Amphotericin B-producing recombinant streptomyces nodocus and application thereof

A technology of amphotericin and streptomyces, applied in the direction of bacteria, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as competition, and achieve the advantages of improving utilization rate, improving overall yield and shortening fermentation cycle. Effect

Active Publication Date: 2019-12-17
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Amphotericin B-producing recombinant streptomyces nodocus and application thereof
  • Amphotericin B-producing recombinant streptomyces nodocus and application thereof
  • Amphotericin B-producing recombinant streptomyces nodocus and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Knockout Vector Construction

[0035] 1. Insertion of homology arm 1

[0036] Using the whole genome of Streptomyces tuberculosis ATCC14899 as a template, design primers TYB1-F and TYB1-R, TYB1-F is the forward primer for homology arm 1, and TYB1-R is the reverse primer for homology arm 1 gene, The homology arm 1 gene was cloned and amplified from the template, and the fragment size was about 3000bp, which was consistent with the target fragment. After sequencing analysis, the results showed that the amplified sequence was identical to the target gene sequence. This fragment was treated with endonucleases XbaI and BamHI After digestion, clean-up this fragment for later use. The vector pJTU1278 is also recovered with the same XbaI and BamHI endonuclease digestion gel, and the recovered gene fragment is connected with the digested pJTU1278 vector to obtain the name of the recombinant plasmid vector is pJTU1278-TYB1.

[0037]The cloning PCR system: add 1 μL of...

Embodiment 2

[0056] Example 2: Resistance Genome Replacement

[0057] Recombinant vector pJTU1278-TYB1-kan-TYB2 conjugative transfer transformation recipient strain Streptomyces tuberculosis A) Preparation of E.coil ET12567 / puz8002 donor strain containing recombinant vector pJTU1278-TYB1-kan-TYB2:

[0058] The constructed recombinant vector pJTU1278-TYB1-kan-TYB2 was introduced into E.coil ET12567 / puz8002 Escherichia coli competent, and ampicillin (Amp + , 50μg / mL), chloramphenicol (Cm + , 50μg / mL), Kanamycin (Kan + , 50 μg / mL) resistance screening, positive transformants were picked, and verified by M13 upstream and downstream primer colony PCR. The results proved that the recombinant vector pJTU1278-TYB1-kan-TYB2 was successfully transformed into E.coil ET12567 / puz8002. The specific operation is as follows:

[0059] E.coil ET12567 / puz8002 Escherichia coli competent preparation method is as follows:

[0060] Take the E.coil ET12567 / puz8002 Escherichia coli liquid from the glycerol cry...

Embodiment 3

[0082] Example 3: Knockout Resistance Tag Restoration

[0083] Recombinant vector pJTU1278-TYB1-TYB2 conjugative transfer transformation recipient strain Streptomyces nodosus ZJB2016050-De5

[0084] A) Preparation of E.coil ET12567 / puz8002 donor bacteria containing recombinant vector pJTU1278-TYB1-TYB2:

[0085] The constructed recombinant vector pJTU1278-TYB1-TYB2 was introduced into E.coil ET12567 / puz8002 Escherichia coli competent, and ampicillin (Amp + , 50μg / mL), chloramphenicol (Cm + , 50μg / mL), Kanamycin (Kan + , 50 μg / mL) resistance screening, positive transformants were picked, and verified by M13 upstream and downstream primer colony PCR. The results proved that the recombinant vector pJTU1278-TYB1-TYB2 was successfully transformed into E.coil ET12567 / puz8002. The specific operation is as follows:

[0086] E.coil ET12567 / puz8002 Escherichia coli competent preparation method is as follows:

[0087] Take the E.coil ET12567 / puz8002 Escherichia coli liquid from the ...

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Abstract

The invention discloses an amphotericin B-producing recombinant streptomyces nodocus and its application. The recombinant streptomyces nodocus is obtained by knockout of competitive branches of amphotericin B in Streptomyces nodule ZJB2016050. Through the introduction of functional genes, the supply quantity of amphotericin B skeleton synthetic precursors is comprehensively increased, the utilization rate of nutrients by bacteria is increased, and the nutrient flow of the target product is changed. The yield of amphotericin B is increased by knocking out the competitive gene clusters. The yield of amphotericin B can be increased by 25%, the utilization rate of nutrient components of a culture medium during the fermentation process is increased, and the fermentation cycle is shortened and then the risk of contamination is reduced.

Description

[0001] (1) Technical field [0002] The invention relates to a recombinant streptomyces tuberculosis producing amphotericin B and application thereof. [0003] (2) Background technology [0004] Amphotericin B (Amphotericin B, AmB) is a polyene broad-spectrum antifungal antibiotic produced by Streptomyces nodosus, and its strain was obtained from soil samples in the Orinoco Delta of Venezuela in 1955. Collect and separate. Launched in 1966, it is the first drug for deep fungal infection and has been used for nearly half a century. AmB Molecular Formula C 47 h 73 NO 17 , AmB belongs to the class of polyene macrolide antibiotics, which has broad-spectrum resistance to fungi, especially for life-threatening systemic fungal infections such as Candida albicans, Aspergillus fungi, etc., and also has effective anti- Drug properties of viruses and parasites, such as prions, Leishmania, etc. Amphotericin B drugs currently in the market include injections, tablets, etc. AmB is yell...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/62C12R1/465
CPCC07K14/36C12P19/62
Inventor 柳志强郑裕国张博黄恺姜圣贤张雨函陈燏
Owner ZHEJIANG UNIV OF TECH
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