CN104928313B - Application of the Avid kyowamycin rex genes in AVM yield is improved - Google Patents
Application of the Avid kyowamycin rex genes in AVM yield is improved Download PDFInfo
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- CN104928313B CN104928313B CN201510313874.7A CN201510313874A CN104928313B CN 104928313 B CN104928313 B CN 104928313B CN 201510313874 A CN201510313874 A CN 201510313874A CN 104928313 B CN104928313 B CN 104928313B
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Abstract
The invention provides a kind of method for improving AVM yield and production bacterial strain, it is to be introduced into the rex genes of Codocyte internal oxidition reduction regulatory factor (redox sensing transcriptional repressor) in Avid kyowamycin in Avid kyowamycin by gene delection carrier, obtain the recombinant bacterium of missing rex genes, it is set no longer to synthesize Rex albumen, so as to improve the yield of AVM so that Avid kyowamycin for limited oxygen condition more resistant to.The genetic engineering bacterium of the present invention can be directly used for the fermenting and producing of AVM, improve the fermentation unit of AVM, reduce production cost.
Description
Technical field
The invention belongs to genetic engineering field, specifically, it is related to a kind of genetic engineering side for improving AVM yield
Method.
Background technology
AVM (avermectins) is to be fermented to produce by Avid kyowamycin (Streptomyces avermitilis)
One group of efficient insecticide 16-membered ring macrolides antibiotic, its natural products have eight components (A1a, A1b,
A2a, A2b, B1a, B1b, B2a and B2b), the insecticidal activity of wherein B1 components is most strong, almost resists all and agriculture relevant nematode
And arthropod, it is widely used in agricultural production.Ivermectin (ivermectin) is existed by raw material of AVERMECTIN B1
C22-C23 hydrogenating reductions are formed, and have identical insecticidal activity with AVERMECTIN B1, but toxicity is lower than AVM 2-3 times,
The preventing and treating of epizoa in animal body is more suitable for, is the effective new anti parasitic antibiotic of a class wide spectrum.AVM and
Ivermectin has unique mechanism of action, is difficult to make insect produce resistance, and degradable, noresidue, to crop, livestock, people
Class and environment high safety, public nuisance-free agricultural chemicals are recommended as by the Ministry of Agriculture of China, with boundless market prospects and apply valency
Value.AVM and ivermectin realize industrialization at home, achieve huge economic and social benefit.Improve AVM hereinafter
The yield of rhzomorph, reduces production cost, has important practical significance.Therefore, AVM hereinafter strepto- is transformed by genetic engineering means
Bacterium is of great significance and value with improving the yield of AVM.
Avid kyowamycin is a kind of aerobic heterotroph actinomyces, and dissolved oxygen is the important ginseng in streptomycete antibiotic fermentation process
Number, is often related to the success or failure of antibiotic fermentation.Therefore, the change of somatic cells quick response redox signal it is survived and
The success or failure of antibiotic fermentation are most important.Find that Avid kyowamycin is very sensitive to dissolved oxygen in the fermentation process of AVM,
The of short duration oxygen supply of fermentation mid-early stage, which is interrupted, can often cause AVM yield to substantially reduce or can hardly synthesize, and this phenomenon exists
It is especially apparent in AVM superior strain.Rex is unable to the oxygen signal in direct feeling environment, and is in response to intracellular NADH/
NAD+Rate of change.Intracellular NAD (H) has a very high turnover rate, but when cell limited oxygen condition or aerobic respiration by
To when suppressing, NADH/NAD+ ratios can be improved rapidly.When cell is suppressed in limited oxygen condition or aerobic respiration, NADH/
NAD+Ratio can be improved rapidly, and internal high-caliber NADH can release Rex and target gene cydABCD and (encode oxygen high-affinity
Cytochrome bd terminal oxidizing ferment) and Rex-hemACD combination, the expression of these operators is activated to tackle the deficient of oxygen
It is weary.
The content of the invention
It is an object of the invention to provide a kind of method for improving AVM yield.
It is a further object of the present invention to provide the genetic engineering bacterium that high yield AVM and hypoxemia are resistant to.
