CN104195094A - Bacillus subtilis for producing N-acetylglucosamine as well as construction method and application of bacillus subtilis - Google Patents
Bacillus subtilis for producing N-acetylglucosamine as well as construction method and application of bacillus subtilis Download PDFInfo
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- CN104195094A CN104195094A CN201410376584.2A CN201410376584A CN104195094A CN 104195094 A CN104195094 A CN 104195094A CN 201410376584 A CN201410376584 A CN 201410376584A CN 104195094 A CN104195094 A CN 104195094A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
Abstract
The invention belongs to the construction and the application of genetically engineered bacterium and particularly relates to bacillus subtilis for producing N-acetylglucosamine as well as a construction method and application of bacillus subtilis. The construction method for the genetically engineered bacterium (bacillus subtilis) comprises the following steps: expressing and coding 6-glucosamine phosphate synthetase gene and 6-glucosamine phosphate acetylase gene in bacillus subtilis to form a complete metabolic pathway from glucose to N-acetylglucosamine; knocking out or inactivating glucosamine 6-phosphate deaminase genes nagB and gamA, an N-acetylglucosamine 6-phosphate deacetylase gene nagA, a glucosamine transport protein gene gamP and an N-acetylglucosamine transport protein gene nagP in an N-acetylglucosamine catabolism pathway in bacillus subtilis. The construction method can be used for solving the problem that N-acetylglucosamine produced in the prior art is poor in safety, low in yield and high in cost. The constructed genetically engineered bacterium has the advantage that N-acetylglucosamine with relatively high concentration can be accumulated, and the industrial utilization value is relatively high.
Description
Technical field
The invention belongs to the construction and application of genetic engineering bacterium, refer to especially a kind of subtilis and construction process and application of producing 2-Acetamido-2-deoxy-D-glucose.
Background technology
2-Acetamido-2-deoxy-D-glucose is the derivative of glucose, and conventionally with beta-1,4-glycosidic link aggregates into chitin.Chitin be occurring in nature next in number only to cellulosic second largest class carbohydrate, be extensively present in mushroom, algae, the shell of shrimp, crab, insect and the cell walls of higher plant etc., therefore, 2-Acetamido-2-deoxy-D-glucose is huge in natural storage.
2-Acetamido-2-deoxy-D-glucose and D-Glucose aldehydic acid form the disaccharide unit repeating and form hyaluronic acid, and hyaluronic acid is the key substance of closing internode lubricate, has important provide protection for osteoarthrosis.Experiment has proved that 2-Acetamido-2-deoxy-D-glucose can be used as treatment and prevents the active drug of middle-aged and old joint diseases, and experimental results show that even heavy dose of 2-Acetamido-2-deoxy-D-glucose is also without any side effects to human body, be a kind of safe and reliable healthcare products and medicine.2-Acetamido-2-deoxy-D-glucose also can produce glucosamine by enzymic hydrolysis or acid hydrolysis, and the latter is also a kind of middle-aged and old joint care product of being used widely, and market is huge.
The production of 2-Acetamido-2-deoxy-D-glucose is mainly to utilize glucosamine and diacetyl oxide to form through chemical condensation reaction at present, raw material glucosamine mainly obtains by acid hydrolysis chitin, this process is accompanied by the discharge of a large amount of acid-bearing wastewaters, and because the chitinous raw material supply of shrimp and crab shells is subject to the restriction of season and place of production factor, cause final 2-Acetamido-2-deoxy-D-glucose production cost too high.Therefore, carried out the work that utilizes Production by Microorganism Fermentation 2-Acetamido-2-deoxy-D-glucose both at home and abroad.
Patent WO2004003175 discloses the colibacillary construction process of a series of metabolic engineerings in (patent families comprises US7332304 and CN101365785 etc.), after fermentation, 2-Acetamido-2-deoxy-D-glucose output is up to 110g/L, patent CN102268399 and CN102286420 disclose two kinds and have built the method for colibacillus engineering, and reach 70g/L by fermentation 2-Acetamido-2-deoxy-D-glucose output.Above-mentioned patent is all the genetic engineering bacterium that obtains energy high yield 2-Acetamido-2-deoxy-D-glucose by genetic modification intestinal bacteria, but intestinal bacteria can produce intracellular toxin during the fermentation, this is restricted the application of its leavened prod in healthcare products and food, therefore adopts the microorganisms producing glucosamine of safety non-toxic or 2-Acetamido-2-deoxy-D-glucose to become industrial needs.
