Bacillus Cry Protein Production via High-Copy Plasmid
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Solution Overview
Problem
Current methods for producing Cry proteins in Bacillus bacterium, such as Bacillus thuringiensis, face challenges in achieving high expression levels due to limitations in plasmid copy number and promoter regulation, resulting in low productivity and inhibition of sporulation, making it difficult to produce large amounts of active Cry proteins.
Innovation Solution
A method involving transformation of Bacillus bacterium with an expression plasmid containing a Cry protein gene linked to a σA-dependent or σH-dependent promoter, along with a replication protein sequence, to enhance intracellular production of Cry proteins, utilizing a high-copy-number plasmid and optimizing culture conditions for high expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a high-copy-number plasmid is used to increase Cry protein production, then productivity is improved, but sporulation is inhibited
Solution Approach 1:
The invention changes the copy number parameter of the plasmid from low-copy (theta-replicating) to high-copy (rolling circle replicating), which directly increases the amount of Cry protein produced while accepting the trade-off of sporulation inhibition. This parameter change enables high-level expression of the cry gene.
2Productivity
If cry gene copy number is increased to enhance expression, then productivity is improved, but physiological equilibrium changes causing sporulation inhibition
Solution Approach 1:
The invention deliberately changes the copy number parameter from natural low-copy to engineered high-copy, which disrupts the natural physiological equilibrium but achieves the goal of high-level Cry protein expression. The system accepts this imbalance as necessary for productivity.
3Stability of the object's composition
If a theta-replicating plasmid is used, then physiological stability is maintained, but Cry protein production amount is limited due to low copy number
Solution Approach 1:
The invention changes the replication mechanism parameter from theta-replication to rolling circle replication, which fundamentally alters the copy number from low to high. This parameter change prioritizes productivity over the physiological stability associated with natural plasmid systems.
4Reliability
If intracellular expression is used, then host cell safety is maintained, but protein forms insoluble aggregates requiring complex recovery procedures
Solution Approach 1:
The invention extracts the Cry protein from the intracellular environment where it forms insoluble aggregates and relocates it to the extracellular space where it remains soluble and active. This extraction approach eliminates the need for complex solubilization and refolding procedures while maintaining host cell safety.
Data Source
AI summary
Provided is a method for intracellularly producing a large amount of a Cry protein in a Bacillus bacterium. A method for producing a Cry protein or a culture product comprising the Cry protein, comprising transforming a Bacillus bacterium with an expression plasmid incorporating a gene encoding the Cry protein operably linked to a regulatory region comprising a σA-dependent promoter or a σH-dependent promoter, and culturing the transformed cell, wherein the expression plasmid comprises a polynucleotide encoding a replication protein consisting of the amino acid sequence set forth in SEQ ID NO: 9 or a protein having an identity of 80% or more or more with the amino acid sequence of the replication protein and involved in replication initiation.