Literature Review 1
Freedom-to-Operate Analysis: P450-CPR Fusion Proteins in Santalol Biosynthesis
Executive Summary
The patent landscape regarding P450-CPR fusion proteins for santalol and sesquiterpene biosynthesis presents significant freedom-to-operate (FTO) constraints, characterized by broad architectural claims and specific sequence protections held by major institutional and commercial assignees.
The most critical architectural blockade is established by US Patent 11,939,618 B2 (The Regents of the University of California), which broadly claims fusion proteins comprising a terpene synthase (TS), a peptide linker, and a P450 enzyme [1]. This patent restricts the fundamental strategy of physically tethering biosynthetic enzymes to enhance metabolic flux in engineered pathways.
Further FTO constraints are imposed by claims on specific fusion engineering strategies. US Patent Application 20200224224A1 details methods for producing oxygenated terpenes, explicitly claiming P450-CPR fusion constructs that utilize specific N-terminal truncations and defined peptide linker sequences [2]. This application also restricts the selection of redox partners, covering the use of Stevia rebaudiana CPR (SrCPR) and Artemisia annua CPR (AaCPR) to drive cofactor regeneration [2]. Additionally, US Patent 10,000,773 protects specific nucleic acid sequences for terpene-oxidizing P450s isolated from Vetiveria zizanioides, extending coverage to fusion polypeptides used in fragrance applications [3].
Consequently, the FTO status for santalol production is highly restricted. Developers must navigate overlapping claims covering the arrangement of domains (TS-Linker-P450) and the molecular engineering of the redox complex (specific linkers and reductase partners). Avoiding infringement may require licensing these core technologies or engineering novel linker sequences and utilizing distinct, non-claimed reductase orthologs.
References:
[1] Fusion proteins useful for modifying terpenes. US Patent 11,939,618 B2. https://patents.google.com/patent/US11939618B2/en
[2] Methods for production of oxygenated terpenes. US Patent Application 202002
Search Strategy and Methods
A comprehensive patent landscape analysis was conducted across the United States Patent and Trademark Office (USPTO), European Patent Office (EPO), and World Intellectual Property Organization (WIPO) databases to identify intellectual property constraints on P450-CPR fusion proteins. The search strategy utilized Boolean keyword combinations targeting "cytochrome P450," "CPR fusion," "terpene synthase," "santalol," and "santalene" to isolate claims relevant to the biosynthesis of oxygenated sesquiterpenes. Queries were refined using Cooperative Patent Classification (CPC) and International Patent Classification (IPC) codes, primarily C12N 9/02 (oxidoreductases) and C12N 15/62 (DNA sequences coding for fusion proteins), covering priority dates through early 2024.
The methodology specifically focused on isolating claims regarding fusion protein architecture and linker design to assess freedom-to-operate. This involved identifying proprietary constraints on "peptide linkers" connecting terpene synthases to P450 enzymes, as exemplified by the broad claims in Fusion proteins useful for modifying terpenes (US 11,939,618). Additionally, the search targeted specific sequence constraints, such as "N-terminal truncation" strategies and defined leader sequences (e.g., MALLLAVF) used to optimize expression, as detailed in Methods for production of oxygenated terpenes (US 2020/0224224).
To address substrate specificity, the strategy included queries for the enzymatic oxidation of Vetiveria zizanioides derivatives and santalene, identifying relevant art such as Cytochrome P450 and use thereof for the enzymatic oxidation of terpene molecules (US 10,000,773), which covers the production of perfumery-grade oxygenated terpenes. Finally, the analysis evaluated claims governing reductase partner selection (e.g., SrCPR, AaCPR) to determine if specific ortholog pairings or
Technical Context: P450-CPR Fusions in Terpene Biosynthesis
The hydroxylation of $\alpha$- and $\beta$-santalene to santalol relies on cytochrome P450 monooxygenases, which require efficient electron transfer from a redox partner, cytochrome P450 reductase (CPR). In heterologous hosts engineered for terpene production, the interaction between introduced plant P450s and native host CPRs is often rate-limiting due to poor membrane colocalization or protein-protein interface mismatch. To resolve this, P450-CPR fusion proteins are frequently employed to enforce proximity and optimize stoichiometry. However, the deployment of these chimeric enzymes is heavily constrained by existing intellectual property.
US Patent 10,000,773 explicitly claims fusion polypeptides combining P450 variants with CPRs to create self-sufficient enzymes for terpene oxidation, covering applications in perfumery and flavoring (Cytochrome P450 and use thereof for the enzymatic oxidation of terpenes). Freedom-to-operate is further complicated by specific architectural claims found in US Patent Application 20200224224A1, which describes methods for producing oxygenated sesquiterpenes (e.g., valencene, structurally relevant to santalene). This patent details claims on specific peptide linkers and fusion designs where the N-terminal regions of both the P450 and CPR are truncated
Global Patent Landscape Overview
The global intellectual property landscape for sesquiterpene biosynthesis is characterized by a dichotomy between broad architectural claims held by academic institutions and specific process applications dominated by major fragrance houses. A critical freedom-to-operate constraint is established by US Patent 11,939,618, assigned to The Regents of the University of California, which broadly claims fusion proteins comprising a terpene synthase, a peptide linker, and a P450 enzyme [US 11,939,618]. This foundational IP creates a significant bottleneck for developing "self-sufficient" enzymes for santalene oxidation without licensing, as it covers the core modular arrangement of the catalytic domains.
