5. Influences of the Escherichia coli strains integrated with pTrc-nanKM gene cassette on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the nagB gene and glmS gene is replaced and/or deleted to transform nanE/pTrc99A plasmid, and the natural endogenous promoter of the nanE gene is replaced by Trc promoter
(392) Carry out a shake-flask fermentation trial with the recombinant strains with different genotypes, obtained form the strains integrated with pTrc-nanKM gene cassette, of which the natural endogenous promoter of the glmS and nagB genes are replaced and/or deleted to transform nanE/pTrc99A plasmid, or the natural endogenous promoter of the nanE gene is replaced by Trc promoter. Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(393) See Table 9 for the output from shake-flask fermentation. The results show that: The output of N-Acetyl-D-Glucosamine may be increased substantially from overexpression of NanE by recombinant strain with NanE promoter replaced by Trc promoter, or with transformed nanE/pTrc99A plasmid; moreover, the output of N-Acetyl-D-Glucosamine is increased more significantly by the recombinant strain with NanE promoter replaced by Trc promoter, than that with transformed nanE/pTrc99A plasmid.
(394) TABLE-US-00010 TABLE 9 Output from Shake-flask Fermentation by Recombinant Strains with Transformed NanE/pTrc99A Plasmid or with NanE promoter replaced by Trc Promoter Output of N-Acetyl-D-Glucosamine species (g/L) AT-011 21.6 ± 1.8 (Reference) AT-014 (AT-011, nanE/pTrc99A) 26.1 ± 1.5 AT-015 29.8 ± 1.5 (AT-011, nanE promotor::Trc promoter) AT-013 21.2 ± 1.6 (Reference) AT-016 (AT-013, nanE/pTrc99A) 26.7 ± 1.6 AT-017 30.9 ± 1.5 (AT-013,
nanE promotor::Trc promoter)
EXAMPLE 7
(395) This implementation example describes Escherichia coli strains integrated with pTrc-nanKM gene cassette, and the gene wecB thereof for overexpression of UDP-N-Acetyl-D-Glucosamine-2-Epimerase as well as its influence on the output of N-Acetyl-D-Glucosamine.
(396) The gene wecB of UDP-N-AcetylGlucosamine-2-Epimerase (WecB) is controlled by Trc promoter to transform strains, or the natural endogenous promoter of the wecB gene is replaced by Trc promoter for overexpression of the enzyme, to strengthen production of ManNAc (N-Acetyl-D-mannosamine, N-Acetyl-D-Mannosamine or N-Acetyl-D-Mannosamine) from UDP-GlcNAc (UDP-N-Acetyl Glucosamine, UDP-N-Acetyl-D-Glucosamine).
(397) 1. Transformation of Escherichia coli Strains Integrated with pTrc-NanKM Gene Cassette by wecB/pTrc99A
(398) (1) Amplify the wecB Gene of Escherichia coli and Insert pTrc99A
(399) According to NCBI, look up the nucleotide sequence SEQ ID No.49 of the wecB gene of Escherichia coli, and its amino acid sequence is SEQ ID No.50.
(400) Primer Design: Forward primer (TrcwecB-F) SEQ ID No.51, and reverse primer (TrcwecB-R) SEQ ID No.52.
(401) Template: AT-001 (Escherichia coli ATCC 27325) Genome.
(402) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(403) Amplification Product Size: 1.13 kb.
(404) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(405) Digest enzymatically the obtained PCR amplification segment and carrier pTrc99A with Nco I and Hind III, separate by agarose gel electrophoresis, purify and recover wecB segment and pTrc99A segment, connect overnight with T4 DNA ligase at 16° C., and carry out identification to obtain wecB/pTrc99A plasmid.
(406) (2) Transformation of Escherichia coli Strains Integrated with pTrc-NanKM Gene Cassette by wecB/pTrc99A
(407) (1) Preparation of Competence: First, inoculate the bacterial suspension of AT-007-02 stored at −20° C. into 10 mL of LB broth medium in a ratio of 1:50-100, and shake-cultivate at 37° C. and 225 rpm for 2-3 h. Then add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation.
(408) (2) Plasmid Transformation: Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of wecB/pTrc99A plasmid, and cultivate at −4° C. for 30 min. Then heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h. Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate. Cultivate overnight (for 12-16 h) at 30° C. Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification. Positive clones are stored for use.
(409) No. of the obtained strain: AT-018 (AT-007-02, wecB/pTrc99A).
(410) 2. The Natural Endogenous Promoter of the wecB Gene of Escherichia coli Strains Integrated with pTrc-nanKM Cassette is Replaced with Trc Promoter
(411) First, amplify Trc promoter sequence segment and fKanrf segment, and splice together. Then design homologous primers, and amplify linear DNA full-length segment for targeting of Red recombination.
(412) (1) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(413) Design of Homologous Arm Primers: According to NCBI, look up NC_000913 to obtain the nucleotide sequence SEQ ID No.53 of the gene promoter of Escherichia coli UDP-N-AcetylGlucosamine-2-Epimerase (WecB), design homologous arm primers with the promoter replaced by Trc promoter: Forward primer (ProwecBpTrc-F) SEQ ID No.54, and reverse primer (Pro wecBpTrc-R) SEQ ID No.55.
(414) Template: Mix Trc promoter PCR segment and fKanrf PCR segment from secondary amplification in the ratio of 1:1.
(415) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(416) Amplification Product: Homologous Arm+fKanrf+Trc Promoter+Homologous Arm.
(417) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(418) (2) Red Recombination Operation
(419) First, pKD46 carrier is introduced into the AT-007-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(420) No. of the obtained strain: AT-019 (AT-007-02, wecB promotor::Trc promoter).
(421) 3. Recombinant strain integrated with pTrc-nanKM gene cassette, and influences of the strain thereof with transformed wecB/pTrc99A plasmid, and of the strain thereof with nanE promoter replaced by Trc promoter on the output of N-Acetyl-D-Glucosamine.
(422) Carry out a shake-flask fermentation trial with the strain integrated with pTrc-nanKM gene cassette, and the recombinant strain produced thereof for overexpression of wecB (including the strain thereof with transformed wecB/pTrc99A and the strain thereof with wecB promoter replaced by Trc promoter). Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of M9 culture solution. The initial OD.sub.600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(423) See Table 10 for the output from shake-flask fermentation. The results show that: Compared with the reference strain AT-007-02, the output of N-Acetyl-D-Glucosamine is increased obviously by recombinant strain with transformed wecB/pTrc99A, and is increased more greatly by recombinant strain with wecB promoter replaced by Trc promoter.
