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

An ansesomycin and gene transcription technology, which is applied in the field of biomedicine, can solve the problems of low transcription level of PKS gene ansa9 and bottleneck of AP-3 biosynthesis, and achieve the effect of increasing yield

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

Based on the analysis of previous transcriptional data, we found that the transcription level of the PKS gene ansa9 in the AP-3 biosynthetic gene cluster is very low, which may be the bottleneck of AP-3 biosynthesis

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] This example is a specific process for preparing mutant strains with overexpression of gene ansa9. Specifically include the following steps:

[0033] Step 1), construct plasmid pLQ593: use plasmid pIB139 as DNA template, use primer ermE-F / R to amplify the erythromycin resistance gene promoter fragment (216 bp) by PCR, and introduce XbaI / BglII restriction site; using Actinomyces precious ATCC 31280 genomic DNA as a template, two sets of primers ansa9-L-F / R and ansa9-R-F / R were used to amplify by PCR to obtain the two sites upstream and downstream of the ansa9 start codon. The segment sequence is used as the homology arm inserted into the promoter, in which, the 5' end and 3' end of the left arm (1445 bp) are respectively introduced into SacI and BglII restriction sites, and the 5' end and 3' end of the right arm (1499 bp) are respectively introduced BglII / XbaI and HindIII restriction sites were introduced, and the correctness of the sequence was confirmed by gene seque...

Embodiment 2

[0050] This example is a fermentation process for biosynthesizing ansamectin by a mutant strain overexpressing the gene ansa9. The specific steps are as follows: spread the overexpressed strain on solid YMG medium for activation, culture it at 30°C for 2 days, pick a small amount of mycelia and inoculate it into the primary seed medium, culture it at 30°C, 220r / min for 24h, and press 4% The inoculum was transferred to the secondary seed medium, and after 24 hours of cultivation at 30°C / 220r / min, 10% of the inoculum was transferred to the fermentation medium. After 7 days, the fermentation broth was collected for extraction and compound detection.

[0051] Table 1 Composition of seed medium and fermentation medium

[0052]

Embodiment 3

[0054] This example is a method for measuring the transcription level of the doubled gene in the gene doubled strain by fluorescent quantitative PCR. Samples 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 12000r / min unless otherwise specified.

[0055] The specific steps are: take 500 mL of the crushed sample, add 100 mL of chloroform, vortex and mix well, centrifuge for 15 minutes, 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. ), 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 for 2 min. Replace with a new collection tube, add 60 mL of DEPC-treated ...

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Abstract

The invention provides a high-yield ansamitocin strain for reinforcing the polyketide synthase gene 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. In the rare actinosynnema pretiosum ATCC31280, the expression level of PKS gene ansa9 is reinforced by utilizing erythrocin resistance gene promoter, the biological synthesizing capacity of actinosynnema pretiosum can be reinforced, and the yield of actinosynnema pretiosum can be increased. The ansamitocin fermenting final yield of engineering strains is increased by about 125 percent in comparison with that of the original strain, and the lab shaking flask level reaches 99mg/L.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically a high-yield ansamitocin strain with enhanced polyketide synthase gene transcription level and its preparation method, mainly by using erythromycin The promotor of the resistance gene enhances the expression level of PKS gene ansa9, thereby enhancing the biosynthetic ability of ansamitocin, thereby increasing the production of ansamitocin. Background technique [0002] Ansamitocin is a microbial-derived maytansinoid molecule, belonging to type I polyketides, produced by Actinosynnema pretiosum, and has strong antitumor activity. Among its components, Ansamitocin P-3 has the strongest activity. At present, a variety of antibody-conjugated molecules of ansamitocin have entered different stages of clinical trials, among which trastuzumabemtansine (ie T-DM1) developed by Roche for the treatment of human breast cancer has been marketed as a drug. The enhancement of ansamicin productio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/90C12P17/18C12R1/01
CPCC12N9/1029C12N15/902C12P17/188
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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