CN114806997A - Construction and application of streptomyces fuscospora for efficient extracellular transport of natamycin - Google Patents
Construction and application of streptomyces fuscospora for efficient extracellular transport of natamycin Download PDFInfo
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- CN114806997A CN114806997A CN202210669080.4A CN202210669080A CN114806997A CN 114806997 A CN114806997 A CN 114806997A CN 202210669080 A CN202210669080 A CN 202210669080A CN 114806997 A CN114806997 A CN 114806997A
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Abstract
The invention discloses a genetic engineering bacterium for producing natamycinS.gilvosporeusF-EX, the genetically engineered bacteriumS.gilvosporeusF-EX comprising two copies of the natamycin extracellular transporter coding genesgnA/BA gene. The invention discloses a gene engineering bacteriumS.gilvosporeusF-EX has good effect, is favorable for transferring natamycin from the fermentation bacteria cell to the outside, and is favorable for promoting the generation and transformation of natamycin in the fermentation liquid; the yield of natamycin is improved, and the production cost is greatly reduced; the invention discloses a gene engineering bacteriumS.gilvosporeusThe construction method of the F-EX is simple, convenient, easy to operate, safe and environment-friendly, and is beneficial to realizing industrialized expanded application.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to construction and application of a strain of streptomyces fuscoporia for efficient extracellular transport of natamycin.
Background
Clinical fungal infections and food fungal contamination are two major factors threatening human health. Natamycin (also known as Pimaricin) which has NO obvious toxic and side effect on mammalian cells is a natural, broad-spectrum and efficient polyene macrolide antifungal agent, the molecular formula of which is C33H47NO13, and the Natamycin is widely applied to clinical fungal infection and tumor treatment and food fungal contamination prevention and treatment. With the intensive research on natamycin and the continuous development of the application value of natamycin, the demand of natamycin is continuously increased. However, the natamycin fermentation technology and the construction and optimization of high-yield strains suffer from bottlenecks at present, and the application of the natamycin fermentation technology and the construction and optimization of high-yield strains in various fields is severely limited.
During the fermentation process of preparing natamycin, ROS (superoxide dismutase) with a certain concentration in the bacterial cells of the fermentation liquor has an important effect on the biosynthesis of antibiotics, natamycin is used as polyene macrolide antibiotics with reducibility, and H with a certain concentration is maintained during the fermentation process 2 O 2 The synthesis speed of the natamycin can be improved; therefore, the accumulation of intracellular natamycin reduces the concentration of ROS (superoxide) in the mycocyte and reduces the synthesis speed of natamycin. Therefore, the extracellular transport of the antibiotic has important significance for inhibiting the accumulation of antibiotic cells, relieving the product inhibition effect and improving the synthesis efficiency of the antibiotic.
The prior published Chinese patents CN103555755A and ZL 201410180186.3 respectively disclose a natamycin genetic engineering strain constructed by utilizing vitreoscilla hemoglobin gene vgb gene and cholesterol oxidase pimE gene and application thereof, and VHb expression is respectively utilized in recombinant bacteria to solve oxygen supply and demand contradiction in natamycin fermentation production and pimE overexpression stimulates the production of natamycin, so that the yield of natamycin is improved. However, the research of using the biological functions of SgnA and SgnB encoded ABC transporters in the natamycin synthesis gene cluster to participate in the intracellular outward transfer of natamycin from antibiotics and synthesis precursors thereof to improve the natamycin synthesis efficiency and the natamycin yield has not been reported in the open.
Therefore, those skilled in the art are dedicated to develop the construction and application of a streptomyces fuscoporia strain with efficient extracellular natamycin transport, so as to solve the above-mentioned deficiencies of the prior art.
Disclosure of Invention
In view of the above-mentioned defects of the prior art, the technical problems to be solved by the present invention are that the efficiency of natamycin transport in the bacteria cells is poor in the natamycin synthesis process disclosed by the prior art, the concentration of ROS (superoxide) in the bacteria cells is reduced, the synthesis of natamycin is not facilitated, the yield of natamycin is low, and the method is not suitable for industrial production.
In order to achieve the above object, the present invention provides, in a first aspect, a genetically engineered bacterium for producing natamycinS. gilvosporeusF-EX (full name)Streptomyces gilvosporeus F-EX)；
The genetically engineered bacteriumS. gilvosporeusThe F-EX classification is named as Streptomyces fuscospora (Streptomyces gilvosporeus)，The accession number is GDMCC No: 62228, the preservation unit is GDMCC, the address of the preservation unit is microbial research institute of Guangdong province academy of sciences of Guangdong province, No. 59 building, No. 5 building, No. 100 institute of Miehuo, Miehu, Guangzhou, the preservation date is 2022 years, 1 month and 20 days;
further, the genetically engineered bacteriumS. gilvosporeusF-EX comprising two copies of the natamycin extracellular transporter coding genesgnA/BA gene;
further, the genetically engineered bacteriumS. gilvosporeusF-EX, including recombinant plasmid pMS-EX; the recombinant plasmid pMS-EX comprises a promoter sequencesgnA/BA gene;
further, thesgnA/BThe PCR amplification primer of the gene is as follows:
an upstream primer EX-F:
5’-AAGCTTGGATCCGATGCTGTTGTGGGCACAATCGTGCCGGTTGGTAGGATCAAGCGGAAGAAGGG AGCGGACATCATCATCACCATCACCATGTGCTGCTCAGCCTTCTGCGAATC -3’
(the lower solid line is marked asHindIII cleavage site, marked by the lower solid lineemrPromoter fragment, underlined wavy line denoted His tag sequence);
the downstream primer EX-R:
5’-GGTACCCATCATCACCATCACCATTCAGGAAAGACTGGGGGAAATGCC-3’
(the lower dashed line is indicated asKpnI restriction site, underlined wavy line labeled His tag sequence).
