Phage-Insensitive Streptococcus Thermophilus via Sedimentation Selection
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Solution Overview
Problem
Streptococcus thermophilus bacteria used in dairy fermentations are highly susceptible to bacteriophage predation, leading to fermentation failures and economic losses, with existing CRISPR systems providing limited stability against phage evolution.
Innovation Solution
A method for constructing bacteriophage insensitive mutants (BIMs) of Streptococcus thermophilus by selecting mutants with increased sedimentation rate and/or chain formation, which confer phage resistance through mechanisms other than CRISPR, ensuring robustness against phage attack.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR systems are used to provide phage resistance in Streptococcus thermophilus, then phage resistance is achieved, but stability against phage evolution is limited
Solution Approach 1:
The patent applies parameter changes by selecting for mutants with altered sedimentation rates and chain formation characteristics, which correspond to fundamental changes in bacterial physiology and morphology. These parameter changes lead to stable phage resistance through mechanisms unrelated to CRISPR, thereby resolving the contradiction between achieving phage resistance and maintaining stability against phage evolution.
Solution Approach 2:
The patent extracts the CRISPR mechanism from the phage resistance pathway by specifically selecting for mutants that exhibit phage resistance through non-CRISPR mechanisms. This is achieved by screening for mutants with increased sedimentation rates and chain formation, thereby removing the instability associated with CRISPR systems while retaining the desired phage resistance phenotype.
2Reliability
If robust starters are selected to reduce susceptibility to phage attack, then fermentation reliability is improved, but selection and characterization complexity increases
Solution Approach 1:
The patent uses sedimentation rate and chain formation as visual and measurable phenotypic markers that serve as proxies for phage resistance. By selecting for mutants with increased sedimentation rates and chain formation, the patent simplifies the selection process by using easily observable physical characteristics rather than requiring complex molecular characterization, thereby resolving the contradiction between improving fermentation reliability and reducing selection complexity.
Solution Approach 2:
The patent employs self-service by using the mutants' own phenotypic characteristics (sedimentation rate and chain formation) as the selection criterion for phage resistance. This eliminates the need for external complexity in selection systems, as the resistant mutants naturally exhibit distinct physical properties that facilitate their identification and selection without requiring additional characterization tools or procedures.
Data Source
AI summary
Bacteriophage Insensitive Mutants (BIMs) of three Streptococcus thermophilus parent strains were generated and characterized for phage sensitivity, sedimentation rate, cell chain length, phage adsorption and CRISPR loci alterations. Several BIMs showed an altered sedimentation phenotype as well as an increase cell chain length, reduced phage sensitivity, reduced phage adsorption and 100% identity in three CRISPR loci. The results show that the derived BIMs have become phage-resistant through a mechanism other than CRISPR.