Patent CN103045527A, the Bacillus subtilus engineering bacteria construction process of a series of product 2-Acetamido-2-deoxy-D-glucoses is disclosed in CN102978149A and CN103060252A, built the subtilis of several strains product 2-Acetamido-2-deoxy-D-glucoses by genetic modification, after fermentation, output reaches respectively 115mg/L, 415mg/L and 1.23g/L, tentatively solve the problem of fermentative Production 2-Acetamido-2-deoxy-D-glucose security, but the highest of above fermentation yield reaches 1.23g/L, the too low production cost that causes of output increases, and is unfavorable for the application of suitability for industrialized production.
Summary of the invention
One of object of the present invention is to provide a kind of subtilis and construction process and application of producing 2-Acetamido-2-deoxy-D-glucose, utilize the genetically engineered subtilis of constructed high yield 2-Acetamido-2-deoxy-D-glucose, by biological fermentation engineering, reaching production can be in the object of the high security 2-Acetamido-2-deoxy-D-glucose of field of health care products application.
Overall technology design of the present invention is:
A subtilis Bacillus subtilis who produces 2-Acetamido-2-deoxy-D-glucose, its deposit number is CCTCC M 2014342.
Above-mentioned bacterial classification applicant submits on July 16th, 2014 the Chinese Typical Representative culture collection center preservation that is positioned at Wuhan City, Hubei Province Wuhan University to, and depositary institution is referred to as CCTCC.
Produce the construction process of the subtilis of 2-Acetamido-2-deoxy-D-glucose; to introduce the high efficient expression of subtilis Host Strains by the 6-phosphorylated amino glucose synthetic enzyme of encoding, 6-phosphorylated amino glucose acetylase, and knock out or inactivation subtilis in 6-phosphorylated amino glucose deaminase gene nagB in 2-Acetamido-2-deoxy-D-glucose catabolic pathway and gamA, 6-phosphoric acid-2-Acetamido-2-deoxy-D-glucose deacetylase gene nagA, glucosamine transporter gene gamP and 2-Acetamido-2-deoxy-D-glucose transporter gene nagP build and form.
Described 6-phosphorylated amino glucose synthase gene and 6-phosphorylated amino glucose acetylase gene are introduced subtilis overexpression; after can these two genes being cloned into respectively on expression vector; with mode tandem expression in subtilis of plasmid, in the present invention, expression vector is selected pHT01 carrier.
Knocking out of described nagB, nagA, nagP, gamA and gamP gene is to complete by homologous recombination system.In five genes, nagA and nagB interlocked arrangement on bacillus subtilis chromosome, can disposablely knock out, and gamA and gamP interlocked arrangement, can disposablely knock out.Therefore, knocking out of above-mentioned 5 genes can complete by three experimental implementation, also can complete respectively by five experiments.The gene knockout work of subtilis can complete by pMAD carrier and molecular biology experiment.
The application of the subtilis of production 2-Acetamido-2-deoxy-D-glucose in 2-Acetamido-2-deoxy-D-glucose, the aseptic culture medium that the subtilis CCTCC M 2014342 that produces 2-Acetamido-2-deoxy-D-glucose is seeded to taking glucose as carbon source, aerobic fermentation 70 hours under 37 DEG C of conditions.
Concrete technical conceive of the present invention also has:
Described construction process comprises following processing step:
A, knock out nagAB gene cluster, gamAP gene cluster and the nagP gene in subtilis respectively, obtain genetic engineering bacterium host;
B, structure GNA1 and glmS dual-expression vector;
C, dual-expression vector is proceeded to 1) gained Host Strains, obtain the subtilis of final production 2-Acetamido-2-deoxy-D-glucose.
Described 6-phosphorylated amino glucose synthase gene derives from subtilis or other and has the microorganism of identical function enzyme.
