CN109929791A - It is a kind of accumulate Glucosamine recombination bacillus coli and its application - Google Patents
It is a kind of accumulate Glucosamine recombination bacillus coli and its application Download PDFInfo
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Abstract
The invention discloses a kind of recombination bacillus coli for accumulating Glucosamine and its applications, belong to genetic engineering technology field.Using Escherichia coli as host expresses; by knocking out 4 genes in Escherichia coli in Glucosamine route of synthesis: acetylglucosamine transports sub- encoding gene nagE, mannose phosphoric acid transports sub- encoding gene manXYZ, deacetylation enzyme gene nagA and Deaminase Gene nagB gene; obtain recombination bacillus coli; and be applied to recombination bacillus coli in the production of Glucosamine, it realizes and blocks product Glucosamine by extracellular to transport pathway intracellular；And by overexpression Glucosamine synthetase-coding gene, Glucosamine acetylase encoding gene, the route of synthesis of Glucosamine is strengthened, to improve the yield of Glucosamine.
Description
Technical field
The invention belongs to genetic engineering technology fields, and in particular to it is a kind of accumulate Glucosamine recombination bacillus coli and
It is applied.
Background technique
Sugar industry is the basic industries of food service industry, and is the original of the multiple products such as papermaking, chemical industry, fermentation, medicine, building materials
Material industry, occupies highly important status in national economy.Function carbohydrates and their derivative is the important composition portion of health industry
Point, it is the rising industry of technical field of food biotechnology, industry development was swift and violent in recent years.Glucosamine (Glucosamine,
2-amino-2-deoxy-D-glucose, GlcN), also known as aminoglucose, gucosamine or aminoglucose are one of glucose
Hydroxyl replaced by amino after compound, be the amino monosaccharide that a kind of important function monosaccharide and first are identified structure.
Currently, the relevant compound of GlcN generally comprises: aminoglucose hydrochloride, aminoglucose sulfate and acetylamino Portugal
Grape sugar (N-acetylglucosamine, GlcNAc).Wherein, GlcN molecular formula is C6H13O5N is commonly called as amino sugar.GlcN is almost
It is present in all organisms, including bacterium, yeast, filamentous fungi, plant and animal body, is glycoprotein and proteoglycans
Main constituents, while being also the main constituents of chitosan and chitin, it in the cell can be by glucose 6-phosphate ammonia
Base metaplasia at.
GlcN and its derivative medicine, food, in terms of have very extensive application, the main life of GlcN
Production method has chitin Hydrolyze method, enzyme biotransformation method and microbe fermentation method.There is raw material in production in chitin Hydrolyze method
Source is limited, easily causes environmental pollution and allergic reaction, it is difficult to realize industrialized production；Enzyme biotransformation method is compared and chitin
The problem of Hydrolyze method, environmental pollution is too small, but long there are the reaction time, low efficiency；Microbe transformation method avoids preceding two
It is determined existing for kind method, and high-efficient, becomes the main method of production GlcN now.
Summary of the invention
Goal of the invention: aiming at the problems existing in the prior art, the purpose of the present invention is to provide a kind of accumulation amino is general
Lead to the recombination bacillus coli of sugar.Another object of the present invention is a kind of answering for recombination bacillus coli for accumulating Glucosamine
With.By improved Escherichia coli the life of ammonia sugar preferably can be carried out using carbon source materials such as glucose compared to wild type
It produces, and its yield is also obviously improved.
Technical solution: to solve the above-mentioned problems, the technical solution adopted in the present invention is as follows:
A kind of recombination bacillus coli accumulating Glucosamine, knocks out Glucosamine route of synthesis in the Escherichia coli
In 4 genes: acetylglucosamine transports sub- encoding gene nagE, mannose phosphoric acid transports sub- encoding gene manXYZ,
Deacetylation enzyme gene nagA and Deaminase Gene nagB gene block product Glucosamine by extracellular to transport intracellular
Approach improves the yield of Glucosamine.
Further, it is Escherichia coli W3110 as the Escherichia coli of host, or has knocked out acetylglucosamine
It transports sub- encoding gene nagE Escherichia coli W3110 or has knocked out mannose phosphoric acid and transport sub- encoding gene manXYZ large intestine
Bacillus W3110 or deacetylation enzyme gene nagA Escherichia coli W3110 is knocked out, or has knocked out Deaminase Gene
NagB Escherichia coli W3110.
Further, it by the Glucosamine synthetase-coding gene GlmS in Glucosamine route of synthesis, is cloned into
Expression vector pTargetF, then converts Escherichia coli, by changing metabolic pathway, realizes the accumulation of Glucosamine.
Further, by the Glucosamine acetylase encoding gene GNA1 in Glucosamine route of synthesis, clone
To expression vector pTargetF, Escherichia coli are then converted, by changing metabolic pathway, realize the accumulation of Glucosamine.
Further, rite-directed mutagenesis is carried out to GlmS gene on pGEM-T-GlmS plasmid, by 64 amino acids-paddy ammonia
Acid-replace with glutamine.
A method of the recombination bacillus coli of building accumulation Glucosamine, comprising the following steps:
(1) clone's acetylglucosamine transports sub- encoding gene, mannose phosphoric acid transports sub- encoding gene, deacetylation
Enzyme gene, Deaminase Gene, are connected on recombinant expression plasmid；
(2) above-mentioned recombinant expression carrier is converted into Escherichia coli, obtains accumulation Glucosamine recombination bacillus coli, it is described
Escherichia coli are Escherichia coli W3110, Escherichia coli W3110 Δ nagE, Escherichia coli W3110 Δ nagE Δ manXYZ, large intestine
Bacillus W3110 Δ nagE Δ manXYZ Δ nagA and Escherichia coli W3110 Δ nagE Δ manXYZ Δ nagA Δ nagB.
A method of using above-mentioned recombination bacillus coli fermenting production Glucosamine, by 200rpm revolving speed 37 DEG C
Overnight incubation in incubator obtains bacterial strain seed liquor；Seed liquor is seeded in the shaking flask equipped with fermentation medium, shaking flask with
The revolving speed of 180rpm cultivates 48h in 30 DEG C of incubator.
