CN104531598B - It is a kind of to recombinate streptomycete, its construction method and the method for improving antibiotic yield - Google Patents
It is a kind of to recombinate streptomycete, its construction method and the method for improving antibiotic yield Download PDFInfo
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- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
- C12P19/623—Avermectin; Milbemycin; Ivermectin; C-076
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Abstract
Streptomycete, its construction method and the method for improving antibiotic yield are recombinated the invention discloses a kind of.The restructuring streptomycete is overexpressed acetolactate synthase small subunit ilvH.Subunit gene is adjusted by the recombinant bacterium overexpression acetolactate synthase, is remarkably improved the yield for the product (such as antibiotic) that cometabolism is carried out using L isoleucines and/or L valines and/or L leucines as precursor.
Description
Technical field
The invention belongs to genetic engineering field, is related to a kind of restructuring streptomycete and its application, and in particular to anti-by transforming
The key gene in the route of synthesis of plain precursor is given birth to improve the yield of antibiotic.
Background technology
Streptomyces Gram positive actinomycetes, streptomyces (Strptomyces) is attributed in systems biology, is had
Complicated life cycle and secondary metabolism approach.Many members of the category can produce a variety of cometabolism productions with bioactivity
Thing.In up to ten thousand kinds of antibiotic being currently known, have 70% above is as caused by streptomycete.In addition, it can also produce and exempt from
Epidemic disease inhibitor, antitumor agent, insecticide and a variety of extracellular hydrolases, such as cellulase, amylase, pectase and protease.
During streptomycete produces secondary metabolite, amino acid can both be used as carbon source, can also be used as nitrogen source,
Also many amino acid are the precursors of some secondary metabolites, such as valine, isoleucine, leucine, their metabolism production
Thing includes acetyl-CoA, propionyl CoA, butyryl CoA and isovaleryl CoA etc., and these materials are macrolide antibiotics biosynthesis
Precursor.Such as biosynthesis (Hafner E W, Holley B W, Holdom K S, the et al.Branched- of AVM
chain fatty acid requirement for avermectin production by a mutant of
Streptomyce avermitilis lacking branched-chain 2-oxo acid dehydrogenase
activity[J].J Antibiot(Tokyo),1991,44(3):349~356), spiramycin streptomyces produce Bitsoft's spiral
Mycin (Li Zhenlin, Jiang Wei, Wang Yonghong, waits influence [J] China of .Val, Ile and Leu to Bitespiramycin biosynthesis anti-
Raw plain magazine, 2007,32 (11):660~668), biosynthesis (the Yoshida T, Katagiri of kinomycin
K.Influence of Isoleucine upon quinomycin biosynthesis by Streptomyces sp.732
[J].J Bacteriol,1967,93(4):1327~1331) etc..
AVM (avermectin) is one kind by AVM streptomycete (Streptomyces avermitilis)
A kind of macrolide antibiotics caused by fermentation, there is extensive expelling parasite and insecticidal activity.AVM streptomycete fermentation liquid
In usually contain 8 kinds of AVMs component A1a, A1b, A2a, A2b, B1a, B1b, B2a, B2b, wherein, insecticidal effect is with B1 groups
Divide most preferably, especially B1a components.The mechanism of action of AVM is unique, and different from conventional chemical pesticides, insecticidal effect is one
As tens times of chemical pesticide, and be not easy to make insect produce resistance.The action target spot of AVM is nematode and arthropod class
The nerve conduction medium γ-aminobutyric acid of parasite.Its toxicity very little to mammal, selectivity are very high, and in plant
Residence time it is very short, the microorganism in soil can be broken down into innocuous substance quickly.Due to the wide spectrum of AVM, height
Effect, noresidue, it is highly safe to animals and plants outstanding advantages of, in agricultural, animal husbandry, medicine and other fields extensive use, have
Wide market prospects and application value.
In addition, using AVM as parent, it further developed that a series of activity are higher, selectivity is stronger, to animals and plants
Safer derived product, commercialized product include doractin, ivermectin, emaricin etc..