The reaction of the synthetic 6-phosphorylated amino glucose of described 6-phosphorylated amino glucose synthetic enzyme catalysis fructose-1, 6-diphosphate and glutamine, this enzyme is subject to the feedback inhibition of reaction product 6-phosphorylated amino glucose, therefore this reaction is the rate-limiting step in 2-Acetamido-2-deoxy-D-glucose route of synthesis, high expression level 6-phosphorylated amino glucose synthetic enzyme contributes to increase the supply of intermediate product 6-phosphorylated amino glucose, thereby can increase the concentration of end product 2-Acetamido-2-deoxy-D-glucose.Described enzyme can derive from subtilis, the acquisition of gene can be synthetic according to the full gene of glmS gene order in subtilis 168 strain gene group sequence GenBank No.NC_000964, or the genomic dna that utilizes subtilis 168 bacterial strains obtains by pcr amplification for template.
Described 6-phosphorylated amino glucose acetylase gene source has the microorganism of identical function enzyme in yeast saccharomyces cerevisiae or other.
Described 6-phosphorylated amino glucose acetylase catalysis 6-phosphorylated amino glucose and acetyl-CoA synthesize 6-phosphoric acid-2-Acetamido-2-deoxy-D-glucose; this enzyme does not exist in subtilis; introduce and high expression level by genetically engineered, thereby realize the object of synthesizing 2-Acetamido-2-deoxy-D-glucose.This enzyme source is in yeast saccharomyces cerevisiae, and the acquisition of gene can be according to GenBank No.NM_001179949 sequence, obtains through full gene is synthetic, or utilizes Saccharomyces Cerevisiae in S 288c strain gene group DNA to obtain by pcr amplification for template.
Described knock out or inactivation subtilis in 6-phosphorylated amino glucose deaminase gene nagB and gamA in 2-Acetamido-2-deoxy-D-glucose catabolic pathway carry out simultaneously.
The substantive distinguishing features that the present invention possesses and the remarkable technical progress obtaining are:
The present invention by carrying out genetically engineered operation in subtilis, build a new metabolic pathway from glucose to 2-Acetamido-2-deoxy-D-glucose, strengthen the rate-limiting enzyme genetic expression in 2-Acetamido-2-deoxy-D-glucose route of synthesis, simultaneously, knock out the gene that causes 2-Acetamido-2-deoxy-D-glucose consumption and backflow, stop backflow and the consumption of 2-Acetamido-2-deoxy-D-glucose, compared to existing technology, the 2-Acetamido-2-deoxy-D-glucose that the engineering strain that the present invention builds can accumulate greater concn (after testing, in fermentation end product, 2-Acetamido-2-deoxy-D-glucose concentration can reach 38.3g/L), having higher industrialized utilization is worth.
The microorganism adopting in the present invention is submitted the center preservation of Chinese Typical Representative culture collection on July 16th, 2014, and depositary institution is referred to as CCTCC.
Brief description of the drawings
Fig. 1 is the associated metabolic approach of producing 2-Acetamido-2-deoxy-D-glucose engineering strain in the present invention.
Embodiment
Below in conjunction with accompanying drawing, embodiments of the invention are described further; but not as a limitation of the invention; the content that protection scope of the present invention is recorded with claim is as the criterion, and any equivalence techniques means of making according to specification sheets are replaced, and all do not depart from protection scope of the present invention.
The plasmid extraction that uses in the present embodiment, genome extracting, PCR reagent etc. adopt commerical prod, and concrete operations are carried out to specifications.Other not marked experimental implementation are carried out according to " the molecular cloning experiment guide third edition " (the yellow training hall of (U.S.) J. Sha nurse Brooker is translated) working method.
Subtilis experimental implementation and pMAD gene knockout principle and method are shown in document (Maryvonne Arnaud et al, New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov.2004, p.6887 – 6891).
Subtilis electricity method for transformation reference literature Meddeb Mouelhi F et al, High transformation efficiency of Bacillus subtilis with integrative DNA using glycine betaine as osmoprotectant, Anal Biochem.2012 May 15; 424 (2): 127-9.
Embodiment 1
One, knock out nagAB gene cluster in subtilis 168 bacterial strains
1, according to subtilis 168 strain gene groups (Genbank No.NC_000964) sequence, design primer: upstream primer F-nagAB-up-BamHI:CT
gGATCCgACTGCAAGATTTCGGCCTGGG (shown in SEQ ID NO.1) and downstream primer R-nagAB:CATAAGTCAGCATGTTCCTTTCACATAGATGATCCGCCTTTCTGG (shown in SEQ ID NO.2).Taking subtilis 168 strain gene group DNAs template, obtain nagAB gene cluster upstream 1000bp fragment through pcr amplification.
