CN106566797A - Method for eliminating actinoplanes spp. producing strain production of component C by using metabolic engineering - Google Patents
Method for eliminating actinoplanes spp. producing strain production of component C by using metabolic engineering Download PDFInfo
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Abstract
A method for eliminating acarbose producing strain actinoplanes spp. production of a component C by using metabolic engineering is provided; through deletion of a malt oligosaccharide based trehalose synthase (TreY)-encoding treY (ACPL-6622) gene in the acarbose producing strain actinoplanes spp., accumulation of the by-product component C in the strain fermentation process is eliminated. An obtained engineering strain QQ-2 does not accumulate the component C, and besides, the acarbose yield is increased by 10.7%.
Description
Technical field
The invention belongs to biological technical field, and in particular to eliminate Acarbose production bacterial strain using metabolic engineering and produce component
The method of C.
Technical background
Acarbose (acarbose) is a kind of false tetrose of C7 cyclitols class, can occur to compete with alpha-glucosidase
Sexual reaction, reduces hydrolysis of the alpha-glucosidase in small intestinal parietal cell to disaccharidase, oligosaccharide and polysaccharide, to reduce to Fructus Vitis viniferae
Generation, absorption and the utilization of sugar and Fructose, so as to reduce the purpose of Patients with NIDDM post-prandial glycemia.As which has
Good pharmacokinetic property and hypotoxicity, and become preferable type Ⅱdiabetes mellitus medicine, its sales quota exceedes
The hypoglycemic medicine such as traditional sulfonylurea and biguanideses.
It is industrial at present mainly to adopt Actinoplanes spp. fermenting and producing Acarbose, it is responsible for which biosynthetic
Gene cluster (acb cluster) is made up of 22 structural genes, is encoded and its biosynthesis and transhipment and α-glucoside metabolism phase
The albumen of pass.On this basis, by molecular biology and genetic engineering means, then it is aided with external enzymatic analysis, isotope mark
Note is fed and mass-spectrometric technique, has parsed the biosynthesis pathway of part Acarbose, has mainly included：C7 cyclitols and 4- amino-
The synthesis and assembling of 4,6- two modules of dideoxy glucose, and the assembling with maltose molecule.
Component C is the Main By product in acarbose sweats, and structure is as shown in figure 1, the main product of phase after fermentation
It is raw, and with the increase of osmotic pressure of fermentation liquor, content is increased substantially.Speculate component C generation may occur mainly in it is extracellular,
Possibly ferment the later stage, the intracellular enzyme catalysiss discharged after cell lysis are generated.Add Validacin (Takeda) in the fermentation medium, have
The analog of the effect acarbose such as oxygen amine A and effective amine can be greatly lowered the generation of component C, while A Kabo can be improved
The yield of sugar.Experiment in vitro proves that Acarbose can be converted into component C by Fructus Hordei Germinatus oligomerization glycosyl trehalose synthetase (TreY).
The content of the invention
The purpose of the present invention, is to provide a kind of utilization metabolic engineering and eliminates the side that Acarbose production bacterial strain produces component C
Method.
In order to realize the purpose of the present invention, technical scheme below is present invention employs：
A kind of method that utilization metabolic engineering eliminates the generation of component C in Acarbose production bacterial strain, be by lack Ah
The treY genes of Fructus Hordei Germinatus oligomerization glycosyl trehalose synthetase are encoded in card ripple sugar production bacterium Actinoplanes spp., eliminating should
The accumulation of class strain fermentation byproduct in process thing component C；Comprise the following steps that：
The first step：Build the recombiant plasmid pLQ756 for treY gene knockouts；
Second step：Recombiant plasmid pLQ756 is imported into Acarbose by way of mycelium engagement transfer and produces bacterial strain
Actinoplanes spp., obtain conjugon；
3rd step：The conjugon of acquisition Jing after culture, lax, spread plate, picking single bacterium colony, and verifying whether as A Bo
Draw mycin responsive type；After the strain culturing with apramycin resistance, genome is extracted, enter performing PCR checking, it is right to obtain
TreY deletion mutation strain QQ-2；
4th step：TreY deletion mutation strain QQ-2 are fermented, HPLC and LC-MS analyses are carried out to fermentation liquid.
The construction method of the recombiant plasmid pLQ756 is：Expanded under the upstream homology arm and 2.56kb of 2.57kb respectively
Trip homology arm, inserts in the replicating vector pLQ752 after BamHI/HindIII enzyme action, obtains recombiant plasmid pLQ756.
