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Method for producing chlorine-free vancomycin

A technology of vancomycin and protoplasts, applied in the field of biology

Inactive Publication Date: 2010-06-09
SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although according to the information obtained by gene sequence alignment, the vcm8 gene encodes a halogenase, it is not certain that chlorine-free vancomycin can be obtained after knocking out the vcm8 gene. For example, in the biosynthetic gene cluster of Balhimycin, according to gene sequence alignment , it was found that there are two halogenase genes related to halogenation, bhaA(FADH 2 -dependent halogenase) and bhp (haloperoxidase / perhydrolase), Oliver Puk et al. found in 2002 that the bhaA knockout strain produced chlorine-free Balhimycin, while the bhp knockout strain did not produce Balhimycin

Method used

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  • Method for producing chlorine-free vancomycin
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  • Method for producing chlorine-free vancomycin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 , Construction of recombinant plasmid pLY02D

[0042] 1.1. Design and synthesis of primers

[0043] According to the gene sequence of Amycolatopsis orientalis ATCC 43491, two pairs of primers PCA1 and PCA2 and PCB1 and PCB2 were designed to amplify the fragments of the left homology arm and the right homology arm, respectively. According to the sequence of pHP45omega-aac, the primer pair PCC1 and PCC2 were designed for the amplification of the apramycin (Apr) resistance gene fragment, and the above sequence and its restriction site are as follows:

[0044] PCA1: GAATTC ATTTCGTCTGGCAGGCCC;

[0045] EcoRI

[0046] PCA2: TCTAGA TTCCCACCATTCCTTTGTCG;

[0047] wxya

[0048] PCB1: GCATGC TGACTTCGCCCGACGGGA;

[0049] SphI

[0050] PCB2: AAGCTT GTCGACAACAACACACGCATCAC;

[0051] Hind III

[0052] PCC1: TCTAGA GGAACTTATGAGCTCAGCCAATCGACT;

[0053] wxya

[0054] PCC2: GCATGC CTGACGCCGTTGGATACACCA.

[0055] ...

Embodiment 2

[0068] Example 2 , blocking the acquisition of mutant Amycolatopsis orientalis dvcm8

[0069] 2.1. Preparation of protoplasts from Amycolatopsis orientalis ATCC 43491

[0070] The mycelia liquid of Amycolatopsis orientalis ATCC 43491 was inoculated into 20 ml of TSB liquid medium, and glycine was added to a final concentration of 2%. Then shake culture at 28° C. for 36-60 hrs, collect the bacterial cells by centrifugation, and wash once with Buffer I buffer solution.

[0071] Use 10ml filter-sterilized lysozyme solution (1mg / ml, prepared with Buffer I buffer) to suspend mycelium, place it at 28°C for 15 minutes, and then use a sterile funnel equipped with degreasing cotton to filter twice to remove Mycelium. The precipitate was collected by centrifugation, washed twice with Buffer I buffer, and resuspended in 1 ml Buffer I buffer to obtain protoplasts of Amycolatopsis orientalis ATCC 43491.

[0072] 2.2. Preparation of DNA for protoplast transformation

[0073] The recom...

Embodiment 3

[0089] Example 3 , Amycolatopsis orientalis dvcm8 fermentation culture

[0090] Amycolatopsis orientalis dvcm8 and Amycolatopsis orientalis dvcm8 were separated naturally to obtain a single colony.

[0091]Pick a single colony and smear it on the Gaoshi No. 1 slant medium, and cultivate it at 28°C for 5 days. 48h. Then, under aseptic conditions, transfer the cultured seed solution into a fermentation shaker flask with an inoculum amount of 8%, and vibrate and culture at 28°C and 220rpm for 115-120h.

[0092] Get the fermentation broth, centrifuge at 12000rpm for 10min, take the supernatant, and carry out HPLC detection, the detection conditions are as follows:

[0093] Mobile phase preparation: use 0.2% triethylamine solution and phosphoric acid to adjust the pH to 3.2 as a buffer. The buffer solution is mixed with acetonitrile and tetrahydrofuran in a ratio of 92:7:1 and shaken as solution A; the buffer solution is mixed with acetonitrile and tetrahydrofuran in a ratio o...

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Abstract

The invention provides a method for producing chlorine-free vancomycin, which comprises the following steps: (i) interdicting a gene of encoding halogenase in amycolatopsis orientalis for producing vancomycin, constructing gene engineering bacteria for producing the chlorine-free vancomycin; and (ii) fermenting the gene engineering bacteria for producing the chlorine-free vancomycin so as to prepare the chlorine-free vancomycin. The invention also provides a method for constructing the gene engineering bacteria for producing the chlorine-free vancomycin and the gene engineering bacteria obtained through the method. The invention opens up a new approach for the preparation of the chlorine-free vancomycin.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for producing chlorine-free vancomycin. Background technique [0002] like figure 1 As shown, the chlorine-free vancomycin is an analogue of vancomycin, only the two chlorine atoms on the vancomycin molecule are replaced by H. Although its in vitro activity is lower than that of vancomycin, it can be used as a precursor of vancomycin derivatives. [0003] C.M.Harris etc. removed two chlorine atoms on the vancomycin molecule by chemical method in 1985, obtained chlorine-free vancomycin, but the cost of using chemical method to produce chlorine-free vancomycin is high, therefore, it is necessary to provide a A cost-effective method for the production of chlorine-free vancomycin. [0004] The applicant disclosed 26 genes in the vancomycin biosynthesis gene cluster in the Chinese invention patent application with the application number 200710036861.5. According t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12P21/02C12N1/21C12R1/01
Inventor 罗敏玉李航黄鹤魏维阮林高杨晟朱丽姜卫红戈梅陈代杰杨志钧夏兴王天娇殷瑜杨天金文翔
Owner SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT
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