(424) TABLE-US-00011 TABLE 10 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-Glucosamine species (g/L) AT-007-02 11.8 ± 1.4 (Reference) AT-018 (AT-007-02, wecB/pTrc99A) 16.3 ± 1.5 AT-019 23.2 ± 2.0 (AT-007-02, wecB promotor::Trc promoter)
EXAMPLE 8
(425) This implementation example describes influences of recombinant strains integrated with pTrc-nanKM gene cassette on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the nanE gene is replaced by Trc promoter to transform wecB/pTrc99A plasmid, or the natural endogenous promoter of the wecB gene is replaced by Trc promoter.
(426) 1. Escherichia coli strains integrated with pTrc-nanKM cassette are used to transform wecB/pTrc99A plasmid, of which the natural endogenous promoter of the nanE Gene is Replaced by Trc Promoter
(427) Preparation of competence: Transform wecB/pTrc99A plasmid with CaCl.sub.2 into Escherichia coli strain AT-009 integrated with pTrc-NanKM gene cassette, of which the natural endogenous promoter of the nanE gene is replaced by Trc promoter, pick up monoclones and cultivate, and pipet plasmid to identify positive clones.
(428) No. of the obtained strain: AT-020 (AT-009, wecB/pTrc99A).
(429) 2. In Escherichia coli strains integrated with pTrc-nanKM Cassette, of which the natural endogenous promoter of the nanE gene is replaced by Trc Promoter, the natural endogenous promoter of the NanE gene is replaced by Trc promoter
(430) First, pKD46 carrier is introduced into the AT-009 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(431) No. of the obtained strain: AT-021 (AT-009, wecB promotor::Trc promoter).
(432) 3. Influences of recombinant strains integrated with pTrc-nanKM gene cassette on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the nanE gene is replaced by Trc promoter to transform wecB/pTrc99A plasmid, and wecB promoter is replaced by Trc promoter
(433) Carry out a shake-flask fermentation trial with the strain integrated with pTrc-nanKM gene cassette, of which the natural endogenous promoter of the nanE gene is replaced by Trc promoter, and the recombinant strain produced thereof for overexpression of wecB (including the strain thereof with transformed wecB/pTrc99A and the strain with wecB promoter replaced by Trc promoter). Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of M9 culture solution. The initial OD.sub.600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(434) See Table 11 for the output from shake-flask fermentation. The results show that: Compared with the reference strain AT-009, the output of N-Acetyl-D-Glucosamine is increased obviously by recombinant strain with transformed wecB/pTrc99A, and is increased more greatly by recombinant strain with wecB promoter replaced by Trc promoter.
(435) TABLE-US-00012 TABLE 11 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-Glucosamine species (g/L) AT-009 19.5 ± 1.5 (Reference) AT-020 (AT-009, wecB/pTrc99A) 26.5 ± 1.8 AT-021 31.6 ± 2.1 (AT-009, wecB promotor::Trc promoter)
EXAMPLE 9
(436) This implementation example describes influences of the Escherichia coli strains integrated with pTrc-nanKM gene cassette on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the glmS gene and nagB gene is replaced and/or deleted to transform nanE/pTrc99A plasmid, and the natural endogenous promoter of the wecB gene is replaced by Trc promoter
(437) 1. Escherichia coli strains integrated with pTrc-nanKM cassette are used to transform wecB/pTrc99A plasmid, of which the natural endogenous promoter of the nagB gene is replaced by Trc promoter, and meanwhile the natural endogenous promoter of the glmS gene is deleted
(438) Preparation of competence: Transform wecB/pTrc99A plasmid with CaCl.sub.2 into Escherichia coli strain AT-011 integrated with pTrc-NanKM gene cassette, of which the natural endogenous promoter of the nagB gene is replaced by Trc promoter and meanwhile the natural endogenous promoter of the glmS gene is deleted, pick up monoclones and cultivate, and pipet plasmid to identify positive clones.
(439) No. of the obtained strain: AT-022 (AT-011, wecB/pTrc99A).
(440) 2. In Escherichia coli strains integrated with pTrc-nanKM cassette, of which the natural endogenous promoter of the nagB gene is replaced by Trc promoter and meanwhile the natural endogenous promoter of the glmS gene is deleted, the natural endogenous promoter of the wecB gene is replaced by Trc promoter
(441) First, pKD46 carrier is introduced into the AT-011 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(442) No. of the obtained strain: AT-023 (AT-011, wecB promotor::Trc promoter).
(443) 3. Escherichia coli strains integrated with pTrc-nanKM cassette are used to transform wecB/pTrc99A plasmid, of which the natural endogenous promoter of the glmS gene is replaced by Trc promoter, and meanwhile the natural endogenous promoter of the nagB gene is deleted
(444) Preparation of competence: Transform wecB/pTrc99A plasmid with CaCl.sub.2 into Escherichia coli strain AT-013 integrated with pTrc-nanKM gene cassette, of which the natural endogenous promoter of the glmS gene is replaced by Trc promoter and meanwhile the natural endogenous promoter of the nagB gene is deleted, pick up monoclones and cultivate, and pipet plasmid to identify positive clones.
(445) No. of the obtained strain: AT-024 (AT-013, wecB/pTrc99A).
(446) 4. In Escherichia coli strains integrated with pTrc-nanKM cassette, of which the natural endogenous promoter of the glmS gene is replaced by Trc promoter and meanwhile the natural endogenous promoter of the nagB gene is deleted, the natural endogenous promoter of the wecB gene is replaced by Trc promoter
(447) First, pKD46 carrier is introduced into the AT-013 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(448) No. of the obtained strain: AT-025 (AT-013, wecB promotor::Trc promoter).
(449) 5. Influences of the Escherichia coli strains integrated with pTrc-nanKM gene cassette on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the nagB gene and glmS gene is replaced and/or deleted to transform wecB/pTrc99A plasmid, and the natural endogenous promoter of the wecB gene is replaced by Trc promoter
(450) Carry out a shake-flask fermentation trial with the recombinant strains with different genotypes, obtained form the strains integrated with pTrc-nanKM gene cassette, of which the natural endogenous promoter of the glmS and nagB genes are replaced and/or deleted to transform wecB/pTrc99A plasmid, or the natural endogenous promoter of the wecB gene is replaced by Trc promoter. Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD.sub.600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(451) See Table 12 for the output from shake-flask fermentation. The results show that: Compared with the reference strains AT-011 or AT-013, the output of N-Acetyl-D-Glucosamine is increased obviously by recombinant strain with transformed wecB/pTrc99A, and is increased more greatly by recombinant strain with wecB promoter replaced by Trc promoter.
