CN104498394A - Recombinant bacillus subtilis increased in yield of acetylglucosamine - Google Patents

Recombinant bacillus subtilis increased in yield of acetylglucosamine Download PDF

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CN104498394A
CN104498394A CN201410709273.3A CN201410709273A CN104498394A CN 104498394 A CN104498394 A CN 104498394A CN 201410709273 A CN201410709273 A CN 201410709273A CN 104498394 A CN104498394 A CN 104498394A
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bacillus subtilis
encoding gene
acetylglucosamine
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recombined bacillus
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CN104498394B (en
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陈坚
刘龙
堵国成
李江华
马文龙
刘延峰
朱妍萩
顾洋
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Jiangnan University
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Abstract

The invention discloses a recombinant bacillus subtilis increased in yield of acetylglucosamine and belongs to the field of genetic engineering. According to the recombinant bacillus subtilis increased in yield of acetylglucosamine, the recombinant bacillus subtilis BSGN6-PxylA-glmS is taken as an original strain, an alpha-acetolactate synthetase encoding gene and an alpha-acetolacetate decearboxylase encoding gene are knocked out by virtue of homologous recombination, and therefore, the approach by which acetoin and butanediol are generated from 2,3-pyroracemic acid in host bacteria is blocked. In the host bacteria from which the alsS and alsD are knocked out, the glucosamine acetylase encoding gene derived from saccharomyces cerevisiae is excessively expressed, and therefore, the route of synthesis of the acetylglucosamine is enhanced, the yield of the acetylglucosamine in the recombinant bacillus subtilis is increased to 38.46g/L, and a foundation is laid for producing the glucosamine by modifying the bacillus subtilis in the metabolic engineering.

