Pro-Region Mutations in Gram-Positive Cells for Protein Secretion
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Solution Overview
Problem
Current methods for protein production in Gram-positive bacterial cells are inefficient, necessitating the development of more effective strategies to enhance the secretion and production of proteins of interest for industrial applications.
Innovation Solution
Introduction of novel pro-region nucleic acid sequences operably linked to DNA sequences encoding proteins of interest in recombinant Gram-positive bacterial strains, including variant pro-region sequences with amino acid substitutions, insertions, and modifications, to improve protein secretion and production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional protein production methods are used in Gram-positive bacterial cells, then the production process is simpler, but the productivity and protein secretion efficiency are low
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at specific positions (1, 2, 3, 4, 6, 14, 16, 19, 20, 23, 36, 37, 38, 39, 42, 43, 44, 49, 50, 64, 65, 67, 68, 71, 79, 83, 84) in the pro-region sequence to optimize protein secretion efficiency. Multiple variant sequences with different amino acid substitutions are generated and tested to identify combinations that maximize productivity
Solution Approach 2:
The patent implements preliminary action by designing and constructing a comprehensive library of pro-region variant sequences before actual protein production. The pro-region is engineered in advance with predicted beneficial mutations, and expression cassettes are prepared with these modified sequences prior to transformation and fermentation experiments
2Productivity
If pro-region sequences are modified to enhance protein secretion, then protein production increases, but the difficulty of constructing recombinant strains increases
Solution Approach 1:
The patent applies segmentation by dividing the pro-region sequence into specific positional segments (individual amino acid positions) that can be independently modified. This allows systematic testing of mutations at each position without requiring complete redesign of the entire sequence, simplifying the construction process
Solution Approach 2:
The patent uses expression cassettes as intermediaries to facilitate the introduction of modified pro-region sequences into Gram-positive bacterial hosts. The cassettes serve as standardized vectors that simplify transformation and reduce the complexity of strain construction
Data Source
AI summary
The instant disclosure is generally related to recombinant polynucleotides comprising novel pro-region DNA sequences. Certain aspects of the disclosure are related to recombinant Gram-positive bacterial strains comprising one or more introduced polynucleotides comprising novel pro-region DNA sequences operably linked to DNA sequences encoding proteins of interest.