AVM has been realized in industrialization at home, achieves huge economic and social benefit.But Avermectin
The problems such as production bacterial strain of element is also low there is fermentation unit, and production cost is high.The yield of AVM how is improved, reduces life
Cost is produced, is an important topic in AVM research.
The content of the invention
For demand of the prior art, the present invention provides a kind of restructuring streptomycete, and the restructuring streptomycete passes through overexpression
Acetolactate synthase small subunit come promote acetolactate synthase to the ILE in the restructuring streptomycete, Valine and/
Or the biosynthesis of L-Leu, so as to improve in streptomycete using ILE, Valine and/or L-Leu as precursor
Carry out the Product yields of cometabolism.The present invention also provides bright with ILE, Valine, L- in a kind of raising streptomycete
Propylhomoserin is the method for product (such as AVM, Bitespiramycin, kinomycin) yield that precursor carries out cometabolism.
According to the first aspect of the invention, there is provided one kind restructuring streptomycete, wherein the restructuring streptomycete overexpression sequence
Acetolactate synthase small subunit ilvH shown in the amino acid sequence 2 of table.
According to a kind of embodiment, by the way that the ilvH gene clonings of the acetolactate synthase small subunit will be encoded to expression
Carrier, then obtained recombinant expression carrier is incorporated into the streptomycete that sets out and obtains the restructuring streptomycete.
The streptomycete that sets out is to produce antibiosis as precursor using ILE and/or Valine and/or L-Leu
The streptomycete of element.Such as can be wild type streptomycete, or through modifying, being mutated, the streptomycete that mutagenesis or genetic recombination obtain.
Preferably, the above-mentioned streptomycete that sets out is selected from AVM streptomycete Streptomyces avermitilis, spiral
A kind of bacterial strain in mycin streptomycete Streptomyces spiramyceticus and streptomycete Streptomyces sp.732.
Most preferably, the streptomycete that sets out is AVM streptomycete Streptomyces avermitilis MA4680
(ATCC31267)。
The expression vector is preferably Escherichia coli-streptomycete shuttle vector.
According to a kind of embodiment, Escherichia coli-streptomycete shuttle vector is pSET152, by promoter and ilvH genes
Fragment inserts pSET152 multiple cloning sites, obtains recombinant expression carrier.
The ilvH genes are most preferably the ilvH genes of encoding acetolactate synthase small subunit in AVM streptomycete,
Nucleotide sequence 1 i.e. in sequence table；The promoter is the erythromycin resistance gene shown in the nucleotide sequence 3 in sequence table
Strong promoter ermE*p.
Most preferably, the restructuring streptomycete is AVM streptomycete Streptomyces avermitilis AV-
2320-8, the China Microbiological bacterium that address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 is preserved on December 08th, 2014
Kind preservation administration committee common micro-organisms center, preserving number is CGMCC 10146.
According to another aspect of the invention, there is provided a kind of method for preparing above-mentioned restructuring streptomycete, be included in the strepto- that sets out
Acetolactate synthase small subunit ilvH in bacterium shown in the amino acid sequence 2 of overexpression sequence table.
According to a kind of embodiment, by the way that the ilvH gene clonings of the acetolactate synthase small subunit will be encoded to expression
Carrier, then obtained recombinant expression carrier is incorporated into the streptomycete that sets out and obtains the restructuring streptomycete.
By the recombinant expression carrier introduce set out streptomycete method can use bioengineering field common method,
Such as Conjugative tiansfer method, electrotransformation, protoplast transformation etc..
Preferably Conjugative tiansfer method.Preferably, expression vector is transformed into the large intestine bar of restriction modification effect defect first
In bacterium ET12567 (pUZ8002), collect resistant clones and do Conjugative tiansfer with the streptomycete that sets out again.
The streptomycete that sets out is to produce antibiosis as precursor using ILE and/or Valine and/or L-Leu
The streptomycete of element.Such as can be wild type streptomycete, or through modifying, being mutated, the streptomycete that mutagenesis or genetic recombination obtain.
