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Biosynthesis gene cluster of Nocathiacins and application thereof

A technology for nocardiazol and biosynthesis, which is applied in the fields of microbial gene resources and genetic engineering, and can solve the problems of complex chemical structure, high production cost, numerous transformation steps, etc.

Active Publication Date: 2011-02-02
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are often difficult to improve the water solubility while maintaining the good antibacterial activity of the original structure.
And the chemical structure of this class of antibiotics is extremely complicated, and people have only completed the synthetic [Tetrahedron Lett.1984,25,2127 of partial module and acidic hydrolyzate; J.Org.Chem.1996,61,4623; J .Org.Lett.2003, 5, 4421; Tetrahedron Lett.1991, 32, 4263; Angewandte Chemie.2005, 117, 3802-3806; Chem.Commun.2008, 591-593]
It was not until recent years that organic synthesis masters Moody and Nicolaou completed the total synthesis of thiazocin A, amythiamycin D, thiostrepton, siomycin A, GE2270A / T, and the synthesis of nocardiazolin Total synthesis has not been reported [Angew.Chem.Int.Ed.2007, 46, 7930-7954]
Moreover, due to the complex polycyclic structure and numerous chiral centers of this class of antibiotics, there are many steps for transformation by simply utilizing the total synthesis method, and the actual production cost is too high

Method used

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  • Biosynthesis gene cluster of Nocathiacins and application thereof
  • Biosynthesis gene cluster of Nocathiacins and application thereof
  • Biosynthesis gene cluster of Nocathiacins and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Extraction of total DNA from Nocardia sp.WW-12651, a nocardiazolin-producing bacterium:

[0145] Inoculate 100 μL of Nocardia sp.WW-12651 (ATCC 202029) mycelia suspension into 3 mL of TSB liquid medium, culture at 30° C., 250 rpm, and shake for about 36 hrs to reach the late logarithmic growth phase. Take 3mL and inoculate into 50mL TSB (containing 5mM MgCl 2 , 0.5% glycine), 30 DEG C, 250rpm, shake culture for about 25hrs and reach the early stage of the stable growth period, it is milky white and turbid, and there are a large number of suspended mycelia. Centrifuge the bacterial solution at 4°C, 3500 rpm for 15 minutes to collect mycelia, wash twice with lysis buffer to obtain about 2.5 mL of mycelia. Add 10 mL of lysis buffer (containing 5 mg / mL of lysozyme) to 2.5 mL of mycelium, vortex until uniform, then add Achromopeptidase (Achromopeptidase) to 3 mg / ml, and mix well. 37°C water bath for 30min. Add 0.1mL proteinase K (20mg / mL, freshly prepared with lysis buffe...

Embodiment 2

[0147] Construction of Genome Library of Nocardia sp.WW-12651, a Nocardiazolin-producing Bacteria:

[0148] First, through a series of dilution experiments to determine the amount of Sau3AI, on this basis, a large number of enzyme-digested DNA fragments slightly larger than 40kb, dephosphorylated. The vector pOJ446 (Gene 1992, 116: 43-49; US 7,109,019) was first cut and dephosphorylated from the middle of the two cos sequences with HpaI, and then cut with BamHI from the multiple cloning site to obtain two tethered arms, and The prepared DNA fragments with a length of about 40kb were ligated overnight. Take out the packaging kit (Promega PackageneExtract) from -80°C and put it on ice. After it just melts, add 5uL of the above connection solution immediately, flick and mix well, be careful not to generate air bubbles, and place at room temperature (about 22°C) for 3 hours. Add 445uL Phage buffer and mix by inverting up and down. Then add 25uL of chloroform, mix well by inverti...

Embodiment 3

[0151] Fermentation, product isolation, purification and identification of Nocardia sp.WW-12651, a nocardiazolin-producing bacterium:

[0152] First, inoculate 300 μL of Nocardia sp.WW-12651 mycelial suspension frozen at -80°C into 25 mL of seed medium (soluble starch 2%, glucose 0.5%, N-Z Case 0.3%, yeast extract 0.2%, fish meat Extract 0.5%, calcium carbonate 0.3%), 32 ℃, 250rpm cultivated for 3 days. Get 2mL therefrom and transfer to 50mL fermentation medium (glucose 2%, HY-yeast 4121%, nutritional soybean 1%), 30 ℃, 250rpm culture 4-5 days. Then the fermentation broth was combined, transferred to a centrifuge tube, centrifuged at 3800rpm for 15min, and the mycelia precipitate was discarded. The supernatant was extracted twice with an equal volume of ethyl acetate, and the organic phases were combined. with anhydrous MgSO 4 or anhydrous Na 2 SO 4 Dry, filter, and concentrate to dryness under reduced pressure at 37°C. The sample was dissolved in a mixed solvent of chlo...

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Abstract

The invention relates to a biosynthesis gene cluster of Nocathiacins and an application thereof, in particular to providing the cloning, the sequencing, the analysis, the functional research of an antibiotic-Nocathiacins biosynthesis gene cluster with good antibiosis activity generated by Nocardia and an application thereof. The integral gene cluster contains 37 genes, wherein the 37genes comprise 8 genes relevant to the biosynthesis of big ring skeletons, 4 genes relevant to the side chain synthesis of indole acid, 6 genes relevant to glycosyl synthesis, 5 modified enzyme genes after the redox of P450, 2 methyl transferase genes, 2 resistance genes, 3 regulator genes, 3 genes with the unknown functions and 4 genes relevant to transcription and translation, and a series of new sulfur peptide antibiotics can be generated by utilizing the heterologous expression of the biosynthesis genes. The genes provided by the invention and proteins thereof can search and discover compounds or the genes and the proteins for medicine, industry and agriculture.

Description

technical field [0001] The invention belongs to the field of microbial gene resources and genetic engineering, and in particular relates to the cloning, sequence analysis, gene function research and application of the biosynthetic gene cluster of the thiopeptide antibiotic Nocathiacins. technical background [0002] Nocathiacins are a class of cyclic peptide antibiotics rich in elemental sulfur and highly modified amino acid residues. As important members of the thiopeptide family, they were initially screened for new penicillin-resistant Staphylococcus aureus (Methicillin-Resistant Staphylococcus aureus, MRSA) and multidrug-resistant Enterococcus faecium (Multi-Drug Resistant Enterococcus faecium, MREF) were found in soil samples during the course of growth-inhibiting antibiotics [J. Antibiot. 1998, 51, 715]. In 1998, Tokushima Research Center took the lead in isolating and purifying MJ347-81F4-A (Nocathiacin Ⅰ) and B from Amycolatopsis sp.MJ347-81F4, and completed their f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/52C12N15/63C12N1/19C12P17/10C12N1/21C12P21/02C12N1/15C12N9/00C12N15/53C12N5/10C12P17/00C12N15/54C12P19/02C12P17/12C12P21/04
CPCC12P19/60C12N15/52
Inventor 刘文丁莹虞沂潘海学
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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