Metabolic Pathway Engineering for Terpenes

Introduction

Terpenes are a diverse class of naturally occurring hydrocarbons built from isoprene units (C₅H₈) and include monoterpenes, sesquiterpenes, diterpenes, and more. Found in plants, fungi, and microbes, they serve essential roles in flavor, fragrance, pharmaceuticals, biofuels, and specialty chemicals. However, traditional extraction from plants is inefficient, seasonal, and land-intensive.

Metabolic pathway engineering provides a sustainable alternative by enabling the production of terpenes in engineered microbes such as E. coli, S. cerevisiae, and cyanobacteria. Through the introduction and optimization of terpene biosynthesis pathways, scientists can convert simple sugars or CO₂ into high-value terpenes, marking a significant leap in the bioeconomy of renewable chemicals.

What Products Are Produced?

• Monoterpenes (C10): Limonene, pinene – fragrances, cleaning agents, jet fuels
• Sesquiterpenes (C15): Farnesene, nerolidol – lubricants, perfumes, insect repellents
• Diterpenes (C20): Taxadiene – precursor to cancer drug Taxol
• Carotenoids & Retinoids: Food colorants, nutraceuticals
• Terpenoid-based jet fuels and lubricants (C10–C16 branched hydrocarbons)

Pathways and Production Methods

1. Isoprenoid Biosynthesis Routes

a) MEP Pathway

• Native to bacteria and plant plastids
• Pyruvate + G3P → IPP + DMAPP → Terpenes
• Hosts: E. coli, cyanobacteria

b) MVA Pathway (Mevalonate Pathway)

• Native to eukaryotes, introduced into bacteria
• Acetyl-CoA → Mevalonate → IPP + DMAPP → Terpenes
• Hosts: S. cerevisiae, E. coli (with heterologous expression)

2. Terpene Synthase Engineering

• Specific synthases (e.g., limonene synthase, amorphadiene synthase) inserted
• Control product selectivity and cyclization of terpenoid scaffolds

3. Precursor Supply Enhancement

• Overexpression of acetyl-CoA, NADPH, and ATP generating genes
• Engineering feedback-resistant enzymes and dynamic regulation circuits

Catalysts and Key Tools Used

• Enzymes:
• DXS, DXR, HMGR – pathway bottlenecks
• Terpene synthases – control terpene diversity
• Prenyltransferases – generate linear terpene precursors (GPP, FPP, GGPP)
• Synthetic Biology Tools:
• CRISPR/Cas9, TALENs, and modular plasmids for pathway integration
• Dynamic control systems to prevent toxic intermediate accumulation
• Protein scaffolds for channeling intermediates between enzymes
• Inducible promoters for tunable expression of rate-limiting steps
• Microbial Hosts:
• E. coli – fast-growing, genetically tractable
• S. cerevisiae – better handling of hydrophobic and toxic terpenes
• Cyanobacteria – photosynthetic production from CO₂

Case Study: Amyris and Farnesene Production

Highlights

• Engineered S. cerevisiae with MVA pathway + farnesene synthase
• Developed industrial-scale fermentation of farnesene from sugarcane
• Farnesene used in diesel, lubricants, emollients
• Pioneered renewable jet fuel (farnesane) approved for aviation

Timeline

• 2008 – Amyris launches synthetic biology farnesene project
• 2011 – First commercial batch from Brazil plant
• 2016 – Jet fuel approved by ASTM
• 2023 – Farnesene production expanded to cosmetics and pharma

Global and Indian Startups Working in This Area

Global

• Amyris (USA) – Farnesene, bisabolol, squalane via yeast
• Ginkgo Bioworks – Terpene pathway design-as-a-service
• Evolva (Switzerland) – Stevia, vanillin, and fragrance terpenoids
• Isobionics (acquired by BASF) – Citrus terpenes in E. coli

India

• IIT Delhi & DBT-ICGEB – Terpene pathway engineering in S. cerevisiae
• Sea6 Energy – Exploring marine biomass-to-terpene platforms
• Bugworks Research – Using synthetic biology for therapeutic terpenoids
• IISc Bangalore – Carotenoid engineering from cyanobacteria

Market and Demand

The global terpene market was valued at USD 7.1 billion in 2023, expected to reach USD 11.2 billion by 2030, with a CAGR of ~6.8%. Bio-terpenes are gaining traction due to their natural origin, renewability, and applications across cosmetics, food, pharma, and fuels.

Major Use Segments:

• Fragrances and flavors (limonene, linalool, nerolidol)
• Biofuels and lubricants (farnesene, pinene)
• Pharmaceuticals (artemisinin, taxol precursors)
• Nutraceuticals and food additives (carotenoids)

Key Growth Drivers

• High consumer demand for natural and renewable ingredients
• Progress in metabolic modeling and gene editing
• Opportunity for carbon-neutral or carbon-negative terpene production
• Diversification of feedstocks (sugarcane, CO₂, lignocellulose)
• Corporate partnerships pushing bio-based materials

Challenges to Address

• Toxicity of terpenes to microbial hosts at high titers
• Low pathway flux and precursor competition with central metabolism
• Complexity of cyclization and functionalization in terpene biosynthesis
• High downstream purification costs for volatile/hydrophobic terpenes
• In India: Need for integrated scale-up infrastructure

Progress Indicators

• 2005–2010 – Core MEP/MVA pathway engineering in E. coli and S. cerevisiae
• 2013–2016 – Commercial-scale production of farnesene and limonene
• 2019 – CRISPR regulation for terpene tuning developed
• 2022 – Indian consortia initiate work on terpene fuels from CO₂
• 2024 – Scalable terpene production integrated into cosmetic supply chains

Bio-terpene production via metabolic engineering is at TRL 8–9 globally, with several products commercially deployed. In India, efforts are at TRL 4–6, with strong academic momentum and pilot-scale work underway.

Conclusion

Metabolic pathway engineering for terpenes is unlocking a future where renewable microbes replace petrochemicals in producing scents, fuels, drugs, and materials. The scalability, tunability, and eco-friendly nature of bio-terpene production make it a cornerstone of the next-generation bioeconomy.

As India deepens its synthetic biology capabilities and global markets shift toward sustainable chemistry, engineered terpenes will lead the way in replacing fossil-derived specialty chemicals with renewable, programmable molecules.

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