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Engineering strain used for heterologously expressing Disorazole Z efficiently, gene cluster for establishing strain and application of strain

A technology of engineering strains and heterologous expression, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of slow growth of Sorangium cellulosum, unsuitable for large-scale fermentation, and difficult cultivation, etc., to shorten the fermentation production cycle and reduce fermentation production Effects of cost and yield improvement

Active Publication Date: 2018-05-18
SHANDONG UNIV
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  • Abstract
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Problems solved by technology

[0006] Aiming at the shortcomings of the wild strain Sorangium cellulosum So ce 427 that currently produces Disorazole Z, which grows very slowly and is not easy to cultivate and is therefore not suitable for large-scale fermentation, the problem to be solved in the present invention is to mine the original strain So ce 427 to provide a Disorazole Z biosynthesis Pathway gene cluster (dis427) and the use of this gene cluster to construct an engineering strain for efficient heterologous expression of Disorazole Z for efficient heterologous biosynthesis of Disorazole Z

Method used

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  • Engineering strain used for heterologously expressing Disorazole Z efficiently, gene cluster for establishing strain and application of strain
  • Engineering strain used for heterologously expressing Disorazole Z efficiently, gene cluster for establishing strain and application of strain
  • Engineering strain used for heterologously expressing Disorazole Z efficiently, gene cluster for establishing strain and application of strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Mining of Disorazole Z biosynthetic gene cluster (dis427)

[0030] Sorangium cellulosum So ce 427 was inoculated into VY / 2 solid medium (5g / L Angel yeast, 1.36g / L calcium chloride dihydrate, 0.5mg / L vitamin B12, 15g / L agar powder, adjusted The pH value is 7.2), and cultured at 30°C until the state of diffuse growth. Scrape the edge bacterial film and transfer to M26 liquid medium (8g / L potato starch, 2g / L soybean peptone, 2g / L yeast extract, 1g / L magnesium sulfate heptahydrate, 1g / L calcium chloride dihydrate, 1mL / L trace element solution, adjust the pH value to 7.2), place in a shaker at 30°C and cultivate to a sufficient amount of bacteria for the preparation of genomic DNA.

[0031] After the cells were collected by centrifugation, they were resuspended in 10 mM Tris-HCl buffer (pH 8.0). Add proteinase K with a final concentration of 1 mg / ml and SDS with a final concentration of 1% to the bacterial suspension, and place in a 50° C. water bath for at lea...

Embodiment 2

[0033] Example 2: Direct cloning of the Disorazole Z biosynthetic gene cluster (dis427)

[0034] Disorazole Z biosynthetic gene cluster (dis427) direct cloning procedure see figure 2 .

[0035] 2.1 Preparation of direct cloning vector of Disorazole Z biosynthetic gene cluster (dis427)

[0036] The specific steps are: restriction endonuclease AvaI digests the plasmid p15A-cm-tetR-tetO-hyg-ccdB to obtain the fragment p15A-cm-tetR-tetO (enzyme digestion and recovery of large fragments, the glue runs to the bottom and then cuts the glue, and the glue is recovered For specific methods, refer to the instructions of the Tiangen kit). Then use p15A-cm-tetR-tetO as a PCR template, use primers p15A-Cm BstBI and AflIIfor dis427-F and p15A-Cm BstBI and AflII for dis427-R for PCR amplification, and obtain the PCR product p15A-cm vector for dis427 Homology arms with sequences at both ends of the Disorazole Z biosynthetic gene cluster (dis427) at the ends.

[0037] The PCR primer sequen...

Embodiment 3

[0057] Example 3: Construction of Disorazole Z biosynthetic gene cluster (dis427) expression plasmid p15A-tnpA-kan-tetR-tetO-dis427

[0058] 3.1 Construction of plasmid p15A-cm-tetR-tetO-disZ427

[0059] For the construction process of plasmid p15A-cm-tetR-tetO-disZ427, see image 3 .

[0060] It has been reported that the constitutive expression of the biosynthetic gene cluster of disorazoles may affect the growth and normal metabolic process of the heterologous host. Therefore, the present invention constructs a plasmid that modifies the promoter of the dis427 gene cluster to strictly regulate its expression.

[0061] The specific steps are as follows: first, use primers Amp-ccdB PCR-F and Amp-ccdB PCR-R to amplify the DNA fragment containing amp-ccdB by PCR, and refer to Example 2.1 for the PCR reaction system and amplification conditions. After the gel was recovered, it was eluted with sterile deionized water, and the concentration was measured by Nanodrop 2000, which wa...

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Abstract

The invention discloses a bio-synthesized gene cluster dis427 of Disorazole Z. The nucleotide sequence of the gene cluster is shown in SEQ ID No.1. The invention further discloses an engineering strain DK1622::Km-Ptet-dis427 used for heterologously expressing Disorazole Z efficiently and established through the gene cluster dis427. The engineering strain is obtained in the mode that Myxococcus xanthus DK1622 is used as an original strain, and the bio-synthesized gene cluster dis427 is integrated on a genome of the original strain with a transposition method. The invention further discloses application of the engineering strain DK1622::Km-Ptet-dis427 in preparation of Disorazole Z. A Disorazole Z bio-synthesis way and an efficient expression method of Disorazole Z in heterologous hosts havehigh research and application value in developing novel anti-tumor or anti-infection medicine and reducing the fermentation production cost.

Description

technical field [0001] The invention belongs to the technical field of microbial gene resources and biosynthesis, and specifically relates to a Disorazole Z biosynthetic gene cluster, an engineering strain for efficiently heterologously expressing Disorazole Z constructed by using the gene cluster and its application. Background technique [0002] Disorazoles are macrocyclic dilactone compounds with novel structures that were first isolated from the fermentation broth of Sorangium cellulosum So ce 12 by Jansen et al. in 1994. So far, 29 derivatives of Disorazoles have been found in Sorangiumcellulosum So ce12, namely Disorazole A1 to Disorazole I. [0003] Studies have shown that disorazoles can inhibit tubulin polymerization and promote tubulin depolymerization, thereby interfering with cell division and inducing cell apoptosis. Biologically active, it is a new type of cell microtubule anti-stabilizer. Disorazole Al and Disorazole C1 are currently the most studied compone...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/74C12P17/18C12N9/02C12N9/04C12N9/88C12N15/53C12N15/60
CPCC12N9/0006C12N9/0051C12N9/0095C12N9/1007C12N9/1025C12N9/1029C12N9/88C12N9/93C12N15/52C12N15/74C12P17/188C12Y108/01007C12Y118/01002C12Y201/01C12Y203/01C12Y203/0104C12Y203/01187C12Y402/01001C12Y402/01059C12Y603/04015
Inventor 张友明李瑞娟高运生涂强王宗杰
Owner SHANDONG UNIV
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