(177y) Statistics Guided Systematic Engineering for High-Yield Production of Terpenoids in Auxotrophic Escherichia coli

Isoprenoids, or terpenoids, constitute possibly the largest group of natural products (>75,000). Structural diversity of terpenoids contributes to wide applications ranging from pharmaceuticals (e.g., artemisinin), nutraceuticals (e.g., astaxanthin), flavors and fragrances (e.g., linalool), polymer molecules (e.g., isoprene) and biofuels (e.g., farnesene). Unlike plant terpenes that are well studied, fungal terpenes and their synthases remain largely untapped. In collaboration with a German group, we have developed an integrated platform for discovery of novel fungal terpene synthases (TPSs). Coupling bioinformatics (all-by-all blast and active site prediction) and experiments (SPME-GCMS), we have successfully predicted several unique clusters of putative isofunctional TPSs (e.g. protoilludene, viridiflorol) and validated by experiments. In addition to terpene synthase discovery, we also developed a multidimensional heuristic process (MHP) and aided by statistical analysis to efficiently synthesize various isoprenoids in Escherichia coli. Built on statistical analysis and modular metabolic engineering methods, MHP is a focused and systematic approach to optimize the performance of biosynthetic pathways, especially effective on complex systems (>10 genes). MHP adopts ‘modular design’ that significantly reduces experimental workload yet without losing the flexibility by simultaneously control different functional dimensions (transcription, translation and enzymes). As proof-of-concept examples, we effectively enhanced the total biosynthesis of the 15-step heterologous biosynthetic route of two tetraterpenoid (C40), astaxanthin, at 320 mg/L and lycopene (1.5 g/L); a monoterpenoid (C10), linalool (0.5 g/L); and two sesquiterpenoids (C15), viridiflorol (~25.7 g/L) and amorphadiene (30 g/L). Evidently, MHP and statistics guided systematic engineering approach is generally applicable for different metabolic pathways and products.

Selected reference (* corresponding author)

1. Shukal, S., Chen, X. & ZHANG, C*. Systematic engineering for high-yield production of viridiflorol and amorphadiene in auxotrophic Escherichia coli. Metabolic Engineering 55, 170–178 (2019).
2. ZHANG, C.*, Seow, V. Y., Chen, X. & Too, H.-P*. Multidimensional heuristic process for high-yield production of astaxanthin and fragrance molecules in Escherichia coli. Nat Commun 9, 1858 (2018).
3. ZHANG, C.*, Chen, X., Lindley, N. D. & Too, H.-P*. A ‘plug-n-play’ modular metabolic system for the production of apocarotenoids. Biotechnol. Bioeng. 115, 174–183 (2018).