Recombinant Cell DNA Amplification via Specific Polynucleotide Mediator
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for amplifying giant size DNA regions in genomes, particularly for biosynthesis of secondary metabolites like antibiotics, are inefficient and unstable, with existing techniques struggling to reproduce high production strains and maintain stability, especially for large gene clusters.
Innovation Solution
A process involving recombinant cells with specific polynucleotides that encode proteins or are functionally equivalent, allowing for efficient amplification of DNA units between 22 to 154 kb, including the kanamycin biosynthetic gene cluster, by culturing these cells under conditions that promote gene amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If high-copy type plasmids are used to increase gene copy number, then the copy number is improved, but the stability is worsened
Solution Approach 1:
The patent introduces a specific polynucleotide sequence (SEQ ID NO: 2) as an intermediary element that mediates between the plasmid vector and the host chromosome. This polynucleotide enables stable integration and amplification of the gene cluster without the instability associated with high-copy plasmids, resolving the contradiction between high copy number and stability.
Solution Approach 2:
The patent employs a copying mechanism where the gene cluster is amplified through repeated replication cycles in the host cell. The specific polynucleotide sequence serves as a template for copying, enabling the generation of multiple copies (36 or more) of the gene cluster while maintaining stability through chromosomal integration rather than plasmid maintenance.
2Reliability
If cosmid vector and BAC vector are used to clone long region DNA, then the stability is improved, but the copy number is worsened
Solution Approach 1:
The patent changes the key parameter of copy number by utilizing a specific polynucleotide sequence that enables amplification to 36 or more copies. This differs from conventional cosmid and BAC vectors that maintain low copy numbers, thereby resolving the contradiction between stability and copy number through parameter optimization.
3Productivity
If mutation process is used for strain improvement, then the productivity is improved, but the labor and time required are worsened
Solution Approach 1:
The patent applies preliminary action by directly introducing the specific polynucleotide sequence (SEQ ID NO: 2) into the host cell before the amplification process begins. This pre-introduction of the amplification mechanism eliminates the need for lengthy mutation screening processes, achieving rapid strain improvement with high productivity.
Solution Approach 2:
The patent replaces the mechanical process of repeated mutagenesis and screening with a molecular biology approach using specific polynucleotide sequences that directly enable amplification. This substitution of methods dramatically reduces the time and labor required while maintaining or improving productivity.
4Productivity
If repeated sorting and breeding improvements are used, then the productivity is improved, but the reproducibility is worsened
Solution Approach 1:
The patent uses copying through the specific polynucleotide sequence that serves as a template for amplification. This molecular copying mechanism produces identical copies of the gene cluster with high reproducibility, eliminating the variability inherent in repeated sorting and breeding processes while maintaining high productivity.
Data Source
Figure 1
Figure 2
AI summary
The present invention relates to a process for efficiently amplifying a giant DNA. More particularly, the present invention relates to a process for amplifying DNA in a cell, comprising amplifying the DNA as the target of amplification in the presence of DNAs selected from the following (i), (ii) and (iii): (i) DNA encoding a protein selected from the following 1), 2) and 3): 1) a protein consisting of the amino acid sequence of SEQ ID NO: 1, 2) a protein comprising an amino acid sequence which has a deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1, and 3) a protein comprising an amino acid sequence which has an identity of 90% or more to the amino acid sequence of SEQ ID NO: 1, (ii) DNA consisting of the nucleotide sequence of SEQ ID NO: 2, and (iii) DNA hybridizing to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions.