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Recombinant streptomyces nodosus with high yield of amphotericin B and applications of recombinant streptomyces nodosus

An amphotericin and Streptomyces technology, applied to recombinant Streptomyces tuberculosis with high production of amphotericin B and its application field, can solve problems such as loss of activity, and achieve the advantages of increasing production capacity and output value, reducing the content of by-products, and shortening fermentation time. Effect

Active Publication Date: 2018-08-24
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

AmB is yellow or orange powder, hygroscopic, almost odorless and tasteless, its structure should be destroyed under light to cause loss of activity

Method used

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  • Recombinant streptomyces nodosus with high yield of amphotericin B and applications of recombinant streptomyces nodosus
  • Recombinant streptomyces nodosus with high yield of amphotericin B and applications of recombinant streptomyces nodosus
  • Recombinant streptomyces nodosus with high yield of amphotericin B and applications of recombinant streptomyces nodosus

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1 Carrier pJTU1278 conjugative transfer transformation recipient strain Streptomyces tuberculosis

[0044] A) Preparation of E.coil ET12567 / puz8002 donor bacteria containing vector:

[0045] The vector pJTU1278 was introduced into E.coil ET12567 / puz8002 competent cells, and ampicillin (Amp + , 100μg / mL), chloramphenicol (Cm + , 25μg / mL), kanamycin (Kan + , 50 μg / mL) resistance screening, positive transformants were picked, verified by M13 upstream and downstream primer colony PCR, and the sequencing results proved that the vector pJTU1278 was transformed into E.coilET12567 / puz8002. The specific operation is as follows:

[0046] E.coil ET12567 / puz8002 competent preparation method is as follows:

[0047] Take the E.coil ET12567 / puz8002 Escherichia coli liquid from the glycerol cryopreservation tube of the strain, draw the line on the LB plate, and cultivate it at 37°C until a single colony grows. Pick a single colony on the plate and transfer it to 2-5 mL of ...

Embodiment 2

[0067] Embodiment 2 Streptomyces tuberculosis genetically engineered bacteria construction carrying ermE*p-AmphRIV gene

[0068] A) Using the Streptomyces tuberculosis CCTCC NO.M 2017426 genome as a template, design primers amphRIV-F and amphRIV-R for the amphotericin gene cluster regulator gene (AmphRIV, GenBank accession No.CP009313.1SNOD_02710), and amphRIV-F is The forward primer for the AmphRIV gene, amphRIV-R is the reverse primer for AmphRIV, and the AmphRIV fragment is cloned and amplified from the template with a size of about 777bp. It is confirmed by sequencing analysis that it matches the target fragment. The nucleotide sequence is as shown in SEQ ID NO .3, gel recovery and purification of this fragment for future use.

[0069] B) Using the strong erythromycin promoter (ermE*p) as a template (GenBank accession No.HM756283.1), design primers ermE*p-F and ermE*p-R, ermE*p-F is the forward primer for the ermE*p gene, ermE*p-R is the reverse primer for ermE*p, ermE*p ...

Embodiment 3

[0077] Embodiment 3 Streptomyces tuberculosis genetically engineered bacteria construction carrying ermE*p-AraC gene

[0078] A) Using the Streptomyces tuberculosis CCTCC NO.M 2017426 genome as a template, design primers araC-F and araC-R for the global regulator gene (AraC, GenBank accession No.CP009313.1SNOD_12635), araC-F is for the AraC gene The forward primer, araC-R is the reverse primer for AraC. The araC fragment was cloned and amplified from the template with a size of 1227bp. It was confirmed by sequencing analysis that it was consistent with the target fragment. The nucleotide sequence is shown in SEQ ID NO.4. Gel recovery and purification of this fragment for future use.

[0079] B) Using the recombinant plasmid vector pJTU1278-ermE*p-AmphRIV as a template, design primers pE-F and pE-R for the strong promoter and pJTU1278 vector, clone and amplify the pJTU1278-ermE*p fragment with a size of 9598bp from the template Left and right, gel recovery and purification of ...

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Abstract

The invention discloses recombinant streptomyces nodosus capable of producing amphotericin and applications of the recombinant streptomyces nodosus. The recombinant streptomyces nodosus is obtained byimporting vitreoscilla hemoglobin (Vhb), S-adenosylmethionine synthetase (Metk), amphotericin synthetic gene cluster regulating factor (AmphRIV), secondary metabolite global regulation factor (AraC)and erythrocin strong promoter ermE*p sequences as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 into streptomyces nodosus ZJB2016050. Through importing and overexpression of the gene, in recombinant streptomyces nodosus, the yield of amphotericin B is improved by about 45%, the yield of the by-product amphotericin A is reduced by 60%, meanwhile, the growth cycle of the thallus during the fermentation process is shortened, and thus the purpose of improving the production efficiency is achieved.

Description

technical field [0001] The invention relates to a method for increasing amphotericin B, reducing amphotericin A and shortening the fermentation period of strains by means of genetic engineering, in particular to a recombinant streptomyces nodosum with high amphotericin B production and application thereof. Background technique [0002] Amphotericin B (Amphotericin B, AmB) belongs to the polyene macrolide antibiotics and has antifungal activity. In the middle of the last century, the producing bacterium, Streptomyces nodosus, was isolated and identified from soil samples in the Orinoco Delta. Recent studies have found that the fungus Penicillium nalgiovense can also produce AmB, but the yield is extremely low and has not been actually used for production. As a heptacene macrolide antibiotic, AmB has the following characteristics in its structure: it has 7 conjugated polyene macrolides, a hydroxyl group at C8, a carboxyl group at C16 and a fucosamine group at C19. AmB is mai...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/62C12R1/465
CPCC07K14/36C07K14/805C12N9/1085C12P19/62C12Y205/01006
Inventor 柳志强张博郑裕国黄恺牛坤周奕腾
Owner ZHEJIANG UNIV OF TECH
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