Preferably, the above-mentioned streptomycete that sets out is selected from AVM streptomycete Streptomyces avermitilis, spiral
A kind of bacterial strain in mycin streptomycete Streptomyces spiramyceticus and streptomycete Streptomyces sp.732.
Most preferably, the streptomycete that sets out is AVM streptomycete Streptomyces avermitilis MA4680
(ATCC31267)。
The expression vector is preferably Escherichia coli-streptomycete shuttle vector pSET152.
The ilvH genes are most preferably the ilvH genes of encoding acetolactate synthase small subunit in AVM streptomycete,
Nucleotide sequence 1 i.e. in sequence table；The promoter is the erythromycin resistance gene shown in the nucleotide sequence 3 in sequence table
Strong promoter ermE*p.
According to another aspect of the present invention, there is provided one kind is improved in streptomycete with ILE, Valine, the bright ammonia of L-
The method that acid carries out the Product yields of cometabolism for precursor, methods described are fermented using above-mentioned restructuring streptomycete
Step.
According to the present invention, with AVM streptomycete (Streptomyces avermitilis MA4680
(ATCC31267))) it is starting strain, obtained restructuring AVM streptomycete, after Shaking culture 240 hours, AVM
Yield can reach 3486.32 ± 497.97 μ g/mL, be 5.13 times of starting strain AVM yield.
Restructuring AVM streptomyces gene engineering bacteria constructed by instantiation of the present invention can be directly used for AVM
The fermenting and producing of streptomycete, make the output increased of AVM, so as to reduce production cost.Those skilled in the art should manage
Solution, method of the invention, by being overexpressed acetohydroxyacid synthase small subunit, it is bright to improve ILE, Valine, L- in streptomycete
The enzyme amount of acetolactate synthase in propylhomoserin route of synthesis, so as to improve the yield of ILE, Valine, L-Leu,
And the Product yields that cometabolism is carried out using these amino acid as precursor are further improved, this method is suitable to those generations secondary generation
The streptomycete by the route of synthesis is needed when thanking to product (such as antibiotic), so as to obtain the high yield recombinant bacterium of target product, and
Therefore the effect for improving target product is obtained.
Brief description of the drawings
With reference to the following drawings, the present invention is better understood with.
Figure 1A and 1B be respectively the recombinant plasmid pH-19 containing ilvH genes and erythromycin resistance gene strong promoter and
PH-152 plasmid map.
Fig. 2 be AVM streptomycete bacterial strain Streptomyces avermitilis MA4680 (ATCC31267) and its
Restructuring AVM streptomycete (CGMCC10146) fermented obtained AVM yield prepared in accordance with the present invention.
Fig. 3 A are Avermectin B1a standard items HPLC collection of illustrative plates, and Fig. 3 B and 3C are respectively the AVM streptomycete that sets out
Streptomyces avermitilis MA4680 (ATCC31267) and restructuring AVM strepto- prepared in accordance with the present invention
The HPLC collection of illustrative plates of Avermectin B1a caused by bacterium.
Embodiment
The invention will be further described with specific preferred embodiment below in conjunction with the accompanying drawings, but the present invention is not limited to
Following examples.
Based on the excellent characteristic of AVM and it is widely applied, the present invention is mainly using AVM streptomycete as embodiment
Studied, by being overexpressed acetolactate synthase small subunit in AVM streptomycete, obtain AVM yield and show
Write the bacterial strain improved.
Likewise, cometabolism is produced as precursor using ILE and/or Valine and/or L-Leu at other
It is also believed that the yield of respective secondary metabolite can be effectively improved in the streptomycete of product.Such streptomycete such as produces bit
The spiramycin streptomyces of spiramvcin, the streptomycete Streptomyces sp.732 for producing kinomycin etc..
AVM is one group of glycosides generated by sixteen-ring lactone and a disaccharides (oleandrose), hexa-atomic interior ten
There are a Spiroketals system and hexahydro benzo furan nucleus system containing 2 hexatomic rings around ester.Glycosyl ligand backbone is by an a branch
Chain fatty acid S (+)-methylbutyryl aliphatic acid or isobutyryl aliphatic acid are starting, and 7 acetates and 5 propionates polymerize end to end
Form.The different substituents of C-25 positions are divided into a, b component, and the dimethylbutanoyl of a components derives from S (+)-methylbutyryl
The isobutyryl of CoA, b component derives from isobutyryl CoA.S (+)-methylbutyryl CoA and isobutyryl CoA is respectively by the different bright ammonia of L-
Acid, Valine by deamination, turn ammonia and decarboxylation is formed.
Acetolactate synthase is living things catalysis ILE, Valine, the reaction of the L-Leu biosynthesis first step
Enzyme, using ILE, Valine, L-Leu as precursor carry out secondary metabolite biosynthesis during,
The activity or enzyme amount of acetolactate synthase are improved, can all improve the yield of respective secondary metabolite.
In AVM streptomycete, acetolactate synthase contains 3 large subunits, 1 small subunit, and large subunit plays catalysis and made
With small subunit plays activation and feedback regulation.The present invention is had found by studying, and is overexpressed the small subunit energy of acetolactate synthase
Enough dramatically increase using ILE, Valine as the AVM of precursor yield.
It is conventional method unless otherwise specified in following embodiments.
The structure of embodiment 1, ilvH expression vectors
1st, the amplification of ilvH structural genes
Primer is designed, is located at AVM streptomycete Streptomyces avermitilis MA4680 for expanding
(ATCC31267) the ilvH structural genes on chromosome [NC_003155.4 (3353885..3354412)], i.e. in sequence table
Sequence 1.
Sense primer Primer F1：ATGAGCAAGCACACCCTCTCCGTCCT,
Anti-sense primer Primer R1：TTGTAAAACGACGGCCAGTGAATTCTTACGCGGATCGGTCCAGCGCGCGC,
Band underscore base is restriction enzyme EcoRI recognition sites, and amplified production should be 557bp.
Using AVM streptomycete Streptomyces avermitilis MA4680 (ATCC31267) STb gene as mould
Plate, Primer F1 and Primer R1 are primer, using the Q5Hot Start High- of New England Biolabs companies
Fidelity DNA Polymerase, enter performing PCR amplification, amplification condition is 98 DEG C, 3min；(98 DEG C, 10s；72 DEG C, 40s) ×
30 circulations；72 DEG C, 2min.Amplified production is entered row agarose gel electrophoresis detection, about have at 500bp one it is specific
Amplified band.
2nd, the amplification of erythromycin resistance gene strong promoter (ermE*p)
Primer is designed, (Li Jia, to the four seas, Yang Xiushan, Yankee is moved, reporter gene method positioned at pZW221 plasmids for expanding
Compare the activity of two kinds of actinomycete promoters, microorganism journal, 2009,49 (11):1454-1458.) on ermE*p genes
(that is, sequence 3).
Sense primer Primer F2：GCATGCCTGCAGGTCGACTCTAGAAGAGCGAGTGTCCGTTCGAGTGGCGGC
TT,
Anti-sense primer Primer R2：GGACGGAGAGGGTGTGCTTGCTCATATGTGGATCCTACCAACCGGCACGGT
T。
With the restriction enzyme site that underscore base is restriction enzyme XbaI, amplified production should be 235bp.
Using pZY125 plasmids as template, Primer F2 and Primer R2 are primer, using New England Biolabs
The Q5Hot Start High-Fidelity DNA Polymerase of company, entering performing PCR amplification, amplification condition is 98 DEG C,
3min；(98 DEG C, 10s；72 DEG C, 30s) × 30 circulations；72 DEG C, 2min.Enter row agarose gel electrophoresis inspection to amplified production
Survey, about there is a specific amplified band at 250bp.