Further, the seed liquid culture medium is LB culture medium.
Further, the formula of the fermentation medium: glucose 40g/L, potassium dihydrogen phosphate 2g/L, ammonium sulfate 17g/L,
Yeast powder 4g/L, calcium carbonate 30g/L, L-threonine 0.2g/L, Thiamine 0.1mg/L, MgSO41g/L、FeSO40.5mg/L、
MnSO45mg/L and ZnSO45mg/L。
The utility model has the advantages that compared with prior art, the invention has the advantages that
(1) present invention provide it is a kind of accumulate Glucosamine Escherichia coli and its application, by improved large intestine bar
Bacterium preferably can carry out the production of ammonia sugar using carbon source materials such as glucose compared to wild type, and its yield also has and significantly mentions
It rises.
(2) present invention realizes by knocking out the gene in Escherichia coli glucosamine route of synthesis and blocks product ammonia
Base glucose, to transport pathway intracellular, realizes the accumulation of Glucosamine by extracellular.
(3) present invention can mitigate product by overexpression GlcN-6-P synthase and Glucosamine acetylase
Feedback inhibition strengthens the route of synthesis of Glucosamine, improves the yield of Glucosamine.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, right combined with specific embodiments below
A specific embodiment of the invention is described in detail.As it is used herein, term " enhancing " refers to increasing by corresponding
The activity of the enzyme of polynucleotide encoding.The expression regulation sequence of the gene in the overexpression or replacement gene group of gene can be passed through
(promoter replacement etc.).
Embodiment 1
Based on the Metabolically engineered of Wild type E. coli strain
(1) knockout of nagE gene
For the partial inactivation for blocking nagE to realize Glucosamine input system, it is made to reduce taking the photograph for Glucosamine
Enter, therefore the nagE gene in wild-type strain is knocked out.
Pass through CRISPR-Cas9 system (Jiang Y, Chen B, Duan C, et al.Multigene Editing in
the Escherichia coli Genome via the CRISPR-Cas9 System[J].Applied&
2015,81 (7): Environmental Microbiology 2506.) edits wild-type e. coli Escherichia
The nagE base of L-Methionine transportation system is encoded in coli W3110 (being purchased from strain Escherichia coli collection CGSC) genome
Cause.By PCR using SEQ ID NO.1 and SEQ ID NO.2 primer and pTargetF carrier (Jiang Y, Chen B,
Duan C, et al.Multigene Editing in the Escherichia coli Genome via the CRISPR-
2015,81 (7): Cas9 System [J] .Applied&Environmental Microbiology 2506.) is used as template structure
Build the pTarget- Δ nagE mutational vector that can express the sgRNA for targeting target gene metI.PCR reaction condition is as follows: 95
℃5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 2min repeat 30 circulations；72 DEG C are continued to extend 10min.By PCR product Dpn
I is in 37 DEG C of processing 3h, and it is strong to be coated on the hydrochloric acid of 50mg/L containing final concentration into E.coli BL21 (DE3) recipient bacterium for conversion after inactivation
On the LB solid plate of miromycin resistance, 37 DEG C of culture 12h.It is strong that random picking single colonie is forwarded to the hydrochloric acid of 50mg/L containing final concentration
In the LB liquid medium of miromycin resistance, 37 DEG C of culture 12h collect thallus and extract plasmid acquisition pTarget- Δ nagE load
Body.
<SEQ ID NO.1>
<SEQ ID NO.2>
The primer of SEQ ID NO.3 and SEQ ID NO.4 are used, by PCR with Escherichia coli Escherichia coli
W3110 genome is that template amplification obtains nagE upstream region of gene homologous fragment, and PCR reaction condition is as follows: 95 DEG C of 5min；95℃
30s, 55 DEG C of 30s, 72 DEG C of 30s repeat 30 circulations；72 DEG C are continued to extend 10min.
It is same that nagE downstream of gene is obtained using the primer amplification of SEQ ID NO.5 and SEQ ID NO.6 in the same way
Source segment, PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purified fragments.
Two DNA fragmentations of recycling are subjected to fusion DNA vaccine, PCR using the primer of SEQ ID NO.3 and SEQ ID NO.6
Reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min repeat 30 circulations；72 DEG C are continued to extend
10min.PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purifies the segment.
By pTarget- Δ nagE carrier and the DNA fragmentation of recycling together electrotransformation to Escherichia coli W3110
Bacterial strain, it is inverted to have pCas9 carrier (Jiang Y, Chen B, Duan C, et al.Multigene Editing in
the Escherichia coli Genome via the CRISPR-Cas9 System[J].Applied&
Environmental Microbiology, 2015,81 (7): 2506.).
<SEQ ID NO.3>
<SEQ ID NO.4>
<SEQ ID NO.5>
<SEQ ID NO.6>
For electroporation, conversion has the Escherichia coli W3110 bacterial strain of pCas9 carrier containing 50mg/L's
It is cultivated at 30 DEG C in the LB culture medium of kanamycins and 10mM L-arabinose, until OD reaches 0.6, bacterium solution is by centrifugation
Obtain thallus.Thallus is washed twice using sterile distilled water, then washed once using 10% glycerol to use.Electroporation
It is carried out at 2.5KV.Bacterium solution after electrotransformation is applied to the kanamycins containing 50mg/L and the spectinomycin hydrochloride of 50mg/L
On the LB plate of resistance, overnight incubation at 30 DEG C.Picking single colonie is as template, with SEQ ID NO.7 and SEQ ID NO.8's
Primer carries out PCR, and there are the DNA bands of 1000bp to confirm nagE gene in 1.0% electrophoresis detection by observing
Missing.
