A Lactose-Based Kluyveromyces lactis Cell-Free Protein Synthesis System

High Resolution Image Download MS PowerPoint Slide Yeasts have long been celebrated for their metabolic prowess, in particular, their ability to transform grain and fruit into valuable food products. In recent decades, humans have exploited them for pharmaceutical applications driven by advances in metabolic engineering and synthetic biology. Through convergence of these disciplines, this study highlights the development of a cell-free protein synthesis (CFPS) platform using Kluyveromyces lactis, a yeast prized for its role in the dairy industry. We present a workflow for preparing K. lactis extracts and incorporate lactose as a sustainable and cost-effective carbon source for biomass generation and as an energy source for CFPS. A semiautomated design-of-experiments (DoE) approach was undertaken, based on a Latin Hypercube experimental design, which tested 128 unique CFPS reaction mix compositions (against a baseline optimized for Pichia pastoris ). The optimized reaction mix was validated by the synthesis of two model proteins: green fluorescent protein (deGFP) and Erythropoietin (EPO), which is a clinically relevant therapeutic. We identified conditions with 4-fold improvement in yield with the optimized reaction producing 54 nM of EPO. By integrating lactose-based growth, protein synthesis, and rational optimization strategies, this study sets the scene for developing yeast-based CFPS platforms tailored for diverse applications, from biosensor development to industrially relevant biopharmaceutical production.