Targeted mutagenesis in spirulina
Inventors
Takeuchi, Ryo • Roberts, James
Assignees
Publication Number
US-10415012-B2
Publication Date
2019-09-17
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
The invention provides techniques for creating stable, targeted mutations in Spirulina (Arthrospira) by introducing vectors with homology arms into Spirulina cells, thereby allowing homologous recombination to modify specific, predetermined genomic loci. This method enables the incorporation of exogenous or endogenous genes, regulatory elements, or other genetic modifications at desired positions in the Spirulina genome, addressing longstanding challenges in achieving stable site-specific genetic modifications in this organism.
Traditional genetic engineering methods have been largely ineffective in Spirulina due to its lack of natural competence and the instability of introduced genetic material, with prior attempts often resulting in random integration or an inability to sustain modifications over time. The disclosed approach overcomes these problems through the use of artificial competence (such as electroporation in the presence of osmotic stabilizers) and vectors with flanking homologous regions, resulting in efficient, stable transformation and targeted mutagenesis.
The disclosed Spirulina with stable, targeted mutations can be engineered for various purposes, including but not limited to gene deletions (knockouts), insertions of exogenous genes, addition of regulatory sequences, or overexpression of endogenous genes. This capability supports the production of Spirulina strains custom-tailored to express desired proteins, metabolic products, or traits, and allows for these modifications to be inherited stably over many generations.
Claims Coverage
There are two independent claims, each covering major inventive features related to producing phycobiliproteins by stable transformant Arthrospira cells using introduced targeted nucleotide mutations.
Production of phycobiliprotein by culturing stable transformant Arthrospira with introduced targeted nucleotide mutation
A method comprising: - Culturing a stable transformant Arthrospira cell that contains an introduced targeted nucleotide mutation incorporated into the cell's genome, with the mutation increasing phycobiliprotein production. - The introduced mutation is adjacent to a region homologous to a nucleotide homology arm in a transforming vector. - The mutation results in the stable transformant Arthrospira cell producing more phycobiliprotein than a wild-type cell. - The method includes recovering the phycobiliprotein.
Production of phycobiliprotein using mutation involving gene disruption or exogenous protein domain addition
A method comprising: - Culturing a stable transformant Arthrospira cell having an introduced targeted nucleotide mutation that increases phycobiliprotein production, with the mutation being either a deletion/disruption of a portion of a gene or an addition of a polynucleotide encoding an exogenous protein domain. - The method specifies that culturing occurs under suitable conditions for phycobiliprotein production.
The inventive features claim culturing genetically modified Arthrospira cells with targeted nucleotide mutations—introduced via vectors with homologous arms—that result in increased phycobiliprotein production, including by gene insertion, deletion, disruption, and/or regulatory modifications.
Stated Advantages
Provides an efficient technique to create stable, targeted mutations in Spirulina, overcoming the difficulties and instability of previous transformation methods.
Allows for targeted introduction of exogenous or endogenous genes, gene regulatory elements, or protein domains into predetermined locations in the Spirulina genome.
Enables the production of Spirulina strains with stably inherited modifications across multiple generations.
Facilitates increased production of desired proteins or metabolic products, such as C-phycocyanin, via targeted gene modifications.
Documented Applications
Production of phycobiliproteins, such as phycocyanin, allophycocyanin, and phycoerythrin, from genetically modified Spirulina.
Manufacture of Spirulina strains for increased production of neutral lipids (wax esters and triglycerides) for biofuel and specialty chemical feedstock.
Engineering Spirulina to reduce glycogen accumulation and redirect carbon flow to lipid or fatty acid biosynthesis.
Modification of Spirulina for altered or increased carotenoid production, including astaxanthin and zeaxanthin, through introduction or knockout of specific genes.
Production of C-phycocyanin-overproducing Spirulina for use in cosmetic, food, and medical imaging industries.
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