CN104928313A - Application of rex gene of streptomyces avermitilis to improvement of avermectins yield - Google Patents

Abstract

The invention provides a method for improving the yield of avermectins and strains producing avermectins. The method is as follows: the rex gene of redox-sensing transcriptional repressor in the encoding cell of streptomyces avermitilis is introduced into streptomyces avermitilis through a gene deletion vector, so that recombinant bacteria without the rex gene is obtained, Rex protein is no longer synthesized, the yield of avermectins is improved, and streptomyces avermitilis is more tolerable of oxygen limiting conditions. The genetically engineered bacterium can be directly used for fermentation production of avermectins, can improve the fermentation unit of avermectins and reduce the production cost.

Description

Avid kyowamycin rex gene is improving the application in Avrmectin output

Technical field

The invention belongs to genetically engineered field, specifically, relate to a kind of gene engineering method improving Avrmectin output.

Background technology

Avrmectin (avermectins) is the 16-membered ring macrolides microbiotic of the one group of efficient insecticide produced by Avid kyowamycin (Streptomyces avermitilis) fermentation, its natural product has eight components (A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b), wherein the insecticidal activity of B1 component is the strongest, almost anti-all nematodes relevant with agricultural and arthropods, be widely used in agriculture production.Ivermectin (ivermectin) is that raw material forms at C22-C23 position hydrogenating reduction with AVERMECTIN B1, with AVERMECTIN B1, there is identical insecticidal activity, but the low 2-3 of toxicity ratio Avrmectin doubly, being more suitable for ectozoic control in animal body, is the effective novel parasiticide microbiotic of a class wide spectrum.Avrmectin and ivermectin have unique mechanism of action, insect is not easily made to produce resistance, and easily degraded, noresidue, to crop, livestock, the mankind and environment high safety, be recommended as public nuisance-free agricultural chemicals by the Ministry of Agriculture of China, there is boundless market outlook and using value.Avrmectin and ivermectin achieve industrialization at home, achieve huge economic and social benefit.Improve the output of Avrmectin, reduce production cost, have important practical significance.Therefore, by genetic engineering means transformation Avid kyowamycin to improve the output of Avrmectin, be of great significance and value.

Avid kyowamycin is a kind of aerobic heterotroph actinomycetes, and dissolved oxygen is the important parameter in streptomycete antibiotic fermenting process, is often related to the success or failure of antibiotic fermentation.Therefore, the somatic cells success or failure of change to its existence and antibiotic fermentation that respond redox signal are fast most important.In the fermenting process of Avrmectin, find that Avid kyowamycin is very responsive to dissolved oxygen, the of short duration oxygen supply of fermentation mid-early stage is interrupted often causing Avrmectin output greatly reduce or almost can not synthesize, and this phenomenon is especially obvious in Avrmectin superior strain.Rex can not oxygen signal in direct feeling environment, but NADH/NAD in response cell ⁺ratio vary.Intracellular NAD (H) has very high turnover rate, but when cell is suppressed at limited oxygen condition or aerobic repiration, NADH/NAD+ ratio can improve rapidly.When cell is suppressed at limited oxygen condition or aerobic repiration, NADH/NAD ⁺ratio can improve rapidly, in body, high-caliber NADH can remove the combination of Rex and target gene cydABCD (the cytochrome bd terminal oxydase of oxygen high-affinity of encoding) and Rex-hemACD, activates the expression of these operons to tackle the scarcity of oxygen.

Summary of the invention

The object of this invention is to provide a kind of method improving Avrmectin output.

Another object of the present invention is to provide the genetic engineering bacterium of high yield Avrmectin and Hypoxic tolerance.

In order to realize the object of the invention, first the present invention provides the application of rex gene in the Avrmectin output improving bacterial classification.

Aforesaid application, namely by lacking rex gene in bacterial classification, to improve Avrmectin output, this engineering strain tolerates more for limited oxygen condition.

Described bacterial classification can be any streptomycete that can produce Avrmectin, such as Avid kyowamycin wild mushroom, or through routine mutagenesis and genetic engineering modified after can produce the bacterial classification of Avrmectin.

As preferably, described bacterial classification is Avid kyowamycin (Streptomyces avermitilis).

Based on this, the invention provides a kind of genetic engineering bacterium lacking the product Avrmectin of rex gene.As previously mentioned, its bacterium that sets out can be any streptomycete that can produce Avrmectin, such as Avid kyowamycin wild mushroom, or can produce the bacterial classification of Avrmectin after genetic engineering modified.

As preferably, its bacterium that sets out is Avid kyowamycin (Streptomyces avermitilis).

