CN104498394B - A kind of recombined bacillus subtilis of acetylglucosamine output increased - Google Patents

A kind of recombined bacillus subtilis of acetylglucosamine output increased Download PDF

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CN104498394B
CN104498394B CN201410709273.3A CN201410709273A CN104498394B CN 104498394 B CN104498394 B CN 104498394B CN 201410709273 A CN201410709273 A CN 201410709273A CN 104498394 B CN104498394 B CN 104498394B
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bacillus subtilis
encoding gene
acetylglucosamine
recombined bacillus
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CN104498394A (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 kind of recombined bacillus subtilis of acetylglucosamine output increased, belong to field of genetic engineering.The present invention is with recombined bacillus subtilis BSGN6 PxylAGlmS knocks out α acetolactate synthestases encoding gene and α acetolactate decarboxylase encoding genes by homologous recombination, has blocked Host Strains to generate 3-hydroxy-2-butanone and 2, the approach of 3 butanediols by pyruvic acid as starting strain.In the Host Strains for having knocked out alsS and alsD; overexpression derives from the Glucosamine acetylase encoding gene of saccharomyces cerevisiae; strengthen acetylglucosamine route of synthesis; improve the yield of acetylglucosamine in recombined bacillus subtilis; reach 38.46g/L, producing Glucosamine for further metabolic engineering bacillus subtilis lays a good foundation.

