Jul 2026· Journal of Biotechnology· 0 citations· 51 references
Medicine
Abstract
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.
Polyketides are a diverse class of natural products with broad pharmacological activities and substantial industrial relevance; however, their scalable biosynthesis is often constrained by inefficient heterologous expression platforms. In this study, a systematic evaluation of multiple microbial hosts identified Aspergillus niger HL-1 as an efficient fungal chassis for polyketide production. Subcellular compartmentalization analysis revealed that cytosolic biosynthesis significantly outperformed peroxisomal localization, leading to a 5.88-fold increase in triacetic acid lactone (TAL) production. To further enhance production, intracellular acetyl-CoA and malonyl-CoA supplies were engineered and integrated with compartmentalized metabolic rewiring to redirect carbon flux toward polyketide biosynthesis. In addition, increasing the copy number of polyketide synthase genes further improved titers. As a result, monacolin J and TAL reached 1.68 g/L and 7.50 g/L in shake-flask cultures, respectively, and were further elevated to 2.61 g/L and 25.08 g/L in a 1.5-L bioreactor. Finally, a versatile type III polyketide synthase (PKS) expression platform was established, enabling the functional expression of ten heterologous PKSs. By further disrupting the endogenous katA gene to decelerate p-coumaric acid consumption, the competitive catabolic pathway was successfully blocked, yielding a naringenin shake-flask titer of 384.91 mg/L from p-coumaric acid. This work establishes a robust and scalable fungal platform for the efficient production of both complex polyketides and flavonoids, providing a promising strategy for biotechnological applications.
Linlin Yao, Rui Huang, Jiaping Liu et al.· Metabolic Engineering· 0 citations
Carcinine is an imidazole dipeptide with potent antioxidant and antiglycation properties, although its chemical synthesis currently relies on severely environmentally harmful processes. In this work, a multi-enzyme cascade biotransformation system comprising 4′-phosphopantetheinyl transferase and non-ribosomal peptide synthetase was constructed. To overcome the limitations arising from stochastic spatial distribution and suboptimal mass transfer associated with independent enzymes, a fusion protein strategy was adopted. The two enzymes were fused via a flexible genetic linker within plasmid pET28a-SFP-L-Ebony, which enabled robust soluble expression in Escherichia coli. Concurrently, the endogenous peptidase genes (pepA, pepB, pepD, and pepN) were systematically knocked out using CRISPR/Cas9-mediated gene editing. This quadruple protease-deficient strain (designated SFP-L-Ebony-ΔpepABDN) effectively suppressed product degradation. Subsequent optimization revealed that optimal catalytic performance occurred at 25 °C and pH 7.0. The highest biotransformation efficiency was achieved using 15 g/L crude enzymes, in the presence of 2 mM ATP and 10 mM MgCl2. Through a fed-batch substrate feeding strategy in a 50 mL reaction system, the final carcinine titer reached 7.0 g/L after 48 h. This study, therefore, provides an efficient and sustainable technological pathway for the green biomanufacturing of carcinine as well as other high-value dipeptides.
Terpenoids are valuable resources for pharmaceutical research, yet their natural supply remains constrained. While the artificial isoprenol utilization pathway (IUP) has emerged as a promising alternative for terpene precursor supply, its full potential is limited by suboptimal pathway flux and substrate tolerance. Here, we report an efficient scaffold-assisted IUP platform tailored for high-throughput terpenoid discovery and scalable production. By recruiting rate-limiting IUP enzymes and prenyltransferases (PTs) onto self-assembling PduA* protein scaffolds displaying CC-Di-B peptides that specifically interact with CC-Di-A-tagged enzymes, we achieved spatial organization of the biosynthetic machinery. Systematic optimization of promoter configuration, fermentation conditions, and enzyme fusion yielded the optimal system ScMKI4-GS, achieving gram-per-liter-scale production (1.1 g/L) of the eunicellane-type diterpene benditerpe-2,6,15-triene in simple shake-flask fermentation-substantially outperforming scaffold-free controls. The platform demonstrated broad applicability across five structurally distinct eunicellane synthases, with each exhibiting enhanced production upon scaffold incorporation. For lycopene biosynthesis, the scaffold-assisted system produced 729.7 mg/L-a 9-fold improvement over the canonical MVA pathway under identical conditions, representing the highest IUP-based lycopene titer reported in Escherichia coli to date. Finally, by systematic substitution of chain-length-specific PTs, we expanded the platform to efficiently generate C10-C35 terpene precursors, facilitating functional characterization of terpene-related genes. Collectively, this versatile scaffold-assisted IUP platform provides a robust tool to expand terpenoid structural diversity and accelerate their scalable overproduction.
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
L-(+)-tartaric acid (L-TA) is a high-value chiral organic acid essential for food and pharmaceuticals. Despite its industrial importance, sustainable green production is constrained by the lack of a fully defined biosynthetic pathway. Here, we report the de novo biosynthesis of L-TA in Saccharomyces cerevisiae through reaction-guided enzyme mining, experimental validation, and Enzyme Commission-specific Catalytic Hybrid Optimizer (ECHO)-assisted enzyme prioritization. We first elucidate the elusive two-step conversion from precursor 5-keto-D-gluconic acid (5-KGA) to L-TA, catalyzed by transketolase (TK) and succinate semialdehyde dehydrogenase (SSDH). To optimize this critical step, we develop the ECHO. This multimodal framework integrates sequence, substrate, and pocket-aware structural information to identify high-performance TK-SSDH pairs. By integrating this pathway with de novo precursor synthesis, cofactor engineering, and semi-rational protein engineering, a final L-TA titer of 6.59 mg L−1 was achieved in a 5-L bioreactor. By connecting computational mining and metabolic assembly through a multi-module engineering strategy, our study establishes a green platform for L-TA production and demonstrates an effective workflow for synthetic pathway design. L-(+)-tartaric acid (L-TA) is a high-value chiral organic acid for food and pharmaceuticals. Here the authors produce L-TA in S. cerevisiae through reaction-guided enzyme mining and Enzyme Commission-specific Catalytic Hybrid Optimizer (ECHO)-assisted enzyme prioritization.
Xuan Zhou, Jiaheng Hou, Zikai Wang et al.· Nature Communications· 0 citations
Natural products constitute a vital source for drug discovery, yet extraction from producers is inefficient and chemical synthesis involves complex, low yield routes, limiting sustainable supply. Reconstructing large scale metabolic pathways (≥10 enzymatic steps) in microbial cell factories using synthetic biology offers a promising solution for efficient and sustainable production of high value natural products. This review systematically analyzes core strategies and cutting edge technologies for reconstructing such pathways, including advanced cloning methods, modular pathway design, self assembly approaches (protein/DNA scaffolds and metabolic channeling), division of labor optimization in co culture systems, and dynamic regulation mechanisms (promoter engineering, riboswitches, biosensor based feedback control). We further explore the emerging roles of systems biology modeling and machine learning in retrosynthetic pathway design, rational enzyme engineering, and precise metabolic flux regulation. By integrating intelligent algorithms with multidisciplinary tools, these approaches hold promise for overcoming bottlenecks in the biomanufacturing of complex natural products, thereby accelerating drug development and enabling green, sustainable production. This review highlights the importance of combining rational design, dynamic control, and modular co culture strategies to address challenges such as metabolic burden, pathway imbalance, and host toxicity, ultimately paving the way for scalable and cost effective biosynthesis of natural product pharmaceuticals.