The mode of the mycelium Conjugative tiansfer, specifically includes following steps：
A. the recombiant plasmid pLQ756 of structure is converted to ET12567, the correct bacterial strain of picking, Jing after 8h cultures, switching
Continue culture 4-5h into fresh LB culture medium, to OD600nmFor 1；
B. Actinoplanes spp. mycelium are lined on Solid media for plates, 30 DEG C are cultivated 3-4 days, inoculation
To mycelium liquid culture medium, after culture 36h, TSB culture medium culturing 8-12h are forwarded to；
C. it is 1 according to thalline quantity by mycelium and the ET12567 containing recombiant plasmid:30 ratio mix homogeneously, coating
On SFM flat boards, it is placed in 30 DEG C of incubators and cultivates 32h, every piece of flat board is covered with 2mg apramycins and trimethoprim respectively
Lid, continues culture 6-7 days, obtains conjugon.
The screening technique of mutant QQ-2 is as follows：Picking conjugon lines the flat board containing apramycin and trimethoprim
On solid medium, after culture 3-4d, it is inoculated in liquid mycelial culture medium, cultivates 36h, is inoculated with according to 10% inoculum concentration
Into fresh SM culture medium, continue culture 48h, after taking ten times of mycelium dilution, filtered with spore filter, filtrate dilution
104-105Afterwards, coat in the STY plating mediums of the 5-flurocytosine containing 50mg/L, cultivate 4-5 days, picking single bacterium colony, training
Genome, PCR checkings are extracted after supporting.
Mutant is lined into Solid media for plates, 30 DEG C of incubator cultures 3-4 days are placed in, it is appropriate with inoculating loop picking
Mycelium, is inoculated in seed culture medium, is placed in 30 DEG C of 220rpm shaking tables and cultivates 30-36h, is inoculated with according to 15% inoculum concentration
To fermentation medium, it is placed in 30 DEG C of 220rpm shaking tables and ferments 7 days, adds within the 3rd day 2% maltose and Fructus Vitis viniferae respectively in fermentation
Sugar, produces Acarbose.
The mass volume ratio formula of the seed culture medium is glucose 1%, maltose 1.5%, glycerol 1%, hot moulding bean
Cake powder 4%, starch 1%, CaCO30.2%, pH are adjusted to 7.2,115 DEG C of high pressure steam sterilization 15min.The quality of fermentation medium
Volume of preparation is maltose 5%, glucose 3%, hot moulding soybean cake powder 1%, FeCl30.05%th, K2HPO40.1%th, glutamic acid
0.3%th, CaCO30.25%, pH are adjusted to 7.2,115 DEG C of high pressure steam sterilization 15min.
The condition that HPLC analyses are carried out to fermentation liquid is as follows：
Pillar：Agilent ZORBAX NH2；
Mobile phase：Acetonitrile：Water=73：27；
Flow velocity：1ml/min；
Detection wavelength：210nm；
Sampling volume：20μL.
The condition that UPLC-Q-TOF-MS analyses are carried out to fermentation liquid is as follows：
Pillar：Agilent ZORBAX NH2；
Mobile phase：Acetonitrile：Water=73：27；
Flow velocity：0.4ml/min；
Detection wavelength：210nm；
Sampling volume：10μL；
Detection pattern：Negative ion mode.
TreY gene orders involved in the present invention are with reference to the Actinoplanes sp.SE50/110 provided in GenBank
Genomic information (Assembly:GCA_000237145.1).
The present invention eliminates Acarbose production bacterium Actinoplanes spp. and was fermenting by knocking out treY genes
The phenomenon of component C is accumulated in journey, while the output increased of Acarbose 10.7%, is the difficulty for reducing Acarbose downstream purification
Degree, saves purification cost, improves Acarbose drug quality and all haves laid a good foundation.
Description of the drawings
The structural formula of Fig. 1 components C；
Fig. 2 is that treY genes are lacked in Actinoplanes spp. to the schematic diagram of acquisition mutant QQ-2；
Fig. 3 is the PCR checkings of mutant QQ-2, wherein, lane1：marker；lane 2：With TV-F/TV-R as primer,
With the PCR primer that the genome of mutant QQ-2 is obtained as template amplification；lane 3：With starting strain SE50/110 genomes it is
The PCR primer that template amplification is obtained；lane 4：With the PCR primer that recombiant plasmid pLQ756 is obtained as template amplification；
Fig. 4 is the HPLC analysis results of starting strain SE50/110 and mutant QQ-2 fermentation liquids；
Fig. 5 is the Acarbose yield of starting strain SE50/110 and mutant QQ-2；
Fig. 6 is the LC-MS analysis results of starting strain SE50/110 and mutant QQ-2 fermentation liquids, wherein, A：A Kabo
The m/z of sugar and component C；B：The m/z of Acarbose and component C is extracted from TIC.