Description

A kind of recombined bacillus subtilis of acetylglucosamine output increased
Technical field
The present invention relates to a kind of recombined bacillus subtilis of acetylglucosamine output increased, especially a kind of method being knocked out alsS and alsD raising recombined bacillus subtilis acetylglucosamine output by homologous recombination, belongs 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 synthesis precursor of glycosaminoglycan disaccharide unit, and it is to reparation and maintain cartilage and joint tissue function and have vital role.Therefore, acetylglucosamine is widely used as medicine and nutritional food and adds and treat and repair joint injury.In addition, acetylglucosamine also has many application at makeup and pharmacy field.At present, acetylglucosamine mainly adopts chitin in acidolysis shrimp shell or crab shell to produce, and the waste liquid environmental pollution that this method produces is comparatively serious, and the product obtained easily causes allergic reaction, and the crowd being not suitable for seafood allergy takes.
Subtilis (Bacillus subtilis) is a kind of production host being widely used as Food enzyme and important nutrient chemistry product, and its product is " generally regarded as safe " (GRAS) level of security by FDA certification.Therefore, using metabolic engineering means to build recombined bacillus subtilis is the effective way of producing aliment security level acetylglucosamine.
The present invention is by blocking the synthesis of acetoin to weaken glycolytic pathway, avoid the acetoin, 2 that the overflow of restructuring B.subtilis central carbon metabolism causes, the by products such as 3-butyleneglycol accumulate in a large number, and then improve ammonia sugar anabolism flux to improve GlcNAc output further.
Summary of the invention
First technical problem that the present invention will solve is to provide a kind of recombined bacillus subtilis of acetylglucosamine output increased, is with subtilis BSGN6-P xylA-glmS is starting strain, knocks out α-acetolactate synthestase encoding gene (alsS) and alpha-acetolactate decarboxylase encoding gene (alsD) simultaneously, and have expressed glucosamine acetylase.
Described subtilis BSGN6-P xylA-glmS is based on subtilis 168, and genotype does following transformation: Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta::lox72, and with wood sugar evoked promoter P xylAregulating and expressing glmS gene.
Described subtilis BSGN6-P xylAthe construction process of-glmS is see Modular pathway engineering ofBacillus subtilis for improvedN-acetylglucosamine production.Yanfeng Liu, et al.MetabolicEngineering, 23 (2014) p42-52.
Described α-acetolactate synthestase encoding gene is as shown in NCBI-Gene ID:936852, and alpha-acetolactate decarboxylase encoding gene is as shown in NCBI-Gene ID:936857.
Described glucosamine acetylase encoding gene (GNA1) derives from yeast saccharomyces cerevisiae.
Another technical problem that the present invention will solve is to provide a kind of method building above-mentioned recombined bacillus subtilis, α-acetolactate synthestase encoding gene (alsS) and alpha-acetolactate decarboxylase encoding gene (alsD) is knocked out by homologous recombination, block Host Strains and generate acetoin and 2 by pyruvic acid, the approach of 3-butyleneglycol, knocking out in the Host Strains of alsS and alsD, 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.
Present invention also offers a kind of method applying described recombined bacillus subtilis fermentative production glycyl glucose, is that seed culture fluid is proceeded to fermention medium, in 30-37 DEG C, cultivate 44-52h under 200-220rpm condition.
Described fermention medium contains by g/L: Dried Corn Steep Liquor Powder 20, yeast powder 20, K2HPO 43H 2o 12.5, KH 2pO 42.5, CaCO 35, micro-15ml/L; Trace element solution (g/L): MnSO 45H 2o 1.0, Cocl 26H 2o 0.4, NaMoO 42H 2o 0.2, ZnSO 47H 2o 0.2, Alcl 36H 2o 0.1, Cucl 2h 2o 0.1, H 3bO 40.05, containing 5M HCl.
Recombined bacillus subtilis provided by the invention can improve acetylglucosamine and accumulate outward born of the same parents, and its concentration can reach 38.46g/L, lays a good foundation for further metabolic engineering subtilis produces glucosamine.Recombined bacillus subtilis construction process provided by the invention is simple, easy to use, has application prospect well.
Embodiment
Seed culture medium (g/L): Tryptones 10, yeast powder 5, NaCl 10.
Fermention medium (g/L): Dried Corn Steep Liquor Powder 20, yeast powder 20, K2HPO 43H 2o 12.5, KH 2pO 42.5, CaCO 35, micro-15ml/L; Trace element solution (g/L): MnSO 45H 2o 1.0, Cocl 26H 2o 0.4, NaMoO 42H 2o0.2, ZnSO 47H 2o 0.2, Alcl 36H 2o 0.1, CuCl 2h 2o 0.1, H 3bO 40.05, containing 5M HCl.
Culture condition: by 37 DEG C, the seed of cultivating 12h under 200rpm proceeds to fermention medium with the inoculum size of 5%, in 37 DEG C, cultivate 48h under 200rpm condition.
The measuring method of acetylglucosamine: high performance liquid chromatography (HPLC) detection method: Agilent 1200, RID detector, NH 2post (250 × 4.6mm, 5 μm), moving phase: 70% acetonitrile, flow velocity 0.75mL/min, column temperature 30 DEG C, sampling volume is 10 μ L.
Embodiment 1 knocks out α-acetolactate synthestase encoding gene (alsS) and alpha-acetolactate decarboxylase encoding gene (alsD)
According to α-acetolactate synthestase encoding gene (alsS) and alpha-acetolactate decarboxylase encoding gene (alsD) the upstream and downstream sequence of the subtilis 168 (ATCC No.27370) that NCBI announces, design knock out frame homology arm amplimer, left arm upstream and downstream primer is respectively: alsSD-L-F:5 '-CCATGTATAGAGTAGGCCATGCTTCTTTAGC-3 ' and
alsSD-L-R：
5 '-AGGATCCCCGGGTACCGAGCTCCACCCTCACTCCTTATTATGCATTTTAAACGTAA AA-3 '; Right arm upstream and downstream primer is respectively: alsSD-R-F:
5 '-GTCGACCTGCAGGCATGCAAGCAAGAAAAAAAGAAAGCCCCTTTTAGCAGGG-3 ' and alsSD-R-R:5 '-CTACTGCGCTGTCAGAAGCAAAATCAG-3 '.Use above-mentioned primer to increase from subtilis (Bacillus subtilis 168) genome and knock out the left arm and right arm that comprise in frame.According to the p7Z6 plasmid sequence (Agricultural University Of Nanjing that NCBI announces, doctor Yan Xin is so kind as to give, NCBI accession no.EU541492), design primer, amplification blasticidin resistance gene (zeo), upstream and downstream primer is respectively: alsSD-Z-F:
5 '-TTTTACGTTTAAAATGCATAATAAGGAGTGAGGGTGGAGCTCGGTACCCGGGGATC CT-3 ' and alsSD-Z-R:5 '-
CCCTGCTAAAAGGGGCTTTCTTTTTTTCTTGCTTGCATGCCTGCAGGTCGAC-3’。By fusion DNA vaccine method, the left and right arm of frame and resistant gene will be knocked out and be fused to and knock out frame.Confirm that alsSD knocks out frame construction success by order-checking, sequence is as shown in SEQ ID NO.9.
The structure of embodiment 2 recombined bacillus subtilis
Frame Transforming B. subtilis BSGN6-P is knocked out by what build xylA-glmS, is verified by blasticidin resistance plate screening, bacterium colony PCR, confirms that α-acetolactate synthestase encoding gene (alsS) and alpha-acetolactate decarboxylase encoding gene knock out successfully, obtains recombined bacillus subtilis BSGN10.
According to glucosamine acetylase encoding gene (GNA1) in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) S288C (ATCC 204508) that NCBI announces, design primer GNA1-F:
5’-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCTTACCCGATGGATTTTATA-3’，GNA1-R：5’-CCCAAGCTTCTATTTTCTAATTTGCATTTCCACG-3’。Above-mentioned primer is used to increase from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae S288C) genome glucosamine acetylase encoding gene (GNA1).Amplified fragments is connected to pP43NMK expression vector after KpnI and HIndIII double digestion.Digestion verification also checks order, and confirms that recombinant plasmid pP43-GNA1 successfully constructs.
By the expression vector pP43-GNA1 Transforming B. subtilis BSGN10 built.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 acetylglucosamine
By 37 DEG C, the seed of cultivating 12h under 200rpm proceeds to fermention medium with the inoculum size of 5%, in 37 DEG C, cultivate 48h under 200rpm condition.Fermentation 48h, in fermented supernatant fluid, acetylglucosamine content reaches 38.46g/L, improves 27.1% than the contrast bacterium 30.25g/L not knocking out alsS, alsD.By knocking out α-acetolactate synthestase encoding gene (alsS) and alpha-acetolactate decarboxylase encoding gene (alsD); and knock out overexpression glucosamine acetylase encoding gene (GNA1) in nagP host, achieve the raising of acetylglucosamine in the outer output of recombined bacillus subtilis born of the same parents.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims (8)

