Synergies in Green Bioprocessing: A Comprehensive Review of Heterologous Protein Expression and the Transformative Potential of Natural Deep Eutectic Solvents

Heterologous protein expression underpins the production of therapeutics, industrial enzymes, and diagnostic reagents, yet persistent challenges remain in enhancing yields, achieving correct folding, and reducing the costs and environmental burdens of down-stream processing. Natural Deep Eutectic Solvents (NADES)—a class of biocompatible, sustainable, and highly tunable solvents—have recently emerged as promising tools to overcome these limitations. This review systematically examines the intersection of recombinant protein production and NADES technology, assessing their applications across the full workflow, from host strain expression to purification and final formula-tion. Literature analysis highlights the potential of NADES as media additives that mitigate cellular stress and improve soluble protein yields, as gentle solubilizing and refolding agents for inclusion bodies, as phase-forming components in aqueous two-phase systems for green purification, and as stabilizing excipients for long-term storage. Key constituents such as betaine, proline, urea, and arginine are identified as functional agents whose eutectic mixtures often deliver synergistic benefits. The inte-gration of NADES into recombinant protein production offers a path toward more sustainable and economically viable biomanufacturing. Critical gaps remain, including in vivo validation and techno-economic assessment. Future opportunities include high-throughput NADES screening and computational design of application-specific solvents.