In order to realize the object of the invention, present invention firstly provides rex genes in the AVM yield for improving strain
Using.
Foregoing application, i.e., by lacking rex genes, to improve AVM yield, the genetic engineering bacterium in strain
Strain for limited oxygen condition more resistant to.
The strain can be it is any can produce the streptomycete of AVM, such as Avid kyowamycin wild mushroom, or
The strain of AVM can be produced after routine mutagenesis and genetic engineering transformation.
Preferably, the strain is Avid kyowamycin (Streptomyces avermitilis).
Based on this, the present invention provides a kind of genetic engineering bacterium of the production AVM of missing rex genes.As it was previously stated, its
It can any can produce the streptomycete of AVM, such as Avid kyowamycin wild mushroom, or through genetic engineering to go out bacterium germination
The strain of AVM can be produced after transformation.
Preferably, it goes out bacterium germination for Avid kyowamycin (Streptomyces avermitilis).
Further, the preparation method of the genetic engineering bacterium is：The deleted carrier of rex genes is built, and this is lacked
Carrier is introduced into the strain of production AVM the recombinant bacterium for obtaining rex missings.
Genetic engineering bacterium can be specifically built by the following method：With PCR expand Avid kyowamycin in rex genes it is upper and lower
Sequence is swum, the deleted carrier of rex genes is built, and by the recombinant vector built conversion production AVM bacterial strain, screening is obtained
Positive restructuring bacterium.Bacterial strain containing deleted carrier is first cultivated into inducing plasmid under 39 DEG C of high temperature and loses acquisition single exchange strains,
It is passed on to progress culture under 28 DEG C, the culture medium without apramycin again and obtains rex gene deletion mutants.Of the invention real
Apply in example, obtain the genetic engineering bacterium of missing rex Avid kyowamycin wild mushroom.
Foregoing method, wherein the carrier that sets out for building rex gene delection carriers contains streptomycete temperature for any one
The Escherichia coli of responsive type replicon-streptomycete shuttle vector.It is preferred that shuttle vector be pKC1139, pGM160, pHZ132 or
PCZA185 etc..
In embodiments of the present invention, using pKC1139 as the vector construction rex gene delection carriers pKCD-rex that sets out.
It is described to be introduced into rex gene delection carriers in the strain of production AVM in foregoing method, can be using biology
The method commonly used in engineering field, such as the protoplast transformation that PEG is mediated, electrotransformation, engagement transfer method, preferably
The protoplast transformation of PEG mediations.
In foregoing method, to improve transformation efficiency, rex gene delection carriers first can be transformed into restriction modification effect and lacked
In sunken Escherichia coli ET12567, therefrom extract plasmid and be transformed into again in the strain of production AVM.
The present invention provides a kind of method for improving AVM yield, and it utilizes the production AVM for lacking rex genes
Genetic engineering bacterium improves the yield of AVM.
The beneficial effects of the present invention are：
The present invention is that the rex genes of encoding transcription inhibiting factor in Avid kyowamycin are passed through into deleted carrier introducing AVM hereinafter chain
In mould, the recombinant bacterium of rex gene delections is obtained, it is no longer synthesized Rex albumen, so as to improve the yield of AVM.This
The genetic engineering bacterium of invention can be directly used for the fermenting and producing of AVM, improve the fermentation unit of AVM, reduction production
Cost.
Brief description of the drawings
Fig. 1 is rex gene delection carriers of embodiment of the present invention pKCD-rex plasmid map.
Fig. 2 is rex deleted carriers pKCD-rex of the embodiment of the present invention and Avid kyowamycin wild-type strain ATCC 31267
The homologous double-crossover schematic diagram of chromosome.
Fig. 3 be Avid kyowamycin wild-type strain of embodiment of the present invention ATCC 31267 and its rex deletion mutations strain Ah
Tie up rhzomorph fermentation unit.
