Production of alpha-(R)-(E)-(+)-ionone in recombinant Saccharomyces cerevisiae
Inventors
Agosin Trumper, Eduardo Esteban • Saitua Perez, Francisco Javier • Lopez Salinas, Javiera Cecilia • Domaradzki, Maciej E • Ibaceta Acevedo, Maximiliano • Arenas Frigolett, Natalia
Assignees
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Publication Number
US-11124809-B2
Publication Date
2021-09-21
Expiration Date
Abstract
This invention provides improved biological synthesis of the apocarotenoid α-ionone in Saccharomyces cerevisiae. The final native step involved in the natural apocarotenoid pathway depends on an endogenous farnesyl pyrophosphate synthase (FPPs). From there, heterologous geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene desaturase (crtI), lycopene ε-cyclase (LycE) and a Carotenoid Cleavage Dioxygenase (CCD1) are required to complete the synthesis of α-ionone. Lycopene ε-cyclase from lettuce (Lactuca sativa) or modified cyclase from Arabidopsis thaliana was used to overproduce lycopene which was then cleaved by the carotenoid cleavage dioxygenase from Petunia hybrida (Ph-CCD1).
Core Innovation
The invention describes engineering Saccharomyces cerevisiae to biosynthesize enantiomerically pure (R)-(E)-(+)-alpha-ionone through an apocarotenoid pathway. The approach leverages mevalonate-derived production of farnesyl pyrophosphate using native nucleic acids encoding enzymes of the mevalonate pathway under constitutive or inducible promoters. The pathway then uses heterologous nucleic acids encoding enzymes to convert farnesyl pyrophosphate into alpha-ionone through a carotenoid cleavage step.
The engineered apocarotenoid pathway includes geranylgeranyl pyrophosphate synthase to form geranylgeranyl pyrophosphate, a bifunctional phytoene synthase/lycopene cyclase to form phytoene, and a phytoene desaturase to convert phytoene to lycopene. The pathway further includes lycopene epsilon-cyclase to cyclize lycopene to form delta-carotene and/or epsilon-carotene. A CCD1 enzyme cleaves delta-carotene and/or epsilon-carotene to produce alpha-ionone.
The described rationale emphasizes cleavage of epsilon-carotene to yield higher alpha-ionone stoichiometry, reported as 2 alpha-ionone per epsilon-carotene. The disclosure includes context of yeast strain characterization and comparative performance of example strains using HPLC/GC-MS, with lycopene accumulation reported up to about 1.61–1.64 g/L in fed-batch fermentation in cited engineering.
Claims Coverage
The independent claim requires a two-part engineered yeast design: mevalonate-pathway enzyme overexpression for farnesyl pyrophosphate supply, and an apocarotenoid pathway with specified enzyme steps that ultimately cleave delta-carotene and/or epsilon-carotene using CCD1 to produce enantiomerically pure (R)-(E)-(+)-alpha-ionone. Dependent claims refine yeast host selection, enzyme source organisms, nucleic-acid expression/integration formats, production level, and peptide sequence constraints via SEQ ID NOs.
Recombinant yeast overexpressing mevalonate pathway for farnesyl pyrophosphate
Constructing recombinant yeast cells that overexpress native nucleic acids, or modified versions thereof, encoding at least one enzyme of the mevalonate pathway for synthesizing farnesyl pyrophosphate, wherein expression of the one or more enzymes is under control of constitutive or inducible promoters.
Apocarotenoid pathway in yeast producing alpha-ionone via CCD1 cleavage
Modifying the recombinant yeast cells to further comprise heterologous nucleic acids that encode enzymes of an apocarotenoid pathway for synthesizing alpha-ionone, wherein the apocarotenoid pathway includes geranylgeranyl pyrophosphate synthase, bifunctional phytoene synthase/lycopene cyclase, phytoene desaturase, lycopene epsilon-cyclase, and CCD1 that cleaves delta-carotene and/or epsilon-carotene to produce alpha-ionone.
Host yeast selection
Performing the method using yeast cells selected from Pichia pastoris, Yarrowia lipolytica, or Saccharomyces cerevisiae.
Geranylgeranyl pyrophosphate synthase source organism
Using geranylgeranyl pyrophosphate synthase sourced from Xanthophyllomyces dendrorhous.
Nucleic-acid delivery and integration formats
Expressing the nucleic acids in yeast using centromeric plasmids, high copy number plasmids, or integration plasmids, and integrating the nucleic acids into specific, stable yeast genomic sites.
Production level constraint for alpha-ionone
Producing alpha-ionone at a level greater than 1 mg per gram of dry cell weight.
Engineered genetic payload sequence identity via SEQ ID NOs
Transforming recombinant yeast cells to introduce nucleic acids encoding peptides with the amino-acid sequences designated by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 10.
Across the independent claim and its refinements, the inventive approach centers on engineered yeast that overexpresses mevalonate-pathway enzymes to supply farnesyl pyrophosphate and that includes a specified apocarotenoid enzyme sequence ending with CCD1 cleavage of delta-carotene and/or epsilon-carotene to produce enantiomerically pure (R)-(E)-(+)-alpha-ionone, with dependent claims specifying host and enzyme sources, expression and integration formats, production threshold, and SEQ ID NO-constrained peptide payloads.
Stated Advantages
Produces enantiomerically pure (R)-(E)-(+)-alpha-ionone.
Higher alpha-ionone stoichiometry is emphasized for epsilon-carotene cleavage (2 alpha-ionone per epsilon-carotene).
Documented Applications
Biosynthesis of enantiomerically pure (R)-(E)-(+)-alpha-ionone in yeast, including Saccharomyces cerevisiae strain characterization with comparative performance of example strains.
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