Modified Plasmid Vector Replication Protein for High Productivity
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Solution Overview
Problem
Current methods for producing proteins using recombinant microorganisms are limited by low productivity and restricted to specific types of proteins, with existing plasmid vectors not adequately addressing the need for high-scale production of industrially valuable proteins.
Innovation Solution
A plasmid vector is developed with modified replication proteins containing specific amino acid substitutions at positions 48, 262, 149, and 198, combined with a promoter and secretory signal region from the alkaline cellulase gene of Bacillus spp., to enhance protein secretion and productivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional plasmid vectors are used for protein production, then the production process is simple, but the productivity is low and protein secretion is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the replication protein (Rep) at positions 48, 149, 198, and 262. These parameter changes in the protein sequence directly improve plasmid copy number and protein secretion efficiency, resolving the contradiction between simple vector structure and low productivity
Solution Approach 2:
The patent creates a composite plasmid vector system by combining the modified replication protein gene (rep*) with promoter regions and secretory signal regions from Bacillus spp. cellulase genes. This composite structure integrates replication control with secretion functionality, achieving high productivity while maintaining reasonable structural complexity
2Productivity
If plasmid copy number is increased to improve productivity, then protein production amount increases, but vector stability may be compromised
Solution Approach 1:
The patent carefully selects specific amino acid positions (48, 149, 198, 262) in the replication protein for modification. These parameter changes are strategically chosen to enhance copy number control while preserving the essential functions that maintain plasmid stability during replication and cell division
Solution Approach 2:
The patent applies local quality changes by modifying only specific critical residues in the replication protein rather than the entire protein sequence. This localized modification approach allows copy number enhancement at specific functional sites while maintaining the overall structural integrity and stability functions of the replication protein
Data Source
AI summary
To provide a plasmid vector having high productivity which can be used for large scale production of an industrially valuable and useful protein, and a transformant using the plasmid vector. A plasmid vector having DNA which encodes a plasmid replication protein in which one or more amino acid residues that are selected from (a) position 48, (b) position 262, (c) position 149 and (d) position 198 in the amino acid sequence represented by SEQ ID NO: 2 are substituted with (a) Ala, Gly, Thr, Arg, Glu, Asn or Gln, (b) Gly, Ser, Thr, Cys or Val, (c) Asn, and (d) Glu, respectively.