Modular Synthetic Biology Platform for Programmable Enzyme Organization and Enhanced Metabolic Pathway Engineering

Background

Monoterpenes are a class of valuable natural products widely used in the fragrance, flavor, pharmaceutical, and biofuel industries. Traditionally, these compounds are sourced from plants through extraction or produced by chemical synthesis, both of which have significant limitations. Plant extraction is constrained by agricultural land use, seasonal variability, and low yields, while chemical synthesis often relies on petrochemical feedstocks and can be environmentally unfriendly. As a result, there is a growing interest in developing sustainable, scalable, and cost-effective microbial production platforms—such as engineered yeast—to biosynthesize monoterpenes. Microbial biosynthesis offers the potential for renewable feedstocks, consistent production, and the ability to tailor product profiles through genetic engineering.

Despite these advantages, current approaches to microbial monoterpene production face significant challenges. The key enzymatic step involves farnesyl pyrophosphate synthase (Erg20), which naturally produces both geranyl pyrophosphate (GPP, the monoterpene precursor) and farnesyl pyrophosphate (FPP, the sesquiterpene precursor), but with a strong preference for FPP. This inherent enzyme specificity diverts metabolic flux away from GPP, severely limiting monoterpene yields and favoring the production of unwanted byproducts. Previous efforts to overcome this bottleneck have focused on mutagenesis of Erg20 to alter its product specificity, but these approaches have yielded only modest improvements and often compromise enzyme stability or activity. Thus, there remains a critical need for new strategies that can efficiently redirect metabolic flux toward GPP and enable high-yield microbial monoterpene production.

Technology

This technology is a modular synthetic biology platform designed to engineer intracellular membraneless organelles, known as biomolecular condensates, with precise control over their composition and function. At its core, the system utilizes “Forever Corelets” (FC)—synthetic scaffolds that self-assemble into stable condensates within yeast cells. These scaffolds can be decorated with a variety of protein-peptide interaction domains (PplDs), enabling the programmable recruitment of specific cargo proteins tagged with cognate peptide ligands. The recruitment process is quantitatively predictable, allowing for fine-tuned stoichiometric control of multiple enzymes or functional proteins within the same condensate. The platform is highly modular and orthogonal, supporting the simultaneous and independent recruitment of diverse cargos, and is underpinned by a robust equilibrium binding model that accurately predicts recruitment dynamics based on domain affinity, cargo concentration, and scaffold expression levels.

What differentiates this technology is its unprecedented ability to decouple and independently tune the assembly of synthetic condensates and the recruitment of functional cargos, overcoming a key limitation of native biomolecular condensates where assembly and composition are tightly intertwined. The system’s modularity enables multiplexed and orthogonal control, allowing for the spatial organization of complex metabolic pathways and precise stoichiometric balancing of pathway enzymes. Additionally, the platform introduces a novel enzyme engineering strategy by fusing short peptides to the C-terminus of key enzymes (such as Erg20), thereby altering their catalytic specificity to favor desired products (e.g., monoterpenes over sesquiterpenes). This dual approach—combining spatial enzyme clustering with engineered enzyme specificity—enables significant enhancements in metabolic flux and product yields, offering a powerful, generalizable toolkit for sustainable bioproduction of high-value chemicals, pharmaceuticals, and biofuels.

Benefits

• Enhanced monoterpene production by engineering Erg20 enzyme to preferentially synthesize geranyl pyrophosphate (GPP) over farnesyl pyrophosphate (FPP).
• Modular synthetic biology platform enabling programmable and tunable recruitment of enzymes and proteins to synthetic biomolecular condensates for spatial organization.
• Orthogonal and multiplexed cargo recruitment using diverse protein-peptide interaction domains, allowing simultaneous control of multiple enzymes or proteins.
• Quantitative equilibrium binding model guiding predictable tuning of cargo recruitment and stoichiometry within condensates.
• Improved metabolic flux control demonstrated by increased production of target compounds (e.g., β-farnesene) through stoichiometric enzyme clustering.
• Versatile and generalizable approach combining enzyme engineering and synthetic condensates for sustainable biosynthesis of valuable natural products including biofuels, flavors, fragrances, and pharmaceuticals.
• CRISPR-Cas9 mediated genomic integration enabling stable expression of engineered enzymes and condensate scaffolds in yeast.

Applications

• Microbial monoterpene fragrance production
• Sustainable cannabinoid biosynthesis
• Biofuel precursor manufacturing
• Pharmaceutical monoterpene synthesis
• Flavor and food additive production

Additional info

This work presents a modular synthetic biology platform utilizing engineered biomolecular condensates (Forever Corelets) for programmable, stoichiometric enzyme clustering. It also introduces a novel enzyme modification method: C-terminal peptide fusion to Erg20, reversing its product specificity to favor geranyl pyrophosphate (GPP). These technologies enable precise metabolic flux control and enhanced monoterpene biosynthesis in yeast.