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Method for constructing gene engineering FK506 high-producing strain and streptomyces tsukubaensis high-producing strain

A technology of engineering strains and genes, applied in the field of biotechnology engineering, can solve the problems of unstable heritage of strains, difficult preservation, low yield, etc.

Active Publication Date: 2013-04-24
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the strains transformed by these methods have problems such as unstable inheritance, difficulty in preservation, and low yield. Therefore, there is an urgent need in this field to develop methods for constructing genetically engineered FK506 high-yielding bacteria to meet clinical needs.

Method used

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  • Method for constructing gene engineering FK506 high-producing strain and streptomyces tsukubaensis high-producing strain
  • Method for constructing gene engineering FK506 high-producing strain and streptomyces tsukubaensis high-producing strain
  • Method for constructing gene engineering FK506 high-producing strain and streptomyces tsukubaensis high-producing strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Gene library construction

[0123]Based on the plasmids pSET 152 and pOJ446, the cosmid used to construct the gene library was transformed and named pJTU2554. Using the cosmid as a vector, the gene library of the starting strain ZJU506-01 was constructed.

[0124] For the structure of pSET152 see image 3 (From: Gene, 116:43-49, 1992): acc(3)IV represents the apramycin resistance gene; intΦC31 represents the phage ΦC31 integrase gene; attPΦC31 represents the DNA attachment site recognized by ΦC31 integrase in lambda phage The base sequence of point; oriT indicates the replication origin required for intergenus conjugative transfer; lacZa indicates the galactose operon gene.

[0125] The cosmid pJTU2554 structure constructed by pSET152 and pOJ446 is shown in Figure 4 . The plasmid contains the ΦC31 integrase gene sequence derived from pSET152, the phage attachment site attP sequence, part of the apramycin resistance gene acc(3)IV, the intergenus conjugative transfer...

Embodiment 2

[0127] Construction of strain CDD506-7G6 containing allylmalonyl-CoA and methoxymalonyl-ACP biosynthetic genes

[0128] The experimental idea of ​​this example is as follows: design screening library primers, screen the gene library of FK506-producing strain ZJU506-01 from the gene library of FK506-producing strain ZJU506-01, and obtain The cosmid pJTU506-7G6 of the malonyl-ACP partial gene cluster → conjugative transfer to obtain gene multiplied zygotes → screening and verification of gene multiplied strains → fermentation verification to obtain high-yield mutants.

[0129] The specific construction steps are as follows:

[0130] 1. Screening of gene multiplication cosmid pJTU506-7G6

[0131] The screening library primers were designed, and the cosmid pJTU2554-7G6 containing the allylmalonyl-CoA and methoxymalonyl-ACP gene clusters related to the synthesis of FK506 was screened by PCR amplification.

[0132] The primer sequence of the left end screening library is:

[0133...

Embodiment 3

[0160] Construction of allylmalonyl-CoA biosynthesis-related gene cluster multiplication strain CDD506-102

[0161] The specific construction steps are as follows:

[0162] 1. Construction of gene multiplication plasmid pSET152-tcsABCD

[0163] The cosmid pJTU506-7G6 containing part of the FK506 biosynthetic gene cluster was used as a template to amplify the target gene fragment related to allylmalonyl-CoA biosynthesis by PCR.

[0164] The primers used to amplify the subgene cluster tcsA-D are:

[0165] Upstream: 5'-TACTAGTCCATCCCGATCATGCCCCTCCTG-3' (SEQ ID NO: 11)

[0166] Downstream: 5'-TTCTAGAAGGCCCTGACGCGGGACTGACC-3' (SEQ ID NO: 12)

[0167] The multiplication subgene cluster fragment was placed under the control of the strong promoter ermE* by restriction enzyme digestion, T4 ligase ligation, etc., and connected to the vector pSET152 to obtain the gene multiplication plasmid pSET152-tcsABCD( Figure 6), ermE* is a strong promoter derived from the erythromycin resistan...

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Abstract

The invention discloses a method for constructing a gene engineering FK506 (tacrolimus) high-producing strain and a streptomyces tsukubaensis high-producing strain. A receptor strain integrated in genome with a multiplication expression box expressing genes associated with biosynthesis of FK506 has the ability of stably producing FK506 in high yield. Specifically, in a site-specific integration mechanism mediated gene recombination way, endogenous genes associated with precursor biosynthesis in an FK506 gene cluster are multiplied to a streptomyces chromosome to directionally reconstruct the streptomyces producing FK506, so as to obtain an FK506 high-producing strain. The invention also discloses the application of the FK506 high-producing strain.

Description

technical field [0001] The invention belongs to the field of biotechnology engineering, and in particular relates to a method for constructing a high-yield strain of genetic engineering FK506 (tacrolimus) and a high-yield strain of Streptomyces tsukuba. Background technique [0002] FK506, also known as tacrolimus (trade name Prograve), is a 23-membered macrolide compound with immunosuppressive activity, which was obtained from Streptomyces tsukubaensis No. Extracted from the metabolites of 9993. It has been widely used clinically since it was approved by the FDA in 1994. Its mechanism of action is that it can inhibit the production of various cytokines such as interleukin-2 and interferon gamma, block T cell activation, and inhibit the proliferation and proliferation of cytotoxic T cells. The expression of interleukin-2 receptor, the activity of FK506 to inhibit IL-2 is 10-100 times that of cyclosporine, so FK506 can not only be used as an immunosuppressant after kidney tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/63C12P17/18C12R1/465
Inventor 刘文徐志南陈丹丹
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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