CN109943513A - A kind of genetic engineering bacterium that phloroglucin biosynthesis yield can be improved and reduces production cost and its construction method and application - Google Patents
A kind of genetic engineering bacterium that phloroglucin biosynthesis yield can be improved and reduces production cost and its construction method and application Download PDFInfo
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Abstract
The invention discloses a kind of genetic engineering bacterium that phloroglucin biosynthesis yield can be improved and reduce production cost and its construction method and applications, it is related to genetic engineering and field of fermentation engineering, the genetic engineering bacterium expression includes the recombinant plasmid of polyketide synthases gene phlD1 and rop gene.Genetic engineering bacterium provided by the invention is used for fermenting and producing phloroglucin, and not only PG output increased, production cost reduce, and also reduces the leakage expression of original pNEW expression vector.This result provides technical support for PG biosynthesis, also provides reference when selecting inducible expression for biosynthesis other chemicals.
Description
Technical field
The present invention relates to genetic engineering and field of fermentation engineering, and in particular to phloroglucin biosynthesis production can be improved in one kind
Measure and reduce genetic engineering bacterium and its construction method and the application of production cost.
Background technique
Phloroglucin (abbreviation PG) is a kind of widely used bulk chemical, is in duplication and textile dyeing
The common precursor of useful chemicals and drug, food additives and Cosmetic Manufacture, therefore its demand at home and abroad is very
Greatly.The method of method for synthesizing phloroglucinol includes chemical synthesis and biological synthesis process, traditional chemical synthesis production technology at present
Fall behind, environmental pollution is serious, and product quality does not catch up with the development of medicinal industry.Bioanalysis method for synthesizing phloroglucinol has green, peace
Entirely, the advantages such as low cost, it has also become the developing direction of phloroglucin synthesis.
Currently, bioanalysis method for synthesizing phloroglucinol has obtained many impressive progresses.Achkar et al. has found in Escherichia coli
In BL21 (DE3) only expression from Pseudomonas fluorescens phlD gene can produce PG (Jihane Achkar, Xian M,
Huimin ZhaoA,et al.Biosynthesis of Phloroglucinol[J].Journal of the American
Chemical Society,2005,127(15):5332-5333.).Zha et al. is by knocking out e. coli bl21 (DE3)
Acetic acid and ethyl alcohol form gene ackA-pta and adhE, and using the pRSFDuet-1 carrier or pACYCDuet-1 of IPTG induction
Carrier is overexpressed the acetyl coenzyme A of acetyl-CoA carboxylase (ACC) and Escherichia coli itself from Corynebacterium glutamicum
Synzyme (ACS) promotes acetic acid to generate acetyl coenzyme A respectively, and acetyl coenzyme A generates malonyl CoA, thus between improving
Benzenetriol yield (Zha W, Rubinpitel S B, Shao Z, et al.Improving cellular malonyl-CoA
level in Escherichia coli via metabolic engineering.[J].Metabolic
Engineering, 2009,11(3):192-198.).Later CaoYj by using the IPTG pET30a carrier induced or
PACYC carrier is overexpressed multiple resistance activity factor marA gene and acetyl-CoA carboxylase ACC, there is phloroglucin yield
Biggish raising (Yujin Cao, Xinglin Jiang, Rubing Zhang, Mo Xian.Improved
phloroglucinol production by metabolically engineered Escherichia coli[J]
.Appl Microbiol Biotechnol,2011, 91:1545–1552.).Rao et al. with the IPTG pET28a induced or
PACYCDuet-1 is carrier, is oriented evolution to phlD gene, improves the thermal stability of PhlD enzyme, keeps PG content wilder
Raw type improves 30% (Rao G. Directed evolution of phloroglucinol synthase PhlD with
increased stability for phloroglucinol production[J].Applied Microbiology&
Biotechnology,2013,97(13):5861-5867.).However, the overwhelming majority is lured using IPTG in existing research
Lead expression PG synthesis related gene.Since IPTG is expensive, phloroglucin production cost will be increased, thus between limiting bioanalysis
The industrialization and popularization and application of benzenetriol.In addition, PG yield is still lower at present, if wanting PG carrying out industrialized production, PG's
There is still a need for further increase for yield.