3rd, recombinant plasmid pH-152 structure
IlvH genes and ermE*p pcr amplified fragment are reclaimed with DNA QIAquick Gel Extraction Kits (OMEGA, D2500-02),
PUC19 carriers are through XbaI and EcoRI digestions, recovery about 2660bp fragment.These three fragments are tried with Gibson Assembly
Agent box (New England Biolabs companies) is attached, and is named as pH-19 (referring to Figure 1A).Show by comparison is sequenced,
The fragment of amplification is ermE*p and ilvH genes really.By the pH-19 carriers containing ermE*p and ilvH genes through XbaI and
EcoRI digestions, the junction fragment of about 710bp ermE*p and ilvH genes is reclaimed, by this fragment and through XbaI and EcoRI digestions
Carrier pSET152 (Bierman M, Logan R, O ' Brien K, et al.Plasmid cloning vector for
the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.Gene,
1992,116:43-49) connect, connection product converts competent cell (the TransGen Biotech of bacillus coli DH 5 alpha respectively
Company), extraction plasmid enters performing PCR and digestion verification from transformant, and correct recombinant plasmid is named as into pH-152 (referring to figure
1B).PH-152 is integrative plasmid, the ilvH genes in this plasmid be placed in erythromycin resistance gene strong promoter ermE*p it
Under.
The conversion of embodiment 2, recombinant plasmid
Acted on due to very strong restriction modification in AVM streptomycete be present, with E.coli DH5 α directly and Avermectin
Plain streptomycete is combined transfer, and transformation efficiency is extremely low, sometimes even cannot get transformant.And made using no restriction modification
E.coli ET12567 (PUZ8002) are combined transfer, and transformation efficiency significantly improves.Therefore, the restructuring that will be built
Plasmid is transformed into E.coli ET12567 (PUZ8002) (Kieser T, Bibb M J, Buttner M J, et
al.Practical Streptomyces Genetics,2000,Norwich:The John Innes Foundation.) in
To obtain the non-DNA to methylate, transfer is then combined.
AVM streptomycete Streptomyces avermitilis MA4680 (ATCC31267) are selected in this example, are made
For starting strain, the bacterium produces canescence spore on flat board.
By the E.coli ET12567 (PUZ8002) containing the ilvH gene expression plasmids pH-152 built in embodiment 1
Transfer is combined with above-mentioned AVM streptomycete, is coated on containing 10mM MgCl2MS flat boards on, 28 DEG C culture 16~
After 20h, with sterilized water uniform folds of the 1mL containing 1000 μ g nalidixic acids and 1000 μ g apramycins, cultivated 5~7 days at 28 DEG C,
The bacterium colony grown is transformant, and transformant carries out the fermentation research of next step after plasmid extraction and PCR checkings are correct.
Gained transformant is the restructuring streptomycete obtained according to the construction method of the present invention.The restructuring streptomycete be named as Ah
Rhzomorph streptomycete Streptomyces avermitilis AV-2320-8 are tieed up, are preserved in Chinese microorganism strain preservation management committee
Member's meeting common micro-organisms center, preserving number CGMCC10146.The bacterial strain optimal culture condition is 28 DEG C, the close neutrality of pH,
Cultivated 5-7 days on solid medium (such as MS), canescence spore can be produced, gas silk lacks, and base silk lacks.
The fermentation research of embodiment 3, recombinant bacterial strain
1st, the shake flask fermentation of AVM streptomycete
AVM streptomycete starting strain AVM streptomycete Streptomyces avermitilis MA4680
(ATCC31267) and embodiment 2 obtain recombinant bacterium abundant spore is grown on slant medium after, to AVM strepto-
The AVM yield level of bacterium transformant is tested.Take inclined-plane lawn 1cm2, accessed sterilized equipped with 40mL
The seed bottle of seed culture medium, 28 DEG C of shaking table cultures 44~48 hours, rotating speed 200rpm, radius of turn 50mm, obtains seed
Nutrient solution.Take above-mentioned seed culture fluid to be inoculated in by the inoculum concentration of 5% (percent by volume) to train equipped with fermentation sterilized 30mL
In the triangular flask for supporting base, 28 DEG C of shaking table cultures 10 days, bottle is put, the fermentation unit of AVM, specific HPLC are determined with HPLC methods
It is analyzed as follows and states described in step 2.
