Isobutanol synthetic bacterium genome dimension metabolic network model and molecular modification method

A genome-scale, metabolic network technology, applied in the field of metabolic engineering molecular transformation of microbial synthetic bacteria

Inactive Publication Date: 2012-11-07
TIANJIN UNIV
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Problems solved by technology

Up to now, there are no relevant patents and literature reports that use the genome-scale metabolic network model of isobutanol-synthesizing bacteria (Bacillus subtilis) to predict key genes and key enzymes, and then guide the molecular transformation of engineering strains

Method used

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  • Isobutanol synthetic bacterium genome dimension metabolic network model and molecular modification method
  • Isobutanol synthetic bacterium genome dimension metabolic network model and molecular modification method
  • Isobutanol synthetic bacterium genome dimension metabolic network model and molecular modification method

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Embodiment 1

[0031] Based on the metabolic network model of the Bacillus subtilis type strain, the characteristic reactions of isobutanol biosynthesis were added. The model uses glucose as the sole carbon source, and other subsystems except central carbon metabolism (glycolysis, pentose phosphate pathway and tricarboxylic acid cycle), such as amino acid synthesis, nucleic acid synthesis, lipid synthesis, energy and coenzyme synthesis, And the linear pathways in cell wall synthesis were combined, and the related transport reactions of substrate absorption and product secretion were added. A total of 130-150 reactions were included to obtain the metabolic network model of isobutanol synthesizing bacteria.

Embodiment 2

[0033] Transform the metabolic network model of isobutanol-synthesizing bacteria into a recognizable mathematical matrix model, and use the elementary pattern analysis algorithm with the help of the METATOOL tool (version 5.1, 2008) to obtain all relevant patterns that can exist independently in the strain. In EXCEL, from 10000-12000 models, it is determined that there is a model for isobutanol synthesis, of which the maximum theoretical yield of isobutanol is 0.6-0.7C-mol / C-mol, and the maximum theoretical value of bacterial cell yield is 0.5-0.6 C-mol / C-mol (eg figure 1 ). After screening for these modes, the linear correlation of each reaction with the target reaction in these modes was calculated. A linear correlation coefficient of 65-80% indicates that the reaction is related to isobutanol synthesis, and a positive correlation indicates that the gene encoding the enzyme that catalyzes the reaction has a positive effect on isobutanol, and vice versa. The standard deviat...

Embodiment 3

[0036] From the two most important key genes obtained in Example 2, calculate the average value, variance and standard deviation coefficient of the relative flux of carbon flow in these modes for each reaction. The relative flux of carbon flow in each reaction is based on the glucose absorption rate. By simulating the genes ldh and pdhC that have the greatest impact on the synthesis of isobutanol, the effect of gene knockout on the flux of each reaction in the central carbon metabolism of the whole cell is determined. influence, such as figure 2 . Compared with the wild strain BSUL03, the central metabolic pathway simulated by the single-gene modification BSUΔldh, including the pentose phosphate pathway and the citric acid cycle pathway, was 40%-70% larger, and the glycolysis pathway was not changed much, only reduced by 4 %, the flux of isobutanol synthesis pathway increased by 1-1.5 times. For the flux distribution simulated by double gene knockout, the pentose phosphate ...

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Abstract

The invention relates to an isobutanol synthetic bacterium genome dimension metabolic network model and a molecular modification method. According to the method, the necessary path for the thallus growth and the isobutanol synthesis is calculated by a network module, the metabolic network model is subjected to the element mode analysis, the standard deviation coefficient of each gene is calculated, and the isobutanol biosynthesis yield of different genes is determined; the two key genes including an L-lactate dehydrogenase gene 1dh and a pyruvate dehydrogenase complex E2 subunit coding gene pdhC which are most important to the isobutanol biosynthesis are predicted according to a principle that the standard deviation coefficient is smaller than 0.35, the standard deviation coefficients of the two genes are respectively 2.5 to 3 and 1.5 to 2; and through the determination of the two genes, the isobutanol yield can be improved, and 0.5-0.6C-mol / C-mol glucose can be reached. The key genes which are most important to the isobutanol biosynthesis are obtained through utilizing the relative flux value and are used as modification target spots, the molecular modification of the isobutanol synthetic bacterium is guided, and the isobutanol yield is improved.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering molecular transformation of microbial synthetic bacteria, and in particular relates to a genome-scale metabolic network model and molecular transformation method of isobutanol synthetic bacteria. Background technique [0002] At present, the energy crisis and environmental problems are imminent, and there is an urgent need to change the status quo of today's social development that is overly dependent on fossil fuels. With the continuous progress and development of biotechnology, research on bioethanol, biodiesel and biobutanol has become increasingly extensive. The development and utilization of green fuel has long-term significance for alleviating the energy crisis, solving environmental problems, and realizing sustainable development. [0003] Isobutanol is a four-carbon platform compound widely used in many industrial production fields such as chemical industry, medicine and ene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G06F19/12C12N15/75C12N1/21C12R1/125
Inventor 闻建平黄笛李珊珊贾晓强
Owner TIANJIN UNIV
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