The pNEW expression vector of ChoiY J et al. building has at low cost, the few spy of dosage using cumate as inducer
Point, so being researcher (Choi Y J, Morel L, Teffanie Le of interestet al.Novel,
Versatile, and Tightly Regulated Expression System for Escherichia coli
Strains[J].Applied and Environmental Microbiology,2010,76(15):5058-5066.).But
It is that pNEW expression carrier leak expression is more serious and less with the example that pNEW carrier synthesizes product, so limiting pNEW expression
The application of carrier.
Summary of the invention
In order to solve the problems, such as PG low output and high production cost, the present invention, which provides one kind, can be improved the conjunction of phloroglucin biology
At yield and reduce genetic engineering bacterium and its construction method and the application of production cost.
Firstly, the present invention provides a kind of genetic engineering for improving phloroglucin biosynthesis yield and reducing production cost
Bacterium, the genetic engineering bacterium expression include the recombinant plasmid of polyketide synthases gene phlD1.
Preferably, the rop base in the recombinant plasmid comprising polyketide synthases gene phlD1 also comprising control copy number
Cause.
It preferably, also include T5 promoter, CymO in the recombinant plasmid comprising polyketide synthases gene phlD1
Operator, T7 terminator, lacI gene.
Preferably, for the rop gene source in pET28a carrier, GeneBank ID is ATU32939.1；The polyketone closes
Pseudomonas fluorescens is derived from enzyme gene phlD1, GeneBank ID is 11830552；The CymO operator comes from
Pseudomonas putida F1, GenBank:MH101728.1.
Preferably, host strain is Escherichia coli, including but not limited to E.coliBL21 (DE3), DH5 α, JM109, Top10
And DH10B etc..
Second, the present invention also provides above-mentioned raising phloroglucin biosynthesis yield and reduce the genetic engineering of production cost
The construction method of bacterium, comprising the following steps:
1) building includes the recombinant plasmid pNEW-phlD1-rop of rop gene and polyketide synthases gene phlD1；
2) pNEW-phlD1-rop that step 1) constructs is transformed into host strain, is improved phloroglucin biosynthesis production
Measure and reduce the genetic engineering bacterium of production cost.
The method specifically:
1) addition controls the rop gene of copy number on pNEW carrier, and is overexpressed polyketide synthases gene phlD1, obtains
Obtain recombinant plasmid pNEW-phlD1-rop；
2) by gained recombinant plasmid pNEW-phlD1-rop imported into host strain i.e. E.coliBL21 (DE3), DH5 α,
In the competent cell of JM109, Top10 or DH10B, obtains and improve phloroglucin biosynthesis yield and reduce production cost
Genetic engineering bacterium.
Step 1) is further specifically: the maneuvering area replacement laboratory of pNEW carrier is had recombinant plasmid pET28a-
The maneuvering area of phlD1 obtains recombinant plasmid pNEW-phlD1-norop, adds on recombinant plasmid pNEW-phlD-norop
After the rop gene for controlling copy number, recombinant plasmid pNEW-phlD1-rop is obtained.
Preferably, also whole comprising T5 promoter, CymO operator, T7 in the recombinant plasmid pNEW-phlD1-rop
Only son, lacI gene；For the rop gene source in pET28a carrier, GeneBank ID is ATU32939.1；The polyketone closes
Pseudomonas fluorescens is derived from enzyme gene phlD1, GeneBank ID is 11830552；The CymO operator comes from
Pseudomonas putida F1, GenBank:MH101728.1.
Third the present invention also provides above-mentioned raising phloroglucin biosynthesis yield and reduces the genetic engineering of production cost
The application of bacterium, the genetic engineering bacterium for improving phloroglucin biosynthesis yield and reducing production cost are raw by shaking flask culture
Produce phloroglucin.
