RESUMEN
Glucose and fatty acids play essential physiological roles in nearly all living organisms, and the pathway that converts glucose into fatty acid is pivotal to the central metabolic network. We have successfully reconstituted a pathway that converts glucose to fatty acid in vitro using 30 purified proteins. Through systematic titration and optimization of the glycolytic pathway and pyruvate dehydrogenase, we increased the yield of free fatty acid from nondetectable to a level that exceeded 9% of the theoretical yield. We also reconstituted the entire pentose-phosphate pathway of Escherichia coli and established a pentose phosphate-glycolysis hybrid pathway, replacing GAPDH to enhance NADPH availability. Our efforts provide a useful platform for research involving these core biochemical transformations.
Asunto(s)
Escherichia coli/metabolismo , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Cromatografía en Capa Delgada , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/análisis , Cromatografía de Gases y Espectrometría de Masas , Glucólisis , NADP/metabolismo , Vía de Pentosa Fosfato , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
Cell-free protein synthesis (CFPS) systems have been widely used for decades as a rapid and efficient tool in fundamental biology. Without the requirements for cell viability and growth, CFPS systems have distinct advantages over in vivo systems for protein production. Recently, great efforts have been made to further optimize CFPS systems to produce proteins at high yields, reduced cost and increased scale, including simplifying extract preparation procedures, developing new energy regeneration systems to protein synthesis, stabilizing substrate supply and promoting protein folding. Nowadays, CFPS systems are emerging as a powerful platform for industrial and high-throughput production of protein therapeutics, providing an alternative solution to solve problems in biopharmaceutical engineering. Moreover, CFPS systems have been successfully applied to high-throughput drug screening, large-scale protein therapeutics production, custom-made anti-cancer vaccines. These achievements highlight that CFPS systems have great potential for a wide range of applications in biopharmaceutical engineering in the future.