By the bacterial strain confirmed by this at 30 DEG C in the LB culture medium of the kanamycins containing 50mg/L and 5mM IPTG
Lower overnight incubation is to remove pTarget- Δ nagE carrier.Then the bacterial strain of pTarget- Δ nagE carrier will be had been removed in LB
In culture medium at 37 DEG C overnight incubation to remove pCas carrier (Jiang Y, Chen B, Duan C, et al.Multigene
Editing in the Escherichia coli Genome via the CRISPR-Cas9 System[J].Applied&
Environmental Microbiology, 2015,81 (7): 2506.).The Strain Designation so constructed is W3110 Δ nagE.
<SEQ ID NO.7>
<SEQ ID NO.8>
(2) knockout of manXYZ gene
Pass through CRISPR-Cas9 system (Jiang Y, Chen B, Duan C, et al.Multigene Editing in
the Escherichia coli Genome via the CRISPR-Cas9 System[J].Applied&
2015,81 (7): Environmental Microbiology 2506.) edits manXYZ in W3110 Δ nagE strain gene group
Gene.By PCR using SEQ ID NO.9 and SEQ ID NO.10 primer and pTargetF carrier (AEM (2015), 81
(7): 2506-14) the pTarget- Δ manXYZ that can express the sgRNA for targeting target gene manXYZ as template building dashes forward
Variable load body.PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 2min repeat 30 circulations；72 DEG C after
It renews and stretches 10min.PCR product is converted after inactivation into E.coli BL21 (DE3) recipient bacterium with DpnI in 37 DEG C of processing 3h,
It is coated on the LB solid plate of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of culture 12h.Random picking single colonie
It is forwarded in the LB liquid medium of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of culture 12h, collects thallus and mentions
Plasmid is taken to obtain pTarget- Δ manXYZ carrier.
<SEQ ID NO.9>
<SEQ ID NO.10>
The primer of SEQ ID NO.11 and SEQ ID NO.12 are used, by PCR with Escherichia coli Escherichia
Coli W3110 genome is that template amplification obtains manXYZ upstream region of gene homologous fragment, and PCR reaction condition is as follows: 95 DEG C
5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s repeat 30 circulations；72 DEG C are continued to extend 10min.It uses in the same way
The primer amplification of SEQ ID NO.13 and SEQ ID NO.14 obtains manXYZ downstream of gene homologous fragment, and PCR product is with 1.0%
Agarose gel electrophoresis detection and gel extraction purified fragments.
Two DNA fragmentations of recycling are subjected to fusion DNA vaccine using the primer of SEQ ID NO.11 and SEQ ID NO.14,
PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min repeat 30 circulations；72 DEG C are continued to extend
10min, PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purifies the segment.By pTarget- Δ manXYZ
Electrotransformation is inverted to have to Escherichia coli W3110 Δ nagE bacterial strain together for carrier and the DNA fragmentation of recycling
PCas9 carrier (Jiang Y, Chen B, Duan C, et al.Multigene Editing in the Escherichia
Coli Genome via the CRISPR-Cas9 System [J] .Applied&Environmental Microbiology,
2015,81 (7): 2506.).
<SEQ ID NO.11>
<SEQ ID NO.12>
<SEQ ID NO.13>
<SEQ ID NO.14>
For electroporation, conversion has the Escherichia coli W3110 Δ nagE bacterial strain of pCas9 carrier containing
It is cultivated at 30 DEG C in the kanamycins of 50mg/L and the LB culture medium of 10mM L-arabinose, until OD reaches 0.6, bacterium solution
Thallus is obtained by centrifugation.Thallus is washed twice using sterile distilled water, then washed once using 10% glycerol to make
With.Electroporation is carried out at 2.5KV.Bacterium solution after electrotransformation is applied to the kanamycins containing 50mg/L and the hydrochloric acid of 50mg/L
On the LB plate of Spectinomycin resistance, overnight incubation at 30 DEG C.Picking single colonie is as template, with SEQ ID NO.15 and SEQ
The primer of ID NO.16 carries out PCR, and there are 1000bp's in the detection of 1.0% agarose gel electrophoresis by observing
The missing of DNA band confirmation manXYZ gene.
By the bacterial strain confirmed by this at 30 DEG C in the LB culture medium of the kanamycins containing 50mg/L and 5mM IPTG
Lower overnight incubation is to remove pTarget- Δ manXYZ carrier.Then the bacterial strain of pTarget- Δ manXYZ carrier will be had been removed
In LB culture medium at 37 DEG C overnight incubation to remove pCas carrier (Jiang Y, Chen B, Duan C, et
al.Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9
System [J] .Applied&Environmental Microbiology, 2015,81 (7): 2506.).The bacterial strain so constructed
It is named as W3110 Δ nagE Δ manXYZ.
<SEQ ID NO.15>
<SEQ ID NO.16>
(3) knockout of nagA gene
Pass through CRISPR-Cas9 system (Jiang Y, Chen B, Duan C, et al.Multigene Editing in
the Escherichia coli Genome via the CRISPR-Cas9 System[J].Applied&
2015,81 (7): Environmental Microbiology 2506.) edits W3110 Δ nagE Δ manXYZ strain gene group
The metJ gene of middle encoding cystathionine γ synzyme.The primer of SEQ ID NO.17 and SEQ ID NO.18 are used by PCR, with
And pTargetF carrier (AEM (2015), 81 (7): 2506-14) can express as template building and target target gene nagA's
The pTarget- Δ nagA mutational vector of sgRNA.PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C
2min repeats 30 circulations；72 DEG C are continued to extend 10min.By PCR product with DpnI in 37 DEG C of processing 3h, converted after inactivation to
In E.coli BL21 (DE3) recipient bacterium, it is coated on the LB solid plate of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration,
37 DEG C of culture 12h.Random picking single colonie is forwarded to the LB liquid medium of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration
In, 37 DEG C of culture 12h collect thallus and extract plasmid acquisition pTarget- Δ nagA carrier.