Specifically includes the following steps:
(1) by the seed containing the genetic engineering bacterium for improving phloroglucin biosynthesis yield and reducing production cost
Liquid is inoculated in culture medium according to the 1%-2% that inoculum concentration is culture volume, and cultivation temperature is 30-37 DEG C, mixing speed 220
Rpm, pH6.0-8.0, culture to OD600 are 0.6-0.8, and inducer IPTG to final concentration 0.2mmol/L is then added, is added eventually
The glucose that concentration is 24g/L is used as substrate, terminates after fed batch fermentation 18h to cultivate；
(2) culture solution for obtaining step (1) carries out centrifuging and taking supernatant, and supernatant extracts 1- using isometric ethyl acetate
3 times；(3) merge and extract product described in (2), vacuum distillation concentration, obtained solid powder is phloroglucin.
Beneficial effect
The present invention is overexpressed phlD1 gene using the pNEW expression vector of new inducer cumate induction, and original
The rop gene of control plasmid copy number is added on pNEW expression vector, the use not only PG output increased of new inducer makes to give birth to
Producing cost reduces, and also reduces the leakage expression of original pNEW expression vector.This result provides technology branch for PG biosynthesis
It holds, also provides reference when selecting inducible expression for biosynthesis other chemicals.
Specifically, the invention has the following advantages that
The price of 1.cuamte is the 1/3 of IPTG, and optimum amount is the 1/7 of IPTG, so being with the cost of cumate
The 1/21 of IPTG.
2. the yield of the PG produced with pNEW expression vector is that the PG of pET28a expression vector production is produced in shaking flask level
2 times of amount.
3. the rop gene of addition control plasmid copy number, not only increases PG yield on original pNEW expression vector
15%, so that the leakage expression of PhlD albumen is reduced half.
Definition and abbreviation
Following abbreviation or abbreviation used herein:
Phloroglucin (Phloroglucinol): PG
Isopropylthiogalactoside: IPTG
Polyketide synthases gene: phlD1
Bacillus coli (Escherichia coli): E.coli
After " overexpression " or " overexpression " refers to intracellular specific gene by various signals-modulatings, it is more than in organism
Previous level expression can be realized by enhancing endogenous expression or introducing foreign gene.
Detailed description of the invention
Fig. 1 pET28a-phlD1 recombinant plasmid structural schematic diagram；
Fig. 2 pNEW-phlD1-rop recombinant plasmid structural schematic diagram；
Fig. 3 pNEW-phlD1-norop recombinant plasmid schematic diagram.
Specific embodiment
The present invention is elaborated below by example.But the present invention is not limited to following embodiments.
It is routine techniques if experimental method involved in following embodiments is without specified otherwise.
If material as used in the following examples, reagent etc. are commercially obtained without specified otherwise.Nothing used
The homologous recombination enzyme of seam clone is purchased from Nuo Weizan biotech company, and plasmid extracts and glue recycling
Used kit is purchased from U.S. OMEGA company, and operating procedure is carried out according to product description；All culture mediums for example without
Special instruction is prepared with deionized water.
Culture medium prescription:
1) LB culture medium: yeast powder 5g/L, NaCl 10g/L, peptone 10g/L, when inoculation, add 50 μ g/ of kanamycins
mL。
2) fermentation medium K2HPO4·3H2O 9.8g/L, Citric acidH2O 2.1g/L, ferric citrate
0.3g/L, (NH4)2SO43.0g/L, glucose 24g/L, MgSO4·7H2O 0.4g/L, 1000 × microelement ((NH4)6Mo7O24·4H2O 3.7g/L；ZnSO4·7H2O 2.9g/L；H3BO324.7g/L；CuSO4·5H2O 2.5g/L； MnCl2·
4H2O 15.8g/L), 50 μ g/mL of kanamycins.
