3,7-Dihydroxytropolone Biosynthetic Gene Cluster and Its Application

A technology for hydroxytropolone and biosynthesis, which is applied in the field of microbial gene resources and genetic engineering, can solve the problems such as the biosynthesis pathway is not well revealed, the research progress is limited, and the yield is limited.

Active Publication Date: 2020-12-29
SHANGHAI JIAOTONG UNIV
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, 3,7-dihydroxytropolone has made very limited research progress in the past 30 years after its discovery due to the limitation of production.
[0004] Compared with the well-studied biosynthetic pathways of tropolones in fungi and plants, the biosynthetic pathways of tropolones in bacteria have not been well revealed
Especially the hydroxylation modification on the seven-membered aromatic ring of 3,7-dihydroxytropolone has not been reported so far, but the hydroxylation modification on the 3,7-dihydroxytropolone is that it chelates metal ions to play antibacterial, Necessary for anticancer and antiviral effects

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 3,7-Dihydroxytropolone Biosynthetic Gene Cluster and Its Application
  • 3,7-Dihydroxytropolone Biosynthetic Gene Cluster and Its Application
  • 3,7-Dihydroxytropolone Biosynthetic Gene Cluster and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Embodiment 1, construction of Streptomyces sp.B222 cosmid library

[0102] (1) Inoculate fresh spores of Streptomyces sp.B222 into 30ml of TSBY medium (with 0.6% glycine added), and culture at 30°C and 220rpm for 16-24h.

[0103] (2) Collect the bacteria by centrifugation, weigh 0.5g of wet bacteria into a 50ml centrifuge tube, add 5ml of SET (75mM NaCl, 25mM EDTA, 20mM Tris, pH 7.5) buffer, shake to break up. Add lysozyme at a final concentration of 1mg / ml, and incubate at 37°C for 10min. After adding 1 / 10 volume of 10% SDS solution, proteinase K with a final concentration of 0.5 mg / ml was added, and incubated at 55° C. for 2 h. Add 1 / 3 volume of 5M NaCl solution, and add one volume of neutral phenolform, invert and mix at room temperature for 30min; centrifuge at 4,000rpm for 30min, draw the supernatant into another new 50ml centrifuge tube, add an equal volume of chloroform Mix again by inversion for 30 min to remove residual protein. After centrifugation at 4,0...

Embodiment 2、3

[0107] Cloning of Example 2, 3,7-Dihydroxytropolone Biosynthetic Gene Cluster

[0108] For the screening method of heterologous expression activity of Streptomyces genomic library, please refer to the literature "Chen L, Wang Y, Guo H, et al. "And literature" Xu M, Wang Y, Zhao Z, et al. Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologoous Expression of a Whole-GenomeBacterial Artificial Chromosome Library in Streptomyces spp. Applied and Environmental Microbiology, 2016, 82(19) :5795-5805."

[0109] (1) Take out the cosmid library (12 pieces of 96-well plates) of Streptomyces ochraceiscleroticus B222 (ie, S. ochraceiscleroticus B222) from the -80°C refrigerator, and place it in a 37°C incubator for 2 hours until it melts completely.

[0110] (2) Add 130 μl of fresh LB medium to a 96-well plate, use a replicator to inoculate the cosmid library of Streptomyces sp.B222 into a 96-well plate, culture at 37°C, 220 rpm for 4-6 hours to OD 6...

Embodiment 3、3

[0116] Embodiment 3, 3, the heterologous expression of 7-dihydroxytropolone

[0117] (1) Pick the 11A11 clone from the cosmid library library of Streptomyces ochraceiscleroticus B222 and put it into 3 ml of fresh LB medium (with 0.1% abramycin added), and culture overnight at 37°C and 220rpm.

[0118] (2) The cosmid DNA extracted from 11A11 was transformed into E. coli E.coli ET12567 electroporation competent cells to obtain the E.coli ET12567 / 11A11 (E.coli ET12567 / pCXF1) strain. For the convenience of description, the subsequent 11A11cosmid plasmids are all represented by pCXF1 .

[0119] (3), using the method of Escherichia coli-Streptomyces three-parent conjugation transfer, with E.coli ET12567 / pCXF1 as the donor bacterium, and S.coelicolor M1154 as the recipient bacterium, with the assistance of E.coli ET12567 / pUB307, the The pCXF plasmid was transferred into S.coelicolor M1154 to obtain the expression strain S.coelicolor M1154 / pCXF1 CGMCC No. of 3,7-dihydroxytropolone,...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a 3,7-dihydroxytropolone biosynthetic gene cluster and an application thereof. The biosynthetic gene cluster totally contains 8 structural genes comprising trlA, trlB, trlC, trlD, trlE, trlF, trlH and trlI, 1 regulatory related gene comprising trlJ, and 1 other functional gene comprising trlG. The trlB and trlJ are key regulatory proteins involved in enhancing a chorismic acid metabolism pathway, and the trlC, trlD and trlE provide tool enzymes which can be used for sequentially oxidizing on a seven-membered aromatic ring of tropolone. The genes and the proteins provided by the invention can be used for discovering or studying new compounds, genes or proteins used in medicine, industry and agriculture. The invention also provides a series of engineered strains capable of high yielding of different kinds of tropolones, such as tropolone, 7-hydroxytropolone, cycloheptatrienone and 1,4,6-cycloheptatriene-1-carboxylic acid.

Description

technical field [0001] The invention belongs to the field of microbial gene resources and genetic engineering, and in particular relates to a biosynthetic gene cluster of antibiotic-3,7-dihydroxytropolone and its application. Background technique [0002] 3,7 dihydroxytropolofaciens is a class of natural products with a rare seven-membered aromatic ring structure, which was first discovered and reported in Streptomyces tropolofaciens No. K611-97 (ATCC 53548) in 1988. 3,7 dihydroxytropolone has a very good broad-spectrum antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, mycobacteria and fungi, and also has a very good anti-tumor effect, especially B16 melanoma Activity and antiviral activity such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes virus (HSV). 3,7 dihydroxytropolone can chelate metal ions due to its special α-hydroxy ketone structure, so it can play the role of metal ion chelating agent and inhibit the activ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N9/00C12N1/21C12P7/26C12R1/465
CPCC12N9/00C12N15/52C12P7/26
Inventor 陶美凤陈雪斐徐敏王业民邓子新
Owner SHANGHAI JIAOTONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap