Targeted mutagenesis in Spirulina
Inventors
Takeuchi, Ryo • Roberts, James
Assignees
Publication Number
US-10131870-B2
Publication Date
2018-11-20
Expiration Date
2035-09-09
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Abstract
This disclosure describes techniques for creating stable, targeted mutations in Spirulina (Athrospiria) and Spirulina having stable, targeted mutations.
Core Innovation
This invention discloses techniques for introducing stable, targeted mutations into the genome of Spirulina (Arthrospira). The methods include contacting Spirulina with an osmotic stabilizer, exposing the cells to a vector containing homology arms that match specific genomic regions, and inducing artificial competence to facilitate transformation. The vector may carry various genetic elements such as exogenous or additional endogenous genes, regulatory elements, or deletions, and is integrated into the Spirulina genome by homologous recombination guided by the homology arms.
The problem addressed is the historic difficulty in achieving stable, targeted genetic modifications in Spirulina. Previous approaches using random integration methods or electroporation resulted in unstable transformations or lacked the ability to target specific genomic locations. As noted in the background, no prior reports described stable transformation or modification of the Spirulina genome at predetermined loci, which has hindered genetic engineering of this organism compared to others like E. coli or yeast.
By enabling the efficient and reliable introduction of mutations at specific sites in the Spirulina genome—including gene insertions, deletions, regulatory changes, or domain additions—this invention provides genetically modified Spirulina strains and methods for producing them. These approaches ensure the genetic changes are stably inherited over multiple generations, thus supporting advanced research or biotechnological applications involving Spirulina.
Claims Coverage
There are three independent claims covering the main inventive features related to modified Spirulina, methods for creating targeted mutations, and genetically altered Spirulina for specific protein or peptide presence or absence.
Stable transformant Spirulina with targeted genome mutation
A non-naturally occurring Spirulina comprising at least one introduced targeted nucleotide mutation incorporated into the genome. The mutation is adjacent to a region homologous to a nucleotide homology arm present in a vector used for transformation via homologous recombination.
Method for creating introduced targeted mutation in Spirulina
A process comprising: 1. Contacting Spirulina with an osmotic stabilizer. 2. Contacting the Spirulina with a vector having a nucleotide homology arm. 3. Inducing artificial competence in the Spirulina. This method enables the introduction of targeted mutations by homologous recombination.
Spirulina lacking at least one protein due to targeted modification
A Spirulina resulting from transformation with a DNA construct containing a sequence homologous to at least part of the target locus and a modification, wherein the DNA is integrated at the locus, and the Spirulina lacks at least one protein but is otherwise capable of functioning in its native manner.
The claims provide broad coverage for genetically engineered Spirulina with site-specific mutations, the procedures for generating such mutations, and resulting strains with altered protein expression. Claims address both the organisms and the transformation protocols.
Stated Advantages
Provides the first technique for targeted, stable transformation of Spirulina, overcoming prior limitations in genetic engineering methods for this genus.
Allows precise, stable introduction of genetic changes—including insertions or deletions—at specific, predetermined locations in the Spirulina genome.
The method creates modifications that are stably inherited across multiple generations of Spirulina.
The technique enables production of Spirulina strains with improved traits, such as altered pigment, lipid, or protein content.
Documented Applications
Creation of Spirulina strains for increased neutral lipid (wax ester and triglyceride) production for use as biofuel feedstock and specialty chemicals.
Engineering Spirulina strains with reduced glycogen production to direct carbon flow toward other products like lipids and fatty acids.
Modification of Spirulina to alter carotenoid biosynthesis, enabling increased synthesis of pigments such as zeaxanthin and astaxanthin.
Generation of Spirulina strains that produce increased levels of phycocyanin and/or phycoerythrin for use as blue and red pigments in food, cosmetics, or medical imaging.
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