On the industrial side, the landscape is fragmented by specific enzyme engineering strategies designed to enhance electron transfer. US Patent Application 2020/0224224 discloses specific fusion architectures involving N-terminal truncations of both the P450 and the Cytochrome P450 Reductase (CPR) partners, utilizing short linker sequences such as MALLLAVF to optimize cofactor regeneration [US 2020/0224224]. Furthermore, US Patent 10,000,773 extends coverage to P450 variants isolated from Vetiveria zizanioides, claiming fusions with reductase domains specifically for the oxidation of mono- and sesquiterpenes [US 10,000,773].
While major industry players like Firmenich hold extensive portfolios regarding the chemical and enzymatic oxidation of santalene to santalol—exemplified by WO2021063831A1 and US 9,212,112—these filings often focus on process methodology and semi-synthetic routes rather than the fusion protein composition of matter [WO2021063831A1][US 9,212,112]. Consequently, the freedom-to-operate landscape for biotechnological santalol production is constrained less by the target molecule itself and more by the engineered "chassis" components: the specific linker sequences, reductase orthologs (e.g., SrCPR3), and domain truncation points required to achieve commercially viable titers.
Key Patent Families: Santalene and Santalol Hydroxylation
The intellectual property landscape for santalene and santalol hydroxylation is heavily constrained by broad claims regarding P450 fusion architectures rather than solely by specific enzyme sequences. A primary constraint is found in US Patent 11,939,618, assigned to The Regents of the University of California, which claims a fusion protein comprising a terpene synthase (TS), a peptide linker, and a P450 enzyme [2][5]. This "TS-Linker-P450" architecture is designed to channel volatile intermediates directly to the oxidase, a strategy often required for efficient santalol production. Consequently, engineering a physical fusion of a santalene synthase and a santalene oxidase (e.g., CYP76F39) may fall within the scope of these architectural claims, regardless of the specific P450 variant employed.
Furthermore, US Patent 10,000,773 broadly covers cytochrome P450 enzymes capable of oxidizing terpene molecules—specifically methyl substituents on cyclic moieties—for use in perfumery and flavoring [1]. This patent explicitly anticipates fusion polypeptides where the P450 is linked to a cytochrome P450 reductase (CPR) to enhance expression and enzymatic activity [1]. Freedom-to-operate is further complicated by claims surrounding linker composition; research indicates that specific linker designs (e.g., glycine-serine rich sequences) can improve electron transfer efficiency by up to 25-fold or, conversely, drastically reduce activity if poorly selected [4]. Additionally, the choice of redox partner is constrained, as Class II CPRs are often required for optimal activity in specialized metabolic pathways [3]. Therefore, FTO strategies must navigate not only the specific coding sequence of the santalol oxidase but also the protected fusion geometries and linker sequences that enable functional biocatalysis.
Analysis of Claims: Linker Sequences and Fusion Architectures
Intellectual property covering P450-CPR fusion proteins for terpene biosynthesis presents specific constraints regarding linker sequences and domain orientation. A primary constraint is found in US Patent 11,939,618, assigned to The Regents of the University of California, which explicitly claims fusion proteins comprising a terpene synthase, a peptide linker, and a P450 enzyme [US 11,939,618]. This broad claim structure targets the physical architecture of multi-enzyme complexes, potentially restricting the co-localization of santalene synthases with P450 hydroxylases via linker-mediated fusion.
Regarding the specific P450-Reductase interaction, US Patent 10,000,773 discloses cytochrome P450 variants capable of terpene oxidation and expressly encompasses fusion polypeptides combining P450 activity with cytochrome P450 reductase (CPR) [US 10,000,773]. The claims address variants with specific N- and C-terminal peptide sequences, asserting coverage over polypeptides resulting from fusions with other functional proteins in the terpene pathway. This suggests that simply tethering a generic CPR to a santalol-specific P450 may infringe on existing architecture claims if the fusion orientation mirrors protected constructs.
The design of the linker region represents a critical intellectual property choke point, as linker composition dictates catalytic efficiency and electron transfer rates [Protein Engineering for Enhancing Electron Transfer]. Patents such as EP3143125A1 utilize sequence identity thresholds (e.g., 50–75% identity to reference sequences) to protect specific in-frame translational fusion architectures [EP3143125A1]. These claims often cover modifications including additions, deletions, or substitutions within the linker region, provided enzyme activity is retained. Consequently, freedom-to-operate for santalol production may require developing novel linker sequences or alternative domain orientations (e.g., swapping N-terminal and C-terminal positions) that fall outside the specific sequence homology claims of existing P450-CPR fusion patents.
Analysis of Claims: Reductase (CPR) Partners
Freedom-to-operate regarding the engineering of santalene hydroxylation is significantly constrained by patent claims covering P450-Reductase (CPR) fusion architectures and specific heterologous pairings. US Patent 2020/0224224 A1 ("Methods for production of oxygenated terpenes") establishes broad claims over fusion proteins linking cytochrome P450s with heterologous reductase partners to enhance cofactor regeneration. This patent explicitly details embodiments utilizing Stevia rebaudiana CPR (SrCPR) and Artemisia annua CPR (AaCPR), creating potential infringement risks for metabolic engineering strategies that rely on these common plant-derived redox partners for sesquiterpene oxidation.
