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Saccharopolyspora spinosa genome scale metabolic network model and construction method and application thereof

A technology of Saccharopolyspora spinosa and genome scale, applied in special data processing applications, instruments, electronic digital data processing, etc.

Active Publication Date: 2014-04-16
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, so far, no one has reported the efficient production of spinosyns by modifying the transhydrogenase pathway in S. spinosa

Method used

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  • Saccharopolyspora spinosa genome scale metabolic network model and construction method and application thereof
  • Saccharopolyspora spinosa genome scale metabolic network model and construction method and application thereof
  • Saccharopolyspora spinosa genome scale metabolic network model and construction method and application thereof

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Embodiment 1

[0028] A method for constructing a genome-scale metabolic network model of Saccharopolyspora spinosa, comprising the steps of:

[0029] According to the annotation information of the genome sequence of Saccharopolyspora spinosa in the KEGG and NCBI databases, the characteristic reactions of spinosyn biosynthesis (see Table 1) and bacterial synthesis reactions (see Table 2) were added:

[0030] Table 1 spinosad biosynthesis reaction

[0031]

[0032]

[0033] Table 2 bacterial synthesis reaction

[0034]

[0035]

[0036] And manually refine the network reaction (removing redundant reactions, adding reaction cofactors, adjusting the reversible direction of the reaction, balancing the reaction, adding transport reactions, adding exchange reactions, analysis to fill in the gaps), manual refining details see (Nature protocols.A protocol for generating a high-quality genome-scale metabolic reconstruction. 2010.) Obtained the genome-scale metabolic network model of Saccha...

Embodiment 2

[0043] The Saccharopolyspora spinosa genome-scale metabolic network model constructed in Example 1 is converted into a computer-recognizable mathematical coefficient matrix, and the Matlab software platform is used to catalyze the reaction with the transhydrogenase PntAB by means of the robustness analysis tool in the COBRA toolbox. In order to control the reaction, the synthesis reaction of spinosyn was taken as the target reaction, and the effect of the gene activity of transhydrogenase PntAB on the synthesis rate of spinosyn was determined. The simulation results were as follows: figure 1 .

Embodiment 3

[0045] Molecular amplification is carried out to the transhydrogenase gene pntAB determined in Example 2, and primers are designed according to the transhydrogenase gene sequence on NCBI (the underline represents the restriction site):

[0046] Upstream primer pntAB-F:CCTA TCTAGA CGCCGGAACAAGGACG (shown in SEQ ID NO.1)

[0047] Downstream primer pntAB-R:GGATC CATATG ACCTCTCCGGAGAGC (shown in SEQ ID NO.2)

[0048] Using this pair of primers, using the Saccharopolyspora spinosa genome as a template, the transhydrogenase PntAB gene was amplified by PCR technology, and the transhydrogenase pntAB gene was inserted into the Streptomyces strong Promoter ermE* on plasmid pOJ260, resulting in plasmid pOJ261 (see figure 2 ), transform Escherichia coli DH5а, extract the plasmid and transfer it into Escherichia coli ET12567 (ATCC product number: BAA-525), and transfer it into Saccharopolyspora spinosa (Saccharopolyspora spinosa ATCC49460) by the combined transfer method, with 50 μg / m...

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Abstract

The invention discloses a saccharopolyspora spinosa genome scale metabolic network model and a construction method and application of the saccharopolyspora spinosa genome scale metabolic network model. The construction method includes the following steps that according to annotation information of saccharopolyspora spinosa genome sequences in KEGG and NCBI databases, a characteristic reaction for spinosad biosynthesis and a thallus sythesis reaction are added, a network reaction is manually refined, and then the saccharopolyspora spinosa genome scale metabolic network model is acquired. Through the saccharopolyspora spinosa genome scale metabolic network model, influences on improvement of the output of spinosad on a gene target spot can be predicted, the modification direction is finally determined, and the bacterial strain path molecule modification method is achieved. Experiments show that through genes predicted by the saccharopolyspora spinosa genome scale metabolic network model, genetically engineered bacteria obtained through modification can make the output of spinosad improved by 86.5% compared with wild bacterial strains. An instruction platform is provided for construction, research and analysis of the bacterial strains for efficiently producing and synthesizing spinosad.

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 genome-scale metabolic network model of Saccharopolyspora spinosa and its construction method and application. technical background [0002] Spinosad is a macrolide antibiotic produced by aerobic fermentation of Saccharopolyspora spinosa. Due to the unique chemical structure and mechanism of action of spinosyn, it can effectively control various Lepidoptera pests such as diamondback moth and beet armyworm, and is not toxic to mammals, birds, etc. (ACS symposium series. Discovery, isolation, and structure elucidation of a family of structurally unique, fermentation-derived tetracyclic macrolides. 1992). Spinosyn is therefore considered to be the most effective biopesticide after abamectin, and has become a hot spot in the research and development of new biopesticides. Since this compound has the advantage...

Claims

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Application Information

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IPC IPC(8): G06F19/12G06F19/18
Inventor 卢文玉王晓阳张传波薛超友
Owner TIANJIN UNIV
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