2, according to subtilis 168 strain gene groups (Genbank No.NC_000964) sequence, design primer: upstream primer F-nagAB:CCAGAAAGGCGGATCATCTATGTGAAAGGAACATGCTGACTTATG (shown in SEQ ID NO.3) and downstream primer R-nagAB-down-NcoI:GCT
cCATGGtAACGTATATACCAATGAAGAG (shown in SEQ ID NO.4).Taking subtilis 168 strain gene group DNAs template, obtain nagAB gene cluster downstream 1000bp fragment through pcr amplification.
3, will after above-mentioned two kinds of PCR product purifications, respectively get 5ul as template, do overlap pcr amplification with primers F-nagAB-up-BamHI (shown in SEQ ID NO.1) and R-nagAB-down-NcoI (shown in SEQ ID NO.4), obtain the each 1000bp of upstream and downstream in nagAB gene cluster, and intercalary deletion the DNA fragmentation of nagAB coding region.
4, the above-mentioned fragment of purifying, utilizes conventional molecule clone technology to be inserted into carrier pMAD, obtains knockout carrier pMAD-Δ nagAB;
5, pMAD-Δ nagAB plasmid is transformed into subtilis 168 bacterial strains, X-gal (the chloro-3-indoles-β-D-of the bromo-4-of 5-galactoside) and erythromycin flat board that coating contains 50ug/mL, 30 degree incubated overnight;
6, the aobvious blue transformant of picking, 30 spend incubated overnight, are forwarded to the LB substratum of 5mL erythromycin resistance by 5% inoculum size, and 30 degree are hatched 2 hours, are warming up to 42 degree and continue to hatch 6 hours, dilution (10
-2-10
-5) be coated with the flat board that contains X-gal and erythromycin, 42 spend incubated overnight;
7, the blue single bacterium colony of picking is hatched 6 hours in 30 degree and non-resistant LB substratum, is warming up to 42 degree and continues to hatch 3 hours, dilution (10
-2-10
-5) be coated with the non-resistant LB flat board that contains X-gal, 42 spend incubated overnight;
8, single bacterium colony of picking whitening spot is respectively in erythromycin and nonreactive LB substratum incubated overnight, select to do bacterium colony PCR checking without single bacterium colony of erythromycin resistance, Primer selection F-nagAB-up-BamHI (shown in SEQ ID NO.1) and R-nagAB-down-NcoI (shown in SEQ ID NO.4), can amplify the big or small bacterium colony for 2kb and be the positive bacterium colony Bs168/ Δ nagAB that has knocked out nagAB gene cluster.
Embodiment 2
Two, continue to knock out the gamAP gene cluster in subtilis
1, according to subtilis 168 strain gene groups (Genbank No.NC_000964) sequence, design primer: upstream primer F-gamAP-up-BamHI:CT
gGATCCaCTGCTCCCCACAGCACTTTTCC (shown in SEQ ID NO.5) and downstream primer R-gamAP:CGCAGCAGGGGGGACTTTTTTACATGTGACACCCCCTCAAAGAG (shown in SEQ ID NO.6).Taking subtilis 168 strain gene group DNAs template, obtain gamAP gene cluster upstream 1000bp fragment through pcr amplification.
2, according to subtilis 168 strain gene groups (Genbank No.NC_000964) sequence, design primer: upstream primer F-gamAP:CTCTTTGAGGGGGTGTCACATGTAAAAAAGTCCCCCCTGCTGCG (shown in SEQ ID NO.7) and downstream primer R-gamAP-down-NcoI:GCT
cCATGGaTACCACTCGTTTGGGACAGCC (shown in SEQ ID NO.8).Taking subtilis 168 strain gene group DNAs template, obtain gamAP gene cluster downstream 1000bp fragment through pcr amplification.
3, will after above-mentioned two kinds of PCR product purifications, respectively get 5ul as template, with primers F-gamAP-up-BamHI (shown in SEQ ID NO.5) and R-gamAP-down-NcoI (shown in SEQ ID NO.8) amplification, obtain the each 1000bp of upstream and downstream in gamAP gene cluster, and intercalary deletion the DNA fragmentation of gamAP coding region.
