Taxol (paclitaxel) is a frontline anticancer drug widely applied for the treatment of breast, ovarian and lung cancers. Currently, its supply mainly relies on the semi-synthesis using baccatin III from Taxus plants. Heterologous biosynthesis of baccatin III in microorganisms offers a promising solution to alleviate global Taxol supply shortage, but remains challenging due to pathway complexity. Here, we report a novel taxusin-mediated biosynthetic pathway for baccatin III production via the identification of C13 deacetylase, elucidation of the exact sequence underlying C1 hydroxylation, and stepwise enzymatic functional validation. Through protein engineering of the promiscuous C1 and C5 hydroxylases, coupled with the distribution of pathway modules in Saccharomyces cerevisiae and Escherichia coli, we achieved the de novo biosynthesis of baccatin III. Collectively, our findings remodel the current biosynthetic framework governing the formation of Taxol precursors and highlight the great potential of microbial cell factories for the production of complex plant-derived therapeutic compounds. Highlights • Discovery of C13 deacetylase reveals a novel biosynthetic route to baccatin III via taxusin • Stepwise verification of the complete biosynthetic route to baccatin III through taxusin and baccatin VI • Single-site mutation reversed the product selectivity of T1OH and converted T5OH into a specific taxoid C5 hydroxylase • Complete biosynthesis of baccatin III in engineered microbes
Hypericin is a promising Phase II/III drug candidate for oncology and psoriasis, yet its sustainable supply is problematic: multi-step synthesis is resource-intensive, and heterologous expression is blocked by the unknown plant pathway. Here, we re-engineer the polyketide biosynthetic pathways in Cladosporium fulvum and overcome substrate inhibition and metabolic inefficiency, achieving scalable and plant-pathway-independent biomanufacturing of hypericin. In particular, we redesigned the catalytic cavity of the critical P450 enzyme RugG to yield a variant (RugGT452P-R160D) with enhanced activity and reduced substrate inhibition. This achievement was combined with the suppression of shunt product formation to redirect metabolic flux toward emodin bianthrone—an indispensable hypericin precursor. These efforts enabled us to construct an engineered strain, ΔclaC-ΔclaH2-rugGT452P-R160D-claA-claE, which produced 1.87 g/L hypericin in a 5-L bioreactor after photocatalytic treatment. Collectively, this work provides an effective strategy for alleviating substrate inhibition and redirecting metabolic flux in the biomanufacturing of complex, high-value natural products.
Qing Cui, Xin Shi, Jia Li et al.· National Science Review· 0 citations
The heterologous production of terpene in microbial hosts is often limited by inefficient and unstable pathway expression, creating a major bottleneck for industrial-scale synthesis. While E. coli as a chassis offers significant advantages, such as rapid growth, ease of cultivation, and genetic tractability. Its endogenous supply of terpenoid precursors remains a critical constraint, fundamentally restricting high-yield production. To address this challenge, we developed a genomically integrated Mevalonate (MVA) pathway from Actinomycetota in E. coli BL21(DE3) to enhance terpene precursor supply. Our approach began with an in silico multi-layer global genome mining analysis of 25,261 Actinomycetota genomes to identify a series of MVA pathway enzymes with potentially high catalytic efficiency, created a high-efficiency chassis E. coli MVA platform (ecMVA-1 and ecMVA-2) for terpene precursor synthesis. Its functionality was validated by testing eight distinct TSs. Among them, the fermentation of artemisinin precursor amorphadiene using a 5-liter bioreactor yielded 947.80 mg/L. These results indicated that E. coli (MVA) is well-suited for TS studies in the laboratory as well as holding significant promise for industrial applications. In addition, this in silico approach offers a new perspective for metabolic engineering and provides potential reservoir of diverse chassis for the industrial production of terpenoid-derived compounds.