The second aspect of the invention also provides a genetic engineering bacterium for producing natamycinS. gilvosporeusThe construction method of F-EX comprises the steps of using S.gilvospreus F607 genome as a template, amplifying an sgNA/B gene sequence with a promoter PemrE by using high-fidelity DNA polymerase, inserting the sgNA/B gene sequence into a streptomycete recombinant plasmid, transforming streptomyces fuscospora by using a conjugal transfer mode, screening antibiotics to obtain recombinant engineering bacteria containing two copies of sgNA/B genes, and screening the genetic engineering bacteria with highest natamycin yield through a shake flask fermentation experimentS. gilvosporeus F-EX；
Further, the promoter is a strong promoter of an erythromycin resistance gene (Emr), and can be efficiently expressed in streptomycete;
further, the genetically engineered bacterium for producing natamycinS. gilvosporeusIn the construction method of F-EX, the recombinant plasmid is pMS-EX, the recombinant plasmid also includes pMD-18T and pMS82 carriers in the process of constructing the recombinant plasmid pMS-EX, wherein pMD-18 is used for preserving sgnA/B gene sequence with erythromycin resistance gene promoter PemrE, pMS82 contains elements required by plasmid transfer and integrase and integration site of phage, the recombinant plasmid pMS-EX is that fusion fragment Pemr-sgnA/B is cut by KpnI, HindIII enzyme and then is connected into pMS82 plasmid to construct;
further, the step of conjugative transfer is specifically to introduce the recombinant plasmid pMS-EX into E.coli ET12567(pUZ8002), and to join the recombinant E.coli withS. gilvosporeus F607 co-culturing on MS solid culture medium, culturing at 30 deg.C for 16-20 h, coating 1mL sterile water (preferably containing 0.5mg nalidixic acid and 1mg hygromycin) to cover the plate, and culturing at constant temperature at 30 deg.C in dark condition until the binder grows out; the zygotes were screened in medium containing 25 mg of nalidixic acid and 30 mg of hygromycin. After at least three rounds of purification of the selected zygotes, PCR was performed to verify the amplification of hygromycin gene and PermE-sgnAFusing; the positive zygospore is the gene engineering bacterium for producing natamycinS. gilvosporeus F-EX；
Further, the shake flask fermentation experiment condition is 29 ℃, the rotating speed is 220rpm, and the fermentation time is 120 h;
further, the gene engineering bacterium for producing natamycinS. gilvosporeusThe construction method of the F-EX specifically comprises the following steps:
Further, the step 1 specifically includes the following steps:
step 1.1, preparing and optimizing small molecules: obtaining a natamycin 3D structure by using software Pubchem; setting natamycin as a ligand micromolecule in the Discovery Studio of the software;
step 1.2, protein receptor preparation: constructing SgnA and SgnB three-dimensional structure MODELs by WISS-MODEL homologous modeling; introducing the protein structure into software Discovery Studio and setting the protein structure as a receptor molecule, deleting water molecules and organic solvents, only keeping the protein and original substrate ligand, setting docking parameters and selecting a docking activity pocket;
step 1.3, molecular docking: performing molecular docking on the ligand and the receptor with the well-defined pocket, and inputting the path of the stored molecular database into a dialog box;
step 1.4, analyzing a docking result; judging the transport effect of the natamycin by judging the strong and weak change of the binding capacity of the natamycin and the SgnA/B site;
further, the step 2 specifically includes the following steps:
step 2.1, according to the Emr gene promoter Pemr sequence recorded in GenBank and downstream fused gene fragment, synthesizing the Emr gene promoter sequence intosgnA/BOn the PCR amplification primer of (1)
Designing upstream and downstream primers:
an upstream primer EX-F:
5’-AAGCTTGGATCCGATGCTGTTGTGGGCACAATCGTGCCGGTTGGTAGGATCAAGCGGAAGAAGGG AGCGGACATCATCATCACCATCACCATGTGCTGCTCAGCCTTCTGCGAATC-3' (drawn solid lines are shown)HindIII cleavage site, marked by the lower solid lineemrPromoter fragment, underlined wavy line denoted His tag sequence);
a downstream primer EX-R:
5’-GGTACCCATCATCACCATCACCATTCAGGAAAGACTGGGGGAAATGCC-3' (dashed lines are indicated asKpnI, enzyme cutting site, and marking a lower wavy line as His tag sequence);
step 2.2. withS. gilvosporeus F607 genome as template, PCR amplification high-fidelity DNA polymerase amplification with erythromycin resistance geneDue to the promoter PemrEsgnA/BGene sequence, cutting gel and recovering, measuring the DNA concentration of recovered fragment, cloning to pMD-18T vector by TA cloning method, and sequencing pMD-EX plasmid;