<SEQ ID NO.17>
<SEQ ID NO.18>
The primer of SEQ ID NO.19 and SEQ ID NO.20 are used, by PCR with W3110 Δ nagE Δ manXYZ bacterial strain
Genome be template amplification obtain nagA upstream region of gene homologous fragment, PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s,
55 DEG C of 30s, 72 DEG C of 30s repeat 30 circulations；72 DEG C are continued to extend 10min.In the same way using SEQ ID NO.21 and
The primer amplification of SEQ ID NO.22 obtains nagA downstream of gene homologous fragment, and PCR product is examined with 1.0% agarose gel electrophoresis
Survey simultaneously gel extraction purified fragments.
Two DNA fragmentations of recycling are subjected to fusion DNA vaccine using the primer of SEQ ID NO.19 and SEQ ID NO.22,
PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min repeat 30 circulations；72 DEG C are continued to extend
10min, PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purifies the segment.PTarget- Δ nagA is carried
Electrotransformation is inverted to have pCas9 carrier to W3110 Δ nagE Δ manXYZ bacterial strain together for body and the DNA fragmentation of recycling
(Jiang Y, Chen B, Duan C, et al.Multigene Editing in the Escherichia coli Genome
Via the CRISPR-Cas9 System [J] .Applied&Environmental Microbiology, 2015,81 (7):
2506.)。
<SEQ ID NO.19>
<SEQ ID NO.20>
<SEQ ID NO.21>
<SEQ ID NO.22>
For electroporation, conversion have the W3110 Δ nagE Δ manXYZ bacterial strain of pCas9 carrier the card containing 50mg/L that
It is cultivated at 30 DEG C in the LB culture medium of mycin and 10mM L-arabinose, until OD reaches 0.6, bacterium solution is obtained by centrifugation
Thallus.Thallus is washed twice using sterile distilled water, then washed once using 10% glycerol to use.Electroporation exists
It is carried out under 2.5KV.The spectinomycin hydrochloride that bacterium solution after electrotransformation is applied to the kanamycins containing 50mg/L and 50mg/L is resisted
On the LB plate of property, 30 DEG C of overnight incubations.Picking single colonie is as template, with drawing for SEQ ID NO.23 and SEQ ID NO.24
Object carries out PCR, and there are the confirmations of the DNA band of 1000bp in the detection of 1.0% agarose gel electrophoresis by observing
The missing of nagA gene.By the bacterial strain confirmed by this in the LB culture medium of the kanamycins containing 50mg/L and 5mM IPTG
Overnight incubation is at 30 DEG C to remove pTarget- Δ nagA carrier.Then the bacterium of pTarget- Δ nagA carrier will be had been removed
Strain in LB culture medium at 37 DEG C overnight incubation to remove pCas carrier (Jiang Y, Chen B, Duan C, et
al.Multigene Editing in the Escherichia coli Genome via the CRJSPR-Cas9
System [J] .Applied&Environmental Microbiology, 2015,81 (7): 2506.).The bacterial strain so constructed
It is named as W3110 Δ nagE Δ manXYZ Δ nagA.
<SEQ ID NO.23>
<SEQ ID NO.24>
(4) knockout of nagB gene
NagB gene in W3110 Δ nagE Δ manXYZ Δ nagA bacterial strain is knocked out.
Pass through CRISPR-Cas9 system (Jiang Y, Chen B, Duan C, et al.Multigene Editing in
the Escherichia coli Genome via the CRISPR-Cas9 System[J].Applied&
Environmental Microbiology, 2015,81 (7): 2506.) in W3110 Δ nagE Δ manXYZ Δ nagA bacterial strain
GNA1 gene carry out be overexpressed transformation W3110 Δ nagE Δ manXYZ Δ nagA strain gene group in encoded homoserine swash
The nagB gene of enzyme is edited.The primer and pTargetF of SEQ ID NO.25 and SEQ ID NO.26 are used by PCR
Carrier (Jiang Y, Chen B, Duan C, et al.Multigene Editing in the Escherichia coli
Genome via the CRISPR-Cas9 System [J] .Applied&Environmental Microbiology, 2015,
81 (7): 2506.) the pTarget- Δ nagB mutation that can express the sgRNA for targeting target gene nagB as template building carries
Body.PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 2min repeat 30 circulations；72 DEG C after reneing
Stretch 10min.By PCR product with DpnI in 37 DEG C of processing 3h, conversion is into E.coli BL21 (DE3) recipient bacterium after inactivation, coating
In on the LB solid plate of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of culture 12h.Random picking single colonie switching
Into the LB liquid medium of the spectinomycin hydrochloride of 50mg/L containing final concentration resistance, 37 DEG C of culture 12h collect thallus and extract matter
Grain obtains pTarget- Δ nagB carrier.
<SEQ ID NO.33>
<SEQ ID NO.34>
The primer of SEQ ID NO.27 and SEQ ID NO.28 are used, by PCR with W3110 Δ nagE Δ manXYZ Δ
The genome of nagA-GNA1 (trc) bacterial strain is that template amplification obtains nagB upstream region of gene homologous fragment, and PCR reaction condition is as follows:
95℃5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s repeat 30 circulations；72 DEG C are continued to extend 10min.In the same way
NagB downstream of gene homologous fragment is obtained using the primer amplification of SEQ ID NO.29 and SEQ ID NO.30, PCR product is used
The detection of 1.0% agarose gel electrophoresis and gel extraction purified fragments.
Two DNA fragmentations of recycling are subjected to fusion DNA vaccine using the primer of SEQ ID NO.27 and SEQ ID NO.30,
PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min repeat 30 circulations；72 DEG C are continued to extend
10min, PCR product is detected with 0.9% agarose gel electrophoresis and gel extraction purifies the segment.PTarget- Δ nagB is carried
Electrotransformation has turned to W3110 Δ nagE Δ manXYZ Δ nagA-GNA1 (trc) bacterial strain together for body and the DNA fragmentation of recycling
Change has pCas9 carrier (Jiang Y, Chen B, Duan C, et al.Multigene Editing in the
Escherichia coli Genome via the CRISPR-Cas9 System[J].Applied&Environmental
Microbiology, 2015,81 (7): 2506.).
