Targeted mutagenesis in spirulina
Inventors
Takeuchi, Ryo • Roberts, James
Assignees
Publication Number
US-10336982-B2
Publication Date
2019-07-02
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
This invention describes techniques for creating stable, targeted mutations in Spirulina (Arthrospira spp.) through the use of genetic engineering. The disclosed methods involve introducing targeted nucleotide modifications into predetermined regions of the Spirulina genome using vectors with homology arms, osmotic stabilizers, and artificial competence induction techniques. The result is Spirulina cells containing specific, heritable genomic changes that are stably maintained for at least 50 generations.
The background highlights that while genetic manipulation of cyanobacteria is generally possible, Spirulina has posed significant challenges due to its resistance to stable transformation and targeted genome modification. Existing methods only permitted random integration of foreign DNA or random mutagenesis, which did not allow for directed changes at specific genomic loci, and transformations were often not stable over time.
The core innovation solves these problems by providing a method to efficiently introduce stable, targeted mutations into the Spirulina genome. This involves contacting Spirulina with an osmotic stabilizer, exposing it to a vector carrying homology arms flanking the mutation, and inducing artificial competence (e.g., via electroporation in the presence of the osmotic stabilizer). The invention further covers modified Spirulina strains with such targeted mutations, including insertions, deletions, gene regulatory elements, or entire genes for applications such as enhanced biosynthesis of specific products.
Claims Coverage
There are two independent claims, each establishing main inventive features concerning stable, targeted genomic modifications in Arthrospira (Spirulina) cells and their use for production of valuable compounds.
Stable transformant Arthrospira cell with introduced targeted nucleotide mutation
A non-naturally occurring, stable transformant Arthrospira cell comprising an introduced targeted nucleotide mutation incorporated into the cell's genome, with the mutation flanked on each side by regions of nucleotides homologous to homology arms contained in a transformation vector, and the mutation being stable for at least 50 generations.
Stable transformant Arthrospira cell engineered for product of interest
A non-naturally occurring, stable transformant Arthrospira cell engineered for production of a product of interest, comprising an introduced targeted nucleotide mutation incorporated into the genome, flanked on each side by regions homologous to vector-derived homology arms, wherein the mutation is stable for at least 50 generations, and the cell is used in the biosynthesis of products such as lipids, wax esters, triglycerides, biofuels, fatty acids, glycerin, enzymes, alcohols, fatty alcohols, glycogen, pigments, carotenoids, peptides, polypeptides, proteins, and phycobiliproteins (including allophycocyanin, phycocyanin, C-phycocyanin, and phycoerythrin).
The inventive features collectively cover the creation of Spirulina cells with precise, stable, targeted genetic changes using homologous recombination, and their utility in producing a diverse range of valuable biological products.
Stated Advantages
Provides a technique to efficiently create stable transformants in Spirulina.
Enables targeted introduction of mutations at specific, predetermined regions of the Spirulina genome.
Allows for the addition, deletion, or modification of genetic material in Spirulina to produce or enhance desired traits or products.
Generates mutations that are stable and heritable over at least 50 generations.
Overcomes prior inability to stably introduce foreign DNA at targeted chromosome locations in Spirulina.
Documented Applications
Increasing production of neutral lipids (wax esters and triglycerides) in Spirulina for biofuels and specialty chemicals.
Reducing glycogen accumulation in Spirulina to redirect carbon flux toward the synthesis of lipids and fatty acids.
Modifying or enhancing carotenoid biosynthesis pathways in Spirulina for production of compounds like astaxanthin and zeaxanthin.
Increasing production of phycocyanin and/or phycoerythrin in Spirulina for use in the food, cosmetic, and medical imaging industries.
Interested in licensing this patent?