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Recombined streptomyces nodosus capable of producing amphotericin B and application thereof

A technology of amphotericin and streptomyces, applied in the direction of enzymes, bacteria, bacterial peptides, etc., can solve the problems of miscellaneous bacteria pollution, no antibiotic resistance, etc., achieve low bacterial contamination rate, strong bactericidal effect, reduce human factors and environment The effect of factor influence rate

Active Publication Date: 2018-04-10
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Most of the existing AmB-producing strains are wild-type strains selected for high-yield strains, and none of them are resistant to antibiotics. Therefore, there will be bacterial contamination in the industrial fermentation process

Method used

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  • Recombined streptomyces nodosus capable of producing amphotericin B and application thereof
  • Recombined streptomyces nodosus capable of producing amphotericin B and application thereof
  • Recombined streptomyces nodosus capable of producing amphotericin B and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, the screening of high-yielding AmB mutant strain

[0045] (1) Streptomyces nodosus (Streptomyces nodosus, Caffrey P, Lynch S, Flood E, Finnan S, Oliynyk M. Amphotericin biosynthesis in Streptomyces nodosus: reductions from analysis of polyketide synthase and late genes. Chem Biol 2001; 8:713-723 .or Carmody M, Byrne B, Murphy B, Breen C, Lynch S et al.Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515transductiontechniques.Gene 2004; 343(1):107-115) inoculated onto GYM plate or slant Culture medium, cultivated at 28°C for 7 days, take gray-black spores, use cotton swab to elute the surface spores into 10mL sterile water, filter the washed spore suspension with a syringe containing cotton, and filter the spores at 12000rpm After centrifuging for 5 minutes, remove the supernatant, add 10 mL of sterile water to resuspend, centrifuge at 12000 rpm for 5 minutes to elute again, and resuspend with 5 mL of sterile water a...

Embodiment 2

[0058] Embodiment 2, the construction of AmB genetically engineered bacteria resistant to kanamycin

[0059] 1. Construction of recombinant vector

[0060] 1. Construction of recombinant vector pJTU1278-Kan

[0061] Using the pEC-XK99E plasmid as a template (the plasmid was purchased from BioVector NTCC Plasmid Vector Strain Cell Gene Collection Center, GenBank accession No. AY219682.1, Kirchner O, Tauch A. Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum. J Biotechnol 2003; 104(1-3):287-299.), design primers 99E-Kan-F and 99E-Kan-R, 99E-Kan-F is the forward primer for Kan resistance-related genes, 99E-kan- R is the reverse primer for Kan resistance-related genes, and the related genes containing kanamycin resistance genes are cloned and amplified from the template, including the kanamycin resistance gene (i.e. aph(3')-IIa gene), Promoter and RBS, the fragment size is about 1100bp, which is consistent with the target fragment. Af...

Embodiment 3

[0100] Embodiment 3, Streptomyces tuberculosis genetically engineered bacteria construction carrying vhb gene

[0101] Using the Vitiligo hyaline hemoglobin (vhb) gene as a template (GenBank accession No. JN418989.1), design primers vhbF and vhbR, vhbF is the forward primer for the vhb gene, vhbR is the reverse primer for the vhb, cloned from the template The vhb was amplified, and the fragment size was about 450bp. It was confirmed by sequencing analysis that it was consistent with the target fragment. The nucleotide sequence is shown in SEQID NO.7. After cutting this fragment with endonuclease BamHI and HindIII, clean-up this fragment for later use. The vector pJTU1278-Kan constructed in Example 2 was also digested with the same BamHI and HindIII endonuclease to recover the gel. The recovered gene fragment was ligated with the pJTU1278-Kan vector after digestion. The obtained recombinant plasmid vector was named pJTU1278-Kan-vhb, and the schematic diagram is shown in Figu...

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Abstract

The invention discloses recombined streptomyces nodosus capable of producing amphotericin B and application thereof. The recombined streptomyces nodosus is obtained by introducing a kanamycin resistance gene sequence showed in SEQ ID NO.1 into streptomyces nodosus ZJB2016050; by introducing the kanamycin resistance gene, produced strains which have no resistance previously have kanamycin resistance, the purity and quality of the strains of seed culture mediums are improved, and strain contamination phenomena can be prevented from occurring during seed cultivation in a lab. By expressing a function gene, namely vitreoscilla hemoglobin (vhb), the yield of AmB is increased by 15%; by expressing a function gene, namely the S- adenosylmethionine synthetase gene (metk), the yield of AmB is increased by about 40%.

Description

(1) Technical field [0001] The invention relates to a production method of amphotericin B, in particular to a recombinant Streptomyces nodosum producing high amphotericin B and application thereof. (2) Background technology [0002] 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. It was collected and isolated and found to have antifungal activity. In 1959, AmB was extracted from the fermentation broth of Streptomyces tuberculosis. Launched in 1966, it is the first drug for deep fungal infection. It has been used for nearly half a century and is still an irreplaceable antifungal drug in clinical practice. AmB belongs to the class of polythrein macrolide antibiotics, which has broad-spectrum resistance to fungi, especially for life-threatening systemic fungal infections such as Candida albicans, Aspergillus fungi, e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/62C12R1/465
CPCC07K14/195C12N1/20C12N9/1085C12P19/62C12Y205/01006
Inventor 郑裕国柳志强张博黄恺周奕腾张海东
Owner ZHEJIANG UNIV OF TECH
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