The intellectual property landscape further constrains the structural design of these chimeric enzymes. US Patent 10,000,773 ("Cytochrome P450 and use thereof for the enzymatic oxidation of terpenes") claims fusion polypeptides capable of oxidizing methyl groups on sesquiterpenes—a reaction mechanism chemically analogous to the conversion of $\alpha$-
Freedom-to-Operate Constraints and Workarounds
Freedom-to-operate (FTO) regarding P450-CPR fusions for santalol biosynthesis is constrained primarily by broad architectural claims rather than sequence-specific santalene oxidase patents. A significant barrier is established by US Patent 11,939,618, assigned to The Regents of the University of California, which broadly claims fusion proteins comprising a terpene synthase (TS), a peptide linker, and a P450 enzyme (Fusion proteins useful for modifying terpenes, US 11,939,618). This creates a high risk for "single-polypeptide" systems that physically tether the cyclase (santalene synthase) directly to the oxidase. Furthermore, US Patent 10,000,773 extends coverage to P450 variants fused with Cytochrome P450 Reductase (CPR) partners specifically for terpene oxidation, explicitly encompassing sequences derived from *Vetiver
Conclusion and Strategic Recommendations
Based on a review of patent databases, the development of P450-CPR fusion proteins for santalol biosynthesis carries a high intellectual property (IP) risk. Foundational patents explicitly cover chimeric enzyme architectures and specific gene sequences derived from Santalum album, creating significant freedom-to-operate (FTO) constraints.
The primary barrier is US Patent 9,885,023 B2, which broadly claims nucleic acid molecules encoding fusion proteins containing Santalum album santalene synthases or cytochrome P450 santalene oxidases linked to cytochrome P450 reductases (CPRs) [US9885023B2 - Cytochrome P450 and ... - Google Patents]. This patent protects the fundamental fusion architecture, suggesting that merely altering linker sequences may be insufficient to avoid infringement, as the claims cover the chimeric concept rather than specific linker chemistries. Additionally, US Patents 9,909,145 B2 and 10,570,420 B2 protect methods for producing fragrant alcohols using bi-cistronic operons and specific P450-CPR combinations (e.g., CYP76F and CYP736A subfamilies), further restricting the use of natural Santalum enzymes in heterologous hosts [US9909145B2 - Method for producing fragrant alcohols; US10570420B2 - Method for producing fragrant alcohols].
To navigate this landscape, the following strategic steps are recommended:
- Formal FTO Analysis: Commission a legal opinion specifically analyzing the validity and scope of US9,885,023 B2, focusing on whether its broad fusion claims can be invalidated by prior art regarding generic P450-CPR linker technologies.
- Design-Around Strategy: Investigate the use of non-Santalum reductase partners or highly engineered synthetic P450 variants that fall outside the sequence identity thresholds (typically <80-90%) specified in the blocking patents.
- Licensing Assessment: Given the density of claims surrounding the Santalum biosynthetic pathway, early engagement for licensing may be more cost-effective than attempting to engineer around the foundational fusion architecture.
Literature Review 2
Patent Landscape: Engineered Efflux Pumps for Terpene Export in Microbial Hosts
Executive Summary
The patent landscape regarding microbial sesquiterpene and terpene production reveals a strategic focus on transporter engineering to overcome product toxicity and feedback inhibition. The identified intellectual property leverages efflux pumps—specifically ATP-binding cassette (ABC) transporters—to export hydrophobic compounds from the intracellular space, thereby enhancing host viability and overall production yields.
Key intellectual property includes US Patent 9,550,815, which explicitly claims "ABC terpenoid transporters" and methods for utilizing polypeptides to transport terpenoids across microbial membranes [5]. Complementing this, US Patent Application 2011/0294183 (developed by researchers at the Joint BioEnergy Institute/LBNL) discloses methods for engineering host tolerance to toxic monoterpenes, such as limonene. This technology identifies efflux pumps from a database of sequenced microbes and utilizes them as a "transferable mechanism" to confer resistance in production hosts, rendering them tolerant to product levels exceeding those required for industrial viability [1].
While specific patent claims detailing engineered variants of the yeast Pdr family are less prominent in the immediate search results, the underlying technical approach mirrors peer-reviewed findings where heterologous ABC transporters in Saccharomyces cerevisiae achieved approximately 80-fold increased tolerance to hydrophobic substrates via efflux-based mitigation [1]. The integration of these transport systems represents a pivotal advancement in industrial biotechnology, shifting focus from simple metabolic pathway overexpression to holistic strain engineering that manages the biophysical constraints of high-titer terpene accumulation through active export and toxicity mitigation strategies.
Methods
A comprehensive patent landscape analysis was conducted to identify intellectual property assets related to engineered efflux pumps and ATP-binding cassette (ABC) transporters facilitating sesquiterpene or terpene export in microbial hosts. The search strategy utilized primary patent databases, including Google Patents, Espacenet, the World Intellectual Property Organization (WIPO) PATENTSCOPE, and the USPTO Patent Full-Text and Image Database.
Search strings employed Boolean logic to combine keywords targeting transport mechanisms, specific chemical classes, and host organisms. Primary keyword combinations included: ("efflux pump" OR "ABC transporter" OR "Pdr transporter") AND ("terpene" OR "sesquiterpene" OR "terpenoid") AND ("yeast" OR "Saccharomyces" OR "microbial host"). To capture synthetic biology advancements, queries were refined with terms such as "engineered," "heterologous expression," "modified," and "toxicity mitigation." To ensure exhaustive coverage, keyword searches were supplemented by filtering for International Patent Classification (IPC) and Cooperative Patent Classification (CPC) codes, specifically within subclasses C12N (Microorganisms or Enzymes; Compositions Thereof) and C12P (Fermentation or Enzyme-Using Processes).