Wherein: K2HPO4·3H2O 9.8g/L, Citric acidH2O 2.1g/L, ferric citrate 0.3g/L,
(NH4)2SO47.0,121 DEG C of pH are adjusted to after 3.0g/L mixing, 20min autoclaving.Glucose liquid storage is 500g/L,
115 DEG C, 20min individually sterilizes, MgSO4·7H2O liquid storage is 200g/L, and 121 DEG C, 20min individually sterilizes, 1000 × micro member
Element uses 0.22 μm of bacteriological filtration film filtration sterilization, and when seed liquor of transferring adds glucose, the MgSO of above-mentioned independent degerming respectively4·
7H2O, 1000 × microelement liquid storage and antibiotic.
1 construction recombination plasmid of embodiment
A. building pNEW-phlD1-norop recombinant plasmid is as control, and detailed process is as follows:
1. with 5 ' ATTGCACGAACCTTGTTTAACTTTAAGAAGGAGATATACCATGGCTTCT 3 '
Polymerase chain reaction is utilized using pUC57-pNEW as template with 5 ' GGGCCCGGGATCCGAT 3 ' for primer
(PCR) method amplifies the nucleic acid fragment of the maneuvering area of pNEW carrier, is named as pNEW-fragment1, and at 5 ' ends and 3 '
End introduces homology arm respectively；
2. with 5 ' ACATGTGAGCAAAAGGCCAGCAAAA3 ' and 5 '
ATTGCACGAACCTTGTTTAACTTTAAGAAGGAGATATACCCATGGCTTCT3 ' is primer, with pET28a-
PhlD1 is template, and amplifying linear pET28a-phlD1 using polymerase chain reaction (PCR) method (does not include maneuvering area
Nucleic acid fragment, be free of rop gene), be named as pET28a-phlD1-fragment1, and introduce respectively at 5 ' ends and 3 ' ends same
Source arm, homology arm of the homology arm with pNEW-fragment1；
3. being completed under the action of recombinase exnase in the seamless Cloning Kit that promise is only praised using homology arm
The connection of pNEW-fragment1 and pET28a-phlD1-fragment1, constructs pNEW-phlD1-norop.
PCR system:
PCR amplification condition:
The reaction system of seamless clone is as follows:
Reaction condition: 50 DEG C, after 30min, 4 DEG C is down to or is immediately placed in cooled on ice, obtains recombinant products.In next step will
Recombinant products are transferred to cloning vector.
4. the competent cell from the E.coli BL21 (DE3) of TAKARA is bought, by recombinant plasmid pNEW-phlD1-norop
Pass through heat shock method cotransformation to the competent cell of above-mentioned preparation.
The preparation of embodiment 2 can be improved phloroglucin (PG) biosynthesis yield and reduce the genetic engineering bacterium of production cost
1) pNEW-phlD1-rop recombinant plasmid is constructed, detailed process is as follows:
1. with 5 ' ATTGCACGAACCTTGTTTAACTTTAAGAAGGAGATATACCATGGCTTCT 3 '
It is primer with 5 ' TGATGCCTCCGTGTAAGGGGTGTCTAGCGCTTGAATTTCGCGT 3 ', with pUC57-pNEW
For template, the nucleic acid fragment of the maneuvering area of pNEW carrier is amplified using polymerase chain reaction (PCR) method, is named as
PNEW-fragment2, and homology arm pNEW-fragment1 and pNEW-fragment2 are introduced respectively at 5 ' ends and 3 ' ends
3 ' homology arm is different；
2. with 5 ' CCCCTTACACGGAGGCATCAGTGAC 3 ' and 5 '
ATTGCACGAACCTTGTTTAACTTTAAGAAGGAGATATACCCATGGCTTCT 3 ' is primer, with pET28a-
PhlD1 is template, and amplifying linear pET28a-phlD1 using polymerase chain reaction (PCR) method (does not include maneuvering area
Nucleic acid fragment, gene containing rop), be named as pET28a-phlD1-fragment2, and introduce respectively at 5 ' ends and 3 ' ends homologous
Arm, the same pNEW-fragment2 of homology arm；
3. being completed under the action of recombinase exnase in the seamless Cloning Kit that promise is only praised using homology arm
The connection of pNEW-fragment2 and pET28a-phlD1-fragment2, constructs pNEW-phlD1-rop.