In this experiment slant medium (glucose 1.5%, beef extract 0.3%, asparagine 0.05%,
KH2PO40.05%th, agar 1.8%), seed culture medium (final concentration of 30g/L cornstarch, final concentration of g/L soyabean cake
Powder, final concentration of 10g/L groundnut meal, final concentration of 4g/L dusty yeast, final concentration of 0.03g/L CoCl2) and fermentation
Culture medium (final concentration of 150g/L cornstarch, final concentration of 28g/L bean cake powder, final concentration of 9g/L dusty yeast, end
Concentration is 0.25g/L (NH4)2SO4, final concentration of 0.02g/L CoCl2, final concentration of 0.022g/L Na2MoO4, eventually it is dense
Spend the MnSO for 0.0023g/L4, final concentration of 4000U/L amylase and final concentration of 0.8g/L CaCO3).(referring to China
Patent application No.200810227639.8, a kind of method and its special strain therefore for preparing AVM, the full text of the patent application
Merged in this article by quoting.)
2nd, the HPLC analyses of tunning
1) sample treatment：1.0mL zymotic fluids are taken, 9.0mL methanol is added, ultrasonic oscillation more than 30 minutes, stands 10-20
Minute；
2) 1mL supernatants are taken, with 0.2 μm of filtering with microporous membrane, obtaining filtrate progress HPLC, (Agilent 1200 is efficient
Liquid chromatograph) analysis.
3) HPLC analysis conditions：The reverse posts of C18 (Agilent), column length 150mm, column internal diameter 4.6mm, mobile phase are methanol：
Water (90:10), flow velocity 1.0mL/min, automatic sampler sample introduction, sample size are 10 μ L, Detection wavelength 245nm.At this
Under part, the retention time of Avermectin B1a standard items (DR, Germany) is 6 minutes or so, when retaining in calculating ferment filtrate
Between be 6 minutes or so the peak areas gone out, calculate Avermectin B1a yield.Experiment sets 3 repetitions, as a result takes the mean.
Fig. 2 fermentation results show that the abamectin fermented unit of the transformant containing ilvH expression vectors is with going out bacterium germination
Strain adds 4 times or so compared to significantly improving.
It should be understood that without departing from the spirit and substance of the case in the present invention, the inventive method, step and condition are done
Modifications or substitutions, belong to the scope of the present invention.
Claims (7)
1. one kind restructuring streptomycete, the restructuring streptomycete is AVM streptomycete (Streptomyces avermitilis)
AV-2320-8, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC
10146。
2. a kind of method of the restructuring streptomycete built described in claim 1, is included in overexpression sequence table in the streptomycete that sets out
Amino acid sequence 2 shown in acetolactate synthase small subunit ilvH, the streptomycete that sets out is AVM streptomycete
(Streptomyces avermitilis)MA4680。
3. according to the method for claim 2, wherein by the way that the ilvH genes of the acetolactate synthase small subunit will be encoded
Expression vector Escherichia coli-streptomycete shuttle vector pSET152 is cloned into, then obtained recombinant expression carrier is incorporated into and set out
Streptomycete obtains the restructuring streptomycete.
4. method according to claim 3, wherein, expression vector is transformed into the Escherichia coli of restriction modification effect defect
In ET12567 pUZ8002, collect resistant clones and do Conjugative tiansfer with the streptomycete that sets out again.
5. method according to claim 3, wherein the ilvH genes are the AVM hereinafters shown in the nucleotide sequence 1 in sequence table
The ilvH genes of encoding acetolactate synthase small subunit in rhzomorph streptomycete, promoter are the institute of nucleotide sequence 3 in sequence table
The erythromycin resistance gene strong promoter ermE*p shown.
6. a kind of improve the product for carrying out cometabolism in streptomycete using ILE, Valine, L-Leu as precursor
The method of yield, the step of methods described is fermented using the restructuring streptomycete described in claim 1.
7. according to the method for claim 6, wherein described entered using ILE, Valine, L-Leu as precursor
The product of row cometabolism is AVM.
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| CN111019965B (en) * | 2018-10-10 | 2022-05-13 | 中国科学院微生物研究所 | Engineering bacterium for genetic modification of neomycin biosynthesis gene cluster and application thereof |
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