Specific implementation method：
Following examples will combine accompanying drawing and subordinate list is further described to the present invention, but these embodiments are not intended to
To any restriction of the invention.
Embodiment 1
Step one：The structure of recombiant plasmid pLQ756
With Actinoplanes sp. genomes as template, with treY-1/treY-2 and treY-3/treY-4 as primer
(primer sequence such as table 1), amplification length is that the homology arm T-1 of 2570bp and 2556bp and T-3, T-1 two ends contain BamHI/ respectively
EcoRI restriction enzyme sites, T-3 two ends restriction enzyme site containing EcoRI/HindIII, by replication form plasmid pLQ752 with BamHI/
After HindIII enzyme action, it is connected with T-1 the and T-3 fragments of enzyme action purification respectively, obtains recombiant plasmid pLQ756.
The structure and checking primer of 1 treY deletion mutation strains of table
Step 2：By the method for mycelium Conjugative tiansfer, recombiant plasmid pLQ756 is imported into Actinoplanes spp.
In, concrete grammar is：
A. by the recombinant plasmid transformed for building to ET12567 (pUZ8002), the correct bacterial strain of picking, Jing after 8h cultures, turns
Continue culture 4-5h in being connected to fresh LB culture medium, to OD600nmAbout 1.
B. the Actinoplanes spp. mycelium of preservation are lined on Solid media for plates, 30 DEG C of culture 3-4
My god, mycelium liquid culture medium is seeded to, after culture 36h, TSB culture medium culturing 8-12h is forwarded to.
C. it is 1 according to thalline quantity by mycelium and the ET12567 containing recombiant plasmid (pUZ8002):30 ratios mix
Uniformly, coat on SFM flat boards, be placed in 30 DEG C of incubators and cultivate 32h, every piece of flat board is respectively with 2mg apramycins and first
The pyridine of oxygen benzyl is covered, and continues culture 6-7 days, obtains conjugon.
Step 3：The screening of mutant
Picking conjugon is lined on the Solid media for plates containing apramycin and trimethoprim, after culture 3-4d,
It is inoculated in liquid mycelial culture medium, cultivates 36h, be seeded in fresh SM culture medium according to 10% inoculum concentration, continues
Culture 48h, after taking ten times of mycelium dilution, is filtered with spore filter, filtrate dilution 104-105Afterwards, coat containing 50mg/L's
In the STY plating mediums of 5-flurocytosine, cultivate 4-5 days, picking single bacterium colony extracts genome, with TV-F/TV-R after culture
Enter performing PCR checking for primer.As shown in Figure 2 and Figure 3, the PCR primer of mutant is 0.7kb, the PCR with recombiant plasmid pLQ756
Primer size is consistent, and meets expection, shows that mutant QQ-2 is correct.
Step 4：The fermentation of mutant
The method of Actinoplanes spp. fermenting and producing Acarbose is：Mutant is lined into flat-plate solid culture
Base, is placed in 30 DEG C of incubator cultures 2-3 days, with the appropriate mycelium of inoculating loop picking, is inoculated in seed culture medium (glucose
1%th, maltose 1.5%, glycerol 1%, hot moulding soybean cake powder 4%, starch 1%, CaCO30.2%, pH are adjusted to 7.2,115 DEG C
High pressure steam sterilization 15min), be placed in 30 DEG C of 220rpm shaking tables and cultivate 30-36h, fermentation is seeded to according to 15% inoculum concentration
Culture medium (maltose 5%, glucose 3%, hot moulding soybean cake powder 1%, FeCl3 0.5、K2HPO40.1%th, glutamic acid
0.3%th, CaCO30.25%, pH are adjusted to 7.2,115 DEG C of high pressure steam sterilization 15min), it is placed in 30 DEG C of 220rpm shaking tables and ferments
7 days, add within the 3rd day 2% maltose and glucose respectively in fermentation.
Step 5：HPLC analyzes the tunning of starting strain and mutant QQ-2
1mL fermentation liquids are taken, 12000rpm centrifugation 10min take fermentation supernatant, after 2 times of dilution, carry out HPLC analyses.Analysis
Condition is as follows：
Pillar：Agilent ZORBAX NH2
Mobile phase：Acetonitrile：Water=73：27
Flow velocity：1ml/min
Detection wavelength：210nm
Sampling volume：20μL
As a result as shown in Figure 4, Figure 5, using new liquid phase pillar, the appearance time of Acarbose is about 40min, component C
Appearance time be about 43.2min, component C for having about 177mg/mL in wild-type strain is produced, and in treY deletion mutations
Can't detect the generation of component C in strain QQ-2, and the output increased of Acarbose 10.7%, the result of study shows, disappearance
TreY genes can reach the purpose for eliminating component C in fermentation liquid.
