Metabolic Engineering of Microbes for Terpene Production

Introduction

Terpenes are a diverse class of hydrocarbons built from isoprene units (C₅H₈), forming the backbone of many bioactive compounds, flavors, fragrances, pharmaceuticals, and even biofuels. Naturally produced by plants and some microbes, terpenes such as limonene, farnesene, pinene, and bisabolene are increasingly seen as sustainable alternatives to petrochemicals and jet fuels.

To meet industrial demand, metabolic engineering of microbes—especially bacteria, yeast, and cyanobacteria—offers a scalable, green route for terpene biosynthesis. By introducing or enhancing specific pathways (e.g., MVA or MEP), microbes can be optimized to produce high yields of target terpenoids, while consuming renewable feedstocks like glucose, glycerol, or CO₂.

What Products Are Produced?

• Monoterpenes (C₁₀) – Limonene, pinene (flavors, biojet fuel)
• Sesquiterpenes (C₁₅) – Farnesene, bisabolene (diesel-range fuels)
• Diterpenes and triterpenes (C₂₀–C₃₀) – Squalene, taxadiene (pharma precursors)
• Isoprenoids – Wide class including rubber, hormones, and fragrances

Pathways and Production Methods

1. MVA and MEP Pathways

• Mevalonate (MVA) Pathway – Common in yeast and engineered E. coli
• Acetyl-CoA → Mevalonate → IPP → Terpenes
• Methylerythritol Phosphate (MEP) Pathway – Native in many bacteria
• G3P + pyruvate → DXP → IPP → Terpenes

2. Pathway Engineering Strategies

• Overexpression of rate-limiting enzymes (e.g., HMGR, DXS, DXR)
• Knockout of competitive pathways to direct carbon to terpenes
• Use of heterologous terpene synthases for specific product formation
• Dynamic control systems (inducible promoters, biosensors)

3. Host Organisms

• Escherichia coli – High-growth, well-studied system for MVA-based production
• Saccharomyces cerevisiae – GRAS status, native MVA pathway
• Synechocystis and Cyanobacteria – Light-driven CO₂-based terpene biosynthesis
• Engineered Bacillus subtilis, Yarrowia lipolytica, and Pichia pastoris

Catalysts and Key Tools Used

• Enzymes:
• Terpene synthases – Pinene synthase, farnesene synthase, limonene synthase
• Precursor enzymes – HMGR (MVA), DXS, DXR (MEP), IDI (isomerization)
• Cytochrome P450s – For hydroxylation and derivatization
• Genetic Engineering Tools:
• CRISPR/Cas9, multiplex gene editing
• Modular DNA assembly platforms (Golden Gate, MoClo)
• Synthetic operon construction and metabolic scaffolds
• Fermentation Systems:
• Fed-batch and two-phase fermentation (to extract volatile terpenes)
• Use of in situ extraction solvents (e.g., dodecane, isopropyl myristate)

Case Study: Amyris – Farnesene Production via Engineered Yeast

Highlights

• Engineered S. cerevisiae to produce farnesene, a sesquiterpene used in diesel, lubricants, and cosmetics
• Introduced optimized MVA pathway and plant-derived farnesene synthase
• Operates large-scale fermentation and extraction facility in Brazil
• Farnesene also converted to renewable jet fuel (farnesane)

Timeline

• 2008 – Engineered farnesene yeast strains demonstrated
• 2013 – Commercial-scale farnesene biofuel production launched
• 2018 – Integrated farnesene into beauty, pharma, and materials segments
• 2023 – Amyris licenses platform to global chemical producers

Global and Indian Startups Working in This Area

Global

• Amyris (USA) – Farnesene, squalene from engineered yeast
• Lygos (USA) – Malonic acid, terpenes via MVA pathway
• Conagen (USA) – Natural flavor terpenes via precision fermentation
• Zymochem (USA) – Carbon-efficient microbial terpene production

India

• Seagull BioSolutions (Mumbai) – Engineered microbes for natural product pathways
• IIT Delhi, CSIR-NCL – Research on microbial limonene and pinene biosynthesis
• NCCS Pune & IISc – Metabolic flux modeling and enzyme engineering for terpene output
• Startup under BIRAC BIG scheme – Early-stage terpene strain development

Market and Demand

The global terpene market is projected to grow from USD 8.2 billion in 2023 to USD 14.1 billion by 2030, at a CAGR of ~8%, driven by bio-based demand in fuel, cosmetics, flavors, and pharma. Microbial terpene production is expected to dominate future sustainable supply chains.

Major End-Use Segments:

• Biofuels (diesel and aviation)
• Flavors and fragrances
• Nutraceuticals and health products
• Agrochemicals (natural pesticides, insect repellents)
• Green solvents and lubricants

Key Growth Drivers

• Increasing demand for renewable jet and diesel fuels
• Rise in plant-derived product bans (sustainability-driven)
• Availability of low-cost sugars and feedstocks
• Advances in strain engineering and fermentation scale-up
• Growing interest in carbon-negative chemical production

Challenges to Address

• Volatility and toxicity of terpenes to host microbes
• Low solubility and accumulation limits inside cells
• Product recovery costs due to separation from aqueous broth
• Balancing growth vs. production trade-offs
• Regulatory hurdles for food- or pharma-grade terpenes

Progress Indicators

• 2005 – Microbial monoterpene synthesis demonstrated
• 2010 – Farnesene commercialized by Amyris
• 2016 – Cyanobacteria engineered to produce terpenes from CO₂
• 2020 – CRISPR-based control of terpene flux pathways
• 2023 – India’s first funded microbial terpene program under BIRAC

Microbial terpene production is at TRL 8–9 for specific products like farnesene and limonene globally, and at TRL 4–6 in India, where academic and biotech labs are actively developing engineered strains for fuels and fragrances.

Conclusion

Metabolic engineering of microbes for terpene production opens the door to a future of sustainable, high-performance fuels and specialty chemicals. With microbes acting as green chemical factories, this approach reduces dependence on petroleum and plants, while enabling scalable, modular, and customizable terpene supply chains.

As India invests in green chemistry, biomanufacturing, and synthetic biology, microbial terpene production offers a promising bio-based industrial pathway—integrating CO₂ capture, renewable feedstocks, and value-added exports across energy and health sectors.

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