Further, the preparation method of described genetic engineering bacterium is: the deleted carrier building rex gene, and this deleted carrier is introduced the recombinant bacterium obtaining rex disappearance in the bacterial classification producing Avrmectin.

Specifically build genetic engineering bacterium by following method: by the upstream and downstream sequence of rex gene in pcr amplification Avid kyowamycin, build the deleted carrier of rex gene, and the recombinant vectors built is transformed product Avrmectin bacterial strain, screening obtains positive recombinant bacterium.Bacterial strain containing deleted carrier is first cultivated under 39 DEG C of high temperature inducing plasmid to lose and obtain single cross and change bacterial strain, then by its 28 DEG C, without the substratum of apramycin under go down to posterity and carry out cultivation acquisition rex gene deletion mutants.In embodiments of the present invention, the genetic engineering bacterium of the Avid kyowamycin wild mushroom of disappearance rex is obtained.

Aforesaid method, the carrier that sets out wherein building rex genetically deficient carrier is that any one contains the intestinal bacteria-streptomycete shuttle vectors of streptomycete responsive to temperature type replicon.Preferred shuttle vectors is pKC1139, pGM160, pHZ132 or pCZA185 etc.

In embodiments of the present invention, with pKC1139 for the vector construction rex genetically deficient carrier pKCD-rex that sets out.

In aforesaid method, described by the bacterial classification of rex genetically deficient carrier introducing product Avrmectin, method conventional in bioengineering field can be adopted, the protoplast transformation, electrotransformation, conjugal transfer method etc. of such as PEG mediation, the protoplast transformation of preferred PEG mediation.

In aforesaid method, be improve transformation efficiency, can first by rex genetically deficient vector in the intestinal bacteria ET12567 of restriction modification effect defect, therefrom extract plasmid be transformed into again produce Avrmectin bacterial classification in.

The invention provides a kind of method improving Avrmectin output, it utilizes the genetic engineering bacterium of the product Avrmectin of disappearance rex gene to improve the output of Avrmectin.

Beneficial effect of the present invention is:

The rex gene of encoding transcription supressor in Avid kyowamycin is introduced in Avid kyowamycin by deleted carrier by the present invention, obtains the recombinant bacterium of rex genetically deficient, makes its not resynthesis Rex albumen, thus improve the output of Avrmectin.Genetic engineering bacterium of the present invention can be directly used in the fermentative production of Avrmectin, improves the fermentation unit of Avrmectin, reduces production cost.

Accompanying drawing explanation

Fig. 1 is the plasmid map of embodiment of the present invention rex genetically deficient carrier pKCD-rex.

Fig. 2 is embodiment of the present invention rex deleted carrier pKCD-rex and the chromosomal homologous double-crossover schematic diagram of Avid kyowamycin wild type strain ATCC 31267.

Fig. 3 is the abamectin fermented unit of embodiment of the present invention Avid kyowamycin wild type strain ATCC 31267 and rex deletion mutantion strain thereof.

Fig. 4 is under different fermentations condition, the abamectin fermented unit of Avid kyowamycin wild type strain ATCC 31267 and rex deletion mutantion strain thereof.In figure: during * representative fermentation, shaking speed is 230rpm (normal shaking speed is 250rpm); * representative fermentation is resumed operation after shaking table when the 5th day has a power failure two hours again.Remarks: the per-cent of digitized representation respective handling group Avrmectin production declining compared with starting strain on histogram.

Embodiment

Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.

The structure of embodiment 1 rex genetically deficient carrier

One, the clone of Avid kyowamycin rex gene

According to the sequence of rex gene [NC_003155.4 (5777053..5777811)] and the sequences Design PCR primer of upstream and downstream thereof in the Avid kyowamycin wild type strain ATCC 31267 that Genebank announces:

Primer rex_1:5 '-CCC aAGCTTcATCACCCAGGACGCGGAG-3 '

Primer rex_2:5 '-CGC gGATCCcTCGGAGGTACAGCGGAAGC-3 '

Primer rex_3:5 '-CGC gGATCCtCACCTCCATCCTGAACTTCG-3 '

Primer rex_4:5 '-CCG gAATTCcCCTCGCCCACGACCATC-3 '

Primer rex_1, rex_2, rex_3 and rex_4 introduce HindIII in two ends respectively, BamHI, BamHI and EcoRI restriction enzyme site (underscore part).With Avid kyowamycin ATCC 31267 genomic dna for template, carry out pcr amplification with rex_1 and rex_2, rex_3 and rex_4 respectively.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 DEG C, 1min.Electrophoresis detection PCR primer, has specific amplified band at 560bp (rex_1 and rex_2) and 573bp (rex_3 and rex_4) place respectively, respectively called after upper arm and underarm.

