Cell‐free protein synthesis enables one‐pot cascade biotransformation in an aqueous‐organic biphasic system

• Published in: Biotechnology and bioengineering Vol. 117; no. 12; pp. 4001 - 4008
• Main Authors: Liu, Wan‐Qiu, Wu, Changzhu, Jewett, Michael C., Li, Jian
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.12.2020
• Subjects: biocatalysis; Biocatalysts; Biotransformation; biphasic system; Cascade chemical reactions; cascade reaction; Cell-Free System; cell‐free protein synthesis; Chemical reactions; Chemical synthesis; Conversion; Enzymes; Escherichia coli - chemistry; Escherichia coli - metabolism; Green Fluorescent Proteins - biosynthesis; Organic solvents; Protein Biosynthesis; Protein synthesis; Proteins; Solvents; Styrene; Substrates; biotransformation; cell-free protein synthesis; cascade reaction; biocatalysis; biphasic system
• ISSN: 0006-3592; 1097-0290; 1097-0290
• DOI: 10.1002/bit.27541

Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this study, we used cell‐free protein synthesis (CFPS) to express enzymes in an aqueous‐organic biphasic system for the construction of an artificial enzymatic pathway. CFPS‐expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to (S)‐1‐phenyl‐1,2‐ethanediol (two steps in one pot) with 100% conversion. In addition, our CFPS system showed great tolerance to different organic solvents, and, importantly, the entire biocatalytic system can be consistently scaled up without a reduction of the substrate conversion rate. We, therefore, anticipate that our cell‐free approach will make a possible cost‐effective, high‐yielding synthesis of valuable chemicals. This study demonstrates the ability to use cell‐free protein synthesis (CFPS) for one‐pot production of (S)‐1‐phenyl‐1,2‐ethanediol from the water‐insoluble substrate, styrene, in an aqueous‐organic biphasic system. CFPS‐expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to (S)‐1‐phenyl‐1,2‐ethanediol (two steps in one pot) with 100% conversion. This cell‐free approach will enable new directions in developing biocatalytic cascades for defined biotransformations, providing a new and feasible avenue for efficiently synthesizing chemicals of pharmaceutical and industrial importance.