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 one kind passes through Homologous recombination knocks out the method that alsS and alsD improves recombined bacillus subtilis acetylglucosamine yield, belongs to hereditary work Journey field.
Background technology
Acetylglucosamine is a kind of monose in organism, be widely present in bacterium, yeast, mould, plant and In animal body.In human body, acetylglucosamine is the synthesis precursor of glycosaminoglycan disaccharide unit, and it is to repairing and maintaining soft Bone and joint tissue function play an important roll.Therefore, acetylglucosamine is widely used as medicine and nutritious food addition To treat and repair joint injury.In addition, acetylglucosamine also has many applications in cosmetics and pharmaceutical field.Mesh Before, acetylglucosamine is mainly produced using chitin in acidolysis shrimp shell or crab shell, and waste liquid caused by the method is dirty to environment Dye is more serious, and obtained product easily causes allergic reaction, and the crowd for being not suitable for seafood allergy takes.
Bacillus subtilis (Bacillus subtilis) is that one kind is widely used as Food enzyme and important nutrient laden The production host of product, its product are " generally regarded as safe " (GRAS) level of security by FDA certifications.Cause This, is the effective way for producing aliment security level acetylglucosamine with metabolic engineering means structure recombined bacillus subtilis Footpath.
The present invention weakens glycolytic pathway by blocking the synthesis of 3-hydroxy-2-butanone, avoids restructuring B.subtilis centers carbon The accessory substance such as 3-hydroxy-2-butanone, 2,3-butanediol largely accumulates caused by overflow metabolism, and then improves ammonia sugar anabolism flux to enter One step improves GlcNAc yield.
The content of the invention
The invention solves first technical problem to be to provide a kind of restructuring of acetylglucosamine output increased withered Careless bacillus, it is with bacillus subtilis BSGN6-PxylA- glmS is starting strain, while knocks out α-acetolactate synthestase Encoding gene (alsS) and alpha -acetolactate decarboxylase encoding gene (alsD), and express Glucosamine acetylase.
The bacillus subtilis BSGN6-PxylA- glmS is based on bacillus subtilis 168, and genotype is made as follows Transformation：ΔnagPΔgamPΔgamAΔnagAΔnagBΔldhΔpta::Lox72, and with xylose evoked promoter PxylAAdjust Control expression glmS genes.
The bacillus subtilis BSGN6-PxylA- glmS construction method referring to：Modular pathway engineering of Bacillus subtilis for improvedN-acetylglucosamine production.Yanfeng Liu,et al.Metabolic Engineering,23(2014)p42-52.
The α-acetolactate synthestase encoding gene such as NCBI-Gene ID:Shown in 936852, α-acetolactic acid decarboxylation Enzyme coding gene such as NCBI-Gene ID:Shown in 936857.
The Glucosamine acetylase encoding gene (GNA1) derives from saccharomyces cerevisiae.
The invention solves another technical problem be to provide a kind of method for building above-mentioned recombined bacillus subtilis, α-acetolactate synthestase encoding gene (alsS) and alpha -acetolactate decarboxylase encoding gene are knocked out by homologous recombination (alsD), block Host Strains to generate 3-hydroxy-2-butanone and the approach of 2,3-butanediol by pyruvic acid, knock out alsS and alsD place In main bacterium, with expression vector pP43NMK overexpressions saccharomyces cerevisiae (Saccharomyces cerevisiae S288C) Glucosamine acetylase encoding gene (GNA1), by transforming metabolic pathway, realize carrying for acetylglucosamine yield It is high.
Present invention also offers a kind of side using the recombined bacillus subtilis fermenting and producing glycyl glucose Method, it is that seed culture fluid is transferred to fermentation medium, 44-52h is cultivated under the conditions of 30-37 DEG C, 200-220rpm.
The fermentation medium contains based on g/L：Dried Corn Steep Liquor Powder 20, dusty yeast 20, K2HPO4·3H2O 12.5, KH2PO42.5, CaCO35, micro- 15ml/L；Trace element solution (g/L)：MnSO4·5H2O 1.0, Cocl2·6H2O 0.4, NaMoO4·2H2O 0.2, ZnSO4·7H2O 0.2, Alcl3·6H2O 0.1, Cucl2·H2O 0.1, H3BO40.05, contain 5M HCl。
It is reachable in extracellular accumulation, its concentration that recombined bacillus subtilis provided by the invention can improve acetylglucosamine To 38.46g/L, produce Glucosamine for further metabolic engineering bacillus subtilis and lay a good foundation.The present invention carries The recombined bacillus subtilis construction method of confession is simple, is easy to use, and has application prospect well.
Embodiment
Seed culture medium (g/L)：Tryptone 10, dusty yeast 5, NaCl 10.
Fermentation medium (g/L)：Dried Corn Steep Liquor Powder 20, dusty yeast 20, K2HPO4·3H2O 12.5,KH2PO42.5 CaCO35, micro- 15ml/L；Trace element solution (g/L)：MnSO4·5H2O 1.0, Cocl2·6H2O 0.4, NaMoO4·2H2O0.2, ZnSO4·7H2O 0.2, Alcl3·6H2O 0.1, CuCl2·H2O 0.1, H3BO40.05, containing 5M HCl。
Condition of culture：The seed that 12h is cultivated under 37 DEG C, 200rpm is transferred to fermentation medium with 5% inoculum concentration, in 37 DEG C, cultivate 48h under the conditions of 200rpm.
The assay method of acetylglucosamine：High performance liquid chromatography (HPLC) detection method：Agilent 1200, RID are examined Survey device, NH2Post (250 × 4.6mm, 5 μm), mobile phase：70% acetonitrile, flow velocity 0.75mL/min, 30 DEG C of column temperature, sampling volume are 10μL。
Embodiment 1 knocks out α-acetolactate synthestase encoding gene (alsS) and alpha -acetolactate decarboxylase encoding gene (alsD)
According to the α for the bacillus subtilis 168 (ATCC No.27370) announced on NCBI-acetolactate synthestase coding Gene (alsS) and alpha -acetolactate decarboxylase encoding gene (alsD) upstream and downstream sequence, design knock out the amplification of frame homology arm and drawn Thing, left arm upstream and downstream primer are respectively：alsSD-L-F：5 '-CCATGTATAGAGTAGGCCATGCTTCTTTAGC-3 ' and
alsSD-L-R：
5’-AGGATCCCCGGGTACCGAGCTCCACCCTCACTCCTTATTATGCATTTTAAACGTAAAA-3’；Right arm Upstream and downstream primer is respectively：alsSD-R-F:
5 '-GTCGACCTGCAGGCATGCAAGCAAGAAAAAAAGAAAGCCCCTTTTAGCAGGG-3 ' and alsSD-R- R:5’-CTACTGCGCTGTCAGAAGCAAAATCAG-3’.With above-mentioned primer from bacillus subtilis (Bacillus Subtilis 168) in genome amplification knock out the left arm and right arm included in frame.According to the p7Z6 plasmid sequences announced on NCBI Arrange (Agricultural University Of Nanjing, doctor Yan Xin give, NCBI accession no.EU541492), design primer, expand rich next mould Plain resistant gene (zeo), upstream and downstream primer are respectively：alsSD-Z-F:
5 '-TTTTACGTTTAAAATGCATAATAAGGAGTGAGGGTGGAGCTCGGTACCCGGGGATC CT-3 ' and alsSD-Z-R:5’-
CCCTGCTAAAAGGGGCTTTCTTTTTTTCTTGCTTGCATGCCTGCAGGTCGAC-3’.By fusion DNA vaccine side Method, will knock out the left and right arm of frame and resistant gene is fused to knock out frame.Confirm that alsSD knocks out frame construction success, sequence by being sequenced As shown in SEQ ID NO.9.
The structure of the recombined bacillus subtilis of embodiment 2
By the knockout frame built conversion bacillus subtilis BSGN6-PxylA- glmS, pass through blasticidin resistance flat board Screening, bacterium colony PCR checkings, confirm α-acetolactate synthestase encoding gene (alsS) and alpha -acetolactate decarboxylase encoding gene Knock out successfully, obtain recombined bacillus subtilis BSGN10.
According to saccharomyces cerevisiae (Saccharomyces cerevisiae) S288C (ATCC 204508) announced on NCBI Middle Glucosamine acetylase encoding gene (GNA1), design primer GNA1-F：
5 '-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCTTACCCGATGGATTTT ATA-3 ', GNA1-R：5’-CCCAAGCTTCTATTTTCTAATTTGCATTTCCACG-3’.Using above-mentioned primer from saccharomyces cerevisiae Glucosamine acetylase encoding gene (GNA1) is expanded in (Saccharomyces cerevisiae S288C) genome. Amplified fragments are connected to pP43NMK expression vectors after KpnI and HIndIII double digestions.Digestion verification is simultaneously sequenced, and confirms restructuring Plasmid pP43-GNA1 is successfully constructed.
By the expression vector pP43-GNA1 built conversion bacillus subtilises BSGN10.Using GNA1-F and GNA1-R Primer selects transformant and carries out bacterium colony PCR, 480bp bands occurs, and checking recombined bacillus subtilis successfully constructs.
The fermenting and producing acetylglucosamine of embodiment 3
The seed that 12h is cultivated under 37 DEG C, 200rpm is transferred to fermentation medium with 5% inoculum concentration, in 37 DEG C, 200rpm Under the conditions of cultivate 48h.Ferment 48h, and acetylglucosamine content reaches 38.46g/L in fermented supernatant fluid, than not knocking out AlsS, alsD control bacterium 30.25g/L improve 27.1%.By knocking out α-acetolactate synthestase encoding gene (alsS) With alpha -acetolactate decarboxylase encoding gene (alsD), and the overexpression Glucosamine acetyl in nagP host has been knocked out Change enzyme coding gene (GNA1), realize raising of the acetylglucosamine in the extracellular yield of recombined bacillus subtilis.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention Enclose being defined of being defined by claims.