step 2.3, the correctly sequenced pMD-EX plasmid and the conjugative transfer vector pMS82 are used respectivelyHindIII andKpni double enzyme digestion, cutting glue, recovering fusion fragment and pMS82, mixing according to the mol ratio of 3: 1-10: 1, adding T4 ligase, connecting overnight at 16 ℃, transforming the connecting product into escherichia coli competent cellE.coliET12567, coating with hygromycin: (Hyg50 mu g/mL), ampicillin (Amp, 100 mu g/mL), chloramphenicol (Cm, 25 mu g/mL) on LB solid plate, selecting transformants to obtain recombinant plasmid pMS-EX;
further, the step 3 specifically includes the following steps:
step 3.1, picking Escherichia coli containing recombinant plasmid pMS-EX E.coliET12567 single colony in LB culture medium (containing hygromycin, ampicillin and chloramphenicol resistance), 37 degrees C shaking culture overnight;
step 3.2, 1% inoculum size, will activate overnight recombinationE.coliET12567 was transferred to fresh LB medium and cultured at 37 ℃ toODCentrifuging the strain at 600 ═ 0.4-0.6, washing the strain twice by using an equal volume of LB culture medium, and suspending the strain in 0.1-time volume of LB culture medium;
step 3.3, culturing the mycelium on the MS solid culture medium for 3-4d, and collecting the mycelium with 2-3mL of 20% glycerol for later use;
step 3.4, taking 200 mu L of recombinationE.coliET12567 heat shock treated with equal volumeS. gilvosporeus F607 mycelium was mixed well and applied by adding 10 mmol/L MgCl 2 Culturing the MS solid plate at 30 ℃ for 16-20 h;
step 3.5, coating a 1mL sterile water (containing 0.5mg nalidixic acid and 1mg hygromycin) cover plate, and continuously culturing at 30 ℃ until a transformant grows out;
step 3.6, selecting transformants to perform subculture and repeated screening on an MS culture medium containing nalidixic acid and hygromycin; activating in liquid culture medium to obtain each transformant, and extractingThe genomic DNA of each transformant was taken and verified by using verification primers V1-F (ATCCTGTTACTTCGACCGTATTG) and V1-R (GGATCGGTGAAGCCGGAGAG) to amplify hgy gene, V2-F (CCGATGCTGTTGGGCAC) and V2-R (GGACCAGCAGCAGCACGAGGGG) to amplify PermE andsgnAa fusion fragment; wherein the positive zygomorph is a genetically engineered bacteriumS. gilvosporeusF-EX (Streptomyces fusciparum);
the present invention also provides the genetically engineered bacterium of any one of the first aspect of the present inventionS. gilvosporeusUse of F-EX for the preparation of natamycin for fermentative preparation;
the present invention also provides the genetically engineered bacterium of any one of the first aspect of the present inventionS. gilvosporeusThe use of F-EX for the preparation of natamycin;
the invention relates to a gene engineering bacteriumS. gilvosporeusF-EX comprising two copies of the natamycin extracellular transporter coding genesgnA/BThe gene promotes the transportation of natamycin to the extracellular part, and improves the yield of natamycin in the process of producing the natamycin by fermentation;
by adopting the scheme, the invention discloses the genetically engineered bacteriumS. gilvosporeusF-EX, has the following advantages:
(1) genetically engineered bacteria of the present applicationS. gilvosporeusF-EX ofsgnA/BThe gene is integrated into a natamycin producing strain bysgnA/BThe overexpression of the natamycin increases the extracellular transport proportion of the natamycin, reduces the resistance degree of the natamycin to intracellular ROS, relieves the product inhibition effect, and is beneficial to improving the yield of the natamycin in production;
(2) the genetically engineered bacterium of the present inventionS. gilvosporeusF-EX, ABC transporters (ATP-binding cassette transporter) SgnA and SgnB coded by sgNA and sgnB in a natamycin synthesis gene cluster, which are favorable for transporting natamycin from inside of a zymocyte to outside of the zymocyte and promoting the generation and transformation of natamycin in fermentation liquor; the yield of natamycin antibiotics is increased by improving the transport efficiency, so that the method has great development value;
(3) the genetically engineered bacterium of the present inventionS. gilvosporeusThe construction method of F-EX is simple and convenient, has mild condition and no safetyHidden troubles, when the method is used for producing natamycin by fermentation, the production cost is low, the efficiency is high, and the method is beneficial to realizing industrialized production and application expansion.
In conclusion, the genetically engineered bacteria disclosed by the inventionS. gilvosporeusF-EX has good effect, is favorable for transferring natamycin from the fermentation bacteria cell to the outside, and is favorable for promoting the generation and transformation of natamycin in the fermentation liquid; the yield of natamycin is improved, and the production cost is greatly reduced; the invention discloses a gene engineering bacteriumS. gilvosporeusThe construction method of the F-EX is simple, convenient, easy to operate, safe and environment-friendly, and is beneficial to realizing industrialized expanded application.