Fig. 4 is Avid kyowamycin wild-type strain ATCC 31267 and its rex deletion mutations strain under the conditions of different fermentations
Abamectin fermented unit.In figure：Shaking speed is 230rpm when * representing fermentation (normal shaking speed is 250rpm)；**
Represent after fermentation has a power failure two hours to shaking table at the 5th day and resume operation again.Remarks：Digitized representation respective handling group on block diagram
The percentage that AVM yield declines compared with starting strain.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
The structure of the rex gene delection carriers of embodiment 1
First, the clone of Avid kyowamycin rex genes
Rex genes [NC_003155.4 in the Avid kyowamycin wild-type strain ATCC 31267 announced according to Genebank
(5777053..5777811)] sequence and its upstream and downstream sequences Design PCR primer：
Primer rex_1：5′-CCCAAGCTTCATCACCCAGGACGCGGAG-3′
Primer rex_2：5′-CGCGGATCCCTCGGAGGTACAGCGGAAGC-3′
Primer rex_3：5′-CGCGGATCCTCACCTCCATCCTGAACTTCG-3′
Primer rex_4：5′-CCGGAATTCCCCTCGCCCACGACCATC-3′
Primer rex_1, rex_2, rex_3 and rex_4 two ends introduce HindIII, BamHI, BamHI and EcoRI enzymes respectively
Enzyme site (underscore part).Using the genomic DNAs of Avid kyowamycin ATCC 31267 as template, respectively with rex_1 and rex_2,
Rex_3 and rex_4 enter performing PCR amplification.PCR reaction conditions：95 DEG C, 5min；(95 DEG C, 50s；60 DEG C, 50s；72 DEG C, 40s) ×
29 circulations；72 DEG C, 10min；25℃,1min.Electrophoresis detection PCR primer, respectively in 560bp (rex_1 and rex_2) and
There is specific amplified band at 573bp (rex_3 and rex_4) place, is respectively designated as upper arm and underarm.
2nd, the structure of rex deleted carriers
Electrophoresis reclaims above-mentioned fragment, after PCR primer Purification Kit, upper arm and underarm respectively through HindIII,
BamHI and BamHI, EcoRI digestion be connected to through HindIII and EcoRI double digestions carrier pKC1139 (Bierman M,
Logan R, O ' Brien K, wait to be used for the plasmid cloning vector genes of the engagement transfer from Escherichia coli to streptomycete DNA,
1992,116:43-49) plasmid pKCD-rex is obtained.Sequencing shows that expanded fragment is the upstream and downstream sequence of rex genes really,
And it is consistent with the sequence announced.PKCD-rex plasmid map is as shown in Figure 1.
The conversion of the recombinant plasmid of embodiment 2
The deletion plasmid carrier pKCD-rex of rex genes is constructed by embodiment 1, is as the original plasmid of control
pKC1139。
It is direct with the plasmid for extracting from E.coli JM109 due to there is very strong restriction modification effect in Avid kyowamycin
Avid kyowamycin is converted, transformation efficiency is extremely low, even cannot get transformant sometimes.And with from no restriction modification act on by
Body bacterium E.coli ET12567 plasmid, its transformation efficiency is significantly improved.Therefore, the recombinant plasmid transformed built is arrived
E.coli ET12567 (Kieser T, Bibb M J, Buttner M J, wait the hereditary handbook of the practical streptomycetes of, and 2000,
Norwich:John Ying Nasi foundations) in obtain the non-DNA methylated, then again with the non-plasmid DNA transformation methylated
The protoplast of Avid kyowamycin.
The present embodiment is used as starting strain from Avid kyowamycin strains A TCC 31267.ATCC 31267 is AVM hereinafter strepto-
Bacterium wild-type strain, produces grey spore.The protoplast of this Avid kyowamycin bacterial strain is prepared, with from E.coli ET12567
On the plasmid conversion protoplast of extraction, the RM14 flat boards for being applied to the not added with antibiotic dried up, after 28 DEG C of culture 16-20h,
The aqueous solution that 1mL contains 1000 μ g apramycins is applied on flat board, continues to cultivate 7-10 days at 28 DEG C, the bacterium colony grown is conversion
Son.Because the transformant of Avid kyowamycin does not produce spore on RM14 regeneration culture mediums, therefore three transformants of each picking, it is inoculated in and contains
On the YMS flat boards of 10-15 μ g/mL apramycins, 28 DEG C of cultures recover production spore for 7-10 days.Transformant is tested through plasmid extraction and PCR
Card is correct.