(452) TABLE-US-00013 TABLE 12 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-Glucosamine species (g/L) AT-011 21.4 ± 1.5 (Reference) AT-022 (AT-011, wecB/pTrc99A) 27.3 ± 1.8 AT-023 31.6 ± 2.2 (AT-011, wecB promotor::Trc promoter) AT-013 21.4 ± 1.6 (Reference) AT-024 (AT-013, wecB/pTrc99A) 27.0 ± 1.7 AT-025 31.9 ± 2.5 (AT-013,
wecB promotor::Trc promoter)
EXAMPLE 10
(453) This implementation example describes influences of the Escherichia coli strains integrated with pTrc-nanKM gene cassette on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the glmS gene and nagB gene is replaced and/or deleted and the natural endogenous promoter of the nanE gene is replaced by Trc promoter to transform nanE/pTrc99A plasmid, or the natural endogenous promoter of the wecB gene is replaced by Trc promoter
(454) 1. Escherichia coli strains integrated with pTrc-nanKM cassette, of which the natural endogenous promoter of the nagB gene is replaced by Trc promoter and the natural endogenous promoter of the glmS gene is deleted, and the natural endogenous promoter of the NanE gene is replaced by Trc promoter, are used to transform wecB/pTrc99A
(455) Preparation of competence: Transform wecB/pTrc99A plasmid with CaCl.sub.2 into Escherichia coli strain AT-015 integrated with pTrc-NanKM gene cassette, of which the natural endogenous promoter of the nagB gene is replaced by Trc promoter and the natural endogenous promoter of the glmS gene is deleted, and the natural endogenous promoter of the nanE gene is replaced by Trc promoter, pick up monoclones and cultivate, and pipet plasmid to identify positive clones.
(456) No. of the obtained strain: AT-026 (AT-015, wecB/pTrc99A).
(457) 2. In Escherichia coli strains integrated with pTrc-nanKM cassette, of which the natural endogenous promoter of the nagB gene is replaced by Trc promoter and the natural endogenous promoter of the glmS gene is deleted, and the natural endogenous promoter of the NanE gene is replaced by Trc promoter, the natural endogenous promoter of the wecB gene is replaced by Trc promoter
(458) First, pKD46 carrier is introduced into the AT-015 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(459) No. of the obtained strain: AT-027 (AT-015, wecB promotor::Trc promoter).
(460) 3. Escherichia coli strains integrated with pTrc-nanKM cassette, of which the natural endogenous promoter of the glmS gene is replaced by Trc promoter and the natural endogenous promoter of the nagB gene is deleted, and the natural endogenous promoter of the NanE gene is replaced by Trc promoter, are used to transform wecB/pTrc99A
(461) Preparation of competence: Transform wecB/pTrc99A plasmid with CaCl.sub.2 into Escherichia coli strain AT-017 integrated with pTrc-NanKM gene cassette, of which the natural endogenous promoter of the glmS gene is replaced by Trc promoter and the natural endogenous promoter of the nagB gene is deleted, and the natural endogenous promoter of the nanE gene is replaced by Trc promoter, pick up monoclones and cultivate, and pipet plasmid to identify positive clones.
(462) No. of the obtained strain: AT-028 (AT-017, wecB/pTrc99A).
(463) 4. In Escherichia coli strains integrated with pTrc-nanKM cassette, of which the natural endogenous promoter of the glmS gene is replaced by Trc promoter and the natural endogenous promoter of the nagB gene is deleted, and the natural endogenous promoter of the NanE gene is replaced by Trc promoter, the natural endogenous promoter of the wecB gene is replaced by Trc promoter
(464) First, pKD46 carrier is introduced into the AT-017 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(465) No. of the obtained strain: AT-029 (AT-017, wecB promotor::Trc promoter).
(466) 5. Influences of the Escherichia coli strains integrated with pTrc-nanKM gene cassette on the output of N-Acetyl-D-Glucosamine, of which the natural endogenous promoter of the nagB gene and glmS gene is replaced and/or deleted and the natural endogenous promoter of the nanE gene is replaced by Trc promoter to transform nanE/pTrc99A plasmid, or the natural endogenous promoter of the wecB gene is replaced by Trc promoter
(467) Carry out a shake-flask fermentation trial with recombinant strains, produced from Escherichia coli strains integrated with pTrc-nanKM cassette, of which the natural endogenous promoter of the glmS gene and nagB gene is replaced or deleted, and the natural endogenous promoter of the NanE gene is replaced by Trc promoter to transform wecB/pTrc99A plasmid, and wecB promoter is replaced by Trc promoter. Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD.sub.600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(468) See Table 13 for the output from shake-flask fermentation. The results show that: Compared with the reference strains AT-015 or AT-017, the output of N-Acetyl-D-Glucosamine is increased obviously by recombinant strain with transformed wecB/pTrc99A, and is increased more greatly by recombinant strain with wecB promoter replaced by Trc promoter.
(469) TABLE-US-00014 TABLE 13 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-Glucosamine species (g/L) AT-015 29.5 ± 1.6 (Reference) AT-026 (AT-015, wecB/pTrc99A) 34.8 ± 1.7 AT-027 39.5 ± 2.5 (AT-015, wecB promotor::Trc promoter) AT-017 30.5 ± 1.7 (Reference) AT-028 (AT-017, wecB/pTrc99A) 35.3 ± 1.6 AT-029 39.9 ± 2.3 (AT-017,
wecB promotor::Trc promoter)
EXAMPLE 11
(470) This implementation example describes a fermentation trial for production of N-Acetyl-D-Glucosamine by 10-L fermentation tank
(471) Carry a fermentation trial for production of N-Acetyl-D-Glucosamine by 10-L fermentation tank, using recombinant engineering strain AT-029 as production strain.
(472) 1. Seed Cultivation
(473) (1) Cultivation of Primary Seed: Pick monoclonal strain freshly cultivated in the LB plate medium, inoculate into 8 mL of LB broth medium, and shake-cultivate at 37° C. and 225 rpm for 8 h.
(474) (2) Cultivation of Secondary Seed: Transfer 6 mL of the primary seed culture solution, inoculate into 1000-mL shake flask containing 200 mL of M9 culture solution, and shake-cultivate at 37° C. and 225 rpm for 16 h, until OD.sub.600 value is 6.0-10, approximately the medium stage of log growth.
(475) (3) Prepare the fermentation medium according to Table 14, where the microelement solution is prepared according to Table 15, and the complex vitamins solution is prepared according to Table 16.
(476) TABLE-US-00015 TABLE 14 Fermentation Medium Amount Ingredients (/L) K.sub.2HPO.sub.4 1.30 g KH.sub.2PO.sub.4 1.00 g MgSO.sub.4•7H.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 4 ml Solution Complex Vitamins 4 ml Solution Glucose 6.00 g NOTE: {circle around (1)} The microelement solution is sterilized separately and then added, and the vitamins solution is filtered and then added; {circle around (2)} Glucose: Concentration 65% (w/v); it is sterilized separately and is added prior to inoculation. Amount to be added: 6.0 g/L; {circle around (3)} The above solutions are combined, and then adjust to pH 7.0 with 10M NH.sub.4OH; {circle around (4)} The fermentation medium is 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%.