The conception, the specific technical solutions and the technical effects produced by the present invention will be further described with reference to the following detailed description so as to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a diagram of computer molecule docking simulation of SgnA/B structure and natamycin docking in example 1;
FIG. 2 shows the restriction enzyme digestion verification of the pMS-EX recombinant vector of example 2;
FIG. 3 is a PCR validation graph of the binding of the sgnA fragment of the positive binder of example 3;
FIG. 4 shows the PCR verification of the hygromycin gene fragment binding to the positive binder of example 3;
FIG. 5 shows the detection result of sgNA/B ultrasonic expression Western-blot;
FIG. 6 is a graph showing the effect of sgnA/B overexpression on natamycin biosynthesis and extracellular transformation.
Detailed Description
The following describes several preferred embodiments of the present invention to make the technical contents thereof clearer and easier to understand. The invention may be embodied in many different forms of embodiments, which are intended to be illustrative only, and the scope of the invention is not intended to be limited to the embodiments shown herein.
If there is an experimental method not specified specific conditions, it is usually carried out according to conventional conditions, such as the relevant instructions or manuals.
The noun explains:
pubchem: a software name, organic small molecule biological activity data, which is a chemical module database used for acquiring a natamycin 3D structure;
discovery Studio: the name of software is used for researching the structure and function of SgnA/B protein;
WISS-MODEL: a web server name for automated SgnA/B protein structure homology modeling service;
SgnA: an adenosine triphosphate binding cassette transporter (ABC transporter) component;
SgnB: an adenosine triphosphate binding cassette transporter (ABC transporter) component;
hygromycin: streptomyces (I), (II)Streptomyces hygroscopious) The produced aminoglycoside antibiotics comprise two components A and B;
example 1 computer molecular docking simulation construction
In this embodiment, a computer is used to simulate and assist in checking the interaction between SgnA/B and natamycin by using a molecular docking calculation method, and the specific steps are as follows:
(1) preparing and optimizing small molecules: obtaining a natamycin 3D structure by using software Pubchem; setting natamycin as a ligand micromolecule in the Discovery Studio of the software;
(2) protein receptor preparation: constructing SgnA and SgnB three-dimensional structure MODELs by WISS-MODEL homologous modeling; introducing an ABC transporter structure consisting of SgnA and SgnB into software Discovery Studio, setting the ABC transporter structure as a receptor molecule, deleting water molecules and organic solvents, only retaining proteins and original substrate ligands, setting docking parameters and selecting a docking activity pocket;
(3) molecule docking: carrying out molecular docking on a natamycin ligand molecule and a receptor with a well-defined pocket; detecting the interaction between SgnA/B and natamycin;
as shown in fig. 1, SgnA/B consists of two subunits (SgnA and SgnB) constituting the ABC transporter, presenting a typical inward-opening structure, controlled by the "ATP switch model" to rearrange into an outward-opening conformation. Natamycin has two sites (Drug-binding Cavity) DBC1 and DBC2 on SgnA/B, natamycin and DBC1 form conventional hydrogen bonds, nonflavable bump and alkyl interaction, and the binding amino acids are Gln347, Ser206, Asn86, Gln350, Leu93, Leu343, Pro90 and Glu354 of SgnB. There are also van der Waals interactions, pi-alkyl interactions between DBC2 and natamycin which form nonflavonable bump interactions with Ser500, Asp503, Thr504 of the SgnA chain and Thr433 of the SgnB chain, respectively, with conventional hydrogen bonding to Phe499, Asp507, Leu502 of the SgnA chain, pi-alkyl interactions with Leu629 of the SgnB chain, and alkyl interactions with Thr 504. These differences in forces in DBC result in a decrease in binding energy and an increase in affinity for DBC2 compared to DBC 1. Therefore, SgnA/B presents strong intracellular substrate affinity, but weak transmembrane region affinity, thus being beneficial to the extracellular natamycin transport of the outward transport protein through the strong and weak binding capacity of natamycin and SgnA/B two sites (Drug-binding capacity) DBC1 and DBC 2.
The analysis shows that the interaction between SgnA/B and natamycin can promote the transport of natamycin, therefore, the method comprisessgnA/BGenetically engineered bacterium of (A)S. gilvosporeusF-EX has a function of transporting natamycin from the inside to the outside of the cell.
Example 2 construction of recombinant plasmid pMS-EX
(1) According to the sequence of Emr gene promoter Pemr recorded in GenBank and downstream fused gene fragment, the sequence of Emr gene promoter is synthesized on the PCR amplification primer of sgnA/B
Designing upstream and downstream primers:
upstream primer EX-F
5’-AAGCTTGGATCCGATGCTGTTGTGGGCACAATCGTGCCGGTTGGTAGG
ATCAAGCGGAAGAAGGGAGCGGACATCATCATCACCATCACCATGTGCTGCTCAGCCTTCTGCGAATC -3’
(the lower solid line is marked as HindIII restriction enzyme site, the lower solid line is marked as emr promoter fragment, and the lower wavy line is marked as His tag sequence);
the downstream primer EX-R:
5’-GGTACCCATCATCACCATCACCATTCAGGAAAGACTGGGGGAAATGCC-3’
(underlined dotted line is indicated as KpnI cleavage site, and underlined wavy line is indicated as His tag sequence).