PCR system:
PCR amplification condition:
The reaction system of seamless clone is as follows:
Reaction condition: 50 DEG C, after 30min, 4 DEG C is down to or is immediately placed in cooled on ice, obtains recombinant products.In next step will
Recombinant products are transferred to cloning vector.
2) competent cell from the E.coli BL21 (DE3) of TAKARA is bought, by recombinant plasmid pNEW-phlD1-rop
Pass through heat shock method cotransformation to the competent cell of above-mentioned preparation.
PET28a-phlD1 plasmid construction method is according to document (Cao Y, Jiang X, Zhang R, et al.Improved
phloroglucinol production by metabolically engineered Escherichia coli[J]
.Applied Microbiology&Biotechnology,2011,91(6):1545-1552.)。
Embodiment 3 utilizes engineering strain fermenting and producing phloroglucin
1, shake flask fermentation is tested
1) culture of primary seed solution: 1 He of embodiment being inoculated with respectively in LB seed liquid culture medium on solid LB plate
3 recombinant bacterial strain single colonies that embodiment 2 is prepared, and final concentration of 50 μ g/mL kanamycins is added, 37 DEG C of growth 12h,
Obtain primary seed solution.
2) primary seed solution that step 1) obtains is forwarded in 250mL fermentation shake flask by 1.6% (wt) inoculum concentration respectively,
Fermentation medium containing 50mL, when seed liquor of transferring, respectively add the MgSO of 200g/L4·7H2100 μ L of O, 500 g/L glucose
2.4mL, 1000 × microelement mother liquor, the 50 μ L (preparation of 1000 × microelement mother liquor: (NH4)6MoO24·4H2O 0.37g；
ZnSO4·7H2O 0.29g；H3BO42.47g；CuSO4·5H2O 0.25g； MuCl2·4H2O 1.58g；It is arrived with distilled water constant volume
100mL), 50 μ g/mL kanamycins (kanamycins is not added in control strain) of final concentration, every kind of bacterial strain 3 parallel controls of setting, 37
DEG C, 180rpm culture.
3) cell OD600 reaches IPTG or the cumate induction of addition 200 μm of ol/L of final concentration between 0.6-1.0.
4) after inducing, 37 DEG C, 180rpm continue culture and collect bacterium solution afterwards for 24 hours, centrifuging and taking supernatant, measurement phloroglucin contains
Amount.The plasmid difference and condition of culture difference such as table 1 of 3 bacterial strains:
The plasmid difference and condition of culture difference of 13 bacterial strains of table
2, the content detection of phloroglucin
Phloroglucin (PG) concentration mensuration: cinnamic acid development process and HPLC.
Using anti-Weisner detection method (Wenjuan Zha, Sheryl B.Rubin-Pitel, Huimin
Zhao.Exploiting genetic diversity by directed evolution:molecular breeding of
type III polyketide synthases improves productivity[J].Molecular BioSystems,
2008,4 (3): 246-248.) measurement fermentation liquid in phloroglucin content, principle is according to phloroglucin and cinnamic acid
Chromogenic reaction, the specific steps are as follows:
1) (cinnamic acid is directly dissolved in concentrated hydrochloric acid/ethanol solution of volume ratio 1:3 to the cinnamic acid developing solution of preparation 10mg/L
In).
2) 1mL cinnamic acid developing solution is added into 1.5mL centrifuge tube, adds 5 μ L fermented liquid supernatants, is mixed by inversion, room
Temperature places 15min.
3) OD446 value is read with 10mm optical path cuvette, OD446 value is stablized in 2h；
4) phloroglucin standard curve is drawn, phloroglucin content is calculated according to standard curve.
HPLC: high performance liquid chromatography.Detection method: Eclipse Plus C18 column, mobile phase is acetonitrile: ddH2O=1:
1, flow velocity 0.25mL/min, Detection wavelength 230nm, 30 DEG C of column temperature.
According to the present embodiment operating procedure, after shaking flask level, Fiber differentiation 18h, BL21 (DE3)/pET28a-phlD1
The PG yield of bacterial strain is 0.2g/L, BL21 (DE3)/pNEW-phlD1-rop or BL21 (DE3)/pNEW-phlD1-norop
Yield is respectively 0.38g/L and 0.35g/L.In shaking flask level BL21 (DE3)/pNEW-phlD1-rop or BL21 (DE3)/
High 2 times of PG yield of yield ratio BL21 (DE3)/pET28a-phlD1 or so of pNEW-phlD1-norop, and BL21 (DE3)/
The PG yield ratio BL21 (DE3) of pNEW-phlD1-rop/pNEW-phlD1-norop phloroglucin concentration improves about 15%.
HPLC testing result is identical as the result of cinnamic acid development process.
Meanwhile with HPLC detection in the case where inducer is not added, the leakage expression of 3 bacterial strains.Inducer is being not added
In the case where, BL21 (DE3)/pET28a-phlD1 PG yield is 0.027g/L, BL21 (DE3)/pNEW-phlD1-rop
PG yield is 0.048g/L, and BL21 (DE3)/pNEW-phlD1-norop PG yield is 0.074g/L.It can thus be seen that
The rop gene of addition control plasmid copy number can reduce leakage expression on pNEW carrier.
The most suitable final concentration of the detection of embodiment 4 cumate
1. shake flask fermentation is tested
1) culture of primary seed solution: embodiment 1 and implementation on LB seed liquor inoculation of medium solid LB plate
The recombinant bacterial strain single colonie that example 2 is prepared, and final concentration of 50 μ g/mL kanamycins is added, 37 DEG C of growth 8-12h.
2) primary seed solution that step 1) obtains is forwarded in 250mL fermentation shake flask by 1.6% (wt) inoculum concentration respectively,
Fermentation medium containing 50mL, when seed liquor of transferring, respectively add 100 μ L of MgSO47H2O, the 500g/L glucose of 200g/L
2mL, 1000 × microelement, 50 μ L, 50 μ g/mL kanamycins (kanamycins is not added in control strain) of final concentration, every kind of bacterial strain are set
Fixed 3 parallel controls, 37 DEG C, 180rpm culture.
3) when cell OD600 reaches 0.6-0.8, cumate is added, its final concentration is made to be kept at 0,10,30,50,
100,200μM。
4) after inducing, 37 DEG C, 180rpm continue culture and collect bacterium solution afterwards for 24 hours, centrifuging and taking supernatant, measurement phloroglucin contains
Amount.
2. detecting the concentration of phloroglucin
The assay of phloroglucin is the same as embodiment 3
As can be seen that the most suitable final concentration of cumate is 0.03mM from the result that shake flask fermentation is tested, and IPTG is most
Suitable final concentration is 0.2mM, so the most suitable final concentration of cumate is the 1/7 of IPTG.In addition, the price of cumate be 54 yuan/
The price of 5g, IPTG are 128 yuan/5g, so the price of cumate is the 1/3 of IPTG.Therefore, on the whole, using cuamte
Cost be using IPTG cost 1/21.
5 plasmid pNEW-phlD1-rop of embodiment is expressed in other competent cells
1. buying the competent cell from E.coli the DH5 α, JM109, Top of Quan Shijin Bioisystech Co., Ltd, will weigh
The competent cell that group plasmid pNEW-phlD1-rop passes through heat shock method cotransformation to above-mentioned preparation.
