Phage-Resistant Lactic Acid Bacteria via Anti-CRISPR Mediation
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Solution Overview
Problem
Current methods for obtaining bacteriophage-insensitive mutants of lactic acid bacteria, such as Streptococcus thermophilus, often rely on CRISPR systems, which are prone to rapid evolution by phages, and result in unstable phage resistance and compromised acidification performance, making it difficult to select for robust and stable phage-resistant strains suitable for food and feed fermentation.
Innovation Solution
A method involving exposure to virulent phages expressing anti-CRISPR proteins to enrich for non-CRISPR bacteriophage-insensitive mutants, which are then cultivated in milk-based media under phage challenge conditions to select for strains with improved phage resistance and maintained acidification properties without genetic modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR systems are used to obtain bacteriophage-insensitive mutants, then phage resistance is achieved, but the resistance is unstable and acidification performance is compromised
Solution Approach 1:
The patent introduces an anti-CRISPR protein as an intermediary substance that temporarily inhibits the CRISPR system during phage exposure. This allows the bacteria to survive initial phage attack while preventing premature CRISPR activation that would lead to unstable resistance. The anti-CRISPR protein acts as a controlled mediator between the phage threat and the CRISPR defense mechanism, enabling selection of mutants with stable non-CRISPR resistance mechanisms.
2Reliability
If CRISPR systems are used to obtain bacteriophage-insensitive mutants, then phage resistance is achieved, but acidification performance is compromised
Solution Approach 1:
The anti-CRISPR protein serves as a temporary mediator that blocks CRISPR activity during the selection process, allowing bacteria to maintain normal metabolic functions including acidification. By controlling when CRISPR is active or inactive through the anti-CRISPR mechanism, the system enables selection of phage-resistant mutants without permanently compromising acidification performance.
3Reliability
If extensive screening is performed to select robust phage-resistant strains, then reliable phage resistance is achieved, but time and resources are consumed
Solution Approach 1:
The patent applies preliminary action by pre-treating the bacterial population with anti-CRISPR protein before phage exposure. This pre-conditioning creates a selective environment where only bacteria with stable, non-CRISPR resistance mechanisms can survive and thrive. The preliminary anti-CRISPR treatment filters out CRISPR-dependent mutants, directly enriching the population with robust strains that have inherent stability, thereby dramatically reducing the screening burden and time required.
4Reliability
If CRISPR mutations are used for phage resistance, then resistance is achieved, but it becomes difficult to identify other desirable mutations
Solution Approach 1:
The anti-CRISPR protein acts as a filtering intermediary that suppresses CRISPR-based resistance mechanisms during the selection process. This allows other types of mutations (such as receptor modifications, cell wall changes, or metabolic adaptations) to manifest and be selected without being overshadowed by CRISPR spacer acquisitions. The anti-CRISPR mediator effectively masks CRISPR activity, making it possible to detect and measure alternative resistance mechanisms that would otherwise be difficult to identify among CRISPR mutations.
Data Source
AI summary
The present invention relates to a method for obtaining a bacteriophage insensitive mutant of a lactic acid bacterium parent strain suitable for food and feed fermentation. Further, the present invention relates to a method for the construction of a bacteriophage insensitive lactic acid bacterium whereby the phage resistance is conferred by a mechanism other than CRISPR. A preferred lactic acid bacterium is Streptococcus thermophilus.