Type III Secretion Strain Engineering for Higher Protein Titers
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Solution Overview
Problem
Existing bacterial protein production systems face challenges such as low secretion titers, high purification costs, and toxicity of heterologous proteins, particularly for products like spider silk, due to traditional intracellular expression strategies and sensitivity of the Type III secretion system to environmental conditions.
Innovation Solution
A recombinant Salmonella strain with deletions or modifications, such as hilE knockout and hilD expression enhancement through 3'UTR insertion, combined with an optimized culture medium containing glycerol and high phosphate buffer, significantly increases protein secretion titer and purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional intracellular expression strategies are used, then bacterial protein production is simple, but product recovery requires complex downstream purification steps and proteins aggregate in inclusion bodies
Solution Approach 1:
The invention extracts the heterologous protein from the intracellular space and directs it to the extracellular environment through the T3SS secretion system. By using a targeting polypeptide fusion, the protein is routed through the type III secretion needle complex to be released into the culture medium, eliminating the need for cell lysis and complex purification steps while maintaining production simplicity.
2Manufacturing precision
If heterologous proteins are overexpressed intracellularly, then initial product purity may be higher, but resolubilization and refolding processes are required for each product
Solution Approach 1:
The invention prevents protein aggregation in inclusion bodies by extracting the heterologous protein from the intracellular environment and secreting it directly into the extracellular space. The T3SS secretion system releases the protein in a soluble, native conformation, eliminating the need for resolubilization and refolding processes and significantly improving production efficiency.
3Ease of operation
If the Type III secretion system is used for protein secretion, then proteins are secreted from cytoplasm to extracellular space in a single step, but secretion titers remain below 1 g/L even with hilA overexpression
Solution Approach 1:
The invention optimizes secretion titers by changing key parameters: (1) Using a strong constitutive promoter (J23100) to drive continuous expression of the targeting polypeptide-heterologous protein fusion, (2) Co-expressing chaperones (IpaD and IpaC) to ensure proper folding and secretion efficiency, (3) Optimizing the targeting polypeptide sequence for efficient T3SS recognition, and (4) Culturing in optimized media conditions. These parameter changes achieve secretion titers exceeding 1 g/L while maintaining the simplicity of the single-step secretion process.
4Quantity of substance
If hilA is overexpressed from a plasmid to achieve high secretion titers, then secretion system function is enhanced, but the system becomes more complex and requires synthetic induction
Solution Approach 1:
The invention merges the expression of the targeting polypeptide-heterologous protein fusion and the T3SS master regulator hilA into a single plasmid system. The plasmid contains both the gene of interest with targeting polypeptide under the control of promoter J23100 and the hilA gene under the control of promoter J23101, along with chaperone genes. This integrated design simplifies the system by eliminating the need for separate plasmids and synthetic induction, while maintaining high secretion titers through constitutive expression.
Data Source
AI summary
The present disclosure provides a recombinant Salmonella strain having a Type III secretion system (T3SS) comprising mutation which enhance protein expression and production. Additionally, methods and kits for using the recombinant Salmonella strain for producing a protein of interest are provided. Additionally, an optimized medium that increases protein expression in a Salmonella strain having a Type III secretion system (T3SS) is provided.