Host cells and methods for reducing isoprenoid precursors and isoprenoids by geranylgeranyl reductase
Inventors
Meadows, Corey • Garabedian, Brett • Mingardon, Florence • Chanal, Angelique • Lee, Taek Soon
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Assignees
University of California San Diego UCSD
University of California, San Diego (UCSD)The University of California, San Diego (UCSD) is a leading public research university located in La Jolla, California. Known for its innovative and interdisciplinary approach, UCSD offers a wide range of undergraduate, graduate, and professional programs across various fields. The university is committed to fostering a diverse and inclusive community, promoting sustainability, and driving social mobility through education, research, and public service. UCSD is recognized for its contributions to research and innovation, particularly in areas such as climate science, health innovation, and artificial intelligence.
The University of California, San Diego (UCSD) is a leading public research university located in La Jolla, California. Known for its innovative and interdisciplinary approach, UCSD offers a wide range of undergraduate, graduate, and professional programs across various fields. The university is committed to fostering a diverse and inclusive community, promoting sustainability, and driving social mobility through education, research, and public service. UCSD is recognized for its contributions to research and innovation, particularly in areas such as climate science, health innovation, and artificial intelligence.
Publication Number
US-12365905-B2
Publication Date
2025-07-22
Expiration Date
2039-12-23
Abstract
The present invention provides for a genetically modified host cell capable of reducing one or more isoprenoid, or precursor thereof, said genetically modified host cell comprising one or more geranylgeranyl reductases (GGRs), or polypeptides comprising an amino acid sequence having at least 70% identity to an amino acid sequence of a geranylgeranyl reductase (GGR), wherein the polypeptide comprises the enzymatic activity for catalyzing one or more GGR catalyzed reactions, and/or reducing one or more C15 or C20 prenyl alcohols or prenyl pyrophosphates.
Core Innovation
The invention provides genetically modified host cells capable of reducing one or more isoprenoids or their precursors by expressing one or more geranylgeranyl reductases (GGRs), or polypeptides with at least 70% amino acid identity to known GGRs. These polypeptides possess enzymatic activity to catalyze GGR reactions, including the reduction of C15 or C20 prenyl alcohols or prenyl pyrophosphates.
The invention addresses the challenge in isoprenoid biomanufacturing of reducing isoprenoid double bonds to decrease reactivity and sensitivity to oxidation, which is necessary to produce superior fuels and chemicals. Enzymatic reduction of unactivated double bonds in prenyl pyrophosphates has been limited to few oxidoreductases from the GGR family, mostly from extremophilic archaea or photosynthetic organisms. This invention expands the known GGR repertoire by identifying and characterizing novel GGRs active under industrially relevant, mesophilic conditions.
The invention also includes vectors encoding GGR polypeptides operatively linked to promoters for expression in host cells, methods of culturing these genetically modified host cells to produce and reduce isoprenoid substrates such as geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), and their corresponding alcohols, and recovering the reduced products. The invention further discloses methods to engineer GGRs by mutation to alter enzymatic activity, enhancing reduction efficiency or product specificity.
Claims Coverage
The patent claims cover a method for enzymatic reduction of specific isoprenoid substrates using genetically modified host cells expressing geranylgeranyl reductase polypeptides. The claims focus on the identity and sequence features of the polypeptides, host cell types, and steps for culturing and recovering reduced isoprenoids.
Use of genetically modified host cells expressing GGR polypeptides with defined sequence identity and conserved amino acids
The method employs genetically modified host cells comprising nucleic acid encoding a polypeptide with geranylgeranyl reductase activity having at least 95% identity to specified SEQ ID NOs and conserved amino acid residues at defined positions, enabling reduction of geranyl pyrophosphate or geraniol.
Culturing and reduction of isoprenoid substrates in yeast or bacterial host cells
The method involves culturing the genetically modified host cell, which may be yeast or specified bacteria, to produce and enzymatically reduce geranyl pyrophosphate or geraniol, followed by recovering the reduced products from the culture.
Specific sequence identity and polypeptide composition
Claims specify polypeptides having at least 99% identity to SEQ ID NOs 1-8 or specific sequences among them, ensuring that high similarity enzymes are covered.
The claims encompass methods utilizing genetically modified yeast or bacterial host cells expressing GGR polypeptides with defined sequence identities and conserved residues to catalyze the reduction of geranyl pyrophosphate or geraniol, including steps of cultivation and product recovery, thereby protecting enzymatic reduction methods employing these specific enzyme variants and host systems.
Stated Advantages
The invention enables enzymatic reduction of isoprenoids and their precursors under industrially relevant conditions (neutral pH and mesophilic temperatures), which is advantageous compared to prior enzymes from extremophiles requiring harsh conditions.
It expands the substrate scope of GGR enzymes to include short-chain isoprenoids (C15 and C20) and their alcohol forms, allowing for broader biosynthetic applications.
The engineered GGR enzymes exhibit increased activity and altered product specificity, enabling tailored production of partially or fully saturated isoprenoids.
The invention provides a biochemical route to produce reduced isoprenoids that can serve as precursors for fuels, polymers, pharmaceuticals, and cosmetics, potentially reducing costs and improving product stability.
Demonstrated capability of GGRs to catalyze novel side reactions, such as phosphoester hydrolysis and farnesyl acetate production, broadening their utility.
Documented Applications
Production of partially or fully saturated isoprenoids as feedstocks for biofuels, rubbers, pharmaceuticals, cosmetics, and chemical feedstocks.
Use in microbial hosts such as Escherichia coli and Saccharomyces cerevisiae for metabolic engineering of terpene biosynthesis pathways to produce reduced isoprenoid compounds.
Potential production of farnesyl acetate for industrial applications in fuels and cosmetics.
Engineering tailored isoprenoid products via integration into mevalonate or deoxyxylulose 5-phosphate biosynthetic pathways in microbes.
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