CN102978149B - Recombination bacillus subtilis with high yield of acetylglucosamine, and application of recombination bacillus subtilis - Google Patents
Recombination bacillus subtilis with high yield of acetylglucosamine, and application of recombination bacillus subtilis Download PDFInfo
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- CN102978149B CN102978149B CN201210570249.7A CN201210570249A CN102978149B CN 102978149 B CN102978149 B CN 102978149B CN 201210570249 A CN201210570249 A CN 201210570249A CN 102978149 B CN102978149 B CN 102978149B
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Abstract
The invention discloses a recombination bacillus subtilis with high yield of acetylglucosamine, and an application of the recombination bacillus subtilis, which belong to the field of genetic engineering. According to invention, bacillus subtilis 168 is taken as an original strain, a translocator encoding gene (nagP) of the acetylglucosamine is knocked out by virtue of homologous recombination, and thus a path for host bacteria to transport the acetylglucosamine from an extracellular part to an intracellular part is blocked. In the host bacteria the nagP of which is knocked out, over-expression is derived from glucosamine acetylase encoding gene (GNA1) of saccharomyces cerevisiae S288C, so that the synthesis path of the acetylglucosamine is improved, and the yield of acetylglucosamine in the recombination bacillus subtilis is enhanced to reach 415mg/L. Therefore, the application lays a foundation for producing glucosamine by improving the bacillus subtilis through metabolic engineering.
Description
Technical field
The present invention relates to a kind of high yield acetylglucosamine recombined bacillus subtilis and application thereof, belong to field of genetic engineering.
Background technology
Acetylglucosamine is a kind of monose in organism, is extensively present in bacterium, yeast, mould, plant and animal body.In human body, acetylglucosamine is the synthetic precursor of glycosaminoglycan disaccharide unit, and it is to repairing and maintaining cartilage and joint tissue function has vital role.Therefore, acetylglucosamine is extensively added to treat and repair joint injury as medicine and nutritional food.In addition, acetylglucosamine also has many application at makeup and pharmacy field.At present, acetylglucosamine mainly adopts in acidolysis shrimp shell or crab shell chitin to produce, and the waste liquid environmental pollution that this method produces is comparatively serious, and the product obtaining easily causes allergic reaction, and the crowd who is not suitable for seafood allergy takes.
Subtilis (Bacillus subtilis) is a kind of production host who is widely used as Food enzyme and important nutrient chemistry product, and its product is " generally regarded as safe " (GRAS) level of security by FDA authentication.Therefore, using metabolic engineering means to build recombined bacillus subtilis is the effective way of producing aliment security level acetylglucosamine.Yet, acetylglucosamine is the preferential carbon source material utilizing of a kind of subtilis, after in substratum, glucose exhausts, the outer acetylglucosamine of born of the same parents is transported in born of the same parents and the utilization that is decomposed, and causes the outer acetylglucosamine concentration of born of the same parents to reduce rapidly.For realizing the accumulation of acetylglucosamine, need to knock out acetylglucosamine translocator encoding gene (nagP), can from born of the same parents, be transported to the process in born of the same parents by blocking-up acetylglucosamine, thereby improve glucosamine output.
Summary of the invention
Technical problem to be solved by this invention is to build a kind of method that can improve recombined bacillus subtilis production acetylglucosamine.
For solving the problems of the technologies described above, technical scheme of the present invention is:
Described recombined bacillus subtilis, for glucosamine acetylase encoding gene GNA1 in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) S288C is cloned into subtilis, knocks out this subtilis acetylglucosamine translocator encoding gene nagP simultaneously and builds the recombined bacillus subtilis forming.
Described glucosamine acetylase encoding gene is as shown in NCBI-Gene ID:850529, and acetylglucosamine translocator encoding gene is as shown in NCBI-Gene ID:938807.
Another technical problem that the present invention will solve is to provide a kind of method that builds above-mentioned recombined bacillus subtilis.
Knock out acetylglucosamine translocator encoding gene (nagP), blocking-up Host Strains is transported acetylglucosamine to the approach in born of the same parents from born of the same parents.In knocking out the Host Strains of nagP; use glucosamine acetylase encoding gene (GNA1) in expression vector pP43NMK overexpression yeast saccharomyces cerevisiae (Saccharomyces cerevisiae S288C); by transformation pathways metabolism, realize the raising of acetylglucosamine output.
