CN-119626320-B - Construction and analysis method of paracoccus denitrificans genome scale metabolic network model

CN119626320BCN 119626320 BCN119626320 BCN 119626320BCN-119626320-B

Abstract

The invention discloses a construction and analysis method of a paracoccus denitrificans genome scale metabolic network model, and belongs to the technical field of system biology. The method comprises the steps of whole genome annotation, obtaining global metabolic reaction data of the paracoccus denitrificans, automatically searching genome information and constructing a model 1 based on species codes of the paracoccus denitrificans in a KEGG database and genome annotation results, identifying homologous proteins in the genome of the paracoccus denitrificans by carrying out homology search on proteins of a target organism based on a pre-trained hidden Markov model, adding additional metabolic reactions, constructing a model 2, and integrating the model 1 and the model 2. The invention can efficiently design and reform the denitrification engineering to realize the precise regulation and control of the nitrogen degradation process, and compared with the existing metabolic engineering reforming method, the invention can effectively reduce the workload of exploratory experiments and has great propulsion on deep knowledge of the nitrogen degradation characteristics of the paracoccus denitrification.

Inventors

DENG YU
ZHOU SHENGHU
CAO WENYAN
XU SHA
CHAI LIJUAN
MAO YIN

Assignees

江南大学

Dates

Publication Date: 20260505
Application Date: 20241119

Claims (5)

1. A method for constructing a paracoccus denitrificans genome-scale metabolic network model, comprising the steps of: Step 1, carrying out whole genome annotation according to the published genome sequencing result of the paracoccus denitrificans; step 2, global metabolic reaction data of the paracoccus denitrificans are obtained, key information of metabolic reaction is extracted, and metabolic reaction and metabolite names are standardized; Step 3, automatically searching genome information and constructing a model 1 based on the species codes and genome annotation results of the paracoccus denitrificans in the KEGG database by using a RAVEN tool box of the MATLAB platform; Step 4, using a RAVEN kit on a MATLAB platform, based on species codes, genome annotation results and a pre-trained hidden Markov model of the paracoccus denitrificans in a KEGG database, identifying homologous proteins in the genome of the paracoccus denitrificans by carrying out homology search on proteins of a target organism, and adding additional metabolic reactions to construct a model 2; Integrating the model 1 and the model 2 which are automatically constructed to obtain a paracoccus denitrificans genome scale metabolic network model, wherein the metabolic network model has a structure that metabolic reaction and metabolites form a stoichiometric matrix on an MATLAB platform, the rows of the matrix represent the metabolites, the columns represent the metabolic reaction, and the numerical values represent the stoichiometric coefficient; the method further includes manually refining and optimizing the model, including: unifying and standardizing metabolic reactions and metabolite name formats, replacing the KEGG reaction names and the metabolite names in the model to be the corresponding reactions and metabolite names of BiGG databases, and increasing the universality of the model; Manually supplementing the lacking chemical formula and name information of the metabolites, preparing a table containing the chemical formula and name of each metabolite of the paracoccus denitrificans, reading the table by using MATLAB script, and updating the metabolite information in the model; Referring to the existing metabolic model to reference the exchange reaction, adding the exchange reaction suitable for the metabolic demand of the paracoccus denitrificans into the model to allow the substances to enter and exit from the environment; adding transport reactions according to the transport protein identification result; Deleting the inappropriate response; Performing mass balance check on all reactions; Setting reversibility of each reaction in the model, and defining upper limit and lower limit of reversible metabolic reaction to be 1,000-1000 mmol g -1 h -1 , setting irreversible reaction to be 0-1000 mmol g -1 h -1 , setting upper limit and lower limit of exchange reaction to be-20-1000 mmol g -1 h -1 ; the step 1 adopts Prokka annotation tools; step 1, downloading a complete paracoccus denitrificans whole genome file from an NCBI database; Step 2, downloading KGML files by using an API provided by the KEGG database; And 3, constructing a model 1 by adopting getKEGGModelForOrganism functions of a RAVEN tool box.
2. A method for analyzing key metabolic pathways affecting nitrogen metabolism, which is characterized by comprising the steps of converting a paracoccus denitrificans genome-scale metabolic network model constructed by the method according to claim 1 into a computer-recognizable mathematical coefficient matrix, calculating metabolic flux distribution of other metabolic pathways by using a flow balance analysis tool in a MATLAB platform RAVEN kit and using nitrogen synthesis maximization as an objective function, and identifying the key metabolic pathways.
3. A bottleneck metabolic reaction analysis method for influencing nitrogen metabolism is characterized by comprising the steps of converting a paracoccus denitrificans genome scale metabolic network model constructed by the method according to claim 1 into a computer-recognizable mathematical coefficient matrix, calculating the minimum and maximum fluxes of other metabolic reactions by using a flux variation analysis tool in a MATLAB platform RAVEN kit, and identifying bottleneck reactions by comparing the widths of the flux variation intervals.
4. An electronic device comprising a memory and a processor; The memory is used for storing a computer program; The processor being adapted to implement the method of claim 1 when executing the computer program.
5. A computer readable storage medium, characterized in that the storage medium has stored thereon a computer program which, when executed by a processor, implements the method according to claim 1.

