Streptovaricin derivative and preparation method and application thereof

A technology for streptothricin and derivatives, which is applied in the field of novel streptomycin derivatives and their preparation, can solve problems such as unclear steps, and achieve the effects of rich diversity and good antibacterial effect

Active Publication Date: 2018-01-05
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, the biosynthetic gene cluster of streptothricin has not been reported, and subsequent modification reactions including hydroxylation, methylation, acetylation and redox steps are still unclear; on the other hand, based on chemical synthesis or semi-synthesis The creation of new active streptothricin derivatives by methods such as combinatorial biosynthesis transformation or metabolic engineering has not been reported yet.

Method used

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  • Streptovaricin derivative and preparation method and application thereof
  • Streptovaricin derivative and preparation method and application thereof
  • Streptovaricin derivative and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] [Example 1] Production of novel streptothricin derivatives (1 and 2) construction of engineering strains

[0090] The engineering strain for producing novel streptothricin derivatives is a streptothricin-producing strain that inactivates or deletes the stvP4 gene.

[0091] (1) stvP4 in-frame knockout recombinant plasmid pWHU2801( figure 2 a)

[0092] 1) Using the chromosomal DNA of Streptomyces XW-1 (preservation number: CCTCC NO: M2017417) as a template, using stvP4-L-F and stvP4-L-R as primers, amplified at the upstream of the stvP4 gene The 1990bp fragment is used as the stvP4 in-frame knockout homologous left arm stvP4-L. Using stvP4-R-F and stvP4-R-R as primers, a 1989bp fragment was amplified downstream of the stvP4 gene as the stvP4 in-frame knockout homologous right arm stvP4-R.

[0093] 2) Ligate the homologous left arm stvP4-L directly to the pEASY vector to obtain the recombinant plasmid pEASY-stvP4-L, after restriction enzyme digestion and sequencing ver...

Embodiment 2

[0098] [Example 2] Production of novel streptothricin derivatives (3 and 4) construction of engineering strains

[0099] The engineering strain for producing novel streptothricin derivatives is a streptothricin-producing strain with inactivation or deletion of stvP5 gene.

[0100] (1) stvP5 in-frame knockout recombinant plasmid pWHU2802( image 3 a)

[0101] 1) Using the chromosomal DNA of Streptomyces XW-1 (preservation number CCTCC M2017417) as a template and using stvP5-L-F and stvP5-L-R as primers, a 2098bp fragment was amplified upstream of the stvP5 gene The homologous left arm stvP5-L was knocked out in frame as stvP5. Using stvP5-R-F and stvP5-R-R as primers, a 1928bp fragment was amplified downstream of the stvP5 gene as the stvP5 in-frame knockout homologous right arm stvP5-R.

[0102] 2) The homologous left arm stvP5-L was directly connected to the pEASY vector to obtain the recombinant plasmid pEASY-stvP5-L. After verification by enzyme digestion and sequencing,...

Embodiment 3

[0107] [Example 3] HPLC-UV analysis of bacterial strain fermentation product

[0108] The high-yield strains constructed above and the control group wild-type XW-1 were subjected to 50mL small-scale parallel fermentation and product extraction, and the fermentation products were detected by HPLC-UV. The ion peaks were extracted and the results were as follows Figure 4 shown.

[0109] The mutant strain ΔstvP4 accumulated streptothricin D as expected, and its production was about 100-fold higher than that of the wild-type XW-1. Alternative metabolites (1 and 2); mutant ΔstvP5 was still able to produce protostreptothricin I, and simultaneously accumulated two distinct intermediates or alternative metabolites of streptothricin biosynthesis (3 and 4 )( Figure 4 ).

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Abstract

The invention discloses a streptozotocin derivative and a preparation method and application thereof, and belongs to the field of biomedicine. According to the invention, a streptozotocin spectabilisXW-1 (preservation number is CCTCC NO: M2017417) is used as an original strain, and P450 genes (stvP4 and stvP5) in the post-modification system of streptozotocin biosynthetic gene cluster are respectively singly inactivated or deleted. Through separation and purification of a crude extract of a fermentation product of the two mutant strains, a novel streptozotocin derivative 1-4 is obtained. Thenovel streptozotocin derivative has good antibacterial effect, provides a new solution for the problem of antibiotic resistance, and provides a basis for structural modification of streptozotocin antibiotic.

Description

technical field [0001] The invention relates to the field of streptothricin preparation, in particular to novel streptothricin derivatives and their preparation methods and applications. Background technique [0002] Since the 1960s, antibiotic resistance has gradually become a serious threat to public health, causing many difficult-to-treat human infections and even death. In particular, Methicillin-resistant Staphylococcus aureus (MRSA) is now considered a "superbug" that causes major human illness and death, due to its high virulence and wide distribution (such as community and hospital environments ), which can cause huge economic losses. Therefore, there is an urgent need to develop new drugs against these pathogens. Natural products have been proven to be important resources for the discovery of anti-MRSA antibiotics such as vancomycin and daptomycin; on the other hand, engineered biosynthesis can complement this traditional time-consuming and labor-intensive drug di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D491/18C07D491/08C07D225/08C12P17/18C12N1/21A61K31/395A61P31/04C12R1/465
CPCA61K31/395A61P31/04C07D225/08C07D491/08C07D491/18C12P17/18C12N1/205C12R2001/465
Inventor 孙宇辉刘源振陈旭邓子新
Owner WUHAN UNIV
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