2. the culture of primary seed solution: the embodiment 1 on LB seed liquor inoculation of medium solid LB plate is prepared into
The recombinant bacterial strain single colonie arrived, and final concentration of 50 μ g/mL kanamycins is added, 37 DEG C of growth 8-12h.
3. the primary seed solution that step 2 obtains is forwarded in 250mL fermentation shake flask by 1.6% (wt) inoculum concentration respectively,
The culture medium of LB containing 50mL, when seed liquor of transferring, respectively add 50 μ g/mL kanamycins of final concentration, every kind of bacterial strain set 3 it is parallel
Control, 37 DEG C, 180rpm culture.
3) when cell OD600 reaches 0.6-0.8, cumate is added, its final concentration is made to be kept at 200 μM.
4) after inducing, 30 DEG C, 180rpm continue culture and collect bacterium solution afterwards for 24 hours, centrifuging and taking supernatant, measurement phloroglucin contains
Amount.
2. detecting the concentration of phloroglucin
The assay of phloroglucin is the same as embodiment 3
PG yield such as table 2 of the recombinant plasmid pNEW-phlD1-rop in E.coli DH5 α, JM109, Top10:
2 pNEW-phlD1-rop of table is in DH5 α, the PG yield of JM109 and Top10
As can be seen that recombinant plasmid pNEW-phlD1-rop is in DH5 α, JM109 and from the result that shake flask fermentation is tested
PG can be produced in Top10, it is only variant in the PG yield of different hosts.DH5 α/pNEW-phlD1-rop and Top10/
The PG yield of pNEW-phlD1-rop is not much different, and the PG yield of JM109/pNEW-phlD1-rop is lower, be DH5 α/
The 62% of pNEW-phlD1-rop or Top10/pNEW-phlD1-rop.
The present embodiment institute column data is the mean values that test is repeated several times.Although showing present invention has disclosed exemplary
Model scheme, but it will be apparent to those skilled in the art that without departing substantially from the spirit of the invention as defined in appended claims and
Under conditions of range, the variation of various forms and details can be carried out, any combination of various embodiments can be carried out.
Claims (10)
1. a kind of genetic engineering bacterium for improving phloroglucin biosynthesis yield and reducing production cost, it is characterised in that: described
Genetic engineering bacterium expression includes the recombinant plasmid of polyketide synthases gene phlD1.
2. the genetic engineering bacterium according to claim 1 for improving phloroglucin biosynthesis yield and reducing production cost,
It is characterized by: the rop gene in the recombinant plasmid comprising polyketide synthases gene phlD1 also comprising control copy number.
3. the genetic engineering bacterium according to claim 2 for improving phloroglucin biosynthesis yield and reducing production cost,
It is characterized by: also including T5 promoter, CymO in the recombinant plasmid comprising polyketide synthases gene phlD1
Operator, T7 terminator, lacI gene.
4. the genetic engineering bacterium according to claim 3 for improving phloroglucin biosynthesis yield and reducing production cost,
It is characterized by: the rop gene source, in pET28a carrier, GeneBank ID is ATU32939.1；The polyketide synthases
Gene phlD1 derives from Pseudomonas fluorescens, and GeneBank ID is 11830552；The CymO operator comes from
Pseudomonas putida F1, GenBank:MH101728.1.
5. the genetic engineering bacterium according to claim 4 for improving phloroglucin biosynthesis yield and reducing production cost,
It is characterized by: host strain is Escherichia coli.
6. a kind of gene described in any one of claims 1-6 for improving phloroglucin biosynthesis yield and reducing production cost
The construction method of engineering bacteria, it is characterised in that: the following steps are included:
1) building includes the recombinant plasmid pNEW-phlD1-rop of rop gene and polyketide synthases gene phlD1；
2) pNEW-phlD1-rop that step 1) constructs is transformed into host strain, is improved phloroglucin biosynthesis yield simultaneously
Reduce the genetic engineering bacterium of production cost.