<SEQ ID NO.27>
<SEQ ID NO.28>
<SEQ ID NO.29>
<SEQ ID NO.30>
For electroporation, conversion has W3110 Δ nagE Δ manXYZ Δ nagA-GNA1 (trc) bacterial strain of pCas9 carrier
It is cultivated at 30 DEG C in the LB culture medium of the kanamycins containing 50mg/L and 10mM L-arabinose, until OD reaches
0.6, bacterium solution obtains thallus by centrifugation.Thallus is washed twice using sterile distilled water, then washs one using 10% glycerol
It is secondary to use.Electroporation is carried out at 2.5KV.By the bacterium solution after electrotransformation be applied to the kanamycins containing 50mg/L and
On the LB plate of the spectinomycin hydrochloride resistance of 50mg/L, 30 DEG C of overnight incubations.Picking single colonie is as template, with SEQ ID
The primer of NO.31 and SEQ ID NO.32 carries out PCR, and is deposited in the detection of 1.0% agarose gel electrophoresis by observing
In the missing of the DNA band confirmation nagB gene of 1000bp.By the bacterial strain confirmed by this in the kanamycins containing 50mg/L
With in the LB culture medium of 5mM IPTG at 30 DEG C overnight incubation to remove pTarget- Δ nagB carrier.Then it will have been removed
The bacterial strain of pTarget- Δ nagB carrier in LB culture medium at 37 DEG C overnight incubation with remove pCas carrier (Jiang Y,
Chen B, Duan C, et al.Multigene Editing in the Escherichia coli Genome via the
CRISPR-Cas9 System [J] .Applied&Environmental Microbiology, 2015,81 (7): 2506.).Such as
The Strain Designation of this building is W3110 Δ nagE Δ manXYZ Δ nagA Δ nagB-GNA1 (trc).
<SEQ ID NO.31>
<SEQ ID NO.32>
Embodiment 2
Metabolically engineered, the GNA1 gene table based on W3110 Δ nagE Δ manXYZ Δ nagA Δ nagB-GNA1 (trc) bacterial strain
Up to enhancing
With the original promoter sequence in trc promoter sequence replacement GNA1 gene to reach enhancing GNA1 gene expression mesh
's.
Pass through CRISPR-Cas9 system (Jiang Y, Chen B, Duan C, et al.Multigene Editing in
the Escherichia coli Genome via the CRISPR-Cas9 System[J]·Applied&
2015,81 (7): Environmental Microbiology 2506.) edits W3110 Δ nagE Δ manXYZ Δ nagA bacterium
The promoter sequence of the GNA1 gene of encoded homoserine dehydrogenase in pnca gene group.By PCR using SEQ ID NO.33 and
Primer and pTargetF carrier (Jiang Y, Chen B, Duan C, the et al.Multigene of SEQ ID NO.34
Editing in the Escherichia coli Genome via the CRISPR-Cas9 System[J].Applied&
2015,81 (7): Environmental Microbiology can 2506.) be expressed as template building and be targeted target gene
The pTarget- Δ PGNA1::Ptrc mutational vector of the sgRNA of GNA1 promoter sequence.PCR reaction condition is as follows: 95 DEG C
5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 2min repeat 30 circulations；72 DEG C are continued to extend 10min.By PCR product DpnI
In 37 DEG C of processing 3h, it is strong to be coated on the hydrochloric acid of 50mg/L containing final concentration into E.coli BL21 (DE3) recipient bacterium for conversion after inactivation
On the LB solid plate of miromycin resistance, 37 DEG C of culture 12h.It is strong that random picking single colonie is forwarded to the hydrochloric acid of 50mg/L containing final concentration
In the LB liquid medium of miromycin resistance, 37 DEG C of culture 12h collect thallus and extract plasmid acquisition pTarget- Δ
PGNA1::Ptrc carrier.
<SEQ ID NO.33>
<SEQ ID NO.34>
The primer of SEQ ID NO.35 and SEQ ID NO.36 are used, by PCR with W3110 Δ nagE Δ manXYZ Δ
The genome of nagA bacterial strain is that template amplification obtains GNA1 gene promoter sequence upstream homologous fragment, the promoter of GNA1 gene
Sequence information be based on (the EcoCyc gene accession number: EG10590) obtained in EcocycE.coli Database database,
PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s repeat 30 circulations；72 DEG C are continued to extend
10min.GNA1 gene promoter is obtained using the primer amplification of SEQ ID NO.37 and SEQ ID NO.38 in the same way
Sequence downstream homologous fragment, PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purified fragments.
Two DNA fragmentations of recycling are subjected to fusion DNA vaccine using the primer of SEQ ID NO.35 and SEQ ID NO.38,
PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min repeat 30 circulations；72 DEG C are continued to extend
10min.PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purifies the segment, in the gene band incited somebody to action
Ptrc promoter sequence is inserted between two homologous fragments.By the DNA fragmentation of pTarget- Δ PGNA1::Ptrc carrier and recycling
Electrotransformation is to W3110 Δ nagE Δ manXYZ Δ nagA bacterial strain together, it is inverted have pCas9 carrier (Jiang Y,
ChenB, Duan C, et al.Multigene Editing in the Escherichia coli Genome via the
CRISPR-Cas9 System [J] .Applied&Environmental Microbiology, 2015,81 (7): 2506.).
<SEQ ID NO.35>
<SEQ ID NO.36>
<SEQ ID NO.37>
<SEQ ID NO.38>
For electroporation, conversion has the W3110 Δ nagE Δ manXYZ Δ nagA bacterial strain of pCas9 carrier containing 50mg/L
Kanamycins and 10mM L-arabinose LB culture medium in cultivated at 30 DEG C, until OD reaches 0.6, bacterium solution pass through from
Gains in depth of comprehension are to thallus.Thallus is washed twice using sterile distilled water, then washed once using 10% glycerol to use.Electricity is worn
Hole is carried out at 2.5KV.The hydrochloric acid that bacterium solution after electrotransformation is applied to the kanamycins containing 50mg/L and 50mg/L is grand mould
On the LB plate of plain resistance, 30 DEG C of overnight incubations.Picking single colonie is as template, with SEQ ID NO.39 and SEQ ID NO.40
Primer carry out PCR, and by observe 1.0% agarose gel electrophoresis detection in there are the confirmations of the DNA band of 700bp
The original promoter sequence of GNA1 gene is replaced by Ptrc promoter sequence.