(477) TABLE-US-00016 TABLE 15 Microelement Solution Amount Ingredients used (g/L) CaCl.sub.2•2H.sub.2O 10 FeCl.sub.3•6H.sub.2O 10 MnSO.sub.4•5H.sub.2O 2.5 AlCl.sub.3•6H.sub.2O 2.5 CoCl.sub.2•6H.sub.2O 1.75 ZnSO.sub.4•2H.sub.2O 0.5 NaMoO.sub.4•2H.sub.2O 0.5 CuSO.sub.4•5H.sub.2O 0.25 H.sub.3BO.sub.3 0.125 pH 3 to 4
(478) TABLE-US-00017 TABLE 16 Complex Vitamins Solution Amount Ingredients (mg/L) Folic Acid 2 Vitamin B.sub.2 Riboflavin 100 Vitamin B.sub.1 Thiamine 1500 HCl Nicotinic Acid 500 Vitamin B.sub.6 Pyridoxine 500 HCl Calcium Pantothenate, 500 Ca-panthothenate Biotin 1 Vitamin B.sub.12 10
(479) 2. Inoculation
(480) Inoculate the secondary seed solution to the fermentation tank in the ratio of 40 mL/L; inoculation size: 2.5-5% (v/v); the initial OD.sub.600 is 0.3-0.5.
(481) 3. Process Parameters
(482) Carry out a high-density fermentation by 10-L self-control fermentation tank, and collect data by software equipped in the machine to realize online control by computer. The control parameters are: The air flow is 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 is maintained stable by addition of saturated ammonia water. Supplement glucose when glucose in the basal medium is consumed up, i.e. dissolved oxygen is risen again. Glucose is supplemented in a speed to control residual glucose concentration is not more than 0.45 g/L. The glucose-supplementing solution contains 65% (w/v) of glucose, is added with 2.5% Sodium Gluconate or 6% Ribose. The fermentation is stopped after 60-72 h. Total loading amount: 75%-80%.
(483) 4. Example (10-L fermentation tank)
(484) (1) Strain No.: AT-029. Batch No.: 0072.
(485) (2) Concentration of Seed Solution: OD.sub.6002.7.
(486) (3) Base Stock: 4 L.
(487) (4) Inoculation Size 200 mL.
(488) (5) Glucose supplementation speed: The residual glucose concentration is controlled as not more than 0.45 g/L.
(489) (6) Glucose-supplementing solution: The solution contains glucose in a concentration of 65% (w/v) and is added with 2.5% sodium gluconate.
(490) (7) Track Indicators: Measure OD.sub.600 and residual glucose content (residual glucose in the fermentation solution).
(491) (8) Product: N-Acetyl-D-Glucosamine. Potency: 72 h, 145.5 g/L.
EXAMPLE 12
(492) This implementation example describes the processing process after separation and purification of N-Acetyl-D-Glucosamine and D-Glucosamine Hydrochloride
(493) 1. Refinement of N-Acetyl-D-Glucosamine
(494) (1) Deactivation: The fermentation solution is placed at 80° C. for 30 min.
(495) (2) Solid-liquid separation: Centrifugate at 4000-8000 rpm, discard the bacterial residue and proteins, and transfer the fermentation solution. It may also be filtered via ceramic membrane.
(496) (3) Decoloration: Product:Water:Activated Charcoal=1:(1.5-3):(0.01-0.1); stir for 0.5-5 h.
(497) (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 is 0.5-1.4 V. It may also be desalted by anion or cation ion exchange resin.
(498) (5) Concentration: The fermentation solution desalted is heated at 50-80° C. under vacuum conditions (0.095 MPa) for 8-15 h until oversaturation, by approximately 4-6 folds.
(499) (6) Concentration: The concentrated fermentation solution is 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. Add anhydrous alcohol (in an amount of approximately 5-20 times the product weight), and stir at 700-1500 rpm for 15 min-1 h.
(500) (7) Washing: Add anhydrous alcohol (same amount as that of the product) and stir for 10-100 rpm, 0.5-2 h.
(501) (8) Drying: 50-100° C., 3-10 h. Purity: 99.93%. The total yield is 91.1%.
(502) 2. Refinement of D-Glucosamine Hydrochloride
(503) (1) Deactivation: The fermentation solution is placed at 80° C. for 30 min.
(504) (2) Solid-liquid separation: Centrifugate at 4000-8000 rpm, discard the bacterial residue and proteins, and transfer the fermentation solution. It may also be filtered via ceramic membrane.
(505) (3) Decoloration: Product:Water:Activated Charcoal=1:(1.5-3):(0.01-0.1); stir for 0.5-5 h.
(506) (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 is 0.5-1.4 V. It may also be desalted by anion or cation ion exchange resin.
(507) (5) Concentration: The fermentation solution desalted is heated at 50-80° C. under vacuum conditions (0.095 MPa) for 8-15 h until oversaturation, by approximately 4-6 folds.
(508) (6) Hydrolysis: Introduce the concentrated fermentation solution to an enamel or glass container, add concentrated hydrochloric acid (37%) to a final concentration of 12%46%, stir thoroughly, and maintain at 70° C. for 90 min. Hydrochloric Acid may be used in a recycling way.
(509) (7) Crystalliation: First cool to 25-35° C. in water at 25° C., and then cool to 4° C. in water at 0° C. for 1-3 h.
(510) (8) Washing: Add anhydrous alcohol (same amount as that of the product) and stir for 10-100 rpm, 0.5-2 h. Centrifugate at 700-1500 rpm for 15-60 min to obtain Glucosamine Hydrochloride; the conversion rate is 90.2%.
(511) (9) Dissolution: Dissolve the washed product in water in an similar volume to that of the original fermentation solution.
(512) (10) Decoloration: Add activated charcoal (in an amount of 1%). Mix for 30 min. Then centrifugate at 700-1500 rpm for 15-60 min. Or filter to obtain a colorless filtrate.
(513) (11) Recrystallisation: Evaporate at 50° C. and 55 cmHg vacuum until oversaturation. Add anhydrous alcohol (in an amount of approximately 5-20 times the product weight), and stir at 700-1500 rpm for 15 min-1 h.
(514) (12) Washing: Add anhydrous alcohol (same amount as that of the product) and stir for 10-100 rpm, 0.5-2 h. Then centrifugate at 700-1500 rpm for 15-60 min.
(515) (13) Drying: 50-100° C., 3-10 h. Purity: 99.91%. The total yield is 83.8%.
EXAMPLE 13
(516) This implementation example describes screening for a gene mutant of N-Acetyl-D-Mannosamine-6-P Epimerase (NanE); the said gene encodes N-Acetyl-D-Mannosamine-6-P Epimerase (NanE) with increased activities.
(517) To further increase synthetic quantity of N-Acetyl-D-Glucosamine by the production strain, screen a gene mutant encoding the enzyme with increased activities. To achieve the purpose, amplify the cloned gene by error-prone PCR technology; used for amplified DNA polymerase, amplify the said gene under conditions leading to high-frequency mismatch, so as to obtain a high-frequency mutation in PCR products.
