Methods for production of oxygenated terpenes
Inventors
KUMARAN, Ajikumar Parayil • Lim, Chin Giaw • Li, Liwei • GHOSH, SOUVIK • Pirie, Christopher • Qualley, Anthony
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11952608-B2
Publication Date
2024-04-09
Expiration Date
Abstract
The present invention relates to methods for producing oxygenated terpenoids. Polynucleotides, derivative enzymes, and host cells for use in such methods are also provided.
Core Innovation
The invention provides methods and engineered components for producing oxygenated sesquiterpenes, including nootkatone, by using Stevia rebaudiana kaurene oxidase (SrKO; CYP701A5; SEQ ID NO:37) and derivatives referred to as valencene oxidase/VO that unexpectedly oxygenate sesquiterpene substrates such as valencene. The disclosure reports that oxygenation with the SrKO/VO enzymes yields distinct product profiles compared with other CYP450s, including the formation of nootkatol and nootkatone as well as additional oxygenated terpene products.
The invention further includes mutational engineering of SrKO/VO to increase valencene oxidase activity, including N-terminal membrane anchor modifications for expression in E. coli and active-site substitutions such as T499N and F231L/F231I, where amino-acid numbering corresponds to the amino-acid sequence of SEQ ID NO:37. In addition, the disclosure describes integrating the engineered oxidases with engineered metabolic pathways in microbial hosts, including MEP/MVA flux to IPP, valencene synthase, and P450 reductase partners, with optional conversion of nootkatol to nootkatone through alcohol dehydrogenase.
Claims Coverage
The document’s relevant independent claim covers one inventive feature set for a sesquiterpene oxidase defined by sequence identity to SEQ ID NO:37 or SEQ ID NO:38 and a T499N substitution aligned to SEQ ID NO:37. Dependent claims add higher sequence identity thresholds and an additional F231L or F231I substitution.
Sesquiterpene oxidase with sequence identity and aligned T499N substitution
A sesquiterpene oxidase comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence SEQ ID NO:37 or SEQ ID NO:38, wherein the amino acid sequence comprises a T499N substitution and the amino acid numbering corresponds to the amino acid sequence of SEQ ID NO:37.
Higher sequence-identity sesquiterpene oxidase
The sesquiterpene oxidase of the sequence-identity framework, wherein the amino acid sequence has at least 85% sequence identity to SEQ ID NO:37 or SEQ ID NO:38.
Further higher sequence-identity sesquiterpene oxidase
The sesquiterpene oxidase of the sequence-identity framework, wherein the amino acid sequence has at least 90% sequence identity to SEQ ID NO:37 or SEQ ID NO:38.
Maximal sequence-identity sesquiterpene oxidase
The sesquiterpene oxidase of the sequence-identity framework, wherein the amino acid sequence has at least 95% sequence identity to SEQ ID NO:37 or SEQ ID NO:38.
T499N plus F231L/F231I sesquiterpene oxidase variant
A sesquiterpene oxidase variant that includes an F231L or F231I amino-acid substitution, with numbering corresponding to SEQ ID NO:37, in addition to the sequence-identity and T499N framework.
Overall, the claim set focuses on a sesquiterpene oxidase engineered by maintaining high sequence identity to SEQ ID NO:37 or SEQ ID NO:38 and including a T499N substitution with numbering aligned to SEQ ID NO:37, with additional dependent coverage for increased sequence identity thresholds and an optional F231L or F231I substitution.
Stated Advantages
Produces oxygenated sesquiterpenes, including nootkatone, by oxygenating sesquiterpene substrates such as valencene.
Generates distinct oxygenated terpene product profiles versus other CYP450s, including formation of nootkatol/nootkatone and additional oxygenated terpene products.
Increases valencene oxidase activity through mutational engineering of SrKO/VO, including the specified substitutions.
Documented Applications
Producing oxygenated sesquiterpenes (notably nootkatone) from sesquiterpene substrates such as valencene using engineered SrKO/VO oxidases.
Engineered metabolic pathway integration in microbial hosts using MEP/MVA flux to IPP, valencene synthase, and P450 reductase partners to support oxygenated sesquiterpene production, including optional alcohol dehydrogenase conversion of nootkatol to nootkatone.
In vivo and cell-free contacting concepts for contacting engineered oxidases with sesquiterpene substrates for oxygenation, supported by product analysis.
Interested in licensing this patent?