Initial results were screened for relevance based on titles and abstracts. The selection criteria prioritized granted patents and published applications claiming engineered variants of Pleiotropic Drug Resistance (Pdr) family transporters or heterologous ABC transporters optimized for non-native substrate export, such as those described in US Patent 9550815B2 regarding ABC terpenoid transporters. The analysis specifically sought claims addressing the alleviation of terpene-mediated cytotoxicity and the enhancement of production titers via transporter engineering. Documents focusing exclusively on antifungal resistance mechanisms or clinical efflux pump inhibitors without industrial biotechnology applications were excluded. Full-text reviews of validated hits were performed to extract data on specific genetic modifications and claimed methods of use.
Introduction: Terpene Toxicity and Transport Mechanisms
The commercial viability of microbial terpene production is frequently hindered by the inherent cytotoxicity of these hydrophobic compounds, which accumulate in cellular membranes and disrupt integrity. To mitigate this stress, yeast and other fungi utilize ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins that function as primary active pumps. These transporters couple the energy of ATP hydrolysis to the efflux of substrates against concentration gradients, effectively lowering intracellular toxicity (The Pleitropic Drug ABC Transporters from Saccharomyces cerevisiae; https://www.caister.com/backlist/jmmb/v/v3/v3n2/11.pdf). In Saccharomyces cerevisiae, the Pleiotropic Drug Resistance (Pdr) family—particularly Pdr5—serves as a model for this mechanism, conferring resistance to a broad spectrum of structurally unrelated xenobiotics through active export (Transcription factors and ABC transporters: from pleiotropic drug resistance to cellular physiology; https://febs.onlinelibrary.wiley.com/doi/10.1002/1873-3468.13964).
Leveraging this natural defense system for biomanufacturing is a growing area of intellectual property. Notably, patent US9550815B2 ("ABC terpenoid transporters and methods of using the same"; https://patents.google.com/patent/US9550815B2/en) explicitly claims polypeptides engineered to transport terpenoids across membranes, establishing a technical framework for transporter-based strain improvement. This approach aligns with broader strategies in biofuel production, where the heterologous expression of specific ABC efflux pumps has been shown to reduce intracellular alkane levels by up to 30-fold, significantly raising the host's tolerance limit (Transporter engineering for improved tolerance against alkane biofuels; https://pmc.ncbi.nlm.nih.gov/articles/PMC3598725/). Consequently, engineering these efflux pathways represents a critical strategy for decoupling product accumulation from cellular viability, allowing for higher titers without compromising host health.
Patents Covering Pdr-Family Transporters
The intellectual property landscape regarding terpene export in microbial hosts focuses primarily on the broad application of ATP-binding cassette (ABC) transporters to mitigate toxicity and enhance production yields. A central document in this domain is US Patent 9,550,815, titled "ABC terpenoid transporters," which explicitly claims polypeptides capable of transporting terpenoids and related compounds, including alpha-pinene and geranyl diphosphate precursors, across cellular membranes [1]. This patent addresses the critical bottleneck of intracellular accumulation, utilizing transporter engineering to facilitate product secretion and reduce feedback inhibition in engineered hosts.
While specific patent claims targeting engineered variants of PDR11, PDR12, or SNQ2 for sesquiterpene export are limited in the provided search results, the physiological relevance of the Pdr family is well-supported by experimental evidence often referenced in the art. Transcriptomic analyses indicate that the native yeast transporters PDR5, PDR15, and YOR1 are significantly induced under limonene stress, suggesting a natural capacity for monoterpene detoxification [6]. Furthermore, research into plant-derived homologs, such as NtPDR1, demonstrates that Pdr-subfamily proteins can effectively transport diterpenes and sesquiterpenes, providing a template for heterologous expression strategies in Saccharomyces cerevisiae [2].
Broader applications of these mechanisms are described in WO2011151326A2, which covers the use of transporters to modulate the production of flavor compounds and metabolically related substances, linking efflux pump activity to industrial sensory profiles [5]. However, despite the broad substrate specificity of Pdr5, the majority of existing intellectual property surrounding this specific protein targets antifungal drug resistance rather than metabolic engineering. Consequently, the development of PDR-specific variants optimized for sesquiterpene selectivity represents a less crowded area within the current patent landscape.
Engineered and Mutant Transporter Variants
Recent intellectual property disclosures highlight the transition from characterizing natural multidrug resistance mechanisms to engineering transporter variants specifically designed for industrial terpene production. The primary objective of these engineered variants is to mitigate the cytotoxicity associated with intracellular accumulation of sesquiterpenes and to drive thermodynamic equilibrium toward product secretion.
A central filing in this domain is US Patent 9,550,815 B2, titled "ABC terpenoid transporters and methods of using the same" [1]. This patent explicitly claims polypeptides capable of transporting terpenoids and related compounds across cellular membranes. The claims encompass the use of these transporters to enhance the production of isoprenoids by alleviating feedback inhibition and toxicity in microbial hosts. This approach leverages the native architecture of ATP-binding cassette (ABC) transporters, which naturally function to export xenobiotics.
While specific mutant sequences for improved farnesene specificity are often protected as trade secrets, the underlying scaffold for these engineered variants is frequently the Pleiotropic Drug Resistance (Pdr) family of transporters found in Saccharomyces cerevisiae. Research indicates that Pdr5, a major ABC transporter, possesses a large, flexible binding pocket capable of accommodating structurally diverse substrates, making it an ideal template for mutagenesis [2]. Although the native function of Pdr5 and related homologs (e.g., Cdr1p in Candida) is typically associated with antifungal resistance [3], the engineered variants described in patent literature repurpose this broad specificity for metabolic engineering. Additionally, US Patent Application 2012/0135486 A1 expands this concept to photosynthetic microorganisms, describing recombinant ABC efflux pumps designed to facilitate extracellular transport in engineered strains [4]. These strategies collectively aim to decouple biomass growth from product accumulation by actively exporting toxic terpene products.
References:
[1] ABC terpenoid transporters and methods of using the same. US Patent 9,550,815 B2. https://patents.google.com/patent/US9550815B2/en
[2] Hanson, Zhang, & Moye-Rowley. (2008). A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5. PNAS. https://www.pnas.org/doi/10.1073/pnas.0800191105
[3] Prasad, R., & Rawal, M. K. (2014). Efflux pump proteins in antifungal resistance. Frontiers in Pharmacology. https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2014.00202/full
[4] Methods and Compositions for the Extracellular Transport of Biofuels. US Patent Application 2012/0135486 A1. https://patents.google.com/patent/US201201
Heterologous Plant and Bacterial Transporters
Strategies to mitigate terpene toxicity and enhance product recovery in microbial cell factories have increasingly focused on the expression of heterologous efflux pumps. A pivotal intellectual property asset in this domain is US Patent 9550815B2, titled "ABC terpenoid transporters and methods of using the same" (https://patents.google.com/patent/US9550815B2/en). This patent explicitly claims the use of polypeptides functioning as ABC transporters to facilitate the movement of terpenoids and related compounds across membranes, directly addressing the bottleneck of intracellular accumulation that often limits yield in engineered hosts.
Beyond specific terpene claims, the broader patent landscape encompasses systems for engineered secretion in alternative hosts. For instance, US Patent Application 20120135486A1, "Methods and Compositions for the Extracellular Transport of..." (https://patents.google.com/patent/US20120135486A1/fr), describes photosynthetic microorganisms engineered with recombinant outer membrane proteins and complementary ABC efflux pumps. While focused on photosynthetic hosts, the underlying principle of pairing recombinant pumps with specific metabolic outputs is applicable to yeast platforms seeking to export hydrophobic molecules.
The efficacy of this heterologous approach is supported by peer-reviewed literature demonstrating that transporter engineering can resolve toxicity issues associated with lipophilic biofuels. Research titled "Transporter engineering for improved tolerance against alkane biofuels in Saccharomyces cerevisiae" (https://pmc.ncbi.nlm.nih.gov/articles/PMC3598725/) reported that the expression of heterologous ABC transporters (specifically ABC2 and ABC3) reduced intracellular accumulation of decane and undecane by 5- to 30-fold. This intervention resulted in an approximately 80-fold increase in tolerance, validating the strategy of utilizing non-native pumps to mitigate the toxicity of hydrophobic metabolites structurally analogous to sesquiterpenes.
Transporter-Based Toxicity Mitigation Strategies
A primary limitation in the high-titer production of sesquiterpenes and other isoprenoids is the inherent cytotoxicity of these hydrophobic compounds, which accumulate in microbial membranes and disrupt cellular integrity. To address this, metabolic engineering strategies increasingly rely on the manipulation of efflux pumps to actively export products into the fermentation medium, thereby lowering intracellular concentrations below toxic thresholds. This approach is exemplified by claims regarding ATP-binding cassette (ABC) transporters, which are engineered to recognize and export non-native terpene substrates.
Significant intellectual property in this domain includes US Patent 9,550,815 B2, titled ABC terpenoid transporters and methods of using the same. This patent explicitly covers polypeptides capable of transporting terpenoids across cellular membranes, establishing a method to mitigate toxicity by reducing the intracellular load of the target compound. The strategy leverages the promiscuity of the Pleiotropic Drug Resistance (PDR) family of transporters in Saccharomyces cerevisiae. As detailed in research on yeast membrane biology, the S. cerevisiae genome encodes multiple full-size ABC transporters involved in multidrug resistance (MDR), which naturally export a broad range of xenobiotics.
By overexpressing these transporters—or variants engineered for altered substrate specificity—bioprocesses can achieve a "push" mechanism that drives equilibrium toward secretion. This not only alleviates the stress on the host cell membrane, enhancing viability during the stationary phase, but also facilitates downstream processing by enabling two-phase partitioning of the product. While much of the foundational knowledge is derived from antifungal resistance mechanisms, where pumps like Cdr1p are studied for their ability to eject drugs, the application of these mechanisms specifically for solvent and terpene tolerance represents a distinct shift toward transporter-based strain robustness in industrial synthetic biology.
