Jeong Chan Joo

The widespread use of indigo (C₁₆H₁₀N₂O₂), a historically significant blue pigment, continues across the textile, pharmaceutical, and cosmetic industries. However, conventional production routes based on petrochemical aniline or plant extraction pose serious environmental and sustainability concerns. Here, we present a simulation-guided, cell-free biomanufacturing platform for indigo synthesis that integrates enzyme kinetics modeling with scalable lysate-based catalysis. Three enzymes–tryptophanase A, flavin-containing monooxygenase, and glucose 1-dehydrogenase–were co-expressed in Escherichia coli, enabling the conversion of L-tryptophan to indigo via NADPH-dependent oxidation. Crude lysates prepared from high-cell-density fermentation retained full catalytic activity post-lyophilization, facilitating long-term storage and industrial deployment. Enzyme kinetic parameters were quantified directly in the crude lysates and incorporated into a Python-based ordinary differential equation model (ODE), enabling in silico optimization for substrate loads, cofactor regeneration dynamics, and feeding strategies. Model-optimized feeding strategies enabled cell-free titers of 21.2 g/L and 9.8 g/L from L-tryptophan and indole, respectively, the highest ever reported for FMO-based crude-lysate platforms. Compared to synthetic or whole-cell-derived indigo, the enzymatically produced dye exhibited a markedly reduced endotoxin content, negligible cytotoxicity, and enhanced anti-inflammatory and UV-protective properties. Furthermore, a preliminary raw-material cost analysis identified NADP⁺ as the dominant cost driver (~44.75% of total raw-material costs), and the kinetic model directly linked indigo yield to NADP⁺ loading and feeding strategy, providing a quantitative tool to optimize cofactor utilization in future process development. This work establishes a modular, simulation-driven framework for the sustainable and scalable biosynthesis of high-value dyes, and paves the way for the broader industrial adoption of cell-free biomanufacturing systems.

Marine algal polysaccharides (MAPs) are multifunctional biopolymers with significant potential in biomedical applications. Derived from brown, red, and green algae, key examples include alginate, agar, carrageenan, fucoidan, ulvan, and laminarin. Their structural diversity underlies a broad range of biological activities, particularly among sulfated polysaccharides, which exhibit antiviral, anticancer, anticoagulant, immunomodulatory, and antioxidant effects. Owing to their biocompatibility and tunable physicochemical properties, MAPs are also valuable in wound healing, tissue regeneration, and drug delivery. Advances in ultrasound-, microwave-, and enzyme-assisted extraction methods have enhanced yield and functionality. This review combines structural, extraction, and biomedical views on MAPs, with a focus on how molecular characteristics relate to their potential as drugs. Future work should focus on scalable green extraction, molecular-level characterization, and clinical validation to develop MAPs-based biomaterials for next-generation drug delivery, wound healing, and tissue engineering.