1. a recombined bacillus subtilis for acetylglucosamine output increased is with subtilis
BSGN6-P xylA-glmS is starting strain, knocks out its α-acetolactate synthestase encoding gene and alpha-acetolactate decarboxylase encoding gene, and expresses glucosamine acetylase.
2. recombined bacillus subtilis according to claim 1, it is characterized in that, described starting strain is based on subtilis 168, and genotype does following transformation: Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta::lox72, and with wood sugar evoked promoter P xylAregulating and expressing glucosamine synthase gene glmS.
3. recombined bacillus subtilis according to claim 1, is characterized in that, described α-acetolactate synthestase encoding gene is as shown in NCBI-Gene ID:936852, and alpha-acetolactate decarboxylase encoding gene is as shown in NCBI-Gene ID:936857.
4. recombined bacillus subtilis according to claim 1, is characterized in that, the gene source of described glucosamine acetylase of encoding is in yeast saccharomyces cerevisiae.
5. one kind builds the method for the arbitrary described recombined bacillus subtilis of claim 1-4; it is characterized in that; α-acetolactate synthestase encoding gene and alpha-acetolactate decarboxylase encoding gene is knocked out by homologous recombination; block Host Strains and generate acetoin and 2 by pyruvic acid; the approach of 3-butyleneglycol; knocking out in the Host Strains of alsS and alsD, use the glucosamine acetylase encoding gene of expression vector pP43NMK overexpression Saccharomyces cerevisiae.
6. application rights requires a method for 1-4 arbitrary described recombined bacillus subtilis fermentative production glycyl glucose, and it is characterized in that, be that seed culture fluid is proceeded to fermention medium, in 30-37 DEG C, cultivate 44-52h under 200-220rpm condition.
7. method according to claim 6, is characterized in that, described fermention medium contains by g/L: Dried Corn Steep Liquor Powder 20, yeast powder 20, K2HPO 43H 2o 12.5, KH 2pO 42.5, CaCO 35, micro-15ml/L; Trace element solution contains by g/L: MnSO 45H 2o 1.0, Cocl 26H 2o 0.4, NaMoO 42H 2o 0.2, ZnSO 47H 2o 0.2, Alcl 36H 2o 0.1, Cucl 2h 2o 0.1, H 3bO 40.05, containing 5M HCl.
8. the application of the arbitrary described recombined bacillus subtilis of claim 1-4 in glycyl glucose production.
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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
CN105176879A (en) * 2015-10-14 2015-12-23 江南大学 Method for improving acetylglucosamine yield of recombinant bacillus subtilis by knocking out argCJBD
CN105176903A (en) * 2015-10-14 2015-12-23 江南大学 Recombinant bacillus subtilis for accumulating acetylglucosamine and application thereof
CN105238724A (en) * 2015-11-10 2016-01-13 江南大学 Method for knocking out pckA to promote synthesis of acetylglucosamine through bacillus subtilis
CN105255803A (en) * 2015-11-10 2016-01-20 江南大学 Recombinant bacillus subtilis for efficiently synthesizing 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
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
CN106868033A (en) * 2017-04-03 2017-06-20 天津大学 The Corynebacterium glutamicum strain of high yield chiral D () 3-hydroxy-2-butanone and structure and application
CN106929499A (en) * 2017-04-26 2017-07-07 扬州日兴生物科技股份有限公司 A kind of Glucosamine synthase mutant of directional transformation and its application
CN107636151A (en) * 2015-05-22 2018-01-26 杜邦营养生物科学有限公司 Acetolactate decarboxylase
CN109971696A (en) * 2019-03-20 2019-07-05 江南大学 A kind of recombinant bacterium of resting cell method high yield N-acetyl-neuraminate and application
CN110713966A (en) * 2019-11-26 2020-01-21 江南大学 Method for promoting N-acetylglucosamine synthesis by utilizing GlcN6P sensing component
WO2021102682A1 (en) * 2019-11-26 2021-06-03 江南大学 Method for promoting synthesis of n-acetylglucosamine by using glcn6p sensing component