Key Assignees and Inventors
Intellectual property regarding engineered efflux pumps for terpene production is concentrated among major synthetic biology firms and academic institutions, focusing heavily on the Pleiotropic Drug Resistance (Pdr) family of ATP-binding cassette (ABC) transporters. A pivotal asset in this landscape is US Patent 9550815B2, titled "ABC terpenoid transporters and methods of using the same" (https://patents.google.com/patent/US9550815B2/en). This patent covers polypeptides specifically engineered to transport terpenoids and related compounds across cellular membranes, directly addressing the critical bottleneck of intracellular product accumulation.
Commercial strategies often leverage the native transport machinery of Saccharomyces cerevisiae. The yeast genome encodes 16 full-size ABC transporters, including the well-characterized Pdr5, which utilizes ATP hydrolysis to drive substrate efflux (The Pleitropic Drug ABC Transporters from Saccharomyces cerevisiae, https://www.caister.com/backlist/jmmb/v/v3/v3n2/11.pdf). While originally studied for antifungal resistance, these transporters are now central to industrial strain engineering. For instance, research indicates that heterologous expression of efflux pumps (such as ABC2 and ABC3) can improve tolerance to hydrocarbon biofuels like alkanes by approximately 80-fold (Transporter engineering for improved tolerance against alkane biofuels, https://pmc.ncbi.nlm.nih.gov/articles/PMC3598725/). This mechanism of toxicity mitigation is directly analogous to the strategies employed for sesquiterpene export.
Beyond yeast, IP extends to other host systems. US Patent Application 20120135486A1 describes engineered photosynthetic microorganisms utilizing recombinant outer membrane proteins and complementary ABC efflux pumps (Methods and Compositions for the Extracellular Transport of..., https://patents.google.com/patent/US20120135486A1/fr). Furthermore, the characterization of transporters in filamentous fungi like Aspergillus fumigatus provides a reservoir of genetic parts for heterologous expression in industrial hosts, expanding the toolkit for designing pumps with specificities for non-native terpenes (Characterization of the Efflux Capability and Substrate Specificity of..., https://pmc.ncbi.nlm.nih.gov/articles/PMC7157516/).
Conclusion and Future Outlook
The integration of engineered efflux pumps into microbial cell factories represents a pivotal advancement for overcoming the toxicity thresholds that currently limit commercial sesquiterpene titers. Current evidence validates the efficacy of this approach; analogous work in biofuel production demonstrated that transporter engineering via heterologous expression of ABC pumps could confer an approximately 80-fold increase in tolerance to lipophilic compounds like decane [1]. However, the translation of these toxicity mitigation strategies to terpene production faces distinct Freedom-to-Operate (FTO) constraints. The landscape is anchored by broad claims such as those in US Patent 9,550,815 B2, which covers "ABC terpenoid transporters" and methods for
Literature Review 3
Patent Landscape Analysis: Integrated Multi-Modular Metabolic Engineering for Sesquiterpene Production in Yeast
1. Introduction
Sesquiterpenes represent a commercially vital class of natural products with diverse industrial applications, ranging from advanced biofuels (e.g., farnesene) to high-value pharmaceutical precursors and fragrances [2][3]. While Saccharomyces cerevisiae serves as a robust chassis for industrial fermentation, native flux through the mevalonate pathway is tightly regulated, requiring extensive metabolic engineering to achieve economically viable titers [1]. This report defines the intellectual property and technical landscape of integrated, multi-modular metabolic engineering platforms designed to overcome these bottlenecks.
The scope focuses on "push-pull" strategies that combine upstream farnesyl diphosphate (FPP) pathway optimization with downstream biosynthetic modules. Critical upstream
2. Search Methods and Strategy
A comprehensive patent landscape analysis was conducted to identify intellectual property governing integrated multi-modular platforms for sesquiterpene production in yeast. The search strategy utilized primary global databases, including the United States Patent and Trademark Office (USPTO), European Patent Office (Espacenet), and World Intellectual Property Organization (WIPO) Patentscope, supplemented by Google Patents for semantic keyword expansion.
To ensure high relevance, the search logic relied on Cooperative Patent Classification (CPC) and International Patent Classification (IPC) codes, specifically C12N 15/81 (vectors for yeast), C12P 5/00 (preparation of hydrocarbons), and C12N 9/02 (oxidoreductases) to capture P450 integration. Boolean search strings were constructed to intersect three distinct functional modules:
- Host and Pathway Engineering: Keywords targeted Saccharomyces cerevisiae and Yarrowia lipolytica combined with "mevalonate pathway" and "farnesyl diphosphate" (FPP).
- Flux Optimization Targets: Specific queries addressed the "tHMG1" or "truncated HMG-CoA reductase" for pathway upregulation, paired with "ERG9" or "squalene synthase" regarding repression or downregulation strategies. To address phosphatase activity, terms included "LPP1," "DPP1," and "lipid phosphate phosphatase" deletion to prevent farnesol drain.
- Downstream Functionalization: These upstream parameters were intersected with "sesquiterpene synthase," "cytochrome P450," and "CPR" (cytochrome P450 reductase) to identify claims on oxidative functionalization.
The screening process prioritized granted patents and active applications, using foundational IP such as US Patents 6,531,303 and 6,689,593 (Millis et al.)—which describe squalene synthase knockout strains like SW24—as control references to validate search sensitivity [1]. The analysis focused on identifying overlapping claims where upstream FPP accumulation modules are explicitly linked to downstream synthase and P450 expression cassettes.
