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Primary pathway transformation method under guidance of FK506 strain genome-scale metabolic network model

A genome-scale, primary pathway technology, which is applied in the field of primary metabolic pathway transformation based on the genome-scale metabolic network model of microbial strains, and can solve problems such as key genes and key enzymes in unseen primary pathways.

Inactive Publication Date: 2015-01-21
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Up to now, there are no relevant patents and literature reports that use the genome-scale metabolic network model of the FK506 strain to predict the key genes and key enzymes of the primary pathway, and then guide the molecular transformation of the primary pathway of engineering strains

Method used

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  • Primary pathway transformation method under guidance of FK506 strain genome-scale metabolic network model
  • Primary pathway transformation method under guidance of FK506 strain genome-scale metabolic network model
  • Primary pathway transformation method under guidance of FK506 strain genome-scale metabolic network model

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Embodiment 1

[0044] According to the genome sequence of Streptomyces tsukuba, after adding the characteristic reaction of FK506 biosynthesis and cell synthesis reaction and manually refining the network reaction (removing redundant reactions, adding reaction cofactors, adjusting the reversible direction of reactions, trimming reactions, adding transport reactions, analysis to fill in the gaps), a genome-scale metabolic network model of the FK506 strain Streptomyces tsukuba was obtained ( figure 1 ). The model mainly includes central carbon metabolism (glycolysis pathway, pentose phosphate pathway, tricarboxylic acid cycle), pyruvate metabolism, glyoxylate cycle metabolism, amino acid synthesis and consumption metabolism, nucleotide metabolism, lipid metabolism, peptidoglycan Sugar synthesis, coenzyme synthesis, nitrogen-sulfur metabolism, porphyrin and other related reactions, in addition to the synthetic pathway reactions of FK506 and its by-products FK520 and FK506D, the network contains...

Embodiment 2

[0046] According to the prediction of the precursor glutamate synthesis pathway gene gdhA and the phosphoenolpyruvate supply pathway gene ppc according to Example 1, the FK506 strain was knocked out. Use the primer pair gdhA-LF and gdhA-LR (Table 1) of the homologous left arm to amplify the left arm of the target gene gdhA to obtain the left arm fragment of the gene gdhA. Use the primer pair gdhA-RF and gdhA-RR (Table 1) of the homologous right arm to amplify the right arm of the target gene gdhA to obtain the right arm fragment of the gene gdhA. The two homologous fragments were sent for sequencing verification, and the two homologous fragments were digested with XbaI-BamHI and KpnI-EcoRI respectively, and ligated to pUC119-kana in sequence. The above constructed vector was digested with XbaI–EcoRI and then connected to the E.coli-Streptomyces shuttle plasmid pKC1139 to obtain the gdhA knockout vector pΔGDH ( figure 2 ). The construction process of gene ppc knockout vector i...

Embodiment 3

[0051] According to Example 1, the precursor shikimate pathway gene dahp, the reducing power balance pathway gene pntAB, the fatty acid synthesis metabolic pathway gene accA2, and the pentose phosphate pathway gene zwf2 were predicted, and the FK506 strain was molecularly modified. Using S. roseosporus genome as template, accA2 gene and dahp gene were amplified with accA2-F and accA2-R, dahp-F and dahp-R primers respectively (Table 1); using S. coelicolor genome as template, pntAB -F and pntAB-R, accA2-F and accA2-R are primers (Table 1) to amplify the pntAB gene and zwf2 gene, and the PCR products include the ribosome binding sites of each gene itself. The accA2 gene, dahp gene, pntAB gene and zwf2 gene PCR products were digested with NdeI-XbaI and then ligated into pIB139 that had been cut with the same enzyme, and the ligated products were transferred into the prepared Escherichia coli competent cells JM109 or DH5α. After transformation, Spread onto a screening plate contai...

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Abstract

The invention discloses a primary pathway transformation method under the guidance of an FK506 strain genome-scale metabolic network model, which comprises the following steps of: adding the characteristic reaction and thallus synthesis reaction of FK506 biosynthesis according to the streptomyces tsukubaensis genome sequence, and performing manual refining on the network reaction to obtain an FK506 strain streptomyces tsukubaensis genome-scale metabolic network model; and analyzing the metabolic network model, and determining the primary pathway precursor key gene of FK506 biosynthesis through a flux equilibrium analysis and minimum metabolic regulation analysis method. According to the method disclosed by the invention, the strain production level after the transformation is improved by 80-100%. The method provides efficient guidance to the establishment, research and analysis of the primary pathway of the FK506 strain, and has great application prospects in the research process of FK506 strain transformation.

Description

technical field [0001] The invention belongs to the technical field of molecular modification of metabolic engineering of microbial strains, and in particular relates to a primary metabolic pathway modification method based on a genome-scale metabolic network model of microbial strains. Background technique [0002] As a 23-membered polyketide macrolide compound with a high degree of immunosuppressive effect, FK506 (also known as tacrolimus) is 100 times more potent than cyclosporine, a structurally dissimilar immunosuppressant, and is therefore widely used in allogeneic Anti-rejection therapy after kidney, liver, bone marrow transplants, and in the management of inflammatory skin diseases and eczema. With the exponential growth of organ transplantation in recent years, FK506 has become an extremely important first-line clinical drug. [0003] Many researchers have done a lot of work on the efficient production process of FK506, which is also based on random mutation and sc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/76C12N1/21C12R1/465
Inventor 闻建平黄笛夏梦雷
Owner TIANJIN UNIV
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