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Biosynthetic gene cluster of sanglifehrin

A technology of biosynthesis and biosynthesis of sanglifehrin, which is applied in the field of microbial genetic resources and genetic engineering, and can solve problems such as hindering application

Active Publication Date: 2010-04-07
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

In addition, because the existing immunosuppressive drugs on the market can produce some serious nephrotoxic and central toxic side effects [J Am Chem Soc2002 124 4257-70], thus hindering the application in some immune disorders (for example, calcineurin is also The immunosuppressive activity and toxicity effector of CsA and FK506), for which Novartis has developed SFA as a new generation of highly effective and low toxicity immunosuppressant

Method used

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  • Biosynthetic gene cluster of sanglifehrin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Extraction of sangliferin-producing bacteria Streptomyces flaveolus total DNA

[0088]1. Inoculate the spore suspension of S.flaveolus A92-309110 refrigerated at -80°C into 3mL YEME medium, incubate at 30°C for 12h, transfer to 1.5mLEP tubes twice to recover the bacteria by centrifugation, and suspend with 500μL STE solution cells, and added lysozyme to a final concentration of 4mg / mL, and water bathed at 37°C for 30-45min.

[0089] 2. When the cells become transparent gelatinous, add 250 μL 2% SDS, 60 μL 5mol / L KAc, shake and mix evenly, freeze at -20°C for 10 minutes, then centrifuge at 12,000 rpm for 10 minutes, transfer the supernatant to a new In EP tube.

[0090] 3. Add 500 μL equal volume of phenol / chloroform mixture, centrifuge at 10,000 rpm for 3 minutes, repeat several times until no white denatured protein appears at the interface, then extract once with chloroform alone, remove the supernatant and add an equal volume of isoform After mixing the p...

Embodiment 2

[0091] Example 2 Establishment of the Genetic Transfer System of Streptomyces flaveolus, a sangliferin-producing bacterium, Streptomyces flaveolus

[0092] 1. First transform the target plasmid to be transferred into E. coli E. coli S17-1, pick a single clone on the plate and inoculate it into a test tube containing 3 mL of LB medium and corresponding selection pressure and incubate overnight at 37°C. Harvest the bacterial liquid the next day, inoculate 1 mL of the bacterial liquid into a 250 mL Erlenmeyer flask containing 50 mL of LB medium and corresponding selection pressure, and incubate on a shaking table at 37°C until the OD600 value reaches about 0.5. Transfer the bacterial solution to a 50mLEP tube and centrifuge the bacterial solution at 16°C at 3800 rpm for 10min to recover the bacterial cells, then add 20mL LB medium to wash twice and centrifuge again under the same conditions to recover the bacterial cells, resuspend in 1mLLB, and set aside.

[0093] 2. Take a tube...

Embodiment 3

[0096] Example 3 Establishment of the sangliferin-producing bacteria Streptomyces flaveolus gene library Small-scale enzyme digestion experiment

[0097] 1. First determine the amount of Sau3AI by a series of dilution experiments, first prepare a 250 μl reaction system (containing 40 μl of genomic DNA, 2.5 μl of BSA (100×)), and then divide the 250 μl reaction system into 1 × 50 μl and 7 × 25 μl and keep on ice. Then take 2 μl of Sau3AI previously diluted to 0.5u / μl. Add 50 μl of the reaction system in the first tube, mix well and transfer 25 μl to the second tube 25 μl, repeat this transfer step 7 times. All systems were inactivated in a water bath at 37°C for 15 minutes, then inactivated at 70°C for 10 minutes, electrophoresed on a 0.4% agarose gel in a cold room at 4°C, and stained with ethidium bromide to detect the quality of enzyme digestion under ultraviolet light or a gel imager.

[0098] A large number of enzyme digestion experiments

[0099] 2. According to the re...

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Abstract

The invention relates to a biosynthetic gene cluster of sanglifehrin, which relates to related information of clone, whole sequencing, sequence analysis, gene function research, in vitro biochemical research and application thereof of a natural product sanglifehrin which is produced by streptomyces flavolus and has immunosuppressive activity. The whole gene cluster can contain 24 genes, namely 1 NRPS gene, 7 type-I linear PKS genes, 1 type-I repeated PKS gene, 8 precursor synthetic genes, 2 regulatory genes and 5 functional unknown genes. The generation of the sanglifehrin can be blocked by operating the genetics of the core functional genes. Genes and protein provided by the invention can be used for research and development of new medicine or research and development of other products with industrial and agricultural application prospects.

Description

[0001] The present invention is a continuation patent for invention with a priority date of September 24, 2008, a priority number of 200810200388.4, and an invention title of "Sanglifellin Biosynthetic Gene Cluster". technical field [0002] The invention belongs to the field of microbial genetic resources and genetic engineering, and in particular relates to the cloning, sequence analysis, in vivo function verification, in vitro biochemical experiment and application of the biosynthetic gene cluster of the immunosuppressant Sanglifehrin. technical background [0003] The immunosuppressant sanglifellin is a polyketide-polypeptide hybrid natural product produced by Streptomyces flavolus DSM 9954 [J Antibiotics 1999 52 466-473, ibid 1999 52474-479]. Among the more than 20 structural analogues of Sanflifehrin that have been isolated so far, sanglifehrin A (SFA) has the highest activity [J Biol Chem 2005 280 21965-21971]. From the structural point of view, the 22-membered macrol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/54C12N15/53C12N15/52C12N15/31C12N9/00C12P17/18A61P37/06C12R1/465
Inventor 刘文瞿旭东姜楠
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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