(518) The specific operating process is provided below:
(519) 1. Amplification of the Gene nanE of N-Acetyl-D-Mannosamine-6-P Epimerase in Escherichia coli by Error-Prone PCR
(520) By means of Taq DNA polymerase without the property of 3′-5′ proofreading function, control the frequency of random mutation under high magnesium ion concentration (8 mmol/L) and different dNTP concentrations (where, the concentration of dATP and dGTP is 1.5 mmol/L; and the concentration of dTTP and dCTP is 3.0 mmol/L), introduce random mutations into the target gene, and construct a mutant library; the template concentration A260 value is 1000 ng/mL, the enzyme concentration is 5 U/μL, and the primer concentration is 100 μM.
(521) Error-prone PCR reaction system (50 μL): 10×PCR reaction buffer 5 μl, dNTP (2.5 mM) 5 μL, MgCl.sub.2 (2.5 mM) 5 μL, Forward primer (nanE-F, SEQ ID No.30) 1 μL, reverse primer (nanE-R, SEQ ID No.31) 1 μL, DNA template (nanE/pUC57) 0.1 μL, Taq DNA polymerase 0.5 μL, and ddH.sub.2O 32.4 μL.
(522) PCR procedure: Predegenerate at 96° C. for 4 min; degenerate at 94° C. for 1 min, anneal at 56° C. for 1 min, extend at 75° C. for 2 min, and repeat for 45 cycles; finally extend at 75° C. for 15 min, recover PCR product (product size: 0.7 kb) by gel recovery method; transfer 5 μL of the product and carry out 1% agarose gel electrophoresis test; the product is store at −20° C. for use.
(523) 2. Construction of the Gene Mutant Library of N-Acetyl-D-Mannosamine-6-P Epimerase
(524) Digest the above PCR product by two enzymes of restriction endonuclease Nco I and Hind III, carry out a ligation reaction with pTrc99A digested by Nco I and Hind III, then transform Escherichia coli AT-005-02 with the mixture of the ligated products to obtain a large amount of cloned transformants, and construct a mutant library of transformed thalli.
(525) 3. Screening for Mutants with High Enzyme Activities
(526) Using the wild type NanE/pTrc99A (AT-005-02) as reference, pick up randomly 350 mutant clones from the mutant library of transformed thalli, inoculate into 5 mL of LB medium containing 50 μg/mL Ampicillin (Amp), shake-cultivate at 37° C. and 150 rpm for 18 h, and then centrifugate at 10000 rpm for 5 mim and collect thalli. Discard the supernatant, then resuspend at 4° C. in 1 mL of PBS solution (pH 7.5, 10 mmol/L), carry out ultrasonicate at a voltage of 300 V for 10 min (ultrasonicate for 3 s and pause for 6 s), centrifugate, transfer the supernatant as crude extract of enzyme, and carry out a method for determination of enzyme activity.
(527) Determination of N-Acetyl-D-Mannosamine-6-P Epimerase Activity: Based on the amount of N-Acetyl-D-Mannosamine-6-Phosphate (ManNAc-6-P) transformed into N-Acetyl-D-Glucosamine-6-Phosphate (GlcNAc-6-P); that is to say, 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 reduced, equivalent to 1 μmol N-Acetyl-D-Mannosamine-6-Phosphate per minute, is defined as one enzyme activity unit (IU). The specific procedure is provided as follows: First, prepare isotope-labelled ManNAc-6-P as substrate. Prepare a reaction solution in a total volume of 225 uL, containing ManNAc Kinase (NanK) crude solution (containing 1-5 mg protein), 20 mM ATP disodium, 60 mM Tris-HCl, pH8.1, 20 mM MgCl.sub.2, 5 mM ManNAc, and 50 nCi [.sup.14C]ManNAc. Incubate at 37° C. for 30 min. Add 350 uL of alcohol to stop the reaction. The product is eluted and lyophilized with water. Secondly, prepare a reaction solution in a total volume of 26.5 uL as the determination system for enzyme activity, containing 1 mM isotope-labelled ManNAc-6-P, 37 mM Tris-HCl, pH 8.0, and 19 mM MgCl.sub.2. Incubate at 37° C. for 30 min, heat the reaction solution to boil for 3 min, then add 0.1 volume of alkaline phosphatase buffer to adjust pH and 20 U of alkaline phosphatase. Incubate at 37° C. for 1 h, draw a sample and add on a dry chromatography paper, and presoak with 1% sodium tetraborate. The used solvent system is a mixture of ethyl acetate:isopropanol:pyridine:water (50:22:14:14). Separate radioactive compounds by paper chromatography. Measure the radioactive intensity by a liquid scintillation counter, and calculate the activity units of N-Acetyl-D-Mannosamine-6-P Epimerase, based on the amount of ManNAc-6-P transformed into GlcNAc-6-P.
(528) The results show that: The enzyme activity of the mutant strain with maximum activity is 72 IU/mL, and the enzyme activity of the reference is 9.5 IU/mL. Transform NanE by error-prone PCR, to obtain a mutant strain with enzyme activity increased greatly. Pick up the mutant strain with maximum enzyme activity and extract plasmids for sequencing. The results show that: The mutant gene sequence of N-Acetyl-D-Mannosamine-6-P Epimerase is shown as SEQ ID No.56, and the corresponding amino acid sequence is shown as SEQ ID No.57. Through gene sequence alignment with the wild type of N-Acetyl-D-Mannosamine-6-P Epimerase, 3 base point mutations occur in total: 198C/T, 397T/C, and 559T/C. There occur 2 missense mutations of amino acids, of which the mutation points are: Q133R (cysteine at Site 133 is replaced by arginine), and Y187H (tyrosine at Site 187 is replaced by histidine). The mutant gene is named as nanEM.
(529) 4. Integration of pTrc-nanEM Gene Cassette into the nagE Gene Site in the Chromosome of Escherichia coli
(530) Using the nagE gene site 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 amplify the nanEM segment (i.e. pTrc-nanEM) of Trc promoter, as well as the kanamycin resistance gene segment with FLP recognition site (FRT site) at its both ends: FRT-Kanr-FRT (fKanrf), and splice them. Then design homologous primers for deletion of the nagE gene sequence, and using the spliced segment of pTrc-nanEM and fKanrf as template, amplify the linear DNA full-length segment for targeting of Red recombination.
(531) The specific operating process is provided below:
(532) (1) PCR Amplification of pTrc-nanEM Segment
(533) Template: nanEM/pTrc99A.
(534) Primer Design: Forward primer (Trcff-F) SEQ ID No.20, and reverse primer (Trcff-R) SEQ ID No.21.
(535) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(536) Product Size: 0.86 kb.
(537) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(538) (2) PCR Amplification of fKanrf Segment
(539) Primer Design: Forward primer (mfKanf-F) SEQ ID No.1, and reverse primer (mfKanf-R) SEQ ID No.2.
