Vancocin biological synthesis gene cluster

A vancomycin, biosynthesis technology, applied in the field of bioengineering, can solve the problem of lack of Amycobacterium pseudomycoides genetic operating system and so on

Active Publication Date: 2007-10-31
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no research on combinatorial biosynthesis in bacteria producing glycopeptide antibiotics such as vancomycin, and the key lies in the lack of a complete genetic operating system for Amycolatopsis.

Method used

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  • Vancocin biological synthesis gene cluster
  • Vancocin biological synthesis gene cluster
  • Vancocin biological synthesis gene cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Construction of Amycolatopsis orientalis Genome Library

[0045] Refer to the method of "Molecular Cloning: Laboratory Manual" to extract the genome of Amycobacterium orientalis, then use Sau3A I enzyme to partially digest the Amycobacterium orientalis genome, and select the Amycobacterium orientalis with a main band of 30-50kb Genomic inserts.

[0046] The Supercosl (Stratagene Company) vector was cut into linear fragments with BamH I enzyme, and then ligated with the above-mentioned partially digested Amycolatopsis orientalis genome fragment at 16° C. for 24 hours with T4 ligase to obtain a ligation product.

[0047] Using the Stratagene Gigapack III XL Packaging Extract kit, according to the operation manual of the kit, the obtained ligation product was packaged into phagemids, and transformed into Escherichia coli to construct the Amycolatopsis orientalis genomic library.

Embodiment 2

[0048] Example 2, hybridization screening of cosmids containing a specific gene (GtfE) in the library

[0049] Using the GtfE gene as a template, the DIG High Prime DNA Labeling and Detection StratterKit I kit (Roche Company) was used to prepare probes, and the positive clones were screened from the Amycolatopsis orientalis genome library.

[0050] The obtained positive clones were cultured, and the positive cosmid was extracted, used as a template, and Ep1 (SEQ ID NO: 28) and Ep2 (SEQ ID NO: 29) were used as specific amplification primers for PCR amplification. Amplification results were agarose electrophoresis to verify the size of the cosmid, and the results are shown in Figure 9.

[0051] According to the results in Figure 9, cosmid No. 311 was selected for sequencing, and the sequencing results were analyzed by Veceor NTI advance 10 (invitrogen).

Embodiment 3

[0052] Embodiment 3, sequencing result analysis

[0053] The result of sequencing shows that the nucleotide sequence of the vancomycin biosynthetic gene cluster of the present invention is shown in SEQ ID NO: 1, including 26 genes in total, and these 26 genes can be classified into 3 categories, specifically as follows:

[0054] 1. Vancomycin precursor synthetase genes: vcm1, vcm17, vcm18, vcm13, vcm23, vcm24, vcm25, vcm26, vcm22, vcm19, vcm21, vcm11, vcm20, vcm14, vcm15 and vcm16, 16 in total;

[0055] 2. Vancomycin synthesis internal genes: vcm2, vcm3, vcm4, vcm9 and vcm10, a total of 5;

[0056] 3. Vancomycin modifying enzyme genes: vcm12, vcm8, vcm11, vcm5, vcm6 and vcm7, a total of 6;

[0057] Among them, genes that catalyze the synthesis of vancomycin precursors can be further subdivided into 4 categories, specifically:

[0058] 1) HPG synthetase genes: vcm1, vcm17, vcm18 and vcm13, a total of 4;

[0059] These four genes encode prephenate dehydrogenase, hydroxymandel...

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Abstract

The invention discloses an oriental quasi-non-branch acid bacteria A. orientalis vancocin biological synthetic gene cluster, which is characterized by the following: comprising twenty six genes; encoding and catalyzing vancocin precursor synthase, vancocin synthase and vancocin modifying enzyme. This invention can be used to constitute biology and synthesize vancocin.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, specifically relates to an antibiotic biosynthesis gene cluster, more specifically relates to a vancomycin biosynthesis gene cluster as a glycopeptide antibiotic. Background technique [0002] Vancomycin, whose structure is shown in Figure 1, is a glycopeptide antibiotic isolated from a strain of Amycolatopsis orientalis in 1956 by Micormick et al. A representative class of antibiotics. [0003] In the 1980s, infections caused by methicillin-resistant Staphylococcus aureus (MRSA) became increasingly prevalent due to the widespread use of β-lactam antibiotics. In this case, vancomycin has attracted more and more people's attention, and it is currently the first-choice drug for the treatment of severe infectious diseases caused by MRSA clinically. The last line of defense" and "ace antibiotics". [0004] With the increasing drug resistance of bacteria and the development of modern biotech...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/52
Inventor 黄鹤戈梅姜卫红朱丽阮林高罗敏玉杨蕴刘魏维杨晟陈代杰
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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