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CN107636151B (en) * 2015-05-22 2023-04-04 杜邦营养生物科学有限公司 Acetolactate decarboxylase
CN107636151A (en) * 2015-05-22 2018-01-26 杜邦营养生物科学有限公司 Acetolactate decarboxylase
CN104928333A (en) * 2015-07-07 2015-09-23 江南大学 Method for knocking out glcK and promoting bacillus subtilis to synthesize acetylglucosamine
US9914949B2 (en) * 2015-07-07 2018-03-13 Jiangnan University Method for enhancing N-acetylglucosamine production through glcK knockout of Bacillus subtilis
US20170009267A1 (en) * 2015-07-07 2017-01-12 Jiangnan University Method for Enhancing N-acetylglucosamine Production through glcK Knockout of Bacillus subtilis
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
CN105176879A (en) * 2015-10-14 2015-12-23 江南大学 Method for improving acetylglucosamine yield of recombinant bacillus subtilis by knocking out argCJBD
CN105176903A (en) * 2015-10-14 2015-12-23 江南大学 Recombinant bacillus subtilis for accumulating acetylglucosamine and application thereof
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
CN105176879B (en) * 2015-10-14 2017-12-12 江南大学 A kind of method that knockout argCJBD improves recombined bacillus subtilis acetylglucosamine yield
CN105255802B (en) * 2015-10-14 2017-11-17 江南大学 The method that one kind expression NAD (P) H oxidizing ferment improves recombined bacillus subtilis acetylglucosamine yield
CN105238724B (en) * 2015-11-10 2017-11-17 江南大学 A kind of method that knockout pckA promotes bacillus subtilis synthesis of acetyl Glucosamine
CN105255803B (en) * 2015-11-10 2017-11-17 江南大学 A kind of recombined bacillus subtilis for efficiently synthesizing acetylglucosamine
CN105255803A (en) * 2015-11-10 2016-01-20 江南大学 Recombinant bacillus subtilis for efficiently synthesizing acetylglucosamine
CN105238724A (en) * 2015-11-10 2016-01-13 江南大学 Method for knocking out pckA to promote synthesis of acetylglucosamine through bacillus subtilis
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
CN106635940B (en) * 2016-10-19 2019-10-18 齐鲁工业大学 One plant of construction method for producing Glucosamine bacillus subtilis and application
CN106868033A (en) * 2017-04-03 2017-06-20 天津大学 The Corynebacterium glutamicum strain of high yield chiral D () 3-hydroxy-2-butanone and structure and application
CN106868033B (en) * 2017-04-03 2020-10-27 天津大学 Corynebacterium glutamicum strain for high yield of chiral D- (-) -acetoin and construction and application thereof
CN106929499A (en) * 2017-04-26 2017-07-07 扬州日兴生物科技股份有限公司 A kind of Glucosamine synthase mutant of directional transformation and its application
CN109971696A (en) * 2019-03-20 2019-07-05 江南大学 A kind of recombinant bacterium of resting cell method high yield N-acetyl-neuraminate and application
CN110713966A (en) * 2019-11-26 2020-01-21 江南大学 Method for promoting N-acetylglucosamine synthesis by utilizing GlcN6P sensing component
CN110713966B (en) * 2019-11-26 2021-05-28 江南大学 Method for promoting N-acetylglucosamine synthesis by utilizing GlcN6P sensing component
WO2021102682A1 (en) * 2019-11-26 2021-06-03 江南大学 Method for promoting synthesis of n-acetylglucosamine by using glcn6p sensing component

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