(2) To be provided withS. gilvosporeusF607 genome as template, PCR amplification high fidelity DNA polymerase amplification with erythromycin resistance gene promoter PemrEsgnA/BA gene sequence. Cutting and recovering the gel, measuring the concentration of the DNA of the recovered fragment, cloning the DNA to a pMD-18T vector by a TA cloning method, and sequencing the pMD-EX plasmid.
(3) The pMD-EX plasmid and the conjugation transfer vector pMS82 with correct sequencing are respectively subjected to double enzyme digestion by HindIII and KpnI, the fusion fragment and pMS82 are recovered by gel cutting, the mixture is mixed according to the molar ratio of 3: 1-10: 1, T4 ligase is added, the mixture is connected at 16 ℃ overnight, and the ligation product is transformed into escherichia coli competent cellsE.coliET12567, coating LB solid plate containing hygromycin (Hyg, 50 mug/mL), ampicillin (Amp, 100 mug/mL) and chloramphenicol (Cm, 25 mug/mL), picking up transformant, extracting plasmid for enzyme digestion verification, obtaining recombinant plasmid pMS-EX;
FIG. 2 is a restriction enzyme digestion verification diagram of pMS-EX recombinant vector, wherein M is DNA marker; lane 1 is blank control; 2-7 are positive transformants 1-6;
as shown in FIG. 2, 2-7 showed significant bright bands, and transformants 1-6 were positive transformants as the obtained recombinant plasmid pMS-EX.
Example 3 construction of recombinant Streptomyces Limoniliformis
The specific construction method of the method adopts a mode of joint transfer between escherichia coli and streptomyces fuscosporioides and comprises the following steps:
(1) picking out Escherichia coli containing recombinant plasmid pMS-EXE.coliET12567 single colonies were cultured in LB medium (with hygromycin, ampicillin and chloramphenicol resistance) with shaking at 37 ℃ overnight.
(2) Activated overnight recombination at 1% inoculum sizeE.coliET12567 was transferred in fresh LB medium; culturing at 37 ℃ until OD600 is 0.4-0.6, centrifuging, washing the thalli twice by using LB culture medium with the same volume, and suspending in LB culture medium with 0.1 time volume;
(3) culturing 3-4d mycelium in MS solid culture medium with 2-3mL 20% glycerolCollecting the oil to obtain recombinant E.coliET12567 for use;
(4) 200. mu.L of the recombinant obtained in step 3 was takenE.coliET12567 heat shock treated with equal volumeS. gilvosporeusF607 mycelium was mixed well and applied by adding 10 mmol/L MgCl 2 Culturing the MS solid plate at 30 ℃ for 16-20 h;
(5) coating 1mL of sterile water (containing 0.5mg of nalidixic acid and 1mg of hygromycin) to cover the plate, and continuously culturing at 30 ℃ until transformants grow out;
(6) selecting transformants to perform subculture and repeated screening on an MS culture medium containing nalidixic acid and hygromycin; activating in liquid culture medium to obtain each transformant, extracting genome DNA of each transformant, amplifying hgy gene by using verification primers V1-F (ATCCTGTTACTTCGACCGTATTG) and V1-R (GGATCGGTGAAGCCGGAGAG), V2-F (CCGATGCTGTTGGGCAC) and V2-R (GGACCAGCAGCAGCACGAGGGG) to obtain PermE andsgnAa fusion fragment. The name of the positive zygoteS. gilvosporeusF-EX, S, gilvosporeus-pMS82 with empty pMS-82 vector as control;
FIGS. 3 and 4 are PCR validation of positive zygotes: m is DNA marker; 1-8 is positive zygote 1-6; 9 is F607 control; 10 is blank control;
as shown in FIGS. 3 and 4, significant bright bands were seen in 1-8 relative to the 9 and 10 controls, indicating that a new positive zygote was obtained, that is, that of the present applicationS. gilvosporeus F-EX。
Test example 4:S. gilvosporeus application test of F-EX in natamycin fermentation
The genetically engineered bacterium obtained in example 3 was usedS. gilvosporeusF-EX, andS. gilvosporeusf607, respectively taking out 1mL of the culture medium from a super clean bench, coating the culture medium on an MS solid plate (the engineering strain needs to be added with Hyg with the final concentration of 25 mu g/mL), culturing at 29 ℃ for 3-4d, scraping spores after the white spores grow, activating in a seed activation medium NTZ, and culturing at 29 ℃ at 220 r/min for about 48 h. Inoculating the culture product in fresh seed culture medium at 5% transfer amount, activating the seed twice, culturing at 29 deg.C and 220rpm for about 24 hr, inoculating 5% into 250 mL triangular conical flask with liquid volume of 30 mL, and addingAdding 6% glucose, fermenting at 29 deg.C and 220 r/min for 120 h. The initial pH of the fermentation was 7.5. The extracellular and intracellular yields of natamycin were determined every 24 h.