The conversion of Escherichia coli, the preparation of Avid kyowamycin protoplast and method for transformation, RM14 and YMS in the present embodiment
The preparation of culture medium referring to《Produce the research of green spore Avid kyowamycin genetic modification and fermentation condition》(the beautiful master's degrees of Cai Yu
Paper, 2006, Beijing：China Agricultural University).
The acquisition of the rex gene deletion mutants of embodiment 3
The correct spore containing pKCD-rex plasmids and control plasmid pKC1139 transformants of above empirical tests is collected respectively
Son, is coated on the YMS flat boards containing apramycin with about 100 spores of every culture dish, is placed in 28 DEG C of culture 48-72h, is worked as bacterium
Fall diameter about 2mm or so, when will form aerial hyphae, after flat board is transferred into 39 DEG C of high-temperature cultivations 7 days, contain pKCD-rex
Some bacterium colonies on there is fan-shaped growth, and the bacterium colony containing pKC1139 moves to not regrowth after 39 DEG C.This is due to
PKC1139 is Escherichia coli-streptomycete shuttle plasmid of temperature sensitive type, and self-replacation is unable to when temperature is higher than 34 DEG C, therefore
It can not be grown on YMS culture mediums containing apramycin；Only when the rex genes entrained by recombinant plasmid pKCD-rex are upper and lower
Swim homology segment on sequence and Avid kyowamycin chromosome and occur homologous recombination, and be incorporated into by single-swap the bacterium on chromosome
Strain could express the resistant gene of apramycin, so as to be grown on the YMS culture mediums containing apramycin.39 DEG C are grown
The single-swap mutant strain of anti-apramycin is passed on the YMS flat boards without antibiotic, after corotation connects 2-4 times, and screening peace is general
The sensitive double crossing over recombinant of mycin.Picking 10 plants of bacterium therein, together with Avid kyowamycin ATCC31267 28 DEG C in YEME
Shaken cultivation 48h, fails therefrom to extract plasmid, illustrates that recombinant plasmid pKCD-rex has been eliminated.Using PCR to AprsIt is prominent
Mutant is verified.Many plants of ATCC 31267 rex deletion mutations strain is obtained respectively.Rex deleted carriers pKCD-rex and AVM hereinafter
The homologous double-crossover schematic diagram of the chromosomes of streptomycete ATCC 31267 is as shown in Figure 2.
The Avid kyowamycin wild-type strain ATCC 31267 of embodiment 4 and rex deletion mutations strain fermenting and producing AVM
First, the shake flask fermentation of Avid kyowamycin
Seed culture medium：Soluble starch 30g, malt extract 2g, soy peptone 2g, CoCl2·6H2O 5mg, plus distillation
Water adjusts pH to 7.0-7.2 to 1L.
Fermentation medium：Soluble starch 50g, dusty yeast 12g, MgSO4·7H2O 0.5g, K2HPO4·3H2O 0.5g,
KCl 4g, CaCO32g, CoCl2·6H2O 5mg, plus distilled water is to 1L, adjusts pH to 7.0-7.2.
2nd, the HPLC analyses of tunning
1. sample treatment：1.0mL zymotic fluids are taken, 4.0mL methanol is added, soaked more than 30 minutes, were vibrated every 10 minutes
Once, 4000rpm is centrifuged 10 minutes, takes supernatant sample introduction to analyze；
2.HPLC analysis conditions：C18Reversed-phase column, column length 150mm, column internal diameter 4.6mm, 40 DEG C of column temperature, mobile phase is methanol：
Water (85:15), flow velocity 1.0mL/min, the μ L of sampling volume 20, wavelength is 246nm.