Step 5：LC-MS analyzes the tunning of starting strain and mutant QQ-2
1mL fermentation liquids are taken, 12000rpm centrifugation 10min take fermentation supernatant, after 2 times of dilution, carry out HPLC analyses.Analysis
Condition is as follows：
Pillar：Agilent ZORBAX NH2
Mobile phase：Acetonitrile：Water=73：27
Flow velocity：0.4ml/min
Detection wavelength：210nm
Sampling volume：10μL
As a result as shown in Figure 6A, as Acarbose is completely the same with the molecular weight of component C, it is C25H43NO18, so,
In the negative ion mode, its m/z is 644.2532 ([M-H]-) and 742.2330 ([M+H2PO4]-), and m/z is 742.2330
([M+H2PO4]-) abundance it is very high, it may be possible to due in liquid phase post participate in phosphate it is relevant.
M/z742.2330 ([M+H are extracted from inside the total ion current of fermented sample analysis2PO4]-), as a result such as Fig. 6 B institutes
Show, the appearance time of Acarbose is 89min, and the appearance time of component C is 100min, sending out in mutant consistent with expection
The generation of component C is can't detect in zymotic fluid.
HPLC and high resolution mass spectrum can't detect the presence of component C in mutant fermentation liquid, show by knocking out treY
Gene, eliminates side components C in Acarbose sweat.
<110>Shanghai Communications University
<120>The method that Acarbose production bacterial strain produces component C is eliminated using metabolic engineering
<160> 6
<210> 1
<211> 21
<212> DNA
<213> treY-1
<400>1
CGCAGGATCC ATGCAGGTCT GGCCTGGTCA C 21
<210> 2
<211> 23
<212> DNA
<213> treY-2
<400>2
CCGAGAATTC GTCGTAGTAG ACCAGCTCGT CGC 23
<210> 3
<211> 30
<212> DNA
<213> treY-3
<400>3
CCGAGAATTC CATCTGATCT GGCAGGTCAC 30
<210> 4
<211> 30
<212> DNA
<213> treY-4
<400>4
CCCTAAGCTT AAACACCATT GAGCGGTACG 30
<210> 5
<211> 28
<212> DNA
<213> TV-F
<400>5
CTGGCTGGTG ATCGAGAAGA TCCTGGAG 28
<210> 6
<211> 27
<212> DNA
<213> TV-R
<400>6
GAGCACTCGT GGAACTCGTC GGTCGAG 27。
Claims (8)
1. a kind of method that utilization metabolic engineering eliminates the generation of component C in Acarbose production bacterial strain, it is characterised in that：Pass through
The treY genes of Fructus Hordei Germinatus oligomerization glycosyl trehalose synthetase are encoded in disappearance Acarbose production bacterium Actinoplanes spp.,
Eliminate the accumulation of such strain fermentation byproduct in process thing component C；Comprise the following steps that：
The first step：Build the recombiant plasmid pLQ756 for treY gene knockouts；
Second step：Recombiant plasmid pLQ756 is imported into Acarbose by way of mycelium engagement transfer and produces bacterial strain
Actinoplanes spp., obtain conjugon；
3rd step：The conjugon of acquisition Jing after culture, lax, spread plate, picking single bacterium colony, and verify whether to draw mould for A Bo
Plain responsive type；After the strain culturing with apramycin resistance, genome is extracted, enter performing PCR checking, obtain and treY is lacked
Lose mutant QQ-2；
4th step：TreY deletion mutation strain QQ-2 are fermented, HPLC and LC-MS analyses are carried out to fermentation liquid.
2. utilization metabolic engineering according to claim 1 eliminates the method that Acarbose production bacterial strain produces component C, and which is special
Levy and be：The construction method of the recombiant plasmid pLQ756 is：Expanded under the upstream homology arm and 2.56kb of 2.57kb respectively
Trip homology arm, inserts in the replicating vector pLQ752 after BamHI/HindIII enzyme action, obtains recombiant plasmid pLQ756.
3. the method that Acarbose production bacterial strain produces component C is eliminated using metabolic engineering described in claim 1, its feature exists
In：The mode of the mycelium Conjugative tiansfer, specifically includes following steps：
A. the recombiant plasmid pLQ756 of structure is converted to ET12567, the correct bacterial strain of picking, Jing after 8h cultures, is forwarded to new
Continue culture 4-5h in fresh LB culture medium, to OD600nmFor 1；
B. Actinoplanes spp. mycelium are lined on Solid media for plates, 30 DEG C are cultivated 3-4 days, are seeded to bacterium
Filament fluid medium, after culture 36h, is forwarded to TSB culture medium culturing 8-12h；
C. it is 1 according to thalline quantity by mycelium and the ET12567 containing recombiant plasmid:30 ratio mix homogeneously, coat SFM
On flat board, it is placed in 30 DEG C of incubators and cultivates 32h, every piece of flat board is covered with 2mg apramycins and trimethoprim respectively, is continued
Culture 6-7 days, obtains conjugon.
4. utilization metabolic engineering according to claim 1 eliminates the method that Acarbose production bacterial strain produces component C, and which is special
Levy and be：The screening technique of mutant QQ-2 is as follows：Picking conjugon lines the flat board containing apramycin and trimethoprim
On solid medium, after culture 3-4d, it is inoculated in liquid mycelial culture medium, cultivates 36h, is inoculated with according to 10% inoculum concentration
Into fresh SM culture medium, continue culture 48h, after taking ten times of mycelium dilution, filtered with spore filter, filtrate dilution
104-105Afterwards, coat in the STY plating mediums of the 5-flurocytosine containing 50mg/L, cultivate 4-5 days, picking single bacterium colony, training
Genome, PCR checkings are extracted after supporting.
5. utilization metabolic engineering according to claim 1 eliminates the method that Acarbose production bacterial strain produces component C, and which is special
Levy and be：Mutant is lined into Solid media for plates, 30 DEG C of incubator cultures 3-4 days are placed in, it is appropriate with inoculating loop picking
Mycelium, is inoculated in seed culture medium, is placed in 30 DEG C of 220rpm shaking tables and cultivates 30-36h, is inoculated with according to 15% inoculum concentration
To fermentation medium, it is placed in 30 DEG C of 220rpm shaking tables and ferments 7 days, adds within the 3rd day 2% maltose and Fructus Vitis viniferae respectively in fermentation
Sugar, produces Acarbose.
6. utilization metabolic engineering according to claim 5 eliminates the method that Acarbose production bacterial strain produces component C, and which is special
Levy and be：The mass volume ratio formula of the seed culture medium is glucose 1%, maltose 1.5%, glycerol 1%, hot moulding soybean cake
Powder 4%, starch 1%, CaCO30.2%, pH are adjusted to 7.2,115 DEG C of high pressure steam sterilization 15min.The mass body of fermentation medium
Product is maltose 5%, glucose 3%, hot moulding soybean cake powder 1%, FeCl than formula30.05%th, K2HPO40.1%th, glutamic acid
0.3%th, CaCO30.25%, pH are adjusted to 7.2,115 DEG C of high pressure steam sterilization 15min.
7. utilization metabolic engineering according to claim 1 eliminates the method that Acarbose production bacterial strain produces component C, and which is special
Levy and be：The condition that HPLC analyses are carried out to fermentation liquid is as follows：
Pillar：Agilent ZORBAX NH2；
Mobile phase：Acetonitrile：Water=73：27；
Flow velocity：1ml/min；
Detection wavelength：210nm；
Sampling volume：20μL.
8. the method that Acarbose production bacterial strain produces component C is eliminated using metabolic engineering described in claim 1, its feature exists
In：The condition that UPLC-Q-TOF-MS analyses are carried out to fermentation liquid is as follows：
Pillar：Agilent ZORBAX NH2；
Mobile phase：Acetonitrile：Water=73：27；
Flow velocity：0.4ml/min；
Detection wavelength：210nm；
Sampling volume：10μL；
Detection pattern：Negative ion mode.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112592878A (en) * | 2020-12-25 | 2021-04-02 | 上海交通大学 | Method for enhancing expression of positive regulatory protein gene to improve acarbose fermentation level |
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2016
- 2016-10-28 CN CN201610962770.3A patent/CN106566797A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 余贞等: "游动放线菌8-22 中treY 基因敲除对于降低阿卡波糖C 组分的作用", 《微生物学通报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112592878A (en) * | 2020-12-25 | 2021-04-02 | 上海交通大学 | Method for enhancing expression of positive regulatory protein gene to improve acarbose fermentation level |
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Application publication date: 20170419 |