Traditional botanical extraction of medicinal anthraquinones like aloe-emodin is limited by low yields, long growth cycles, and environmental dependence. Microbial biosynthesis provides a sustainable alternative, but de novo reconstruction of aloe-emodin remains difficult due to unresolved pathway steps and poor metabolic flux distribution in engineered hosts. In this study, we engineered Aspergillus nidulans for the de novo biosynthesis of aloe-emodin via modular assembly of an artificial pathway. We first established an emodin-producing chassis by reconstructing a minimal mdp-derived upstream module and enhancing precursor supply, yielding 86.93 mg/L emodin. Introducing the heterologous cytochrome P450 CYP-H6231 effectively catalyzed the hydroxymethylation of emodin to citreorosein. We then extended the pathway by incorporating the reductive tailoring enzymes MdpK, MdpC, and MdpB to form aloe-emodin. While feeding assays supported a citreorosein-linked route to aloe-emodin, constitutive expression of this downstream module caused unbalanced flux and high accumulation of the competing byproduct chrysophanol. To address this, we applied temporal induction to mdpK. Delaying its expression shifted flux partitioning, significantly reducing chrysophanol formation and improving aloe-emodin selectivity. The final engineered strain achieved 278.06 mg/L citreorosein and 70.97 mg/L aloe-emodin during 1-L fed-batch fermentation. This study establishes a de novo microbial route to aloe-emodin and highlights temporal flux control as a practical strategy for assembling complex plant-derived anthraquinone pathways in fungal cell factories.
What does microbial robustness mean in the context of industrial bioprocesses, and which cellular design principles are required to maintain strain performance from laboratory to industrial scale? This review addresses these questions by presenting an integrated systems metabolic engineering perspective on microbial chassis design. Robust production hosts must maintain viability, metabolic function, production capacity, and population stability throughout the entire process trajectory, including the seed train, biomass formation, production, product accumulation, and late-stage stress exposure. The relevant engineering target is therefore not an isolated gene or pathway, but the coordinated cellular system as a whole. Genome architecture, expression control, stress regulation, metabolic flux distribution, membrane physiology, product export, and population stability must be aligned with the intended process and evaluated under dynamic, process-relevant conditions. Accordingly, robustness engineering should begin with the industrial objective in mind while remaining centered on the microbial host.
A comprehensive understanding of the growth–production trade-off under heat stress is essential for developing high-yield thermotolerant chassis. Here, the effects of elevated temperature on growth and erythritol biosynthesis were systematically investigated in YL (A high-erythritol-producing industrial Yarrowia lipolytica strain). Notably, a heat-induced abnormal growth-surge effect (AGSE) was observed and identified, in which high dissolved oxygen conditions were associated with altered ATP-related metabolic activity, oxidative stress responses, and increased accumulation of stress-protective metabolites. Based on this, iterative dissolved-oxygen optimization under high-temperature conditions enabled the development of a robust erythritol-producing chassis, YL-EV-18-G5, which exhibited stable growth at 35°C with erythritol yield of 0.526 g/g glucose. Further, a hybrid erythritol-responsive bidirectional promoter (Pery-BidR (5AB)) was applied for random genomic integration, thereby coupling erythritol synthesis with the expression of growth-related genes, yielded a growth-production-coordinated strain, YL-EV-18-G5-GPcEs4-F12 (F12). Compared with parental strain YL, F12 exhibited a 179.3% increase in erythritol titer, reaching a maximum concentration of 225.50 g/L, and the yield increased by 35.34% to 0.656 g/g glucose. Mechanistically, the high productivity of F12 is associated with optimized stress-protective metabolite synthesis, reduced single-cell erythritol production heterogeneity, and adaptive genomic changes accumulated during evolutionary engineering. These findings advance the understanding of thermotolerance in oleaginous yeasts and provide a potential strategy for engineering heat-resistant microbial cell factories.
Redox-balance-based growth coupling links oxidoreductase activity to cellular fitness and can enable high-throughput enzyme selection and strain evolution. However, because growth reflects the fitness of the whole cell, metabolic adaptation can circumvent the intended selective pressure. Here, we investigated the evolutionary stability of an aerobic NADPH redox-balance selection strain of Escherichia coli engineered to accumulate NADPH during growth on glucose. Adaptive evolution first selected mutations predicted to relax cofactor specificity in NADH-dependent quinone oxidoreductases, allowing NADPH-derived reducing equivalents to enter respiratory electron flow. Removing major NADH-to-quinone oxidoreductases did not eliminate the capacity for evolutionary escape; in one successfully evolving lineage, adaptive evolution uncovered a latent lactate-mediated route for electron transfer into the quinone pool. These results reveal strong selective pressure to reconnect soluble nicotinamide redox metabolism with energy-conserving respiration and demonstrate that E. coli can access multiple pathways for restoring electron flux between these systems. Overall, this work outlines design principles for aerobic, growth-coupled strains which rely upon redox balance, illustrating that successful designs must limit respiratory rewiring while preserving sufficient carbon and energy flux to support growth and pathway selection.
Porphyrin-derived high-value chemicals are important for food, medicine, and fine chemicals manufacturing, yet the physiological complexity of tetrapyrrole metabolism constrains efficient bioproduction. Here, we systematically engineered the cytochrome-rich bacterium Shewanella oneidensis MR-1 for porphyrin production. A modular coproporphyrin III biosynthetic pathway was reconstructed and progressively optimized through hemB homolog selection, antioxidant defense engineering, attenuation of competing tetrapyrrole branches, genome-scale model-guided target mining, plasmid-backbone reassignment, and ribosome-binding site tuning. The resulting strain achieved a remarkable coproporphyrin III production of 1525.5 ± 37.3 mg L−1 and was further raised to 1906.8 ± 39.2 mg L−1 under 5-aminolevulinic acid supplementation condition. Integrated metabolomic analyses identified a riboflavin-associated redox response linked to improved production physiology, while extracellular product accumulation enabled efficient acetone-assisted crude recovery. The platform was further extended to protoporphyrin IX and heme production with titers of 612.7 ± 4.8 mg L−1 and 107.4 ± 3.3 mg L−1, respectively. This work demonstrates an integrated metabolic engineering strategy combining pathway reconstruction, physiological stress mitigation, model-guided target selection, and multilevel expression balancing for developing S. oneidensis as a porphyrin-producing cell factory.