3rd, ATCC 31267 and its fermentation results of difference rex deletion mutations strain
Avid kyowamycin wild-type strain ATCC 31267 and its rex deletion mutations strain grow abundant on YMS culture mediums
Spore after be inoculated in seed culture medium (loading amount be 100mL/500mL triangular flasks), 28 DEG C of shaking table cultures, 24 hours (rotating speeds
230rpm, eccentric throw 2.5cm).It is inoculated in by 5% inoculum concentration in fermentation medium (loading amount is 50mL/250mL triangular flasks), 28
DEG C culture 10 days (rotating speed 250rpm, eccentric throw 2.5cm), put bottle, extracted with methanol-HPLC methods determine AVM fermentation list
Position, as a result as shown in Figure 3.
Fig. 3 fermentation results show that the deletion mutation strain of rex genes is compared with starting strain, abamectin fermented unit
The substantially increase compared with starting strain, the AVERMECTIN B1 component yield of deletion mutation strain is 3 times of starting strain or so.
Rex gene delections remarkably promote effect to AVM synthesis.
4th, the fermentation results of ATCC 31267 and its rex deletion mutations strain under the conditions of different fermentations
Fig. 4 fermentation results are shown, under 230rpm speed conditions, the deletion mutation strain of rex genes and starting strain
Compare, the reduction amplitude of abamectin fermented unit significantly diminishes, and the range of decrease only has 1/5 of starting strain or so；In fermentation to the 5th
It when shaking table have a power failure under conditions of processing 2h, the deletion mutations of rex genes strain is compared with starting strain, abamectin fermented unit
Reduction amplitude equally diminish.Rex gene delections make Avid kyowamycin for limited oxygen condition more resistant to.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (6)
1. the rex genes of Codocyte internal oxidition reduction regulatory factor are improving the AVM yield of strain in Avid kyowamycin
In application, it is characterised in that by the missing rex genes in the Avid kyowamycin (Streptomyces avermitilis),
To improve AVM yield.
2. the genetic engineering bacterium of the production AVM of a kind of missing rex genes, it is characterised in that it goes out bacterium germination for Avid kyowamycin
(Streptomyces avermitilis), the rex gene codes Avid kyowamycin cellular redox regulatory factor.
3. genetic engineering bacterium according to claim 2, it is characterised in that the preparation method of the genetic engineering bacterium is：Build
The deleted carrier of rex genes, and the deleted carrier is introduced into the strain of production AVM to the missing recombinant bacterium for obtaining rex.
4. genetic engineering bacterium according to claim 3, it is characterised in that build the carrier that sets out of rex gene delection carriers
Contain Escherichia coli-streptomycete shuttle vector of streptomycete responsive to temperature type replicon for any one.
5. genetic engineering bacterium according to claim 4, it is characterised in that the shuttle vector is：pKC1139、pGM160、
PHZ132 or pCZA185.
6. a kind of method for improving AVM yield, it is to utilize the genetic engineering bacterium life described in claim any one of 2-5
Produce AVM.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110396521A (en) * | 2019-07-03 | 2019-11-01 | 中国农业大学 | SAV6604 gene is improving the application in avermectin yield |
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| CN110551784A (en) * | 2019-09-18 | 2019-12-10 | 宁夏泰益欣生物科技有限公司 | Fermentation method for increasing content of abamectin B1a |
Citations (1)
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|---|---|---|---|---|
| CN101613712A (en) * | 2009-07-30 | 2009-12-30 | 中国农业大学 | Improve the method for Avrmectin and/or ivermectin output and produce bacterial strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101613712A (en) * | 2009-07-30 | 2009-12-30 | 中国农业大学 | Improve the method for Avrmectin and/or ivermectin output and produce bacterial strain |
Non-Patent Citations (2)
| Title |
|---|
| cloning and expression of the redox-sensing transcriptional repressor rex and in vitro dna-binding assay of the rex and rex operator in streptomyces rimosus m4018;jingshen et al.;《acta microbiologica sinica》;20120131;38-43 * |
| 阿维链霉菌调节基因aver1,aver2缺失对阿维菌素产量的影响;王晓芳等;《中国生物农药研讨会》;20041231;摘要 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110396521A (en) * | 2019-07-03 | 2019-11-01 | 中国农业大学 | SAV6604 gene is improving the application in avermectin yield |
| CN110396521B (en) * | 2019-07-03 | 2022-05-17 | 中国农业大学 | Application of SAV6604 gene in increasing yield of abamectin |
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