Development of <i>Komagataella phaffii</i> Cell‐Free Protein Synthesis Using Cell From Basic Salt Medium

ABSTRACT Cell‐free protein synthesis (CFPS) offers a promising option of enzyme production for food bioengineering, with the advantages of eliminating the steps of recombinant microbial cell construction, rapid protein synthesis process, and high throughput of various proteins simultaneously. Komagataella phaffii CFPS is highlighted for its high level of protein synthesis, successfully harnessing the native strong alcohol oxidase 1 promoter in the absence of T7 RNA polymerase. Currently, K. phaffii CFPS uses cell extract from complex medium cultivated K. phaffii cells, which is still high‐cost and limits large‐scale cell extract preparation industrially. This study developed a K. phaffii CFPS using chemically defined medium through optimization of protein concentration in cell extracts, a new cell extract preparation method S603, which simplified the process and enhanced the protein concentration in cell extract, and optimization of methanol induction time. Finally, K. phaffii CFPS using cell extract from chemically defined medium cultivation exhibited 90.8 mg/mL total protein in cell extract and synthesized 600.0 mg/L GFP, which was the highest value until now. This successful K. phaffii CFPS provided a cost‐effective and high‐level expression platform for enzyme production for food bioengineering.