7. the genetic engineering bacterium according to claim 6 for improving phloroglucin biosynthesis yield and reducing production cost
Construction method, it is characterised in that: the method specifically:
1) addition controls the rop gene of copy number on pNEW carrier, and is overexpressed polyketide synthases gene phlD1, is weighed
Group plasmid pNEW-phlD1-rop；
2) by gained recombinant plasmid pNEW-phlD1-rop imported into host strain i.e. E.coliBL21 (DE3), DH5 α, JM109,
In the competent cell of Top10 or DH10B, the gene work for improving phloroglucin biosynthesis yield and reducing production cost is obtained
Journey bacterium.
8. the genetic engineering bacterium according to claim 7 for improving phloroglucin biosynthesis yield and reducing production cost
Construction method, it is characterised in that: in the recombinant plasmid pNEW-phlD1-rop also comprising T5 promoter, CymO operator,
T7 terminator, lacI gene；For the rop gene source in pET28a carrier, GeneBank ID is ATU32939.1；It is described poly-
Ketone synthase gene phlD1 derives from Pseudomonas fluorescens, and GeneBank ID is 11830552；The CymO operator comes
From Pseudomonas putida F1, GenBank:MH101728.1.
9. a kind of gene described in any one of claims 1-6 for improving phloroglucin biosynthesis yield and reducing production cost
The application of engineering bacteria, it is characterised in that: the genetic engineering for improving phloroglucin biosynthesis yield and reducing production cost
Bacterium produces phloroglucin by shaking flask culture.
10. the genetic engineering bacterium according to claim 9 for improving phloroglucin biosynthesis yield and reducing production cost
Application, it is characterised in that: the following steps are included:
(1) seed liquor containing the genetic engineering bacterium for improving phloroglucin biosynthesis yield and reducing production cost is pressed
It is inoculated in culture medium according to the 1%-2% that inoculum concentration is culture volume, cultivation temperature is 30-37 DEG C, mixing speed 220rpm,
PH6.0-8.0, culture to OD600 are 0.6-0.8, and inducer IPTG to final concentration 0.2mmol/L is then added, final concentration is added
For 24g/L glucose as substrate, terminate culture after fed batch fermentation 18h；
(2) culture solution for obtaining step (1) carries out centrifuging and taking supernatant, and supernatant is extracted 1-3 times using isometric ethyl acetate；
(3) merge and extract product described in (2), vacuum distillation concentration, obtained solid powder is phloroglucin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201910250966.3A CN109943513A (en) | 2019-03-29 | 2019-03-29 | A kind of genetic engineering bacterium that phloroglucin biosynthesis yield can be improved and reduces production cost and its construction method and application |
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Citations (3)
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| CN101724662A (en) * | 2008-10-29 | 2010-06-09 | 青岛生物能源与过程研究所 | Method for synthesizing phloroglucinol by microbial catalysis |
| US8623652B2 (en) * | 2009-04-06 | 2014-01-07 | Lucigen Corporation | Host-vector system for cloning and expressing genes |
| CN104988172A (en) * | 2015-07-30 | 2015-10-21 | 中国科学院青岛生物能源与过程研究所 | Construction method and application of high-yield phloroglucinol gene engineering bacterium |
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| CN101724662A (en) * | 2008-10-29 | 2010-06-09 | 青岛生物能源与过程研究所 | Method for synthesizing phloroglucinol by microbial catalysis |
| US8623652B2 (en) * | 2009-04-06 | 2014-01-07 | Lucigen Corporation | Host-vector system for cloning and expressing genes |
| CN104988172A (en) * | 2015-07-30 | 2015-10-21 | 中国科学院青岛生物能源与过程研究所 | Construction method and application of high-yield phloroglucinol gene engineering bacterium |
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| CAO,YUJIN等: "Production of phloroglucinol by Escherichia coli using a stationary-phase promoter", 《BIOTECHNOLOGY LETTERS》 * |
| CHOI, YOUNG J等: "Novel, Versatile, and Tightly Regulated Expression System for Escherichia coli Strains", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
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