Modified Bacillus Cells for Increased Protein Yield
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Solution Overview
Problem
Current methods for optimizing protein production in Bacillus host cells, such as Bacillus licheniformis and Bacillus subtilis, face limitations in achieving significant increases in protein yield and secondary metabolite production, despite their use as microbial factories due to their excellent fermentation properties.
Innovation Solution
The development of mutant Bacillus cells with modified glcT genes encoding variant GlcT proteins, specifically with a phenylalanine substitution at amino acid position 67, and the introduction of restored rghR2 genes, along with modified 5'-untranslated regions, to enhance protein production capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional Bacillus host cells are used for protein production, then fermentation properties are excellent, but protein yield increases are limited
Solution Approach 1:
The patent applies parameter changes by modifying the glcT gene at the molecular level, specifically changing amino acid position 67 from leucine to phenylalanine. This genetic parameter modification transforms the Bacillus host cell to achieve up to 9% increased protein production while preserving the organism's inherent fermentation stability and safety profile.
2Productivity
If genetic modifications are introduced to increase protein production, then protein yield increases, but genetic modification complexity increases
Solution Approach 1:
The patent applies local quality by implementing a targeted point mutation at a specific location within the glcT gene (amino acid position 67). Rather than complex genome-wide modifications, only a single nucleotide change is introduced, creating a localized genetic modification that achieves enhanced protein production with minimal complexity. The modification is precisely localized to change leucine to phenylalanine at position 67.
3Productivity
If mutant Bacillus cells with modified glcT genes are used, then protein production increases by up to 9%, but manufacturing process complexity increases
Solution Approach 1:
The patent utilizes parameter changes by modifying a single genetic parameter (the glcT gene sequence at position 67) to achieve improved protein production. This minimal parameter change approach maintains manufacturing simplicity because the modification can be stably inherited by daughter cells during fermentation, avoiding the need for continuous process adjustments or complex manufacturing protocols.
Data Source
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AI summary
The instant disclosure is generally related to novel Bacillus sp. mutants capable of producing increased amounts of industrially relevant proteins of interest. Certain embodiments of the disclosure are related to modified Bacillus sp. cells comprising an introduced polynucleotide encoding a variant GlcT protein. Other embodiments are related to methods and compositions for producing endogenous and/or heterologous proteins of interest in the modified Bacillus sp. (daughter) cells, whereas certain other embodiments are directed to nucleic acid sequences, particularly polynucleotide open reading frame (ORF) sequences, vectors thereof and DNA expression constructs thereof, encoding variant GlcT proteins of the disclosure.