Description

Construction and analysis method of paracoccus denitrificans genome scale metabolic network model Technical Field The invention relates to a construction and analysis method of a paracoccus denitrificans genome scale metabolic network model, and belongs to the technical field of system biology. Background Paracoccus denitrificans is a gram-negative bacterium which is widely existing in soil and sludge, and can utilize nitrate to carry out denitrification reaction under anaerobic or aerobic condition to generate gaseous nitrogen. Because of the good nitrogen degradation and environmental protection characteristics, the strain can be used as a potential biological denitrification strain and applied to biological repair and sewage treatment. However, naturally occurring paracoccus denitrificans generally grow at a relatively slow rate, and the degradation rate of nitrogen is affected by a variety of factors, failing to meet the demands of industrial denitrification. Along with the development of biotechnology, researchers are mainly focused on traditional metabolic engineering transformation to improve the denitrification capability of paracoccus denitrificans, and the gene of a nitrogen degradation pathway is subjected to partial knockout or overexpression transformation by utilizing a genetic engineering means. However, the transformation of the local level has a certain blindness, the accurate regulation and control of the high-efficiency degradation of nitrogen can not be realized, and the transformation result still is difficult to meet the actual requirements. Therefore, the paracoccus denitrificans needs to be comprehensively recognized systematically, and the denitrification function is designed and modified efficiently on the basis of the recognition. Disclosure of Invention In order to improve the efficiency of denitrification engineering design and transformation and realize the accurate regulation and control of the nitrogen degradation process of paracoccus denitrificans, the invention provides a construction and analysis method of a paracoccus denitrificans genome scale metabolic network model, which comprises the following technical scheme: The construction method of the paracoccus denitrificans genome scale metabolic network model comprises the following steps: Step 1, carrying out whole genome annotation according to the published genome sequencing result of the paracoccus denitrificans; step 2, global metabolic reaction data of the paracoccus denitrificans are obtained, key information of metabolic reaction is extracted, and metabolic reaction and metabolite names are standardized; Step 3, automatically searching genome information and constructing a model 1 based on the species codes and genome annotation results of the paracoccus denitrificans in the KEGG database by using a RAVEN tool box of the MATLAB platform; Step 4, using a RAVEN kit on a MATLAB platform, based on species codes, genome annotation results and a pre-trained hidden Markov model of the paracoccus denitrificans in a KEGG database, identifying homologous proteins in the genome of the paracoccus denitrificans by carrying out homology search on proteins of a target organism, and adding additional metabolic reactions to construct a model 2; and 5, integrating the two automatically constructed models 1 and 2 to obtain the paracoccus denitrificans genome scale metabolic network model. Optionally, the method further comprises manually refining and optimizing the model, including adding exchange reactions with reference to existing metabolic models, adding transport reactions based on transporter identification results, deleting duplicate reactions, mass balance checks, and reversibility settings. Optionally, the step 1 uses Prokka annotation tools. Optionally, step 1 downloads the complete paracoccus denitrificans whole genome file from the NCBI database. Optionally, step2 downloads the KGML file using an API provided by the KEGG database. Optionally, the step 3 constructs the model 1 using getKEGGModelForOrganism functions of the RAVEN toolbox. The invention provides a key metabolic pathway analysis method for influencing nitrogen metabolism, which comprises the steps of converting a paracoccus denitrificans genome scale metabolic network model constructed by any one of the above methods into a mathematical coefficient matrix which can be identified by a computer, and calculating metabolic flux distribution of other metabolic pathways by using a flow balance analysis tool in a MATLAB platform RAVEN tool kit and using nitrogen synthesis maximization as an objective function to identify key metabolic pathways. The invention provides a bottleneck metabolic reaction analysis method for influencing nitrogen metabolism, which comprises the steps of converting a paracoccus denitrificans genome scale metabolic network model constructed by any one of the above methods into a mathematical coefficient matrix which can be identified by a com