4, the above-mentioned fragment of purifying, utilizes conventional molecule clone technology to be inserted into carrier pMAD, obtains knockout carrier pMAD-Δ gamAP;
5, pMAD-Δ gamAP plasmid is transformed into the subtilis Bs168/ Δ nagAB bacterial strain in embodiment 1, adopt step similar to the above to screen positive bacterium colony, Primer selection F-gamAP-up-BamHI (shown in SEQ ID NO.5) and R-gamAP-down-NcoI (shown in SEQ ID NO.8), can amplify the big or small bacterium colony for 2kb and be the positive bacterium colony Bs168/ Δ nagAB/Δ gamAP that has knocked out gamAP gene.
Embodiment 3
Three, continue to knock out the nagP gene in subtilis
1, according to subtilis 168 strain gene groups (Genbank No.NC_000964) sequence, design primer: upstream primer F-nagP-up-BamHI:CT
gGATCCcAAGACCTCCTCGTACAGAATAATG (shown in SEQ ID NO.9) and downstream primer R-nagP:GGTTGCCCTCTCCGCTTTTTTACATACCCATCCCCCTCATACCC (shown in SEQ ID NO.10).Taking subtilis 168 strain gene group DNAs template, obtain nagP upstream region of gene 1000bp fragment through pcr amplification.
2, according to subtilis 168 strain gene groups (Genbank No.NC_000964) sequence, design primer: upstream primer F-nagP:GGGTATGAGGGGGATGGGTATGTAAAAAAGCGGAGAGGGCAACC (shown in SEQ ID NO.11) and downstream primer R-nagP-down-NcoI:GCT
cCATGGtTCCGGCGATTCTGAAGTCTAAG (shown in SEQ ID NO.12).Taking subtilis 168 strain gene group DNAs template, obtain nagP gene downstream 1000bp fragment through pcr amplification.
3, will after above-mentioned two kinds of PCR product purifications, respectively get 5ul as template, with primers F-nagP-up-BamHI (shown in SEQ ID NO.9) and R-nagP-down-NcoI (shown in SEQ ID NO.12) amplification, obtain the each 1000bp of upstream and downstream in nagP gene, and intercalary deletion the DNA fragmentation of nagP coding region.
4, the above-mentioned fragment of purifying, utilizes conventional molecule clone technology to be inserted into carrier pMAD, obtains knockout carrier pMAD-Δ nagP;
5, pMAD-Δ nagP plasmid is transformed into the subtilis Bs168/ Δ nagAB/ Δ gamAP bacterial strain in embodiment 2, adopt step similar to the above to screen positive bacterium colony, Primer selection F-nagP-up-BamHI (shown in SEQ ID NO.9) and R-nagP-down-NcoI (shown in SEQ ID NO.12), can amplify the big or small bacterium colony for 2kb and be the positive bacterium colony Bs168/ Δ nagAB/ Δ gamAP/ Δ nagP that has knocked out nagP gene.
Embodiment 4
Four, the structure of GNA1 and glmS Gene Double expression vector
1, according to the complete synthesis GNA1 gene of yeast saccharomyces cerevisiae GNA1 gene order GenBank No.NM_001179949, two ends add BamHI and XbaI site (as shown in underscore), concrete sequence (SEQ ID NO.13) as follows.
ggatccatgagcttacccgatggattttatataaggcgaatggaagagggggatttggaacaggtcactgagacgctaaaggttttgaccaccgtgggcactattacccccgaatccttcagcaaactcataaaatactggaatgaagccacagtatggaatgataacgaagataaaaaaataatgcaatataaccccatggtgattgtggacaagcgcaccgagacggttgccgctacggggaatatcatcatcgaaagaaagatcattcatgaactggggctatgtggccacatcgaggacattgcagtaaactccaagtatcagggccaaggtttgggcaagctcttgattgatcaattggtaactatcggctttgactacggttgttataagattattttagattgcgatgagaaaaatgtcaaattctatgaaaaatgtgggtttagcaacgcaggcgtggaaatgcaaattagaaaatag
tctaga
2, utilize conventional Protocols in Molecular Biology that GNA1 gene is connected on the BamHI and XbaI site of pHT01 carrier (MoBiTec company, Germany), obtain carrier pHT01-GNA1.