By the bacterial strain confirmed by this at 30 DEG C in the LB culture medium of the kanamycins containing 50mg/L and 5mM IPTG
Lower overnight incubation is to remove pTarget- Δ PGNA1::Ptrc carrier.Then pTarget- Δ PGNA1::Ptrc will be had been removed
The bacterial strain of carrier in LB culture medium at 37 DEG C overnight incubation to remove pCas carrier (Jiang Y, Chen B, Duan C, et
al.Multigene Editing in the Escherichia coli Genome via the CRJSPR-Cas9
System [J] .Applied&Environmental Microbiology, 2015,81 (7): 2506.).PCas carrier will be removed
Bacterial strain afterwards carries out PCR amplification using primer SEQ ID NO.39 and SEQ ID NO.38, and PCR reaction condition is as follows: 95 DEG C
5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1.5min repeat 30 circulations；72 DEG C are continued extension 10min and survey PCR product
Sequence verifying, sequencing result by BLAST sequence alignment confirm GNA1 gene promoter sequence in situ by Ptrc promoter at
Function replacement.The Strain Designation of building is W3110 Δ nagE Δ manXYZ Δ nagA-GNA1 (trc).
<SEQ ID NO.39>
<SEQ ID NO.40>
Embodiment 3
The acquisition of GlmS gene with feedback-inhibition resistance
With wild-type e. coli Escherichia coli W3110 (being purchased from strain Escherichia coli collection CGSC)
Genome carries out PCR amplification as template, together with the primer of SEQ ID NO.49 and SEQ ID NO.50.PCR reaction condition: pre-
95 DEG C of 5min, 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min are denaturalized, totally 30 circulations, last 72 DEG C of extensions 10min.PCR product is used
Simultaneously gel extraction purifies the segment to the detection of 1.0% agarose gel electrophoresis, is held using Taq archaeal dna polymerase to segment 5 ' and introduces alkali
Base A.The segment is attached with pGEM-T carrier under T4DNA connection enzyme effect, obtains cloning recombinant plasmids pGEM-T-
GlmS.Rite-directed mutagenesis is carried out to GlmS gene on pGEM-T-GlmS plasmid, 64 amino acids-glutamic acid-is replaced with into paddy ammonia
Amide (E64Q), the primer used are SEQ ID NO.51 and SEQ ID NO.52, and using pGEM-T-GlmS plasmid as template,
It is introduced and is mutated by PCR, PCR response procedures are as follows: 98 DEG C of 3min；98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 3min repeat 30 and follow
Ring；72 DEG C are continued to extend 10min.PCR product is converted after inactivation to E.coli BL21 (DE3) with DpnI in 37 DEG C of processing 3h
In recipient bacterium, it is coated on the LB solid plate of the ampicillin sodium resistance of 50mg/L containing final concentration, after 37 DEG C of culture 12h, with
Machine picking single colonie carries out sequencing analysis, obtains GlmS mutated gene.
<SEQ ID NO.49>
<SEQ ID NO.50>
<SEQ ID NO.51>
<SEQ ID NO.52>
Embodiment 4
The Enhanced expressing of GlmS releasing feedback inhibition gene
With wild-type e. coli Escherichia coli W3110 (being purchased from strain Escherichia coli collection CGSC)
Genome carries out PCR amplification as template, together with the primer of SEQ ID NO.55 and SEQ ID NO.56.PCR reaction condition: pre-
95 DEG C of 5min, 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1.5min are denaturalized, totally 30 circulations, last 72 DEG C of extensions 10min.PCR product
With the detection of 1.0% agarose gel electrophoresis and gel extraction purifies the segment, is held and is introduced to segment 5 ' using Taq archaeal dna polymerase
Base A.The segment is attached with pGEM-T carrier under T4DNA connection enzyme effect, obtains cloning recombinant plasmids pGEM-T-
GlmS。
By the recombinant plasmid transformed into e. coli jm109, being coated on containing concentration is 50 μ g/mL ampicillin sodiums
The LB plate of resistance, random picking positive colony carry out sequencing analysis.Expression primer SEQ ID is designed based on the analysis results
NO.57 and SEQ ID NO.58 (is respectively provided with Kpn I restriction enzyme site and the restricted digestion position Hind III in primer
Point), obtain the GlmS gene order of 1257bp.Enzyme is carried out to amplified fragments using Kpn I and Hind III restriction enzyme
Processing is cut, is connected the segment with the pTrc99A-GlmS11 of identical restriction enzyme enzymatic treatment using T4 DNA ligase
It connects, construction of expression vector pTrc99A-GlmS11-GlmS, and is named as pA11y.
<SEQ ID NO.55>
<SEQ ID NO.56>
<SEQ ID NO.57>
<SEQ ID NO.58>
Embodiment 5
The building of experimental strain
(1) building of bacterial strain
By coli strain W3110 Δ nagE Δ manXYZ Δ nagA Δ nagB-GNA1 (trc), the W3110 of building
ΔnagEΔmanXYZΔnagA-GNA1(trc)、W3110 ΔnagEΔmanXYZΔnagAΔnagB-GNA1-thrA
(trc) it is prepared into competent cell, and is converted the plasmid pA11y constructed in embodiment 4 to above-mentioned by chemical transformation
In the competent cell of preparation, it is coated on the LB plate containing concentration for 50 μ g/mL ampicillin sodium resistances, is contained
Bacterial strain after three kinds of pA11y plasmid are Metabolically engineered.
