Cell-free protein synthesis: Recent advances in bacterial extract sources and expanded applications

Highlights

• New CFPS systems have been developed from different bacterial extract sources.

• Applications of CFPS for next-generation biomanufacturing.

• Future challenges and opportunities for the CFPS technology.

Abstract

Cell-free protein synthesis (CFPS) is a technology for the production of proteins in vitro, which has emerged as a robust platform for biotechnology and synthetic biology applications. Recently, the renewed scientific interest in CFPS technology has driven the rapid development of cell-free biotechnology. In this review, we focus on the very recent advances and applications of CFPS with a time frame from 2016 to the middle of 2018. First, we introduce newly developed CFPS systems from different microorganisms including Streptomyces species, Bacillus subtilis, Pseudomonas putida, and Vibrio natriegens. Then, we summarize emerging applications of CFPS for next-generation biomanufacturing. With that, we discuss the future perspectives of CFPS and its potential for cost-effective, high-yielding production of therapeutics, chemicals, and materials.

Introduction

Cell-free protein synthesis (CFPS), also known as in vitro transcription-translation, has emerged as a powerful platform for the production of recombinant proteins without the use of living, intact cells. While the CFPS system was first used to elucidate the genetic code in the 1960s [1], it has shown substantial utility as a protein production technology during the past two decades [[2], [3], [4], [5], [6], [7]]. CFPS systems use crude cell extracts [8] or purified components [9] to synthesize a wide variety of proteins that include therapeutic proteins [[10], [11], [12]], membrane proteins [[13], [14], [15]], metalloproteins [[16], [17], [18]], and proteins modified with non-standard amino acids [[19], [20], [21]]. Recently, a renaissance in CFPS systems not only expands the protein synthesis toolkit, but also leads to wide and exciting applications in the field of synthetic biology, for example, the prototyping of genetic circuits and metabolic pathways [22,23], designing of medical diagnostics and biosensors [24,25], biosynthesis of natural products [26,27], and engineering of microfluidic biochip devices [28,29], among others [[30], [31], [32]]. Overall, pioneering efforts by scientists and engineers have created simple, robust, cost-effective, and efficient CFPS platforms for the rapid synthesis, study, and engineering of proteins, as well as for compelling applications in synthetic biology and biotechnology.

In recent years, with the fast development and great progress of CFPS technology, some outstanding reviews have been published, which summarize the advancement of CFPS systems and their broad applications [[33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44]]. For example, Jewett and colleagues comprehensively described the technological advances in CFPS and its emerging applications for the production of protein libraries, personalized medicines, evolved proteins, and protein-based biomaterials [34,35]. Different CFPS systems, which are generated from various prokaryotic (e.g., Escherichia coli) and eukaryotic (e.g., wheat germ and Chinese hamster ovary) organisms, have also been reviewed, showing their versatile biological and application-based goals [[36], [37], [38], [39]]. Although multiple CFPS systems are available, each one has its own advantages and disadvantages, which are intensively summarized and compared in details by Kubick and co-workers [40]. In addition, several reviews document the combination of CFPS systems with other high-throughput devices like microfluidics and lab-on-a-chip for the development of new application platforms [43,44]. More recently, the renewed scientific interest in CFPS technology has driven the rapid development of cell-free biotechnology, which stimulates the establishment of novel CFPS platforms, as well as other new research areas like cell-free metabolic engineering, natural product biosynthesis, and portable, on-demand biomolecular manufacturing. These new results are exciting and, therefore, it is necessary to summarize the recent achievements in an effort to give the reader an updated overview of the current state-of-the-art CFPS technology.

In this review, we aim to highlight the very recent advances and applications of CFPS (Fig. 1), covering from 2016 to the middle of 2018. First, we introduce newly developed CFPS systems from different microorganisms. Then, we summarize emerging applications of CFPS for next-generation biomanufacturing. Finally, we discuss the future prospects of CFPS for synthetic biology and biotechnology applications.