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Deleted high-yield ansamitocin strain and preparation method thereof

A technology of ansamitocin and high-yield strains, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problems of formation affecting the yield of target products, etc.

Pending Publication Date: 2018-04-06
辽宁斯韦尔生物科技有限公司 +1
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Problems solved by technology

However, the N-glycosylation reaction catalyzed by the glycosyltransferase ansa30 is a competing pathway for the N-methylation reaction, resulting in the branch product-carbamylated ansamicin glycoside ACGP-3 in the ATCC31280 liquid fermentation broth (4″-O-carbamoyl-ansamitocinoside P-3) accumulation
The formation of by-products affects the yield of the target product

Method used

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  • Deleted high-yield ansamitocin strain and preparation method thereof
  • Deleted high-yield ansamitocin strain and preparation method thereof
  • Deleted high-yield ansamitocin strain and preparation method thereof

Examples

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Embodiment 1

[0033] This example is the specific process of preparing the mutant strain NXJ-22 with the interruption and deletion of gene ansa30. Specifically include the following steps:

[0034] Step 1), construct plasmid pLQ578: use the genomic DNA of Actinomyces fasciatus ATCC31280 as a template, and use two sets of primers DEL30-LF / DEL30-LR and DEL30-RF / DEL30-RR to obtain ansa30 interrupted region by PCR amplification The homology arm of 1440bp on the left and the homology arm of 1465bp on the right were confirmed by gene sequencing. Insert a 1440bp PCR fragment (SpeI / EcoRI) from the left side of the interrupted region and a 1465bp PCR fragment (EcoRI / HindIII) on the right side of the interrupted region into the SpeI / HindIII site of plasmid pJTU1278. The plasmid pLQ578 for the inactivation of ansa30 gene was obtained, and the restriction enzyme digestion verification showed that the plasmid was constructed correctly.

[0035] *Step 1) PCR system and conditions used in the preparation of ...

Embodiment 2

[0052] The present example is the fermentation process of ansa30 interrupting the biosynthesis of ansamicin by the deleted mutant strain. Specifically, it includes the following steps: coating the mutant strain (NXJ-22) on solid YMG medium for activation, after culturing at 30°C for 2 days, picking a small amount of mycelium to inoculate the primary seed medium, and culturing at 30°C 220r / min for 24h; Transfer 4% of the inoculum to the secondary seed culture medium, culture at 30℃ 220r / min for 24h; transfer to the fermentation medium for fermentation according to the 10% inoculum volume, collect the fermentation broth for extraction after 7 days, and use Agilent The Agilent 1200 series performs HPLC analysis on the extracted compounds. Among them, the mobile phase of HPLC is methanol and aqueous solution.

[0053] Table 1 Composition of seed medium and fermentation medium

[0054]

[0055] image 3 The ansa30 gene deletion strain NXJ-22 and wild-type strain ATCC31280 ansamicin f...

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Abstract

The invention provides a deleted high-yield ansamitocin strain and a preparation method thereof, belonging to the technical field of biological medicine. The ansamitocin fermenting level is improved based on the N-glycosyltransferase expression gene of inactivated ansamitocin, and the N-glycosyltransferase expression gene ansa30 inactivated in rare actinosynnema pretiosum ATCC31280, which has 97%homology with the asm25 gene in the rare actinosynnema pretiosum ATCC31565, so that the distribution of ansamitocin bio-synthesis by the method can be reduced, and the ansamitocin yield can be increased. The fermentation final yield of the mutant strain NXJ-22 ansamitocin is increased by 68 percent in comparison with that of a wild strain, and the lab shake flask level reaches 74mg / L.

Description

Technical field [0001] The present invention relates to the field of biomedicine, in particular to a strain with high ansamicin production and a method for preparing the strain. The method mainly involves knocking out the expression of the N-position glycosyltransferase in the genome of Actinomyces fascicularis ATCC31280 Gene ansa30, in order to eliminate the competitive effect of N-position glycosylation on the N-position methylation pathway, so that the accumulation of carbamylated anserin glycoside ACGP-3 disappears, and the metabolic flow flows more to the target product, thereby increasing The production of Ansamicin. Background technique [0002] Ansamicin (also known as Ansamycin or Ansamycin) is a macrolide antibiotic produced by Actinosynnemapretiosum. It is a maytansine molecule derived from microorganisms. The components are P-0, P-1, P-2, P-3, P-3', P-4 and P-4', of which Ansamcin P-3 (AP-3, 1) is the main The fermentation product, Ansamidin can bind to the β subuni...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P17/18C12R1/465
CPCC12N1/20C12N9/1048C12P17/188
Inventor 于丽洁白林泉宁新娟郭舒扬
Owner 辽宁斯韦尔生物科技有限公司
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