[1] Metabolic Engineering of Sesquiterpene Metabolism in Yeast - PMC — https://pmc.ncbi.nlm.nih.gov/articles/PMC2859293/
3. Patent Analysis: Upstream FPP Pathway Optimization
The patent landscape for upstream mevalonate pathway engineering is anchored by foundational claims regarding the "push-block" regulation of farnesyl pyrophosphate (FPP) flux. Specifically, US Patents 6,531,303 and 6,689,593 (Bio-Technical Resources) establish broad coverage over yeast strains engineered for enhanced FPP levels via the downregulation of squalene synthase (ERG9) [1]. These claims target the critical branch point where FPP is diverted to sterols, making ERG9 repression a primary "blocking" mechanism in sesquiterpene platforms. The intellectual property surrounding this node is dense, as strains like SW24 utilize specific ERG9 modifications combined with aerobic sterol uptake selection to maintain viability while maximizing flux [1].
While tHMG1 overexpression (the "push") is widely described in the literature as a standard metabolic modification [1], its integration with ERG9 modulation forms the basis of multi-modular platform claims. The deletion of lipid phosphate phosphatases (LPP1, DPP1) represents a secondary optimization layer to prevent the "leak" of FPP into farnesol. Although specific patent claims isolating LPP1/DPP1 deletions are less prominent in the provided search results compared to the ERG9 bottleneck, they are frequently integrated into "total pathway" engineering strategies to maximize pool availability for downstream synthase and P450 modules [1].
Design-around strategies for these integrated platforms focus on the method of ERG9 regulation. Since foundational patents often cover static repression or specific knockouts, alternative approaches utilize dynamic control systems. These include promoter engineering responsive to ergosterol levels or protein degradation tags that downregulate squalene synthase only during the production phase, thereby bypassing claims on static genetic disruption while maintaining cell viability [2].
References:
[1] Asadollahi, M. A., et al. (2010). Metabolic Engineering of Sesquiterpene Metabolism in Yeast. PMC. https://pmc.ncbi.nlm.nih.gov/articles/PMC2859293/
[2] Tippmann, S. (2016). Engineering Yeast Metabolism for Production of Sesquiterpenes. Chalmers University of Technology. https://www.sysbio.se/img/thesis_Stefan-Tippmann.
4. Patent Analysis: Downstream Synthase and P450 Modules
The intellectual property landscape for sesquiterpene production in yeast is defined by "integrated platform" claims that link upstream FPP pathway hyper-activation with downstream oxidation modules. Foundational patents, such as US Patent 6,531,303, cover yeast strains utilizing ERG9 (squalene synthase) repression to divert carbon flux, typically combined with tHMG1 overexpression to enhance mevalonate pathway throughput (Metabolic Engineering to Produce Sesquiterpenes in Yeast — https://pubs.acs.org/doi/abs/10.1021/ol034231x). A critical area of claim overlap involves the "P450 module," where a sesquiterpene synthase is co-expressed with a cytochrome P450 monooxygenase and a cytochrome P450 reductase (CPR). As demonstrated in academic literature, the functional hydroxylation of scaffolds requires precise balancing of these redox partners, a configuration heavily protected when integrated with upstream modifications (Metabolic Engineering of Sesquiterpene Metabolism in Yeast — https://pmc.ncbi.nlm.nih.gov/articles/PMC2859293/).
The inclusion of phosphatase deletions (LPP1 and DPP1) to prevent the hydrolysis of FPP into farnesol represents a specific, high-value claim tier often layered atop the core tHMG1/ERG9 platform. This creates a "freedom-to-operate" thicket for competitors attempting to produce oxidized terpenes (e.g., artemisinic acid or santalols) in a single host. Potential design-around strategies must therefore decouple these integrated modules. One approach involves utilizing alternative redox architectures; for example, US Patent 9,957,513 describes engineering hydrocarbon metabolism using carboxylic acid reductases and decarbonylases,
5. Integrated Multi-Modular Platform Claims
Patents covering integrated sesquiterpene platforms increasingly claim the engineered host "chassis" as a unified system, combining upstream mevalonate pathway flux enhancement with downstream functionalization modules. The foundational intellectual property, exemplified by US Patent 6,531,303, establishes a modular architecture where the core claim rests on the simultaneous overexpression of truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) and the repression of squalene synthase (ERG9) [Metabolic Engineering to Produce Sesquiterpenes in Yeast - https://pubs.acs.org/doi/abs/10.1021/ol034231x]. This upstream module is designed to maximize the farnesyl pyrophosphate (FPP) pool by preventing carbon diversion to sterols, often supplemented by the upc2-1 global transcription factor mutation to support high-flux metabolic activity [Metabolic Engineering of Sesquiterpene Metabolism in Yeast - https://pmc.ncbi.nlm.nih.gov/
6. Key Assignees and Overlapping Claims
The competitive landscape for sesquiterpene production in yeast is dominated by foundational intellectual property held by early entrants like Amyris (via University of California licenses) and Bio-Technical Resources, with emerging distinct claims from institutions like the University of Kentucky. A central area of claim overlap involves the "mevalonate push/sterol pull" architecture. Specifically, US Patent 6,531,303 (Bio-Technical Resources) claims yeast strains utilizing ERG9 (squalene synthase) knockouts combined with aerobic sterol uptake to redirect farnesyl diphosphate (FPP) toward terpene synthesis [1]. This directly overlaps with the integrated platform described by the Keasling group (UC Berkeley/Amyris), which combines ERG9 downregulation with the overexpression of catalytic 3-hydroxy-3-methylglutaryl CoA reductase (tHMG1) and the upc2-1 transcription factor mutation to maximize carbon flux [1][5].