(2) shake flask fermentation is tested
Fermenting experiment test is carried out in shaking flask to above-mentioned thallus, to produce the energy of ammonia sugar between more each genotype strain
Power.Shake flask fermentation experiment is carried out by following scheme: each bacterial strain streak inoculation is being contained 50 μ g/mL ampicillin sodium resistances
LB plate on, 37 DEG C in the incubator overnight incubation, picking single colonie be seeded in the LB culture medium of 5mL, and with
The revolving speed of 200rpm is in 37 DEG C of overnight incubations in the incubator.
Add the fermentation medium that shows in the table 1 of 20mL in the shaking flask of 500mL, and by the seed of every kind of bacterial strain of 1mL
Liquid is seeded in the culture medium of shaking flask.Then shaking flask cultivates 48 hours in the incubators of 30 speed with the revolving speed of 180rpm, finally
Compare the amount for carrying every kind of bacterial strain acquisition ammonia sugar of plasmid.The results are shown in Table 1, glucose 40g/L, potassium dihydrogen phosphate 2g/L,
Ammonium sulfate 17g/L, yeast powder 4g/L, calcium carbonate 30g/L, L-threonine 0.2g/L, Thiamine 0.1mg/L, MgSO41g/L、
FeSO40.5mg/L、MnSO45mg/L and ZnSO45mg/L。
The experiment of the Metabolically engineered bacterial strain shake flask fermentation of table 1
According to table 1, pass through Metabolically engineered and carrying carrier W3110 Δ nagE Δ manXYZ Δ nagA- for three plants
GNA1(trc)/pA11y、W3110 ΔnagEΔmanXYZΔnagAΔnagB-GNA1(trc)/pA11y、W3110 ΔnagE
Δ manXYZ Δ nagA Δ nagB-GNA1-thrA (trc)/pA11y bacterial strain all has the ability of production ammonia sugar, can weaken it
To the feedback inhibition of GlmS gene, to play the good activity of the enzyme.
(3) L-threonine additive amount is tested
To L-threonine auxotrophic strain W3110 Δ nagE Δ manXYZ Δ nagA Δ nagB-GNA1-thrA
(trc)/pA11y has carried out the experiment of L-threonine additive amount, 0 is added respectively in the fermentation medium without L-threonine~
The L-threonine of 1.6g/L various concentration gradient, and the seed liquor of every kind of bacterial strain of 1mL is seeded in the culture medium of shaking flask.Then
Shaking flask is cultivated 48 hours in 30 DEG C of incubator with the revolving speed of 180rpm.Final examination W3110 Δ nagE Δ manXYZ Δ
NagA Δ nagB-GNA1-thrA (trc)/pA11y bacterial strain production ammonia sugar ability.
The fermenting experiment of L-threonine, W3110 Δ nagE Δ manXYZ Δ nagA Δ nagB-GNA1- are added by external source
ThrA (trc)/pA11y bacterial strain production ammonia sugar yield level is 25g/L.For L-threonine auxotrophic strain,
No external source add L-threonine when, the growth of bacterial strain is restricted, metabolism production ammonia sugar ability also therewith by
To limitation.When L-threonine is excessively added, it cannot not only promote thalli growth, can also play negative shadow to the accumulation of ammonia sugar
It rings.
Embodiment 6
The detection of concentration of glucose is analyzed using SBA-40C type lactic acid-glucose biological sensing analysis instrument.Specific behaviour
Make as follows: fermentation liquid is centrifuged 10min through 8000r/min, takes supernatant suitably to be diluted with deionized water, makes data measured 100
Within, to keep good linear relationship.25 μ L of glucose solution standard sample, which is drawn, with micro syringe injects injection port rapidly,
Multiple sample introduction draws the 25 μ L of sample handled well, sample introduction simultaneously records until screen data is 100 and sample introduction lamp is always on again at this time
Lower data (concentration of glucose is represented when display data on the screen is 100 as 1g/L).Concentration of glucose in fermentation liquid show data/
100 × extension rate.
Glc NAc is detected with HPLC method, Agilent 1200, RID detector, Alltech C18 column (250 ×
4.6mm, 5 μm), mobile phase: 70% acetonitrile, flow velocity 0.7mL/min, 30 DEG C of column temperature, sampling volume is 10 μ L.The survey of acetic acid concentration
It is fixed: HPLC method.Chromatographic column is Zorbax SB-C18 4.6mm × 150mm；Mobile phase 0.1%H3PO4Solution；Flow velocity 1.0mL/
min；Detection wavelength 210nm；30 DEG C of column temperature；5 μ L of sample volume.
The fermentation of ammonia sugar is carried out on 5L fermentor, fermentation liquid initial volume is 2L, and glucose initial concentration is 27g/L,
With F (μ set=0.2h after 2h after inoculation-1) glucose for adding 300g/L is flowed, since the 10h that ferments, with the flow velocity stream of 32mL/h
Add the glucose of 300g/L to fermentation ends.Stage by stage glucose feeding strategy fermentation experimental result: DCW reaches most in 16h
Big value 20.44g/L, Glucosamine and acetylglucosamine reach peak 35.10g/L and 34.56g/L in 16h, and
Acetic acid concentration is only 1.59g/L at this time.With do not flow plus when glucose compared with, Glucosamine total output peak reaches
69.66g/L, less stream added-time improve 1.59 times.Glucosamine and acetylglucosamine production intensity are respectively
58.7% and 51.6% is respectively increased in 1.46g/ (Lh) and 1.44g/ (Lh), less stream added-time.
Embodiment 7
The fermentation that ammonia sugar is carried out on 5L fermentor, is added fermentation medium 2L, controlled at 37 DEG C, adjusts ventilatory capacity
And revolving speed, keeping dissolved oxygen is respectively 10%, 20%, 30%, 40% to ferment.
Under the conditions of not controlling dissolved oxygen level, Glucosamine batch fermentation is carried out in 5L fermentor, research is found: molten
Oxygen level continues to decline in the 8h of beginning, and minimum 17% is reached when 8h, is hereafter gradually increasing, and is reaching 89% for 24 hours.?
