CN-121999875-A - Rapid excavation method of epimerase gene

Abstract

The invention relates to the field of bioengineering and genetic engineering, and particularly provides a rapid excavation method of isomerase genes. The method is based on big data analysis and bioinformatics technology, combines the means of sequence comparison, gene expression analysis, function prediction and the like, realizes rapid and accurate excavation of isomerase genes, and provides powerful technical support for development and application in the field of enzyme engineering.

Inventors

• LI JINSHAN
• TAN MING
• CHEN LU
• XU JIANYONG

Assignees

• 天工生物科技（天津）有限公司

Dates

Publication Date

20260508

Application Date

20241104

Claims (6)

1. 1. A rapid mining method for isomerase genes is characterized by comprising the following steps of collecting relevant information of the isomerase, constructing a data mining model, screening candidate genes by using a sequence comparison module, determining expressed active genes by using a gene expression analysis module, performing functional prediction by using a functional prediction module, verifying the accuracy of a prediction result, and finally determining target isomerase genes.
2. 2. The rapid mining method of isomerase genes according to claim 1, wherein the data mining model comprises a sequence alignment module, a gene expression analysis module, and a functional prediction module.
3. 3. The rapid mining method of isomerase genes according to claim 1 or 2, wherein the sequence alignment module is used for large-scale sequence alignment in a public database to screen candidate genes having similar sequences to known isomerase genes.
4. 4. The rapid mining method of isomerase genes according to any one of the preceding claims, wherein the gene expression analysis module is used to analyze the expression levels of the candidate genes selected to determine which genes have expression activity in the actual organism.
5. 5. The rapid excavation method of isomerase genes according to any one of the preceding claims, wherein the function prediction module is used for performing function prediction on genes having expression activity, and verifying accuracy of prediction results in combination with in vitro enzyme activity assay experiments.
6. 6. The rapid prototyping method of isomerase genes according to any one of the preceding claims, further comprising the step of optimizing and engineering the prototyped isomerase genes to improve the catalytic activity and stability of the enzyme.

Description

Rapid excavation method of epimerase gene Technical Field The invention relates to the fields of bioengineering and genetic engineering, in particular to a high-efficiency excavation and screening method for psicose 3-epimerase (Allulose-epimerase) genes. Background Psicose is a natural rare sugar and has the characteristics of low calorie and no rise of blood sugar, so that the psicose has wide application prospect in the fields of food, medicine and the like. Psicose 3-epimerase is a key enzyme for synthesizing psicose, and has important significance for large-scale biological production of psicose. However, the current excavation and identification of psicose 3-epimerase genes still face the problems of low efficiency, long screening period and the like. Disclosure of Invention The invention provides a rapid and efficient psicose 3-epimerase gene excavation method, which comprises the following steps of: data collection and pretreatment: Genomic sequence data of the target organism is collected, including but not limited to humans, E.coli, saccharomyces cerevisiae, and the like. The genomic sequence is quality controlled and filtered to remove repetitive and low quality sequences. Gene identification and classification: gene prediction software such as GENEMARKS, AUGUSTUS or the like is used to identify gene sequences. The identified genes are classified into known functional genes and unknown functional genes according to sequence characteristics. Homology search and cluster analysis: a homology search is performed on unknown functional genes using BLAST and other tools to find sequences that have similarity to known psicose 3-epimerases. Sequences with similarity are clustered using a clustering algorithm such as CD-HIT to form a candidate set of isomerase genes. Functional domain identification and verification: the active sites and domains in the candidate sequence are identified by a database such as Pfam, interPro. The function of candidate genes is verified by gene knockout, overexpression or in vitro enzyme activity experiments. Analysis of Gene expression Pattern: the expression pattern of candidate genes at different tissues and developmental stages was analyzed using real-time quantitative PCR (qPCR) or RNA sequencing (RNA-seq) techniques. And (3) verifying the activity of the isomerase: the catalytic activity and substrate specificity of the enzyme encoded by the candidate gene was determined by in vitro enzymatic experiments. The isomerase gene mining method of the invention has the following advantages: Based on big data analysis and bioinformatics technology, rapid and accurate excavation of isomerase genes is realized; the accuracy and the reliability of the mining result are improved by combining means of sequence comparison, gene expression analysis, function prediction and the like; the method is suitable for large-scale genome data mining, and provides abundant gene resources for isomerase development and application; The excavated isomerase gene can be optimized and modified according to actual demands, and the catalytic performance of the enzyme is improved. Detailed Description The present invention will be described in detail with reference to specific examples. Examples Target organism human Data Source NCBI RefSeq database By BLAST search, 10 sequences having homology of 90% or more with the known human psicose 3-epimerase gene were identified. By qPCR analysis, 3 genes were found to be expressed significantly higher in liver tissue than in other tissues. Examples Coli Data Source E.coli K-12 MG1655 genome sequence 100 Unknown functional genes are screened out by GENEMARKS predicted genes. 5 Candidate genes clustered with the known AKR genes are screened out through homology cluster analysis. In vitro enzyme activity experiments show that 2 candidate genes have remarkable psicose 3-epimerase activity. Examples Target organism Saccharomyces cerevisiae Data Source Saccharomyces CEREVISIAE S288c genomic sequence 20 Candidate genes with psicose 3-epimerase active sites were identified by the InterPro database. Through RNA-seq analysis, the expression level of 3 genes is found to be significantly up-regulated under a high sugar environment. The gene knockout experiments prove that the deletion of the 3 genes significantly affects the metabolism of saccharides by the yeast. Examples Target organism Arabidopsis thaliana Data Source Arabidopsis thaliana genomic sequence 200 Unknown functional genes are screened out by utilizing Augustus software predicted genes. 10 Candidate genes with psicose 3-epimerase signature domains were identified by Pfam database analysis. Through transgenic plant experiments, 1 candidate gene can improve tolerance of plants to sugar stress. Example 5 genome data mining and bioinformatics analysis procedure keyword searches were performed using the disclosed microbial genome database, and potential A3-RE genes were identified in combination with Hidden Markov M