3, according to glmS primers in subtilis 168 strain gene groups (Genbank No.NC_000964) sequence, forward primer F-glmS-XbaI:CGC
tCTAGAgGAGGAAGAAAAATATGTGTG (shown in SEQ ID NO.14) and reverse primer R-glmS-AatII:GTA
gACGTCtTACTCCACAGTAACACTCTTCGC (shown in SEQ ID NO.15), the two ends in this primer have increased XbaI and AatII site;
4, taking total DNA of subtilis 168 bacterial strains as template, obtain the DNA fragmentation of glmS gene and upstream ribosome bind site thereof with above-mentioned primer (shown in SEQ ID NO.14-15) pcr amplification, by conventional clone technology, fragment is inserted between the XbaI and AatII site of the above-mentioned carrier pHT01-GNA1 building, obtained dual-expression vector pHT01-GNA1-glmS.
Embodiment 5
Five, the structure of final engineering bacteria
The subtilis Host Strains Bs168/ Δ nagAB/ Δ gamAP/ Δ nagP that the dual-expression vector pHT01-GNA1-glmS electricity transformation of above-mentioned structure is crossed, coating paraxin (10mg/L) LB flat board, 37 degree overnight incubation, obtain engineering strain Bs168/ Δ nagAB/ Δ gamAP/ Δ nagP/pHT01-GNA1-glmS, be the subtilis that can synthesize 2-Acetamido-2-deoxy-D-glucose, called after BsNAG01.
Embodiment 6
Six, engineering strain fermentation
1, seed and fermention medium:
Seed culture medium is LB substratum (composition is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L); When solid or liquid culture, add paraxin to final concentration 10mg/L.
Fermention medium is made up of following component: peptone 1-10g/L, and yeast powder 1-5g/L, dipotassium hydrogen phosphate 1-5g/L, potassium primary phosphate 1-8g/L, ammonium sulfate 1-5g/L, glucose 5-20g/L, surplus is water.Supplemented medium is that mass percent is the glucose solution of 40-60%.
2, fermenting process:
First incubated overnight seed culture medium, by the inoculum size of 2-5%, subtilis CCTCC M2014342 is forwarded to fermention medium, 33-37 DEG C of aerobic cultivation, pass through air flow, tank pressure and stirring keep dissolved oxygen to maintain more than 20%, with ammoniacal liquor control pH=6.8-7.0 supplementary nitrogenous source, initial glucose has consumed rear beginning and has added carbon source according to 3-6g/Lh speed, fermented liquid OD600 reaches sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) inducible protein of 15 rear interpolation 0.01-0.05mM to express, continue to be cultured to fermentation ends, sampling utilizes high performance liquid chromatography (HPLC) to detect.
Concrete testing conditions is:
Test column: Bio-Rad AMINEX HPX 87H Organic Analysis Column (300 × 7.8 mm);
Column temperature: 60 DEG C;
Moving phase: 6mM sulfuric acid, flow velocity: 0.6ml/min;
Detect wavelength: 210 nm.
After testing, after fermentation in 72-84 hour, more than in fermented liquid, 2-Acetamido-2-deoxy-D-glucose concentration can reach 38.3g/L.
Claims (7)
1. produce the subtilis (Bacillus subtilis) of 2-Acetamido-2-deoxy-D-glucose for one kind, it is characterized in that its deposit number is CCTCC M 2014342.
2. produce the construction process of the subtilis of 2-Acetamido-2-deoxy-D-glucose, it is characterized in that by the 6-phosphorylated amino glucose synthetic enzyme of encoding, 6-phosphorylated amino glucose acetylase is introduced the high efficient expression of subtilis Host Strains, and knock out or inactivation subtilis in 6-phosphorylated amino glucose deaminase gene nagB and gamA in 2-Acetamido-2-deoxy-D-glucose catabolic pathway, 6-phosphoric acid-2-Acetamido-2-deoxy-D-glucose deacetylase gene nagA, glucosamine transporter gene gamP and 2-Acetamido-2-deoxy-D-glucose transporter gene nagP build and form.
3. the construction process of the subtilis of production 2-Acetamido-2-deoxy-D-glucose according to claim 2, is characterized in that described construction process comprises following processing step:
A, knock out nagAB gene cluster, gamAP gene cluster and the nagP gene in subtilis respectively, obtain genetic engineering bacterium host;
B, structure GNA1 and glmS dual-expression vector;
C, dual-expression vector is proceeded to A gained Host Strains, obtain the subtilis of final production 2-Acetamido-2-deoxy-D-glucose.
