Targeted mutagenesis in spirulina
Inventors
Takeuchi, Ryo • Roberts, James
Assignees
Publication Number
US-12065638-B2
Publication Date
2024-08-20
Expiration Date
2035-09-09
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
This disclosure describes techniques for creating stable, targeted mutations in Spirulina (Arthrospira) and Spirulina having stable, targeted mutations.
Core Innovation
The invention provides techniques for introducing stable and targeted mutations into the genome of Spirulina (including Arthrospira species) by using vectors with homology arms and inducing competence in Spirulina cells. The methods utilize artificial competence, such as electroporation in the presence of an osmotic stabilizer, to allow Spirulina cells to take up exogenous genetic material and integrate it into specific chromosomal locations through homologous recombination. This allows for precise modification at pre-determined loci rather than random insertion or mutagenesis.
The background highlights that prior techniques for genetic manipulation of Spirulina were either incapable of achieving targeted integration of DNA into the Spirulina chromosome or resulted in unstable or random insertions. Previous attempts, such as random mutagenesis or non-targeted transformation, failed to create stable modifications at specific genome sites. There was a recognized need for efficient methods to stably introduce foreign DNA into exact locations of the Spirulina genome.
This disclosure details methods that overcome these limitations, describing the use of specific vectors with homology arms, pH balancers, and osmotic stabilizers to facilitate targeted integration and ensure stability of the introduced modification over multiple generations. The invention encompasses both the modified Spirulina strains containing stable targeted mutations and the processes for producing such strains. Additionally, these techniques enable the use of the modified Spirulina strains for the manufacture of products of interest.
Claims Coverage
The patent contains two independent claims, each defining a main inventive feature relating to methods of targeted mutagenesis in Spirulina and to Spirulina cells with stable, heritable targeted mutations.
Method for creating an introduced targeted mutation in Spirulina
This inventive feature is defined by steps involving: 1. Inducing competence in a Spirulina cell. 2. Contacting the competent Spirulina cell with at least one vector comprising a nucleotide sequence homologous with at least a portion of a targeted genome locus. 3. Introducing a targeted mutation into the genome of the Spirulina cell. The feature encompasses artificial induction of competence, use of homologous nucleic acid sequences for targeting, and introduction of specific mutations at pre-determined loci within the Spirulina genome.
Non-naturally occurring stable transformant Spirulina with introduced targeted mutation
This inventive feature is characterized by: - A Spirulina strain comprising at least one introduced targeted mutation in its genome. - The introduced targeted mutation being adjacent to a nucleotide sequence complementary to a sequence contained in a vector used to introduce the mutation. - The targeted mutation is stable and inherited for at least 50 generations. This feature covers Spirulina cells modified with stable and heritable targeted mutations precisely introduced through vector-directed homologous recombination.
In summary, the main inventive features cover both the procedure for targeted genetic modification of Spirulina using vector-mediated homologous recombination and competence induction, as well as the resulting Spirulina strains having stably inherited and locus-specific genome modifications.
Stated Advantages
The techniques enable efficient creation of stable transformants in Spirulina, overcoming previous challenges in stable and targeted genetic modification.
The methods allow for targeted introduction of mutations in the Spirulina genome, providing specificity instead of random mutagenesis.
Stable targeted mutations in Spirulina can be inherited for many generations, ensuring long-term persistence of the genetic modification.
The invention facilitates addition, deletion, or alteration of specific genes or regulatory sequences, enabling the engineering of Spirulina for diverse biotechnological uses.
Documented Applications
Increasing production of neutral lipids (triglycerides and wax esters) in Spirulina for use as biofuel feedstocks and specialty chemicals.
Reducing glycogen production in Spirulina to redirect carbon towards lipid or fatty acid biosynthesis.
Modifying carotenoid biosynthesis in Spirulina to alter production levels or types of carotenoids (e.g., zeaxanthin, astaxanthin).
Increasing the production of C-phycocyanin and/or phycoerythrin pigments in Spirulina for applications in cosmetic, food, or medical imaging industries.
Interested in licensing this patent?