Claims (5)

1. a kind of recombined bacillus subtilis of acetylglucosamine output increased, it is characterised in that be with bacillus subtilis Bacterium BSGN6-PxylA- glmS is starting strain, knocks out its α-acetolactate synthestase encoding gene and alpha -acetolactate decarboxylase Encoding gene, and express Glucosamine acetylase；The starting strain is the gene based on bacillus subtilis 168 Type makees following transformation：ΔnagPΔgamPΔgamAΔnagAΔnagBΔldhΔpta::Lox72, and induced and started with xylose Sub- PxylARegulating and expressing Glucosamine synthase gene glmS；The α-acetolactate synthestase encoding gene such as NCBI- Gene ID:Shown in 936852, alpha -acetolactate decarboxylase encoding gene such as NCBI-GeneID:Shown in 936857.
A kind of 2. method for building recombined bacillus subtilis described in claim 1, it is characterised in that knocked out by homologous recombination α-acetolactate synthestase encoding gene and alpha -acetolactate decarboxylase encoding gene are knocked out, blocks Host Strains to be generated by pyruvic acid The approach of 3-hydroxy-2-butanone and 2,3-butanediol, it is excessive with expression vector pP43NMK in the Host Strains for having knocked out alsS and alsD Express the Glucosamine acetylase encoding gene of Saccharomyces cerevisiae.
3. a kind of method using recombined bacillus subtilis fermenting and producing glycyl glucose described in claim 1, it is special Sign is, is that seed culture fluid is transferred into fermentation medium, and 44-52h is cultivated under the conditions of 30-37 DEG C, 200-220rpm.
4. according to the method for claim 3, it is characterised in that the fermentation medium contains based on g/L：Dried Corn Steep Liquor Powder 20, dusty yeast 20, K2HPO4·3H2O 12.5, KH2PO42.5, CaCO35, micro- 15ml/L；Trace element solution presses g/L Meter contains：MnSO4·5H2O 1.0, Cocl2·6H2O 0.4, NaMoO4·2H2O 0.2, ZnSO4·7H2O 0.2, Alcl3· 6H2O 0.1, Cucl2·H2O 0.1, H3BO40.05, HCl containing 5M.
5. application of the recombined bacillus subtilis described in claim 1 in glycyl glucose production.
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MX2017014595A (en) * 2015-05-22 2018-03-21 Dupont Nutrition Biosci Aps Acetolactate decarboxylase.
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
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
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
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
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
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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
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