Two, the structure of rex deleted carrier

Electrophoresis reclaims above-mentioned fragment, after PCR primer Purification Kit, upper arm and underarm are respectively through HindIII, BamHI and BamHI, EcoRI enzyme cuts carrier pKC1139 (Bierman M, the Logan R be connected to through HindIII and EcoRI double digestion, O ' Brien K, Deng. for from intestinal bacteria to the plasmid cloning vector of streptomycete DNA conjugal transfer. gene, 1992,116:43 – 49) plasmid pKCD-rex.Order-checking shows that increased fragment is the upstream and downstream sequence of rex gene really, and consistent with announced sequence.The plasmid map of pKCD-rex as shown in Figure 1.

The conversion of embodiment 2 recombinant plasmid

Constructed the deletion plasmid carrier pKCD-rex of rex gene by embodiment 1, original plasmid is in contrast pKC1139.

Owing to there is very strong restriction modification effect in Avid kyowamycin, directly transform Avid kyowamycin with extraction from the plasmid of E.coliJM109, transformation efficiency is extremely low, sometimes even can not get transformant.And with from the plasmid of recipient bacterium E.coli ET12567 not having restriction modification effect, its transformation efficiency significantly improves.Therefore, by the recombinant plasmid transformed that builds to E.coliET12567 (Kieser T, Bibb M J, Buttner M J, Deng. practical streptomycete heredity handbook, 2000, Norwich: John Ying Nasi foundation) in obtain non-methylated DNA, and then with the protoplastis of non-methylated plasmid DNA transformation Avid kyowamycin.

The present embodiment selects Avid kyowamycin strains A TCC 31267 as starting strain.ATCC 31267 is Avid kyowamycin wild type strains, produces grey spore.Prepare the protoplastis of this Avid kyowamycin bacterial strain, with the Plastid transformation protoplastis extracted from E.coli ET12567, be applied on the RM14 flat board of the not added with antibiotic dried up, after 28 DEG C of cultivation 16-20h, flat board is coated with the aqueous solution of 1mL containing 1000 μ g apramycins, continue to cultivate 7-10 days at 28 DEG C, the bacterium colony grown is transformant.Because the transformant of Avid kyowamycin does not produce spore on RM14 regeneration culture medium, therefore each picking three transformants, be inoculated on the YMS flat board containing 10-15 μ g/mL apramycin, cultivate for 28 DEG C and within 7-10 days, recover to produce spore.Transformant is verified correct through plasmid extraction and PCR.

The preparation of the preparation of colibacillary conversion in the present embodiment, Avid kyowamycin protoplastis and method for transformation, RM14 and YMS substratum see " producing the research of green spore Avid kyowamycin genetic modification and fermentation condition " (Cai Yujuan. master thesis, 2006, Beijing: China Agricultural University).

The acquisition of embodiment 3 rex gene deletion mutants

Collect the spore containing pKCD-rex plasmid and control plasmid pKC1139 transformant that above empirical tests is correct respectively, coat on the YMS flat board containing apramycin with every culture dish about 100 spores, be placed in 28 DEG C and cultivate 48-72h, when colony diameter is about about 2mm, when being about to form aerial hyphae, flat board is transferred to 39 DEG C of high-temperature cultivation after 7 days, occurs fan-shaped growth containing on some bacterium colonies of pKCD-rex, and not regrowth after moving to 39 DEG C containing the bacterium colony of pKC1139.This is because pKC1139 is the intestinal bacteria-streptomycete shuttle plasmid of temperature sensitive type, when temperature is higher than can not self-replacation when 34 DEG C, therefore cannot grow on the YMS substratum containing apramycin; Only have when homology segment generation homologous recombination in the rex gene upstream and downstream sequence entrained by recombinant plasmid pKCD-rex and Avid kyowamycin karyomit(e), and change by single cross the resistant gene that the bacterial strain be incorporated on karyomit(e) could express apramycin, thus grow on the YMS substratum containing apramycin.The single cross of the anti-apramycin grown 39 DEG C is changed mutant strain not adding on antibiotic YMS flat board and is gone down to posterity, after corotation connects 2-4 time, and the double exchange recombinant chou of screening apramycin sensitivity.Picking 10 strain bacterium wherein, together with Avid kyowamycin ATCC31267 in YEME 28 DEG C of shaking culture 48h, all fail therefrom to extract plasmid, illustrate that recombinant plasmid pKCD-rex is eliminated.Utilize PCR to Apr ^smutant strain is verified.Obtain the rex deletion mutantion strain of many strains ATCC 31267 respectively.The chromosomal homologous double-crossover schematic diagram of rex deleted carrier pKCD-rex and Avid kyowamycin ATCC 31267 as shown in Figure 2.

