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Method for producing norvancomycin

A technology of norvancomycin and vancomycin, applied in the field of genetic engineering, can solve problems such as indeterminate and indeterminate

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 vcm12 gene encodes N-methyltransferase, it cannot be determined whether norvancomycin is expressed after knocking out the vcm12 gene, for example, in the biosynthetic gene cluster of Balhimycin, according to Gene sequence comparison revealed that there were 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 could produce chlorine-free Balhimycin, while the bhp knockout strain did not produce Balhimycin
Therefore, whether the derivatives of vancomycin can be obtained by knocking out the vcm12 gene, and whether the obtained derivatives of vancomycin are the expected norvancomycin, cannot be determined

Method used

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  • Method for producing norvancomycin
  • Method for producing norvancomycin
  • Method for producing norvancomycin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 , Construction of recombinant plasmid pLY01D

[0042] 1.1. Design and synthesis of primers

[0043] According to the gene sequence of Amycolatopsis orientalis ATCC 43491, two pairs of primers, PNA1 and PNA2 and PNB1 and PNB2, 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 PNC1 and PNC2 were designed for the amplification of the apramycin (Apr) resistance gene fragment, and the above sequence and its restriction site are as follows:

[0044] PNA1: TCTAGA AAGATCAAGGAAGACCTG;

[0045] wxya

[0046] PNA2: CTGCAG CAGGACAACCTCCAGGTC;

[0047] PstI

[0048] PNB1: CTGCAG TGAGCGCGGCGAGCCTGC;

[0049] PstI

[0050] PNB2: AAGCTT CATCATCGCCGACATCATCGCC;

[0051] Hind III

[0052] PNC1: CTGCAG GGAACTTATGAGCTCAGCCAATCGACT;

[0053] PstI

[0054] PNC2: CTGCAG CTGACGCCGTTGGATACACCA.

[005...

Embodiment 2

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

[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 with Buffer I buffer solution.

[0071] Suspend the mycelium with 10ml of lysozyme solution (1mg / ml, prepared with Buffer I buffer) after filter sterilization, keep it at 28°C for 15 minutes, and then use a sterile funnel equipped with degreasing cotton to remove it by continuous filtration twice. 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 transfor...

Embodiment 3

[0087] Example 3 , Amycolatopsis orientalis dNMT strain fermentation culture

[0088] Amycolatopsis orientalis dNMT and Amycolatopsis orientalis dNMT were separated naturally to obtain a single colony.

[0089] 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.

[0090] Get the fermentation broth, centrifuge at 12000rpm for 10min, take the supernatant, and carry out HPLC detection, the detection conditions are as follows:

[0091] 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 ra...

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Abstract

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

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a method for producing norvancomycin. Background technique [0002] Such as figure 1 As shown, N-terminal demethylvancomycin is an analogue of vancomycin, which has only one less methyl group than vancomycin on the amino group of one amino acid. Its antibacterial spectrum is basically the same as that of vancomycin, but its in vitro activity is about 20% higher than that of vancomycin. It is mainly used for the treatment of endometritis, pneumonia, osteomyelitis, sepsis and soft tissue infection caused by Staphylococcus (including methicillin-resistant bacteria). [0003] North China Pharmaceutical Group Co., Ltd. once screened and obtained the strains producing norvancomycin through the traditional method of strain selection and breeding. However, the traditional strain selection method has strong randomness and heavy workload, and it is difficult to obtain the e...

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 戈梅李航黄鹤魏维阮林高杨晟朱丽姜卫红陈代杰罗敏玉杨志钧夏兴齐晓丹王天娇殷瑜金文翔
Owner SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT
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