Endogenous Type I-B CRISPR-Cas Editing for Clostridium Butanol
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for butanol production in Clostridia strains are economically unfeasible due to high costs, low yields, and the presence of undesirable byproducts such as fatty acids and acetone, with conventional CRISPR-Cas systems facing toxicity issues that hinder efficient genome editing.
Innovation Solution
Utilizing an endogenous Type I-B CRISPR-Cas system in Clostridium tyrobutyricum, with identified PAM sequences and a lactose-inducible promoter, to enable multiplex genome engineering, introducing an adhE2 gene and inactivating the native cat1 gene, resulting in a strain that produces high levels of butanol with reduced byproducts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If conventional CRISPR-Cas systems are used for genome editing in Clostridia, then genome modification capability is improved, but host cell toxicity increases severely
Solution Approach 1:
The patent extracts and utilizes the endogenous Type I-B CRISPR-Cas system already present in Clostridium tyrobutyricum, rather than introducing heterologous Cas proteins. This approach removes the toxicity problem associated with conventional CRISPR-Cas9 systems while maintaining genome editing capability through the native system's Cas proteins and CRISPR arrays.
Solution Approach 2:
The invention enables the Clostridium tyrobutyricum strain to edit its own genome using its endogenous CRISPR-Cas system. By designing CRISPR arrays with specific spacers that target desired genomic loci, the system performs self-editing without requiring external Cas protein delivery, thereby avoiding toxicity while achieving precise genome modification.
2Ease of operation
If batch fermentation is used for butanol production with currently available Clostridia strains, then production process is simple, but yield is low and costs are high
Solution Approach 1:
The patent performs preliminary genome engineering modifications to Clostridium tyrobutyricum strains before fermentation, using the endogenous CRISPR-Cas system to optimize metabolic pathways for butanol production. This preliminary genetic optimization enables the strains to achieve high butanol yields during subsequent simple batch fermentation processes, resolving the contradiction between operational simplicity and productivity.
3Productivity
If ABE fermentation pathway is modified to increase butanol yield, then butanol production is improved, but unwanted byproducts are generated
Solution Approach 1:
The patent applies local quality modification by specifically targeting and modifying particular genes in the ABE fermentation pathway through CRISPR-Cas mediated genome editing. By making precise local changes to specific metabolic genes rather than global pathway alterations, the system increases butanol yield while minimizing the generation of unwanted byproducts such as fatty acids and acetone.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The modified strain achieves a record-high butanol yield of 26.2 g/L, demonstrating enhanced production efficiency and reduced byproduct formation, making it a robust workhorse for biobutanol production.
Implementation Method 1
CRISPR-Cas systems can be classified into two classes and six types based on the signature Cas proteins and the architecture of CRISPR-cas loci. A complex of multiple Cas proteins are involved in degrading the invading genetic elements
Implementation Method 2
The type I-B CRISPR array is under the control of a lactose inducible lac promoter
Implementation Method 3
The exogenous nucleic acid further comprises nucleic acid sequences that are homologous to sequences flanking the target protospacer sequence to facilitate the modification of the target genome loci through homologous recombination
Data Source
AI summary
A system for modifying the genome of Clostridium strains is provided based on a modified endogenous CRISPR array. The application also describes Clostridium strains modified for enhanced butanol production wherein the modified strains are produced using the novel CRISPR-Cas system.