4. the construction process of the subtilis of production 2-Acetamido-2-deoxy-D-glucose according to claim 2, is characterized in that described 6-phosphorylated amino glucose synthase gene derives from subtilis or other and have the microorganism of identical function enzyme.
5. the construction process of the subtilis of production 2-Acetamido-2-deoxy-D-glucose according to claim 2, is characterized in that described 6-phosphorylated amino glucose acetylase gene source has the microorganism of identical function enzyme in yeast saccharomyces cerevisiae or other.
6. the construction process of the subtilis of production 2-Acetamido-2-deoxy-D-glucose according to claim 2, it is characterized in that described knock out or inactivation subtilis in 6-phosphorylated amino glucose deaminase gene nagB and gamA in 2-Acetamido-2-deoxy-D-glucose catabolic pathway carry out simultaneously.
7. the application of the subtilis of production 2-Acetamido-2-deoxy-D-glucose in 2-Acetamido-2-deoxy-D-glucose, it is characterized in that the aseptic culture medium that the subtilis CCTCC M 2014342 that produces 2-Acetamido-2-deoxy-D-glucose is seeded to taking glucose as carbon source, aerobic fermentation 70 hours under 37 DEG C of conditions.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410376584.2A CN104195094A (en) | 2014-08-01 | 2014-08-01 | Bacillus subtilis for producing N-acetylglucosamine as well as construction method and application of bacillus subtilis |
| PCT/CN2015/073498 WO2016015469A1 (en) | 2014-08-01 | 2015-03-02 | Bacillus subtilis for producing n-acetylglucosamine, construction method therefor and uses thereof |
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| CN104928333A (en) * | 2015-07-07 | 2015-09-23 | 江南大学 | Method for knocking out glcK and promoting bacillus subtilis to synthesize acetylglucosamine |
| CN105255802A (en) * | 2015-10-14 | 2016-01-20 | 江南大学 | Method for increasing yield of recombinant bacillus subtilis acetylglucosamine through expression of NAD(P)H oxidases |
| CN105274081A (en) * | 2015-08-04 | 2016-01-27 | 南京农业大学 | D-acetylglucosamine deacetylase heterologous expression and application |
| WO2016015469A1 (en) * | 2014-08-01 | 2016-02-04 | 张帆 | Bacillus subtilis for producing n-acetylglucosamine, construction method therefor and uses thereof |
| CN106635940A (en) * | 2016-10-19 | 2017-05-10 | 齐鲁工业大学 | Construction method and applications of bacillus subtilis with high yield of glucosamine |
| CN107699533A (en) * | 2017-10-12 | 2018-02-16 | 江南大学 | A kind of recombined bacillus subtilis of acetylglucosamine output increased |
| CN108998402A (en) * | 2018-08-28 | 2018-12-14 | 江南大学 | A kind of recombined bacillus subtilis and its construction method and application |
| CN110195036A (en) * | 2019-05-23 | 2019-09-03 | 江南大学 | A kind of recombination Corynebacterium glutamicum of high yield acetylglucosamine and its application |
| CN110669708A (en) * | 2019-07-11 | 2020-01-10 | 北京化工大学 | Genetically engineered bacterium for synthesizing N-acetylglucosamine and application thereof |
| CN110760622A (en) * | 2019-12-17 | 2020-02-07 | 大自然生物集团有限公司 | Material supplementing method for producing glucosamine through fermentation of bacillus subtilis |
| CN110885870A (en) * | 2019-12-09 | 2020-03-17 | 山东润德生物科技有限公司 | Fermentation production method of N-acetylglucosamine |
| WO2022016641A1 (en) * | 2020-07-24 | 2022-01-27 | 上海交通大学 | Strain for producing n-acetylglucosamine, and construction method therefor and use thereof |
| CN114107157A (en) * | 2021-12-01 | 2022-03-01 | 中国药科大学 | Construction and application of genetically engineered bacterium for producing N-acetylglucosamine |
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| CN110885870A (en) * | 2019-12-09 | 2020-03-17 | 山东润德生物科技有限公司 | Fermentation production method of N-acetylglucosamine |
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| CN110760622B (en) * | 2019-12-17 | 2023-06-27 | 大自然生物集团有限公司 | Feeding method for producing glucosamine by fermenting bacillus subtilis |
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| CN114107157A (en) * | 2021-12-01 | 2022-03-01 | 中国药科大学 | Construction and application of genetically engineered bacterium for producing N-acetylglucosamine |
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