Promoter And 5'-UTR Mutations for Higher Bacillus Protein Expression
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Solution Overview
Problem
There is a need for improved promoter and 5'-untranslated region (UTR) nucleic acid sequences to enhance protein expression in Gram-positive bacterial cells, particularly in Bacillus species, which are commonly used in industrial biotechnology for producing proteins like enzymes and antibodies, as existing promoters do not adequately increase production levels.
Innovation Solution
Introduction of variant promoter/5'-UTR sequences, such as those described by SEQ ID NO: 8 through SEQ ID NO: 46, operably linked to DNA sequences encoding protein signal sequences and pro-region sequences, which are introduced into Gram-positive bacterial cells like Bacillus sp. to enhance protein production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional promoters are used in Gram-positive bacterial cells, then the expression system is simple and well-established, but protein production levels are insufficient
Solution Approach 1:
The patent applies parameter changes by modifying specific nucleotide positions in the promoter and 5'-UTR sequences. Multiple point mutations were introduced at positions such as -35, -10, and within the 5'-UTR region to optimize RNA polymerase binding affinity and ribosome binding efficiency, thereby increasing protein production levels without fundamentally changing the promoter architecture
Solution Approach 2:
The patent implements local quality by making targeted modifications to specific regions of the promoter and 5'-UTR rather than redesigning the entire sequence. The mutations are concentrated in functionally critical areas such as the -35 box, -10 box, Shine-Dalgarno sequence, and 5'-UTR secondary structure regions, leaving other portions of the sequence unchanged
2Productivity
If promoter mutations are introduced to enhance expression, then protein production increases, but the risk of disrupting essential promoter functions increases
Solution Approach 1:
The patent employs feedback through systematic screening and validation of mutant promoters. Multiple candidate mutations were tested for their ability to drive expression of reporter genes and target proteins, with only those mutations demonstrating enhanced expression while maintaining proper promoter function being selected for further development
Solution Approach 2:
The patent applies preliminary action by using computational analysis to predict the impact of mutations on promoter function before experimental implementation. In silico modeling of RNA polymerase binding affinity and 5'-UTR secondary structure changes guided the selection of mutations that are likely to enhance expression without disrupting essential functions
Data Source
AI summary
The present disclosure is generally related to the fields of microbial host cells, molecular biology, protein engineering, fermentation, protein production, and the like. Certain aspects of the disclosure are related to novel promoter and 5′-untranslated region a nucleic acid (DNA) sequences.