High-yield ansamitocin strain for reinforcing transcriptional level and preparation method thereof

An ansamitocin and transcription-level technology, which is applied in the field of biomedicine and can solve the problem of low efficiency of N-position methylation

Pending Publication Date: 2018-04-06
辽宁斯韦尔生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the glycosyltransferase expression gene can be interrupted and deleted, the obtained mutant strain NXJ-22 can eliminate the competition between N-glycosylation and N-methylation pathway, but the efficiency of N-methylation is still low

Method used

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  • High-yield ansamitocin strain for reinforcing transcriptional level and preparation method thereof
  • High-yield ansamitocin strain for reinforcing transcriptional level and preparation method thereof
  • High-yield ansamitocin strain for reinforcing transcriptional level and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] This example is a specific process for preparing a mutant strain with overexpression of the gene asm10. Specifically include the following steps:

[0029] Step 1) Plasmid pLQ586 was constructed: Genomic DNA of Actinomyces spp. precious ATCC 31565 was used as a template, and the asm10 fragment (885 bp) was amplified by PCR using primers asm10-F / R, and SpeI / EcoRI were introduced at both ends of the sequence site, and insert the RBS sequence (ATCTGAGTTGAAGAGGTGACGTC) before the start codon. Asm10 (SpeI / EcoRI)) was inserted at the SpeI / EcoRI site of plasmid pDR3-K* to obtain plasmid pLQ586.

[0030] * Each endonuclease recognition site (enzyme cutting site) involved in the present invention is as follows:

[0031]

[0032] The primer sequence used in step 1) is:

[0033]

[0034] *PCR system and conditions used in gene fragment preparation in step 1):

[0035] PCR reaction system: DNA template 30 ng, primer 30 pmol, 50% DMSO 3 mL, 25 mM Mg 2+ 2 mL, buffer 3 mL, ...

Embodiment 2

[0043] This example is a fermentation process for biosynthesizing ansamitocin by a mutant strain overexpressing the gene asm10. The specific steps are as follows: spread the asm10 overexpressed strain on solid YMG medium for activation, and after culturing at 30°C for 2 days, pick a small amount of mycelium and inoculate it into the primary seed medium, at 30°C, 220 r / min 24 h under high temperature; transfer to secondary seed medium at 4% inoculum size, and cultivate at 30°C, 220 r / min for 24 h; transfer to fermentation medium at 10% inoculum amount, at 25°C, 220 After 7 days of fermentation under the condition of r / min, the fermentation broth was collected for extraction and compound detection.

[0044] Table 1 Composition of seed medium and fermentation medium

[0045]

Embodiment 3

[0047] This example is a method for measuring the transcription level of the doubled gene in the gene doubled strain by fluorescent quantitative PCR. (The samples used for RNA extraction are generally stored in Redzol solution. The RNA extraction process requires low temperature, and the centrifugation process is carried out at 4°C and 12000 r / min unless otherwise specified.)

[0048] The specific steps are: take 500 mL of the crushed sample, add 100 mL of chloroform, vortex and mix well, centrifuge for 15 min, draw the supernatant, add 100 mL of absolute ethanol and mix well, then suck the sample into the spin column (Beijing Saibaisheng Gene Technology Co., Ltd. Technology Co., Ltd.), let stand for 2 min, centrifuge for 1 min, discard the liquid, rinse twice with washing buffer (Washing Buffer, Beijing Saibaisheng Gene Technology Co., Ltd.), discard the liquid, put the spin column in the collection tube and continue centrifuging 2 min. Replace with a new collection tube, ad...

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Abstract

The invention provides a high-yield ansamitocin strain for reinforcing transcriptional level and a preparation method thereof, belonging to the technical field of biological medicine. The yield of ansamitocin can be increased by improving the rate-limiting enzyme gene expression level in biosynthetic pathway. The gene asm10 is modified in the deleted mutant strain NXJ-22 of the glycosyl transferase expression gene of rare actinosynnema pretiosum ATCC31280 after overexpression of strong promoter kasOp*, the methyltransferase activity can be reinforced, and the yield of ansamitocin can be improved. The ansamitocin fermenting final yield of engineering strains is increased by about 173 percent in comparison with that of a control strain, and the lab shaking flask level reaches 141.8mg / L.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically a high-yield ansamitocin strain with enhanced transcription level and its preparation method, mainly through the interruption deletion mutation of the expression gene of glycosyltransferase in Actinomyces spp. precious ATCC31280 In strain NXJ-22, the gene asm10 was modified after overexpression of the strong promoter kasOp* to enhance the methyl transfer efficiency of ansamitocin, improve the biosynthesis ability of ansamitocin, and then increase the production of ansamitocin. Background technique [0002] Ansamitocin is a macrocyclic lactam antibiotic produced by Actinosynnema pretiosum, which can bind to the β subunit of tubulin to hinder the assembly of microtubules, thereby inhibiting tumor cell division. Chari et al. from ImmunoGen, Inc., formed a DM1 molecule formed by linking a disulfide bond on the C-3 ester chain, which can be linked with different antibodies to form antib...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P17/18C12R1/01
CPCC12N9/1007C12N15/74C12P17/188C12Y201/01
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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