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Gene epoO of sorangium cellulosum and its use in epothilone synthesis

A technology of Helicobacter cellulosum and Epothilone, which is applied in the field of microorganisms, can solve the problems such as difficulty in heterologous expression to improve yield, low yield, high cost and the like

Inactive Publication Date: 2013-07-10
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the high cost and low yield of the chemical synthesis method, the production of epothilone in the world currently adopts the microbial fermentation method, but due to the genetic characteristics of the production strain Sonocystis cellulosus, it is currently difficult to improve its heterologous expression Therefore, in addition to the optimization of fermentation conditions and the improvement of traditional strains, the genetic modification of the production strain itself also has important application value
Internationally it has been reported that the responsible epothilone The synthetic gene cluster is a heterozygous type I polyketide synthase gene cluster (NRPS / PKS) (attached figure 2 ), but there are almost no relevant reports on its related regulatory genes. If important epothilone Synthesizing regulatory genes and effectively modifying them can significantly improve epothilone Yield

Method used

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  • Gene epoO of sorangium cellulosum and its use in epothilone synthesis
  • Gene epoO of sorangium cellulosum and its use in epothilone synthesis
  • Gene epoO of sorangium cellulosum and its use in epothilone synthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example of knocking out some fragments (front-end neck loop structure):

[0027] Using the So0157-2 genome of Sonocystis cellulosus as a template, primers EOF1 and EOR1 were used:

[0028] EOF1: 5'-CCCCTCGAGCCACAGCATCAGG-3';

[0029] EOR1: 5'-CGAGGTCGGGATCCAACCACGC-3', prepare the PCR amplification system according to Table 1, and carry out PCR amplification as shown in Table 1, to obtain the regulatory gene epo The DNA fragment of O, as attached image 3 shown.

[0030] Table 1 Contents of each component in PCR amplification system

[0031] Additives volume wxya 2 o 10.4μL 2×GC buffer I 12.5μL So0157-2 genome DNA (10ng / μL) 0.5μL Primer up EOF1 (62.5μM) 0.2 μL Primer down EOR1 (62.5μM) 0.2 μL dNTP (10mM / each) 1μL Pfu (2.5U / μL) 0.2 μL total 25 μL

[0032] Table 2. Regulatory gene PCR amplification conditions

[0033]

[0034] Using the S. cellulosus genome as a template, primer pairs 157e...

Embodiment 2

[0060] Examples of partial base modification:

[0061] Using the So0157-2 genome of Sonocystis cellulosus as a template, primers EOF2 and EOR1 were used:

[0062] EOF1: 5'-CCCaTCGAtCCACAGCATCAGG-3';

[0063] EOR2: 5'-CGAGGTCGGGATCtAACCACGC-3', (the lowercase part is the base substitution introduced during primer design) Prepare the PCR amplification system according to Table 2, and perform PCR amplification as shown in Table 3, and then the regulatory gene can be obtained epo The DNA fragment of O, as attached image 3 shown.

[0064] Using the S. cellulosus genome as a template, the primer pair 157epo5111-6114F and 157epo5111-6114D were used to amplify the 1 kb homology arm fragment upstream of the regulatory gene, and the primer pair 157epo6137-7680F and 157epo6137-7680D were used to amplify the 1.5 kb homologous arm fragment downstream of the regulatory gene. source arm fragment, two PCR products were Xba After enzyme digestion and ligation, the obtained recombinant ...

Embodiment 3

[0071] Example of replacing with other promoter genes:

[0072] Using the So0157-2 genome of Sonocystis cellulosus as a template, using primers EOF3 and EOR3:

[0073] EOF3: 5'-CCCCTCGAGCCACAGCATCAGG-3';

[0074] EOR3: 5'-CGAGGTCGGGATCCAACCACGC-3', prepare the PCR amplification system according to Table 2, and carry out PCR amplification as shown in Table 3, to obtain the regulatory gene epo DNA fragments of O, such as image 3 shown.

[0075]Using the S. cellulosus genome as a template, primer pairs 157epo5111-6114F + 157epo5111-6114D and 157epo6137-7680F + 157epo6137-7680D were used to amplify the homology arm fragments at both ends of the regulatory gene, including the upstream 1kb and downstream 1.5kb recombinant homology arm fragments , for PCR products Xba I Digest and then connect with other promoters. In this example, a 189bp lysine decarboxylase promoter (GeneBank NO. S61972.1) derived from Streptomyces was used to replace it. The resulting recombinant fragment...

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Abstract

The invention belongs to the field of microbes and relates to a gene epoO in a genome of sorangium cellulosum and its use in epothilone synthesis. The gene epoO of the sorangium cellulosum is a regulatory gene and is characterized in that the gene epoO has a nucleotide sequence shown in the formula of SEQ ID NO.1. The gene epoO of the sorangium cellulosum is discloses first and is closely related to epothilone fermentation synthesis based on sorangium cellulosum. Through a molecular biology technology, gene internal structure reconstruction or direct replacement of a promoter is realized so that an epothilone fermentation yield is obviously improved.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a method derived from Cystis cellulosus ( Sorangium cellulosum ) genes in the strain genome epo O and its use in the synthesis of epothilones. Background technique [0002] Epothilone ( Epothilone ) is a class of 16-membered macrolide compounds produced by the myxobacteria S. cellulosum, which has the activity of promoting microtubule polymerization, and its mechanism of action is similar to paclitaxel ( Taxol ?) (attached figure 1 ), a variety of epothilone Structural analogues, related invention patents have exceeded 800, epothilones It has become one of the exciting research hotspots in biomedicine. It is considered to be an updated product of paclitaxel anti-tumor compounds. Compared with paclitaxel, it has obvious advantages: ①Simple molecular structure, better water solubility; ② Epothilone The thiazole ring structure and the microtubule interaction ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/74C12P17/18C12R1/01
Inventor 赵林刘新利孙欣李越中黎志凤
Owner QILU UNIV OF TECH
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