(540) Template: pPic9K.
(541) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(542) fKanrf size: 1.28 kb. Its nucleotide sequence is SEQ ID No.3.
(543) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(544) (3) Amplification of fKanrf Spliced with pTrc-nanEM
(545) Primer Design: Forward primer (fKanf-F) SEQ ID No.22, and reverse primer (fKanf-R) SEQ ID No.23.
(546) Template: fKanrf.
(547) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(548) fKanrf size from secondary amplification: 1.3 kb.
(549) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(550) (4) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(551) 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.
(552) Template: Mix pTrc-nanEM PCR segment, and fKanrf PCR segment from secondary amplification in the ratio of 1:1.
(553) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(554) Amplification Product: Homologous Arm+pTrc-nanEM-fKanrf+Homologous Arm
(555) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(556) (5) Red Recombination Operation
(557) First, pKD46 carrier is introduced into the AT-004-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(558) 1) Transformation of pKD46 Plasmid
(559) {circle around (1)} Preparation of Competence: First, inoculate 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-cultivate at 37° C. and 225 rpm for 2-3 h. Then add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation.
(560) {circle around (2)} Plasmid Transformation: Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of pKD46 plasmid, and cultivate at −4° C. for 30 min. Then heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h. Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate. Cultivate overnight (for 12-16 h) at 30° C. Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification. Store the positive strain for use.
(561) 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone
(562) Preparation of Electrotransformed Competence: Inoculate the AT-004-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultivate at 250 rpm, on the next day, inoculate in a ratio of 1% into LB medium containing Amp, and cultivate at 30° C.; when OD600 reaches approximately 0.2, add 0.2% L-Arabinose, and induce at 30° C. for 35 min until OD.sub.600 reaches approximately 0.4. Cool on an ice bath. Wash once with ultrapure water, wash twice with 10% glycerin, and finally resuspend with 10% glycerin; the amount of glycerin used is to produce a final concentration of the thalli concentrated by 500-1000 folds.
(563) {circle around (2)} Transformation by electric shock: Take out a 2-mm electrotransformation cup from 70% ethanol, wash with sterilized ultrapure water, and irradiate by UV lamp for 30 min. Precool at 4° C. for 30 min. Transfer 90 μL of finally resuspended cells to a precooled centrifuge tube, add 5 μL (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (4), suction gently by a gun, and maintain on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 200Ω, 25 μF.
(564) {circle around (3)} Resuscitate and screen positive clones: add 1 mL of LB broth medium, and cultivate at 37° C. and 100 rpm for 1 h. Then smear one kanamycin (Kan) plate with every 200 μL, 5 plates in total. Smear evenly and allow to air-dry. Cultivate at 30° C. for 24 h. Pick up clones grown under kanamycin resistance, and carry out PCR identification to screen positive clones.
(565) No. of the obtained strain: AT-031-01 (AT-004-02, nagE::pTrc-nanEM-fKanrf).
(566) As described above, prepare the strain AT-030-01 (AT-004-02, nagE::pTrc-nanE-fKanrf).
(567) (6) Removal of the Resistance Gene
(568) Introduce pCP20 into the above kanamycin-resistant clones, cultivate at 30° C. for 8 h, then increase to 42° C. and cultivate overnight, and thermally induce to express FLP recombinase; the plasmids are lost gradually. Streak the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, pick up grown monoclones and dot on the kanamycin-resistant plate; those that cannot grown are clones of which the kanamycin resistance gene has been removed by FLP recombination. Carry out PCR with identification primer to identify clones losing kanamycin resistance.
(569) No. of the obtained strain: AT-031-02 (AT-004-02, nagE::pTrc-nanEM).
(570) As described above, prepare the strain AT-030-02 (AT-004-02, nagE::pTrc-nanE).
(571) 5. Influence of the Integration of pTrc-nanE and pTrc-nanKM Gene Cassettes on the Output of N-Acetyl-D-Glucosamine
(572) Carry out a shake-flask fermentation trial with the recombinant strains AT-030-02 and AT-031-02, of which the nagE gene site in the chromosome is integrated with pTrc-nanE and pTrc-nanEM gene cassettes, and the reference strain. Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(573) See Table 17 for the output from shake-flask fermentation. The results show that: The output by the reference strain AT-005-02 is very low and is not detected, while the output by the recombinant strain AT-031-02 integrated with pTrc-nanEM gene cassette is increased obviously, and is also increased significantly than that by the unmutant reference strain AT-030-02.
(574) TABLE-US-00018 TABLE 17 Output from Shake-flask Fermentation by the Recombinant Strain integrated with pTrc-nanEM Gene Cassette Output of N-Acetyl-D-Glucosamine species (g/L) AT-005-02 (AT-004-02, nagE) Not detected (Reference) AT-030-02 (AT-004-02,
nagE::pTrc-nanE) 2.6 ± 0.5 AT-031-02 5.9 ± 0.8 (AT-004-02,
nagE::pTrc-nanEM)
(575) The above results show that: The output of N-Acetyl-D-Glucosamine may be increased obviously by overexpression of N-Acetyl-D-Mannosamine-6-P Epimerase; moreover, the output of N-Acetyl-D-Glucosamine may also be greatly by the mutant screened by error-prone PCR technology, due to increased activities of the obtained mutant of the epimerase.
EXAMPLE 14
(576) This implementation example describes screening for a gene mutant of UDP-N-AcetylGlucosamine-2-Epimerase (WecB); the said gene encodes UDP-N-AcetylGlucosamine-2-Epimerase (WecB) with increased activities.
(577) To further increase synthetic quantity of N-Acetyl-D-Glucosamine by the production strain, screen a gene mutant encoding UDP-N-AcetylGlucosamine-2-Epimerase with increased activities. To achieve the purpose, amplify the cloned gene by error-prone PCR technology; used for amplified DNA polymerase, amplify the said gene under conditions leading to high-frequency mismatch, so as to obtain a high-frequency mutation in PCR products.
(578) The specific operating process is provided below:
(579) 1. Amplification of UDP-N-AcetylGlucosamine-2-Epimerase Gene wecB by Error-Prone PCR.
(580) By means of Taq DNA polymerase without the property of 3′-5′ proofreading function, control the frequency of random mutation under high magnesium ion concentration (8 mmol/L) and different dNTP concentrations (where, the concentration of dATP and dGTP is 1.5 mmol/L; and the concentration of dTTP and dCTP is 3.0 mmol/L), introduce random mutations into the target gene, and construct a mutant library; the template concentration A260 value is 1000 ng/mL, the enzyme concentration is 5 U/μL, and the primer concentration is 100 μM.
(581) Error-prone PCR reaction system (50 μL): 10×PCR reaction buffer 5 μl, dNTP (2.5 mM) 5 μL, MgCl.sub.2 (2.5 mM) 5 μL, Forward primer (TrcwecB-F, SEQ ID No.51) 1 μL, reverse primer (TrcwecB-R, SEQ ID No.52) 1 μL, DNA template (wecB/pUC57) 0.1 μL, Taq DNA polymerase 0.5 μL, and ddH.sub.2O 32.4 μL.
(582) PCR procedure: Predegenerate at 96° C. for 4 min; degenerate at 94° C. for 1 min, anneal at 56° C. for 1 min, extend at 75° C. for 2 min, and repeat for 45 cycles; finally extend at 75° C. for 15 min, recover PCR product (product size: 1.13 kb) by gel recovery method; transfer 5 μL of the product and carry out 1% agarose gel electrophoresis test; the product is store at −20° C. for use.
(583) 2. Construction of the Gene Mutant Library of
(584) UDP-N-AcetylGlucosamine-2-Epimerase
(585) Digest the above PCR product by two enzymes of restriction endonuclease Nco I and Hind III, carry out a ligation reaction with pTrc99A digested by Nco I and Hind III, then transform Escherichia coli AT-005-02 with the mixture of the ligated products to obtain a large amount of cloned transformants, and construct a mutant library of transformed thalli.
(586) 3. Screening for Mutants with High Enzyme Activities
(587) Using the wild type WecB/pTrc99A (AT-005-02) as reference, pick up randomly 640 mutant clones from the mutant library of transformed thalli, inoculate into 5 mL of LB medium containing 50 μg/mL Ampicillin (Amp), shake-cultivate at 37° C. and 150 rpm for 18 h, and then centrifugate at 10000 rpm for 5 mim and collect thalli. Discard the supernatant, then resuspend at 4° C. in 1 mL of PBS solution (pH 7.5, 10 mmol/L), carry out ultrasonicate at a voltage of 300 V for 10 min (ultrasonicate for 3 s and pause for 6 s), centrifugate, transfer the supernatant as crude extract of enzyme, and carry out a method for determination of enzyme activity.
(588) Determination of UDP-N-AcetylGlucosamine-2-Epimerase Activity: Based on the amount of UDP-N-Acetyl-D-Glucosamine transformed into N-Acetyl-D-Mannosamine. That is to say, the reduced amount of UDP-N-Acetyl-D-Glucosamine is used as test marker. Definition of Enzyme Activity Unit: Under the enzymatic reaction conditions, the enzyme amount reduced, equivalent to 1 μmol UDP-N-Acetyl-D-Glucosamine per minute, is defined as one enzyme activity unit (IU). The specific procedure is provided as follows: Transfer 20 mL of the reaction system as the system for determination of enzyme activity, which contains 45 mmol/L phosphate buffer (pH7.5), 10 mM MgCl2 and 100 nCi of UDPGlcNAc, and 5 mg crude enzyme solution. Carry out the enzyme activity reaction by incubating on a water bath at 37° C. for 30 min. Add alcohol to stop the reaction. Separate radioactive compounds by paper chromatography. Measure the radioactive intensity by a liquid scintillation counter. The solvent system used is a mixture of n-propanol:1M sodium acetate, pH 5.0:water (7:1:2). Calculate the activity units of UDP-N-AcetylGlucosamine-2-Epimerase, based on the amount of UDPGlcNAc transformed into ManNAc.
(589) The results show that: The enzyme activity of the mutant strain with maximum activity is 653 IU/mL, and the enzyme activity of the reference is 21.0 IU/mL. Transform WecB by error-prone PCR, to obtain a mutant strain with enzyme activity increased greatly. Pick up the mutant strain with maximum enzyme activity and extract plasmids for sequencing. The results show that: The mutant gene sequence of UDP-N-AcetylGlucosamine-2-Epimerase is shown as SEQ ID No.58, and the corresponding amino acid sequence is shown as SEQ ID No.59. Compared with gene sequence of the wild type UDP-N-AcetylGlucosamine-2-Epimerase, 5 base point mutations occur in total: 101G/C, 433C/G, 677G/T, 734T/G, and 1038T/C; There occur 4 missense mutations of amino acids, of which the mutation points are: C34S (cysteine at Site 34 is replaced by serine), H145D (histidine at Site 145 is replaced by aspartate), C226F (cysteine at Site 226 is replaced by phenylalanine), and V245G (valine at Site 245 is replaced by glycine). The mutant gene is named as wecBM.
(590) 4. Integration of pTrc-wecBM Gene Cassette into the nagE Gene Site in the Chromosome of Escherichia coli
(591) Using the nagE gene site 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 amplify the wecBM segment (i.e. pTrc-wecBM) of Trc promoter, as well as the kanamycin resistance gene segment with FLP recognition site (FRT site) at its both ends: FRT-Kanr-FRT (fKanrf), and splice them. Then design homologous primers for deletion of the nagE gene sequence, and using the spliced segment of pTrc-wecBM and fKanrf as template, amplify the linear DNA full-length segment for targeting of Red recombination.
(592) The specific operating process is provided below:
(593) (1) PCR Amplification of pTrc-wecBM Segment
(594) Template: wecBM/pTrc99A.
(595) Primer Design: Forward primer (Trcff-F) SEQ ID No.20, and reverse primer (Trcff-R) SEQ ID No.21.
(596) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(597) Product Size: 1.3 kb.
(598) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(599) (2) PCR Amplification of fKanrf Segment
(600) Primer Design: Forward primer (mfKanf-F) SEQ ID No.1, and reverse primer (mfKanf-R) SEQ ID No.2.
(601) Template: pPic9K.
(602) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(603) fKanrf size: 1.28 kb. Its nucleotide sequence is SEQ ID No.3.
(604) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(605) (3) Amplification of fKanrf Spliced with pTrc-wecBM
(606) Primer Design: Forward primer (fKanf-F) SEQ ID No.22, and reverse primer (fKanf-R) SEQ ID No.23.
(607) Template: fKanrf.
(608) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(609) fKanrf size from secondary amplification: 1.3 kb.
(610) The PCR product is separated by 1% agarose gel electrophoresis, and purified to recover the segment.
(611) (4) Preparation of Linear DNA Full-Length PCR Segment for Targeting of Red Recombination
(612) 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.
(613) Template: Mix pTrc-wecBM PCR segment, and fKanrf PCR segment from secondary amplification in the ratio of 1:1.
(614) PCR reaction conditions: Step 1: Degenerate at 94° C. for 1 min; Step 2: Incubate at 94° C. for 30 s, at 55° C. for 30 s, and at 72° C. for 40 s, and carry out 30 cycles; Step 3: Extend at 72° C. for 10 min.
(615) Amplification Product: Homologous Arm+pTrc-wecBM-fKanrf+Homologous Arm
(616) The PCR product is separated by agarose gel electrophoresis, and purified and recovered to obtain 100 ng/μL linear DNA full-length PCR segment for targeting of Red recombination.