The SgnA/B expression analysis procedure was as follows:
S. gilvosporeusf607, with plasmid pMS-82 unloadedS. gilvosporeuspMS82 andS. gilvosporeusafter the F-EX strain is cultured for 72 hours in a shake flask fermentation mode, 10 mL of fermentation liquor is respectively centrifuged for 20min at 10000 r/min, supernatant TCA is concentrated for 100 times, protein electrophoresis is carried out, PVDF is converted, the supernatant is sealed in a sealing solution for 60 min, a primary antibody (CW 0286, CWBIO, China) is incubated overnight at 4 ℃, a secondary antibody (CW 0102, CWBIO, China) is incubated for 60 min at 37 ℃, a PBS solution is decolorized at room temperature and washed on a bed for three times, and a developing solution is subjected to color development analysis, wherein the result is shown in figure 5.
In FIG. 5, M is Western marker; 1 isS.gilvosporeusF607; 2 isS.gilvosporeu-pMS 82; 3 isS.gilvosporeus F-EX；
As shown in fig. 5, 3 shows two distinct bands; 1-2 bands are not shown;
shows that 1 and 2S.gilvosporeusF607 andS.gilvosporeu-no ABC transporter SgnA/B in pMS82 is normally expressed; clear color band indication in 3S. gilvosporeus The ABC transporter SgnA/B with a 6 XHis tag was normally expressed in the F-EX strain.
Test example 5: method for measuring natamycin yield:
taking 1mL of fermentation liquid obtained in test example 4, adding 9mL of methanol, performing ultrasonic extraction for 20min, centrifuging (1000rpm,10min), filtering the supernatant with 0.22 μm filter membrane, performing HPLC analysis, and detecting the starting strain at ultraviolet 303nmS.gilvosporeus F607、S. gilvosporeuspMS82 and recombinant strainsS. gilvosporeusNatamycin production by F-EX;
the results are shown in FIG. 6, since it can be seen from FIG. 6 thatsgnA/BThe recombinant strain has the yield increased by about 12.5 percent compared with the original strain, and the strain is loaded with empty plasmidsS. gilvosporeusNatamycin production for pMS82 was not different from that of the original strain F607;
show, contrast, andsgnA/Bsuper expression bacteriumThe extracellular/intracellular ratio of the strain natamycin is found that, in the logarithmic natamycin synthesis phase,S. gilvosporeusthe extracellular/intracellular ratio of F-EX is significantly higher than that of F607 strain.
The above results confirm the usesgnA/BIs overexpressedS. gilvosporeusF-EX for the fermentative production of natamycin with a higher total natamycin production than when using the original strain F607 and the empty plasmid-loaded strainS. gilvosporeusThe yield of natamycin fermentative production by the bacterium pMS 82;
it is shown that the light-emitting element constructed by the embodiments 2 to 3 of the present inventionsgnA/BIs overexpressedS. gilvosporeusF-EX promotes the natamycin to be transported from the interior of the mycocyte to the exterior of the mycocyte when the natamycin is fermented and produced; the total yield of the natamycin is improved;
other embodiments of the inventionsgnA/BIs overexpressedS. gilvosporeusF-EX has similar beneficial effects as described above.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Sequence listing
<110> Shandong first medical university (Shandong province medical science institute)
Jiangnan University
<120> construction and application of streptomyces fuscospora for efficient extracellular natamycin transport
<141> 2022-06-10
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1716
<212> DNA
<213> Streptomyces fuscoporicus (Streptomyces gilvosporus)
<400> 1
gtgctgctca gccttctgcg aatccatctg cggccccagg ggcgctccgt cgcccggctg 60
gggcttttgc aactggtgca gatcctggcc actttggccc tgccgacact gggcgccgcg 120
gtcatcgaca acggcgtggt cagggccgac agcggctata tcacccggac cggcctggcc 180
atgctggccg tggcgctggt gcagatcgcg gcgtccgtgg ccgcggtggc gctgggtgcc 240
cgtacggcca tggcgctggg ccgcgacctg cgctcggccg tcttccgccg ggtgctggac 300
ttctcggccc gcgaggtcgg gcagttcggc acgccgtcgc tgatgacgcg gaccgtcaac 360
gatgtgcagc aggtgcagtt gctcgccgtg tccgcgttcg gcgtcgtggt gtcggcgccc 420