Embodiment 4 Avid kyowamycin wild type strain ATCC 31267 and rex deletion mutantion strain fermentative production Avrmectin

One, the shake flask fermentation of Avid kyowamycin

Seed culture medium: Zulkovsky starch 30g, malt extract 2g, soy peptone 2g, CoCl ₂6H ₂o 5mg, adding distil water, to 1L, adjusts pH to 7.0-7.2.

Fermention medium: Zulkovsky starch 50g, yeast powder 12g, MgSO ₄7H ₂o 0.5g, K ₂hPO ₄3H ₂o 0.5g, KCl 4g, CaCO ₃2g, CoCl ₂6H ₂o 5mg, adding distil water, to 1L, adjusts pH to 7.0-7.2.

Two, the HPLC of tunning analyzes

1. sample preparation: get 1.0mL fermented liquid, adds 4.0mL methyl alcohol, soaks more than 30 minutes, and every vibration in 10 minutes once, centrifugal 10 minutes of 4000rpm, gets the analysis of supernatant liquor sample introduction;

2.HPLC analysis condition: C ₁₈reversed-phase column, column length 150mm, column internal diameter 4.6mm, column temperature 40 DEG C, moving phase is methyl alcohol: water (85:15), flow velocity 1.0mL/min, and sampling volume 20 μ L, wavelength is 246nm.

Three, the fermentation results of ATCC 31267 and different rex deletion mutantion strain thereof

Be inoculated in seed culture medium (loading amount is 100mL/500mL triangular flask) after Avid kyowamycin wild type strain ATCC 31267 and rex deletion mutantion strain thereof grow abundant spore on YMS substratum, 28 DEG C of shaking tables are cultivated 24 hours (rotating speed 230rpm, eccentricity 2.5cm).Be inoculated in fermention medium (loading amount is 50mL/250mL triangular flask) by 5% inoculum size, cultivate 10 days (rotating speed 250rpm, eccentricity 2.5cm), put bottle for 28 DEG C, measure the fermentation unit of Avrmectin by methanol extraction-HPLC method, result as shown in Figure 3.

The fermentation results display of Fig. 3, the deletion mutantion strain of rex gene is compared with starting strain, and abamectin fermented unit all obviously increases compared with starting strain, and the AVERMECTIN B1 component output of deletion mutantion strain is about 3 times of starting strain.Rex genetically deficient has remarkable promoter action to Avrmectin synthesis.

Four, ATCC 31267 and the fermentation results of rex deletion mutantion strain under different fermentations condition thereof

The fermentation results display of Fig. 4, under the speed conditions of 230rpm, the deletion mutantion strain of rex gene is compared with starting strain, and the reduction amplitude of abamectin fermented unit significantly diminishes, and the range of decrease only has about 1/5 of starting strain; Under the condition of fermentation to shaking table power failure process 2h when the 5th day, the deletion mutantion strain of rex gene is compared with starting strain, and the reduction amplitude of abamectin fermented unit diminishes equally.Rex genetically deficient makes Avid kyowamycin more tolerate for limited oxygen condition.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (9)

1. The application of 1.rex gene in the Avrmectin output improving bacterial classification.
2. 2. application according to claim 1, is characterized in that, by lacking rex gene in bacterial classification, to improve Avrmectin output.
3. 3. application according to claim 1 and 2, is characterized in that, described bacterial classification is Avid kyowamycin (Streptomyces avermitilis).
4. 4. one kind lacks the genetic engineering bacterium of the product Avrmectin of rex gene.
5. 5. genetic engineering bacterium according to claim 4, is characterized in that, its bacterium that sets out is Avid kyowamycin (Streptomyces avermitilis).
6. 6. genetic engineering bacterium according to claim 4 or 5, is characterized in that, the preparation method of described genetic engineering bacterium is: the deleted carrier building rex gene, and this deleted carrier is introduced the disappearance recombinant bacterium obtaining rex in the bacterial classification producing Avrmectin.
7. 7. genetic engineering bacterium according to claim 6, is characterized in that, the carrier that sets out building rex genetically deficient carrier is that any one contains the intestinal bacteria-streptomycete shuttle vectors of streptomycete responsive to temperature type replicon.
8. 8. genetic engineering bacterium according to claim 7, is characterized in that, described shuttle vectors is: pKC1139, pGM160, pHZ132 or pCZA185.
9. 9. improve a method for Avrmectin output, it utilizes the genetic engineering bacterium described in any one of claim 4-8 to produce Avrmectin.