Wenchao Liu, Xueying Tian, W. Wong et al.· Metabolic Engineering· 0 citations
Polyketides are among the most structurally diverse and therapeutically important classes of natural products, serving as antibiotics, anticancer agents, agrochemicals, and industrial pigments. Their structural complexity and limited natural availability have driven the development of microbial biosynthetic platforms as scalable and sustainable alternatives to traditional extraction or chemical synthesis. Yarrowia lipolytica, a non-conventional and metabolically versatile yeast, has emerged as a promising alternative chassis for polyketide biomanufacturing, owing to its streamlined central metabolism, high acetyl-CoA availability, and exceptional physiological robustness. Recent advances in metabolic engineering and synthetic biology have enabled extensive rewiring of Y. lipolytica metabolism to support the high-yield production of complex, high-value polyketides. This review summarizes state-of-the-art strategies, from classical metabolic rewiring to emerging approaches such as organelle engineering and subcellular compartmentalization. We further highlight representative case studies of polyketide biosynthesis in Y. lipolytica, critically assess current limitations, and explore future directions to establish this organism as a programmable, industrially viable platform for polyketide production.
Pentostatin is a potent adenosine deaminase inhibitor, yet its industrial application is hindered by low extraction yields and complex chemical synthesis. Here, we report an efficient de novo biosynthesis platform for pentostatin in Saccharomyces cerevisiae. Starting with the heterologous expression of cns3 from Cordyceps militaris, we optimized the cell factory via promoter engineering, multicopy integration, and AAH1 knockout. This integration strain achieved a maximum pentostatin titer of 16.28mg/L in shake-flask cultivation, representing a 19.38-fold improvement over our initial production. Separately, to alleviate severe product toxicity, we implemented flux balance analysis (FBA)-guided transporter engineering; the engineered strain expressing the episomal efflux pump Cns4 yielded a titer of 8.27mg/L while significantly accelerating the production process. Molecular docking revealed a distinct binding cavity where key residues (e.g., Asp296, Ala292) capture pentostatin via specific hydrogen bonds and hydrophobic interactions. Furthermore, transcriptomics demonstrated that Cns4 globally reprograms carbon and energy metabolism to boost precursor supply and cellular robustness. This work integrates structural insights with systems metabolic engineering, providing a generalizable paradigm for biosynthesizing toxic nucleoside natural products.
Ming-Zhe Bai, Zhiyi Liu, Chao-Zhong Wang et al.· Journal of Biotechnology· 0 citations
The biosynthetic potency of CPT and their molecular regulatory processes to sustain the CPT productivity by fungi are unraveled by exploring the rate-limiting enzymes, epigenetic regulators and transcriptional factors of CPT biosynthesis.
Ashraf S. A. El-Sayed, Marwa A. Yassin, Ashraf Farag El‐Baz· Discover Applied Sciences· 0 citations
Monoterpenoids are an important class of plant volatile natural products with broad applications in the food, fragrance, pharmaceutical, and agricultural industries. However, their conventional production largely relies on plant extraction, which is often constrained by low efficiency, high cost, and limited sustainability. Structurally, monoterpenoids can be classified into acyclic, monocyclic, and bicyclic types, and their structural diversity is closely associated with differences in biosynthetic routes and regulatory mechanisms. Their biosynthesis depends on precursor supply from the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, followed by terpene synthase (TPS)-mediated scaffold formation and subsequent modification reactions. In addition, monoterpenoid accumulation is regulated by multiple factors, including environmental cues, phytohormone signaling, transcriptional regulation, and epigenetic or post-transcriptional control. Meanwhile, substantial progress has been made in the heterologous production of monoterpenoids in microbial platforms such as Escherichia coli and Saccharomyces cerevisiae through metabolic engineering and synthetic biology. This review summarizes recent advances in monoterpenoid biosynthesis, multilevel regulation, and heterologous production, with particular emphasis on major bottlenecks and optimization strategies for sustainable and efficient biomanufacturing.
Jun-Chi Zhang, Jiale Cui, Shang Li et al.· Natural Products and Biopros...· 0 citations
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