Fermentation to 16h, DCW, Glc N and Glc NAc concentration respectively reaches peak 15.48g/L, 25.42g/L and 27.89g/L, Portugal
Grape sugar concentration continues to decline, and is reaching 10g/L for 24 hours.
By controlling different dissolved oxygen levels (10%, 20%, 30%, 40%) during the fermentation, it is had studied to amino
The influence of glucose batch fermentation, and compared with the fermentation situation for not controlling dissolved oxygen level.It is respectively in dissolved oxygen level
10%, 20%, 30%, 40%DCW peak is respectively 9.45,15.44,13.14 and 11.98g/L, illustrates 20% dissolved oxygen water
Flat to be suitable for thalli growth, when dissolved oxygen level is respectively 10%, 20%, 30%, 40%, Glc N concentration peak is respectively
18.80,25.69,25.19 and 21.88g/L；Glc NAc concentration peak is respectively 15.30,26.01,25.11 and 22.48g/
L.When dissolved oxygen level is increased to 40% from 10%, glucose is averaged specific consumption rate from 0.07h-1It is increased to 0.11h-1；Molten
When oxygen level is 20%, the production intensity of Glc N and Glc NAc respectively reach peak 1.07 and 1.08g/ (Lh), are higher than
The largest production intensity of Glc N and Glc NAc under other 3 kinds of dissolved oxygen levels；However under different dissolved oxygen levels, the highest of acetic acid
Concentration is controlled in dissolved oxygen level in 40% highest, is 2.03g/L, this, which illustrates to control thallus when water containing high dissolved oxygen is flat to ferment, will generate more
More acetic acid, this is unfavorable for the growth of recombinant bacterium and the expression of foreign protein, and DCW is lower under this puts down from water containing high dissolved oxygen can
Out.
By in 5L fermentor convection current add glucose concentration and fed-batch mode optimization and dissolved oxygen levels it is excellent
Change the production capacity for further increasing strain.The optimization for having carried out dissolved oxygen level control, proposes molten stage by stage on this basis
Oxygen level control strategy, i.e., it is 30%, 8-12h control that control DO level, which is 20%, 2-8h control DO level, in the preceding 2h of fermentation
DO level processed is that 40%, 12-18h controlled level DO is 30%, with this condition, the yield of Glucosamine and production intensity compared with
It is significantly improved when not controlling dissolved oxygen.
Claims (9)
1. a kind of recombination bacillus coli for accumulating Glucosamine, which is characterized in that knock out Glucosamine in Escherichia coli and close
At 4 genes in approach: acetylglucosamine transports sub- encoding gene nagE, mannose phosphoric acid transports sub- encoding gene
ManXYZ, deacetylation enzyme gene nagA and Deaminase Gene nagB, improve the yield of Glucosamine.
2. the recombination bacillus coli of accumulation Glucosamine according to claim 1, which is characterized in that as the big of host
Enterobacteria is Escherichia coli W3110, or has knocked out acetylglucosamine and transported sub- encoding gene nagE Escherichia coli
It W3110 or has knocked out mannose phosphoric acid and transports sub- encoding gene manXYZ Escherichia coli W3110 or knocked out deacetylated
Base enzyme gene nagA Escherichia coli W3110, or knocked out Deaminase Gene nagB Escherichia coli W3110.
3. the recombination bacillus coli of accumulation Glucosamine according to claim 1 or 2, which is characterized in that by amino Portugal
Glucosamine synthetase-coding gene GlmS in grape sugar route of synthesis, is cloned into expression vector pTargetF, then converts
Escherichia coli.
4. a kind of recombination bacillus coli for accumulating Glucosamine according to claim 1 or 2, which is characterized in that by ammonia
Glucosamine acetylase encoding gene GNA1 in base glucose route of synthesis, is cloned into expression vector pTargetF, so
After convert Escherichia coli.
5. the recombination bacillus coli of accumulation Glucosamine according to claim 3, which is characterized in that in pGEM-T-
Rite-directed mutagenesis is carried out to GlmS gene on GlmS plasmid, 64 amino acids glutamic acid are replaced with into glutamine.
6. a kind of method of the recombination bacillus coli of any accumulation Glucosamine of building claim 1-5, feature
It is, comprising the following steps:
(1) clone's acetylglucosamine transports sub- encoding gene, mannose phosphoric acid transports sub- encoding gene, deacetylase base
Cause, Deaminase Gene, are connected on recombinant expression carrier；
(2) above-mentioned recombinant expression carrier is converted into Escherichia coli, obtains accumulation Glucosamine recombination bacillus coli, the large intestine
Bacillus is Escherichia coli W3110, Escherichia coli W3110 Δ nagE, Escherichia coli W3110 Δ nagE Δ manXYZ, large intestine bar
Bacterium W3110 Δ nagE Δ manXYZ Δ nagA and Escherichia coli W3110 Δ nagE Δ manXYZ Δ nagA Δ nagB.
7. a kind of method using any recombination bacillus coli fermenting production Glucosamine of claim 1-5, special
Sign is, by recombinant escherichia coli strain in 200rpm revolving speed, 37 DEG C of incubators overnight incubation, obtain bacterial strain seed liquor；It will
Seed liquor is seeded in the shaking flask equipped with fermentation medium, and shaking flask cultivates 48h in 30 DEG C of incubator with the revolving speed of 180rpm.
8. the method according to claim 7 using recombination bacillus coli fermenting production Glucosamine, which is characterized in that
The seed liquid culture medium is LB culture medium.
9. the method according to claim 7 using recombination bacillus coli fermenting production Glucosamine, which is characterized in that
The formula of the fermentation medium: glucose 40g/L, potassium dihydrogen phosphate 2g/L, ammonium sulfate 17g/L, yeast powder 4g/L, calcium carbonate
30g/L, L-threonine 0.2g/L, Thiamine 0.1mg/L, MgSO4 1g/L、FeSO4 0.5mg/L、MnSO45mg/L and
ZnSO4 5mg/L。
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