Mutated Lpp Fusion Strain for Extracellular Protein Secretion
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Solution Overview
Problem
Current bacterial strains used for recombinant protein production, particularly 'leaky' strains, suffer from early cell lysis, leading to reduced protein yield, increased medium viscosity, and complex purification processes, which are costly and inefficient.
Innovation Solution
A bacterial strain with a modified Lpp fusion protein, encoded by a 2xLpp gene, is developed, which includes specific mutations in the N-terminal and C-terminal portions of the Lpp protein, preventing post-translational modifications and enhancing extracellular protein secretion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If leaky strains with altered lipoprotein components are used to increase protein release into the medium, then the amount of target protein in the fermentation medium is improved, but cell lysis occurs early and extensively leading to reduced productivity
Solution Approach 1:
The invention applies parameter changes by modifying the lpp gene to create specific mutations (lpp1, lpp3, Δlpp) that alter the Braun's lipoprotein structure. These parameter changes in the lipoprotein components allow controlled protein leakage into the medium while preventing the extensive cell lysis that occurs with conventional leaky strains, thus resolving the contradiction between protein release and productivity
Solution Approach 2:
The invention segments the approach to protein secretion by using targeted mutations in specific regions of the lpp gene rather than complete deletion or random mutations. The mutations are localized to specific amino acid positions (e.g., Gly14Asp substitution in lpp3, or deletion of specific residues in Δlpp), allowing precise control over the degree of membrane permeability and protein release while maintaining cell integrity
2Ease of operation
If leaky strains are used to release proteins into the medium, then extracellular production is achieved, but the production phase is shortened due to early cell lysis
Solution Approach 1:
The invention uses parameter changes in the lpp gene structure (specific mutations at defined positions) to create a balanced state where the outer membrane remains sufficiently intact to maintain cell viability throughout the production phase, while still allowing adequate protein secretion. This resolves the contradiction by adjusting the membrane permeability parameters to prevent premature cell lysis
3Quantity of substance
If leaky strains are used for protein production, then protein release into medium is improved, but medium viscosity increases due to DNA release from cell lysis complicating purification
Solution Approach 1:
The invention applies parameter changes to the lpp gene that control the degree of cell membrane permeability, allowing protein release while preventing the extensive cell lysis that releases DNA into the medium. By carefully adjusting the mutation parameters in the lipoprotein structure, the system achieves protein secretion without the harmful side effect of DNA contamination that complicates purification
Data Source
Figure 1A~1C
Figure 2
AI summary
The invention relates to a bacterial strain containing at least one gene encoding a recombinant protein, characterized in that it contains an open reading frame consisting of (i) a DNA fragment encoding an N-terminal signal peptide which mediates the translocation of the protein into the periplasm, linked to (ii) a following DNA sequence (lpp(N)) encoding a mutated lipoprotein (Lpp(N)), which exhibits a difference in at most ten amino acids in comparison with the lipoprotein Lpp encoded by the wild-type lpp gene, and (iii) a further DNA sequence (lpp(C)) encoding a mutated lipoprotein (Lpp(C)), which exhibits a difference in at most ten amino acids in comparison with the lipoprotein (Lpp) encoded by the wild-type lpp gene. The invention further relates to a method for the fermentative production of recombinant proteins using the bacterial strain according to the invention. In this way, it is possible to achieve increased amounts, compared to the prior art, of recombinant protein in the culture medium.