ctgatgtgtc tgggcagcat tgcgctcgca cttcagcagg acatcccgct gtccctgctt 480
ctggtggcgc tgatggtggc cgtcggaatg tccttcggcc tcattctcgg ccgcaccgat 540
ccgttctacg ctcgtatgca gaaacacctg gaccgcatca acgggctgct gcgcgaacgc 600
attaccggcg tccgcgtcgt acgggctttt gtgcgcgacg cccatgaagg cgcgagattc 660
ggccgcacca attccgaatt gcgtgacatc tcgctgcgcg tcggccggct gctggccacc 720
gtcatccccc tcgtgctgct ggtcctcaat gccttcatgg cggccgtggt gtggttcggc 780
gcccaccgca tcgacgccgg ggcgatgcgg ttcggtgcgc tcagcgcgtt tctgagctat 840
ctgacgctga tcacgatgtc ggtggtgatg gtgaccttcg tatgcctggc gatgccgcgg 900
gcgagggtct cggccgagcg catccagcag gtgctggcgg ccgagccgag cgtggcggcg 960
ccgagcgaac cggtccgcgc ggtgcccgcg gccggccaac tggcgttgta cggagcgggg 1020
ttccgctatc cgggagcgat ggatccggtg ctgcgcgata tcgatctgag cgtcgggccc 1080
ggcgagacgg tggcggtcct cggcagcacc ggcagcggca agaccaccct gctcaacctc 1140
gtgctgcggc acttcgacgc caccgacggc tccgtccatg taggcggcgt cgacgtccgc 1200
acgctggaca cggaggtgct gcgccgcacc gtcggcttcg taccgcagcg gccgtacctc 1260
ttctccggca ccgtcgccag caatctgcgc ttcggcgacc cggacgccac cgacgacgaa 1320
ctctggcgcg tactggagat cgcccaggcg cgcgatttcg tacggcacct gcccgggggg 1380
ctcgacgcgc cgatcaccca gggcggcacc aatgtgtccg gcgggcagcg gcaacgcctg 1440
gcgatcgccc gcaccctgct gcgcaggccg gacatctatc tcttcgacga ctgcttctcc 1500
gcgctggact acgccacgga cgccgcgctg cggaccgccc tggagccgga gatctcccgg 1560
gcgacggtgg tcaccgtcgc ccagcgcgtc agcaccgtcc gccacgcggg gcgcatcgtg 1620
gtcctggacg cggggcgggt ggccgccacc ggcacgcatg aggagctgct gcgcaccagt 1680
acgacgtacc gggaaatcgc ccggtcccaa ctcacc 1716
<210> 2
<211> 1897
<212> DNA
<213> Streptomyces fuscoporicus (Streptomyces gilvosporus)
<400> 2
gcgctcaccg gcgaccacgg cggatccggc gcggacgccg cggccgccac gacccttcag 60
gaggctgtcc atggtctcga cggacagcgc taggcatgcg cggcgcggcc ccgcccgccg 120
cacgctggga ctgctgcgcc cgcaccgcgc ccgggtgacc gcggcggtcg ccctcggcat 180
cggcggcgtc gcgctcaacg cggcgggccc gatgctcctg ggcagggcca ccgacctcct 240
cttcgacggc atccggcaca cccacggatc caggggagtc gacttcgacg gcatcgcccg 300
tgtgctgctg acggcgctcg cgctcttcgc cgccgccgcc ttcctcaccc tcgtccaggg 360
gcggctggtc acggccgtcg tccagcgtgt ggtcttcgca ctgcgccaat ccgtggagac 420
gaaactcgcc cgcctgccgc tgcggtactt cgaccgccac ccggccggcg aggtgctcag 480
ccgggtgacc aacgacgtcg acaacctcca gctgaccctc cagcagaccc tcagccagct 540
gatcacggcg gcgttctccg tgctgaccat ggtggtgctg atgttcgcca tctcgccgcc 600
gctggcactg atcatgctgg cctgcgtacc ggtgtcggcc gtggtggcgg cccggatcag 660
caagcgcgca cagcctcggt tcacccagca gtggtccgcc accggagcgc tcaacgccca 720
tgtcgaggag gtctacaccg ggcacgcgct ggtcaagggc ttcgggcggc gcgagcaggc 780
cgagcgggtc ttcgacgagc acaacgacgc cctgtaccgc gccggcgcac gggcccagtt 840
cgtctccggc gccatcgagc cgtcgatgat gttcgtctcc aacctcggct atgtcgtcgt 900
ggcggtcgtc ggcgcgctgc gcgtggtctc cggggccctg tccatcggcg acgtccaggc 960
gttcatcctc tactcccggc agttcagcca gccgatcgtg gaggtcgccg gcatcgccgc 1020
gcgcctccag tccgccctcg cctccgcaca gcgggtccac gacctcctgg acgccgacga 1080
acaaggcccc gaccccgagc ggcccgcgcg cctcccccgg gcccgtggac acgtccggtt 1140
cgacaacgtc ggtttccgct actcccccga catccccctc atcgaggacc tctgcctcac 1200
cgtcgaaccc ggccggaccg tggcggtcgt cggtccgacc ggcgcgggga agaccaccct 1260
gggcaacctc ctcatgcggt tctacgagac cgacgcgggg cgcattttcc tcgacggcac 1320
cgacatcacc gccctgaccc gcgaggacct gcgctcccac atcggcctgg tgctccagga 1380
cgcctggctg ttctccggca ccatcgccga gaacatcgcc tacgggcgcc ccgacgcgac 1440
gcgcgaggag atcgtggccg ccgcccgcgc cacctgcgcc gaccgcttca tccgcaccct 1500
gccggacggc tacgacaccg tcctggacga ggagtcgggc aacgtcagcg cgggcgagaa 1560
gcagctcatc accattgccc gggccttcct ggcccggccc tcgatcctgc tcctggacga 1620
ggcgaccagc tccgtcgaca cccgcacgga agtcctcatc cagcgcgcga tgcactcgct 1680
gcgtgcgggc cgtaccagct tcgtcatcgc ccaccggctc tccaccatcc gcgatgcgga 1740
cctcatcctc gtcatggagg acggccgcat cgtcgaacgc ggcacccacg accagctgct 1800
ggccgccgag ggggcctacg cccggctgca tgcgaccgcg gggaccggag cgacggcggc 1860
gccgcacacg tctggcattt cccccagtct ttcctga 1897
Claims (10)
1. Genetic engineering bacterium for producing natamycinS. gilvosporeusF-EX, wherein the genetically engineered bacteriumS. gilvosporeusF-EX comprising two copies of the natamycin extracellular transporter coding genesgnA/ BA gene.
