Modified Signal Sequences for Bacillus Protein Secretion
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Solution Overview
Problem
Current methods for producing proteins in Gram-positive bacterial cells, such as Bacillus, face limitations in achieving high yields and efficient secretion, with existing signal sequences often leading to poor translation, secretion, and folding of heterologous proteins, resulting in reduced productivity and cellular stress.
Innovation Solution
The development of recombinant Bacillus cells using modified signal sequences, such as the modified SacB and Bli03445 signal sequences, which are operably linked to expression cassettes and introduced into the cells to enhance protein secretion and production, specifically by integrating these sequences into the lysA and serA loci of Bacillus licheniformis strains deficient in native proteases, thereby improving protein yield and secretion efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If native signal sequences are used for protein expression in Bacillus cells, then the expression system is simple and easy to construct, but protein secretion efficiency and translation quality are poor, resulting in reduced productivity
Solution Approach 1:
The patent modifies the native SacB signal sequence by changing specific amino acid residues (such as substituting hydrophobic residues with polar or charged residues) to optimize protein secretion. These parameter changes in the signal sequence structure improve translation efficiency and secretion kinetics without requiring complete redesign of the expression system, thus resolving the contradiction between productivity improvement and system complexity.
Solution Approach 2:
The patent creates modified versions of the native SacB signal sequence by copying its core structure and optimizing specific regions. The modified signal sequences (e.g., SacBΔ1-24, SacBΔ1-27 variants) retain the essential function of the native sequence while improving secretion efficiency, allowing the system to achieve higher productivity without entirely abandoning the original simple construction approach.
2Productivity
If modified signal sequences are introduced to enhance protein secretion, then protein yield and secretion efficiency improve, but the construction and integration process becomes more complex
Solution Approach 1:
The patent performs preliminary optimization of the signal sequence in silico by identifying and modifying key amino acid positions based on known secretion determinants. This preliminary action allows the selection of optimized signal sequences before actual cell construction, reducing the need for extensive trial-and-error experiments and simplifying the overall manufacturing process despite the complexity of signal sequence modification.
Solution Approach 2:
The modified SacB signal sequence acts as an intermediary element between the heterologous protein and the Bacillus secretion machinery. By optimizing this intermediary component, the patent improves the interaction between the expressed protein and the secretion system, enhancing secretion efficiency without requiring complex modifications to the entire expression system or host cell physiology.
3Reliability
If existing signal sequences are used for heterologous protein expression, then the expression system is straightforward, but protein folding and secretion quality are compromised, leading to cellular stress
Solution Approach 1:
The patent applies local quality optimization by modifying specific regions of the SacB signal sequence (such as the hydrophobic core region and the n-region) while maintaining the overall structure. These localized modifications improve protein folding guidance and secretion quality without requiring complete redesign of the signal sequence, thus achieving better reliability with controlled complexity.
Solution Approach 2:
The patent systematically changes physical-chemical parameters of the signal sequence, such as hydrophobicity, charge distribution, and amino acid composition, to optimize protein folding and secretion. By adjusting these parameters in a controlled manner, the patent improves protein quality and reduces cellular stress while maintaining a relatively simple expression system design.
Data Source
AI summary
The present disclosure is generally related to recombinant microbial cells expressing heterologous proteins of interest. Certain aspects of the disclosure are therefore related to, inter alia, recombinant Bacillus cells having enhanced protein production capabilities, novel protein signal sequences, recombinant polynucleotides encoding heterologous proteins of interest, and related compositions and/or methods thereof. Thus, as exemplified herein, the recombinant Bacillus cells of the instant disclosure are particularly suitable for use in the expression, production and secretion of heterologous proteins.