Thermostable phycobiliproteins produced from recombinant arthrospira
Inventors
Roberts, James • Takeuchi, Ryo • KHUONG, Nhi • Stoddard, Barry L • KUESTNER, Rolf
Assignees
Publication Number
US-12252513-B2
Publication Date
2025-03-18
Expiration Date
2039-07-16
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Abstract
Provided herein are novel thermostable phycobiliproteins. These proteins may be stabilized by the introduction of disulfide bonds which stabilize the protein. Modified cells expressing these thermostable phycobiliproteins and methods of making them are also provided.
Core Innovation
The invention provides novel thermostable phycobiliproteins, such as phycocyanin and allophycocyanin, that are stabilized through the introduction of covalent disulfide bonds. These bonds are formed by replacing specific amino acid residues (such as alanine, aspartic acid, or serine) with cysteine in the protein sequence, resulting in increased resistance of the protein to elevated temperatures and acids. The thermostable phycobiliproteins can be produced from recombinant Spirulina cells that express these engineered proteins.
The problem addressed by this invention is that naturally occurring phycobiliproteins are sensitive to high temperatures and denature during manufacturing processes, leading to color loss and limiting their use in dye manufacturing and other applications. Current sources do not provide sufficient thermostability for industrial processes involving elevated temperatures.
The disclosed solution includes engineering disulfide bonds either within a subunit (such as between alpha-helices of the CpcA subunit) or between subunits (such as between CpcA and CpcB). Methods for creating these thermostable proteins in Spirulina involve gene modifications to introduce cysteine substitutions at specific positions, enabling the formation of such stabilizing bonds. The patent further describes compositions of modified Spirulina cells containing these thermostable phycobiliproteins and methods for their production and recovery.
Claims Coverage
The patent includes two independent claims, each covering distinct inventive features related to thermostable phycobiliproteins and compositions comprising recombinant Spirulina cells.
Thermostable phycobiliprotein with engineered disulfide bonds for thermostability
A phycobiliprotein modified for enhanced thermostability by forming covalent disulfide bonds, wherein: - Disulfide bonds are introduced by replacing one or more alanine, aspartic acid, or serine residues with cysteine residues within the protein sequence. - The disulfide bonds are located between: - Alpha-helices α2 and α7 within the phycocyanin alpha subunit, or - An alpha-helix α1 of the phycocyanin alpha subunit and the N-terminal region of the phycocyanin beta subunit upstream of alpha-helix α1. - The positions for these modifications are determined by alignment with sequences selected from SEQ ID NO: 35-36 or 41-42 (alpha subunit) and SEQ ID NO: 37-40 (beta subunit). The claim also specifies the exact residues for these alpha helices by sequence numbers.
Composition comprising modified Spirulina cells expressing thermostable phycobiliprotein
A composition that contains modified Spirulina cells engineered to express the thermostable phycobiliprotein as defined above, specifically those featuring the introduced disulfide bonds between designated amino acid residues for increased thermostability.
In summary, the claims cover engineered thermostable phycobiliproteins with disulfide bonds introduced at specific residue positions for enhanced temperature stability, and compositions of recombinant Spirulina cells expressing these proteins.
Stated Advantages
The thermostable phycobiliproteins possess increased resistance to temperature and acids compared to wild type or codon-optimized phycobiliproteins.
The modified phycobiliproteins can be stable at elevated temperatures, including stability over 60°C, 65°C, 70°C, and 75°C.
These engineered phycobiliproteins can exhibit more than a two-fold, ten-fold, or even fifty-fold increase in stability at elevated temperatures relative to the corresponding wild type protein.
Compositions and lysates containing the thermostable phycobiliproteins can contain higher ratios of stable phycobiliprotein compared to endogenous versions.
The invention enables the recovery of thermostable phycobiliproteins through modified production methods in Spirulina.
Documented Applications
Use of thermostable phycobiliproteins as sources of natural dye, especially in manufacturing processes that require resistance to elevated temperatures.
Production of thermostable phycobiliproteins for potential industrial and pharmaceutical uses as indicated by their ability to withstand temperatures greater than 45°C.
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