Lactococcus lactis Strains with Inhibited Surface Proteolysis
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Solution Overview
Problem
Gram-positive bacteria such as Lactococcus lactis and Streptococcus thermophilus exhibit high surface proteolysis, leading to degradation of proteins of interest during production, resulting in reduced yield and altered protein structure and activity.
Innovation Solution
Development of bacterial strains with decreased or abolished expression and activity of endogenous surface proteases, specifically targeting Ster-1612, HtrA, and PrtS proteases, through mutagenesis and gene inactivation, to reduce surface proteolysis and enhance protein production efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild type bacterial strains are used for protein production, then natural proteolytic activity helps in protein processing, but surface proteases degrade heterologous proteins leading to reduced yield and altered structure
Solution Approach 1:
The invention removes the harmful surface proteases (PrtS, HtrA, and Ster-1612) from the bacterial system through genetic deletion. This extraction of the harmful element eliminates the degradation of heterologous proteins while preserving the beneficial functions of the bacterium for protein production.
Solution Approach 2:
The invention converts the harmful proteolytic activity into a benefit by selectively removing only the surface proteases responsible for degradation, while maintaining other cellular functions. The resulting strain has improved productivity because the harmful proteolysis is eliminated without compromising essential bacterial processes.
2Reliability
If multiple proteases are deleted to reduce surface proteolysis, then protein degradation is reduced, but genetic modification complexity increases
Solution Approach 1:
The invention segments the proteolytic system into three distinct components (PrtS, HtrA, and Ster-1612) and selectively removes each through separate genetic deletions. This segmentation allows precise control over which proteases are eliminated, enabling systematic optimization of protein stability while tracking the impact of each deletion.
Solution Approach 2:
The invention combines multiple genetic deletions (prtS, htrA, and ster-1612) into a single integrated strain. By merging these modifications, the bacterium achieves cumulative protection against protein degradation, with each deletion contributing additively to the overall stability of heterologous proteins.
3Manufacturing precision
If surface proteases are inactivated to prevent protein degradation, then protein structure is preserved, but proteolytic processing of native proteins may be affected
Solution Approach 1:
The invention applies local quality by specifically targeting surface-localized proteases (PrtS, HtrA, and Ster-1612) for deletion while leaving cytoplasmic and other cellular proteases intact. This localized approach preserves the structural integrity of secreted and surface proteins without compromising the bacterium's overall proteolytic processing capabilities for native protein maturation.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Significantly decreases surface proteolysis, leading to improved yield and stability of heterologous proteins, with strains like S. thermophilus LMD9 and L. lactis IL1403 showing reduced degradation of proteins like IL-10 and elafin, resulting in higher production levels and increased enzymatic activity.
Implementation Method 1
surface proteolysis leads to a degradation of these proteins during and/or after their export
Implementation Method 2
One, a serine protease of the subtilisin family, called PrtS in Streptococcus thermophilus and PrtP in Lactococcus lactis
Data Source
AI summary
The present invention relates to a bacterium, in which the expression and/or the activity of surface proteases is/are inhibited, to its preparation process and to the uses of this bacterium.