(617) (5) Red Recombination Operation
(618) First, pKD46 carrier is introduced into the AT-004-02 strain of Escherichia coli. Then, the prepared linear DNA segment for targeting is electrotransformed, and positive clones are screened. Finally, the resistance gene is removed.
(619) 1) Transformation of pKD46 Plasmid
(620) {circle around (1)} Preparation of Competence: First, inoculate 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-cultivate at 37° C. and 225 rpm for 2-3 h. Then add the culture solution to a 10-mL centrifuge tube, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL 0.1M CaCl.sub.2 on an ice bath for 5 min. Finally, centrifugate at 4000 g×5 min, discard the supernatant, and suspend with 5 mL of 0.1M CaCl.sub.2 on an ice bath. Allow to stand at −4° C. for 12 h for spontaneous sedimentation.
(621) {circle around (2)} Plasmid Transformation: Transfer 250 μL of spontaneously sedimented thalli, add 5 μL of pKD46 plasmid, and cultivate at −4° C. for 30 min. Then heat on a water bath at 42° C. for 1.5 min, add 0.7 mL of SOC medium, and shake at 30° C. for 2 h. Transfer 0.2 mL of the bacterial suspension, and smear on a penicillin plate. Cultivate overnight (for 12-16 h) at 30° C. Pick up monoclone, add 5 mL of LB broth medium and cultivate, and withdraw plasmid for identification. Store the positive strain for use.
(622) 2) Electrotransform Linear DNA Segment for Targeting, and Screen Positive Clone
(623) {circle around (1)} Preparation of Electrotransformed Competence: Inoculate the AT-004-02 strain of Escherichia coli, containing pKD46, into a test tube of LB medium containing Ampicillin (Amp), and shake-cultivate at 250 rpm, on the next day, inoculate in a ratio of 1% into LB medium containing Amp, and cultivate at 30° C.; when OD.sub.600 reaches approximately 0.2, add 0.2% L-Arabinose, and induce at 30° C. for 35 min until OD.sub.600 reaches approximately 0.4. Cool on an ice bath. Wash once with ultrapure water, wash twice with 10% glycerin, and finally resuspend with 10% glycerin; the amount of glycerin used is to produce a final concentration of the thalli concentrated by 500-1000 folds.
(624) {circle around (2)} Transformation by electric shock: Take out a 2-mm electrotransformation cup from 70% ethanol, wash with sterilized ultrapure water, and irradiate by UV lamp for 30 min. Precool at 4° C. for 30 min. Transfer 90 μL of finally resuspended cells to a precooled centrifuge tube, add 5 μL (more than 100 ng) of the full-length PCR segment (linear DNA) obtained in Step (4), suction gently by a gun, and maintain on an ice bath for 30 min. Electrotransformation Parameters: 2500V, 200Ω, 25 μF.
(625) {circle around (3)} Resuscitate and screen positive clones: add 1 mL of LB broth medium, and cultivate at 37° C. and 100 rpm for 1 h. Then smear one kanamycin (Kan) plate with every 200 μL, 5 plates in total. Smear evenly and allow to air-dry. Cultivate at 30° C. for 24 h. Pick up clones grown under kanamycin resistance, and carry out PCR identification to screen positive clones.
(626) No. of the obtained strain: AT-043-01 (AT-004-02, nagE::pTrc-wecBM-fKanrf).
(627) As described above, prepare the strain AT-042-01 (AT-004-02, nagE::pTrc-wecBM-fKanrf).
(628) (6) Removal of the Resistance Gene
(629) Introduce pCP20 into the above kanamycin-resistant clones, cultivate at 30° C. for 8 h, then increase to 42° C. and cultivate overnight, and thermally induce to express FLP recombinase; the plasmids are lost gradually. Streak the plate of antibiotics-free culture medium by an inoculating loop dipped in the bacterial suspension, pick up grown monoclones and dot on the kanamycin-resistant plate; those that cannot grown are clones of which the kanamycin resistance gene has been removed by FLP recombination. Carry out PCR with identification primer to identify clones losing kanamycin resistance.
(630) No. of the obtained strain: AT-043-02 (AT-004-02, nagE::pTrc-wecBM).
(631) As described above, prepare the strain AT-042-02 (AT-004-02, nagE::pTrc-wecBM-fKanrf).
(632) 5. Influence of the Integration of pTrc-wecB and pTrc-wecBM Gene Cassettes on the Output of N-Acetyl-D-Glucosamine
(633) Carry out a shake-flask fermentation trial with the recombinant strains AT-042-02 and AT-043-02, of which the nagE gene site in the chromosome is integrated with pTrc-wecB and pTrc-wecBM gene cassettes, and the reference strain. Transfer the monoclonal strain freshly cultivated in culture medium of the LB plate, inoculate into a test tube (13×150 mm) containing 3 mL of the LB broth culture medium, and shake-cultivate at 30° C. for 8 h. Then transfer the seed culture solution, inoculate in 3% into a 250-mL shake-flask containing 50 mL of the fermentation culture solution (M9 culture solution). The initial OD.sub.600 is approximately 0.5; shake-cultivate at 37° C. and 225 rpm. The fermentation cycle is 72 h. At 24 h and 48 h, adjust to pH 7.0 with 10M NaOH. According to glucose consumption in the fermentation broth, add 65% glucose solution to maintain a glucose concentration of 20 g/L. After completion of fermentation, transfer 1 mL of the fermentation broth and centrifugate. Measure the content of N-Acetyl-D-Glucosamine by HPLC method.
(634) See Table 18 for the output from shake-flask fermentation. The results show that: The output by the reference strain AT-005-02 is very low and is not detected, while the output by the recombinant strain AT-043-02 integrated with pTrc-wecBM gene cassette is increased obviously, and is also increased significantly than that by the unmutant reference strain AT-042-02.
(635) TABLE-US-00019 TABLE 18 Output from Shake-flask Fermentation by the Recombinant Strain integrated with pTrc-wecBM Gene Cassette Output of N-Acetyl-D-Glucosamine species (g/L) AT-005-02 (AT-004-02, nagE) Not detected (Reference) AT-042-02 7.1 ± 0.8 (AT-004-02,
nagE::pTrc-wecB) AT-043-02 (AT-004-02,
nagE::pTrc-wecBM) 10.9 ± 0.9
(636) The above results show that: The output of N-Acetyl-D-Glucosamine may be increased obviously by overexpression of UDP-N-AcetylGlucosamine-2-Epimerase; moreover, the output of N-Acetyl-D-Glucosamine may also be greatly by the mutant screened by error-prone PCR technology, due to increased activities of the obtained mutant of the epimerase.
(637) Although this Invention is described detailedly by common explanations and specific implementation schemes, it may be revised or improved on top of this Invention; this point is as plain as the nose on your face for a technician in this field. Therefore, such revisions or improvements that are not deviated from the spirits of this Invention still fall in the range under protection required by this Invention.