2. The genetically engineered bacterium of claim 1S. gilvosporeusF-EX, wherein the genetically engineered bacteriumS. gilvosporeusF-EX includes recombinant plasmid pMS-EX.
3. The genetically engineered bacterium of claim 2S. gilvosporeusF-EX, characterized in that said recombinant plasmid pMS-EX comprises a promoter sequencesgnA/BA gene.
4. The genetically engineered bacterium of claim 1S. gilvosporeusF-EX, characterized in thatsgnA/BThe PCR amplification primer of the gene is as follows:
an upstream primer EX-F:
5’-AAGCTTGGATCCGATGCTGTTGTGGGCACAATCGTGCCGGTTGGTAGGATCAAGCGGAAGAAGGGAGCG GACATCATCATCACCATCACCATGTGCTGCTCAGCCTTCTGCGAATC -3’ ；
the downstream primer EX-R:
5’-GGTACCCATCATCACCATCACCATTCAGGAAAGACTGGGGGAAATGCC-3’。
5. genetically engineered bacterium for producing natamycinS. gilvosporeusThe construction method of F-EX is characterized in that S.gilvospreus F607 genome is taken as a template, and high fidelity DNA polymerase is used for amplifying sgnA/B with a promoter PemrEThe gene sequence is inserted into streptomycete recombinant plasmid, streptomycete fuscoporia is transformed by means of conjugal transfer, recombinant engineering bacteria containing two copies of sgnA/B gene are obtained by antibiotic screening, and the genetic engineering bacteria with highest natamycin yield are screened by shake flask fermentation experimentS. gilvosporeus F-EX。
6. The method of claim 5, wherein the recombinant plasmid is pMS-EX, and the recombinant plasmid pMS-EX further comprises pMD-18T and pMS82 vectors in the process of constructing the recombinant plasmid pMS-EX; pMD-18 was used to preserve the sgnA/B gene sequence with the erythromycin resistance gene promoter PemrE, pMS82 contains the elements required for plasmid transfer as well as the integrase and integration site of the phage.
7. The method of claim 6, wherein the recombinant plasmid pMS-EX is constructed by digesting a fusion fragment Pemr-sgNA/B with KpnI and HindIII and ligating the digested fragment into a pMS82 plasmid.
8. The method of claim 5,
the gene engineering bacterium for producing natamycinS. gilvosporeusThe construction method of the F-EX specifically comprises the following steps:
step 1, computer molecule docking simulation construction and verification of genetic engineering bacteriaS. gilvosporeusThe function of F-EX for transporting natamycin from the inside to the outside of the cell;
step 2, constructing a recombinant plasmid pMS-EX;
step 3, adopting a mode of joint transfer of escherichia coli-fusonospora to carry out gene engineering bacteriaS. gilvosporeusConstruction of F-EX (Streptomyces fuscospora).
9. The genetically engineered bacterium of any one of claims 1 to 4S. gilvosporeusF-EX, the gene engineering bacteria obtained by the construction method of any one of claims 5-8S. gilvosporeusUse of F-EX in the fermentative preparation of natamycin.
10. Genetically engineered bacterium obtained by the construction method of any one of claims 5 to 8S. gilvosporeusF-EX, characterized in that it comprises two copies of the natamycin extracellular transporter coding genesgnA/BThe gene(s) is (are),sgnA/Bthe gene promotes the transportation of natamycin to the outside of the cell, and the yield of natamycin is improved in the process of producing natamycin by fermentation.
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| CN103923870A (en) * | 2014-04-30 | 2014-07-16 | 山东大学 | Genetically engineered bacterium for producing natamycin as well as construction method and application of genetically engineered bacterium |
| CN105907778A (en) * | 2015-11-30 | 2016-08-31 | 天津科技大学 | Streptomyces gilvosporeus recombinant expression plasmid, and engineering bacterium and application thereof |
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| CN103923870A (en) * | 2014-04-30 | 2014-07-16 | 山东大学 | Genetically engineered bacterium for producing natamycin as well as construction method and application of genetically engineered bacterium |
| CN105907778A (en) * | 2015-11-30 | 2016-08-31 | 天津科技大学 | Streptomyces gilvosporeus recombinant expression plasmid, and engineering bacterium and application thereof |
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