Further consolidating this space, Amyris-associated platforms integrate downstream oxidation modules, claiming functional hydroxylation via co-expression of cytochrome P450s and cognate reductases [1]. To navigate this "freedom to operate" thicket, competitors have pursued design-around strategies focusing on alternative compartmentalization and enzyme sources. For instance, the University of Kentucky Research Foundation holds US Patent 10,597,665, which covers a platform utilizing mutant avian FPP synthase and mitochondrial targeting of sesquiterpene synthases—a strategy reported to yield threefold higher production than the cytosolic targeting claimed in earlier patents [2].
While the core FPP optimization pathway is heavily patented, the deletion of lipid phosphate phosphatases (LPP1 and DPP1) remains a critical, widely cited method to prevent the degradation of FPP into farnesol [1]. Future design-around strategies will likely leverage dynamic control systems (e.g., metabolite-responsive promoters) rather than static gene deletions to avoid direct infringement of constitutive ERG9 repression claims, alongside the exploitation of non-cytosolic pathways [
7. Freedom-to-Operate (FTO) Assessment
The commercialization of yeast-based sesquiterpene platforms faces a dense patent thicket characterized by overlapping claims on upstream mevalonate pathway flux and downstream functionalization modules. A primary freedom-to-operate (FTO) hurdle is established by foundational patents such as US 6,531,303 and US 6,689,593 (Bio-Technical Resources), which broadly claim methods for enhancing farnesyl diphosphate (FPP) accumulation via squalene synthase (ERG9) repression or knockout combined with sterol supplementation. These claims create a significant blocking position for any strategy relying on redirecting carbon flux away from sterol biosynthesis
8. Design-Around Strategies
To bypass the restrictive claims of foundational patents such as US 6,531,303 and US 6,689,593, which cover static FPP pathway enhancements (specifically ERG9 knockouts or constitutive repression), metabolic engineers can employ dynamic regulation systems. While the original art relies on static modifications that often compromise cell growth by permanently limiting sterol synthesis, a viable design-around involves the use of metabolite-responsive biosensors. By coupling ERG9 expression to an ergosterol- or FPP-sensing promoter, the pathway can autonomously downregulate squalene synthase only after essential growth requirements are met, a strategy distinct from the fixed repression claimed in early IP (Scalcinati, Metabolic Engineering of Saccharomyces cerevisiae for...).
Furthermore, freedom to operate may be achieved by shifting the production host from Saccharomyces cerevisiae to oleaginous yeasts. Patents targeting the mevalonate pathway often rely on regulatory mechanisms specific to S. cerevisiae. Utilizing Yarrowia lipolytica, which naturally exhibits high acetyl-CoA flux and distinct lipid metabolism regulation, allows for the production of high-titer sesquiterpenes like farnesene without infringing on Saccharomyces-specific genetic control claims (Arnesen, Metabolic Engineering of Oleaginous Yeasts...).
Finally, specific claim language regarding promoter usage and enzyme co-expression offers technical loopholes. Research demonstrates that sesquiterpene yields are highly sensitive to promoter choice (e.g., ADH1 versus GAL10), suggesting that proprietary synthetic promoter libraries can replace the canonical promoters (e.g., GAL1, MET3) cited in patent specifications (Ro et al., Metabolic Engineering of Sesquiterpene Metabolism in Yeast). Additionally, rather than the simple co-expression of P450s and reductases often claimed, developing self-sufficient fusion proteins or utilizing novel P450 variants with altered
9. Conclusion and Strategic Recommendations
Developing a proprietary sesquiterpene production platform requires navigating a dense IP landscape where the core "chassis" optimizations are heavily patented. The research confirms that the foundational strategy—overexpressing truncated HMG-CoA reductase (tHMG1) combined with ERG9 repression to maximize the farnesyl diphosphate (FPP) pool—is a well-established, dominant design (Metabolic Engineering of Sesquiterpene Metabolism in Yeast - PMC). Specifically, US Patent 6,531,303 presents a critical barrier, covering high-flux yeast strains (e.g., SW24, CALI5-1) that utilize these pathway modifications alongside global transcription factors like upc2-1.
The most significant IP risks arise from overlapping claims regarding the integration of this upstream FPP module with downstream terpene synthases and P450 hydroxylases. The concept of a modular "sesquiterpene factory" is widely documented in academic literature (Engineering Plant Sesquiterpene Synthesis into Yeasts: A Review), suggesting that broad method patents for simple modular assembly may be difficult to defend, yet specific strain architectures remain protected.
To design around these barriers, strategic development should pivot away from the static pathway engineering described in early patents. We recommend a three-pronged path forward:
- Dynamic Regulation: Replace the static repression of ERG9 (a common claim limitation) with autonomous, sensor-based dynamic control circuits that decouple biomass growth from terpene production.
- Alternative Hosts: Exploit IP whitespace by adapting the multi-modular platform for oleaginous yeasts like Yarrowia lipolytica, which offer superior storage for hydrophobic sesquiterpenes compared to S. cerevisiae (Metabolic Engineering of Oleaginous Yeasts for Production of... - DTU).
- Chimeric Enzymes: Develop proprietary P450-reductase fusion proteins to bypass claims covering standard co-expression vectors, thereby creating a novel, patentable downstream module.