Recombined bacillus subtilis seed culture and fermentation:
Seed culture medium (g/L): Tryptones 10, yeast powder 5, NaCl10.
Fermention medium (g/L): glucose 20, Tryptones 10, yeast powder 5, NaCl10.
Culture condition: the seed of cultivating 12h under 37 ° of C, 200rpm is proceeded to fermention medium with 5% inoculum size, cultivate 30h under 37 ° of C, 200rpm condition.
The measuring method of acetylglucosamine:
High performance liquid chromatography (HPLC) detection method: Agilent1200, RID detector, NH
2post (250 * 4.6mm, 5 μ m), moving phase: 70% acetonitrile, flow velocity 0.75mL/min, 30 ° of C of column temperature, sampling volume is 10 μ L.
Recombined bacillus subtilis provided by the invention can improve acetylglucosamine and accumulate outward born of the same parents, and its concentration can reach 415mg/L, for further metabolic engineering subtilis produces glucosamine, lays a good foundation.Recombined bacillus subtilis construction process provided by the invention is simple, easy to use, has application prospect well.
Embodiment
Embodiment 1 knocks out acetylglucosamine translocator encoding gene (nagP)
According to upper subtilis (the Bacillus subtilis 168 announcing of NCBI, purchased from U.S.'s representative microbial preservation center, ATCCNo.27370) acetylglucosamine translocator encoding gene (nagP) upstream and downstream sequence, design knocks out frame homology arm amplimer, and left arm upstream and downstream primer is respectively: nagP-L-F:5 '-AATGAGATGCCTGTGTCGGAATA-3 ' and nagP-L-R:
5 '-TCCTGTGTGAAATTGTTATCCGCTCATCCACTCTCCAAACGAGTTGATAC-3 '; Right arm upstream and downstream primer is respectively: nagP-R-F:
5 '-ACGTCGTGACTGGGAAAACCCTGGCCGCGGTCTTAACCGGGTTA-3 ' and nagP-R-R:
5’-TACGACAACGCCCAGCTTC-3’。Use above-mentioned primer to increase and knock out left arm and the right arm comprising in frame from subtilis (Bacillus subtilis 168) genome.According to the upper p7Z6 plasmid sequence (Agricultural University Of Nanjing, doctor Yan Xin is so kind as to give, NCBI accession no.EU541492) of announcing of NCBI, design primer, amplification bleomycin resistant gene (zeo), upstream and downstream primer is respectively: nagP-Z-F:
5 '-GTATCAACTCGTTTGGAGAGTGGATAGCGGATAACAATTTCACACAGG-3 ' and nagP-Z-R:
5’-TAAAGATAACCCGGTTAAGACCGCGGCCAGGGTTTTCCCAGTCAC-3’。By merging PCR method, will knock out the left and right arm of frame and resistant gene and be fused to and knock out frame.By order-checking, confirm that nagP knocks out frame construction success.
The structure of embodiment 2 recombined bacillus subtilis
The frame that knocks out building is transformed to subtilis (Bacillus subtilis 168), by the screening of bleomycin resistant panel, bacterium colony PCR, verify, confirm that acetylglucosamine translocator encoding gene (nagP) knocks out successfully, obtains recombined bacillus subtilis BSGN1.
According to upper yeast saccharomyces cerevisiae (the Saccharomyces cerevisiae S288C announcing of NCBI; purchased from U.S.'s representative microbial preservation center; numbering ATCC204508) glucosamine acetylase encoding gene (GNA1) in; design primer GNA1-F:5 '-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCTTACCCGATGGATTTT ATA-3 ', GNA1-R:5 '-CCCAAGCTTCTATTTTCTAATTTGCATTTCCACG-3 '.Use the above-mentioned primer glucosamine acetylase encoding gene (GNA1) that increases from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae S288C) genome.Amplified fragments is connected to pP43NMK expression vector (Virginia, US Polytechnics, doctor Zhang Xiaozhou is so kind as to give) after KpnI and HIndIII double digestion.Enzyme is cut and is verified and check order, and confirms that recombinant plasmid pP43-GNA1 successfully constructs.
The expression vector pP43-GNA1 building is transformed to subtilis BSGN1.Adopt GNA1-F and GNA1-R primer to select transformant and carry out bacterium colony PCR, occur 480bp band, checking recombined bacillus subtilis successfully constructs.
Embodiment 3 fermentative production acetylglucosamines
The seed of cultivating 12h under 37 ° of C, 200rpm is proceeded to fermention medium with 5% inoculum size, under 37 ° of C, 200rpm condition, cultivate 30h.Fermentation 30h, in fermented supernatant fluid, acetylglucosamine content reaches 415mg/L.By knocking out acetylglucosamine translocator encoding gene (nagP); and overexpression glucosamine acetylase encoding gene (GNA1) in knocking out nagP host, has realized the raising of acetylglucosamine output outside recombined bacillus subtilis born of the same parents.
Claims (4)
1. a high yield acetylglucosamine recombined bacillus subtilis, it is characterized in that glucosamine acetylase encoding gene GNA1 in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) S288C to be cloned into subtilis, knock out this subtilis acetylglucosamine translocator encoding gene nagP simultaneously and build the recombined bacillus subtilis forming; Glucosamine acetylase encoding gene is connected on expression vector pP43NMK; Described subtilis is Bacillus subtilis168.
2. recombined bacillus subtilis according to claim 1; it is characterized in that; described glucosamine acetylase encoding gene is as shown in NCBI-Gene ID:850529, and acetylglucosamine translocator encoding gene is as shown in NCBI-Gene ID:938807.
3. the construction process of recombined bacillus subtilis described in claim 1, is characterized in that comprising the steps:
1) knock out acetylglucosamine translocator encoding gene
Build acetylglucosamine translocator encoding gene and knock out frame, by homologous recombination, the bleomycin resistant gene zeo knocking out in frame is substituted to acetylglucosamine translocator encoding gene nagP in subtilis (Bacillus subtilis) 168 genomes;
2) build and produce acetylglucosamine recombined bacillus subtilis
The glucosamine acetylase encoding gene of clone's yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) S288C, is connected on plasmid pP43NMK; Recombinant plasmid transformed has been knocked out to acetylglucosamine translocator encoding gene subtilis, obtained producing acetylglucosamine recombined bacillus subtilis.
4. application rights requires the method for the arbitrary described recombined bacillus subtilis fermentative production acetylglucosamine of 1-2, the recombined bacillus subtilis of cultivating 12h under 37oC, 200rpm is proceeded to fermention medium with 5% inoculum size, in 37 ℃, 200rpm condition bottom fermentation 30h.
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Cited By (2)
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| CN108330095A (en) * | 2018-03-01 | 2018-07-27 | 江南大学 | It is a kind of accumulation N-acetyl-neuraminate recombination Corynebacterium glutamicum and its application |
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| CN104498394B (en) * | 2014-11-27 | 2018-03-16 | 江南大学 | A kind of recombined bacillus subtilis of acetylglucosamine output increased |
| CN105039193B (en) * | 2015-01-27 | 2016-08-17 | 安徽正方生物科技有限公司 | A kind of fermentable produces bacterial strain and the method for glucosamine |
| CN104928333B (en) * | 2015-07-07 | 2017-09-22 | 江南大学 | A kind of method that knockout glcK promotes bacillus subtilis synthesis of acetyl Glucosamine |
| CN105176903B (en) * | 2015-10-14 | 2018-03-16 | 江南大学 | A kind of recombined bacillus subtilis for accumulating acetylglucosamine and its application |
| CN107699535B (en) * | 2017-11-08 | 2021-07-06 | 光明乳业股份有限公司 | Recombinant bacillus subtilis for induced synthesis of guanosine diphosphate fucose and construction method and application thereof |
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Cited By (4)
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| CN104498517A (en) * | 2014-11-29 | 2015-04-08 | 滨州市金朗生物科技有限公司 | Construction and application methods of escherichia coli with high yield of producing N-acetylglucosamine |
| CN104498517B (en) * | 2014-11-29 | 2017-04-12 | 滨州市金朗生物科技有限公司 | Construction and application methods of escherichia coli with high yield of producing N-acetylglucosamine |
| CN108330095A (en) * | 2018-03-01 | 2018-07-27 | 江南大学 | It is a kind of accumulation N-acetyl-neuraminate recombination Corynebacterium glutamicum and its application |
| CN108330095B (en) * | 2018-03-01 | 2020-12-29 | 江南大学 | Recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof |
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