Cell-free synthetic biology: Engineering in an open world

Abstract

Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells. It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system. This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels, including protein engineering, metabolic engineering, and artificial cell engineering. In cell-free protein engineering, the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins, toxic proteins, membrane proteins, and novel proteins with unnatural amino acids. Cell-free systems offer the ability to design metabolic pathways towards the production of desired products. Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

“…Not possible to add supplements externally during translation MPs have to be purified and reconstituted into liposomes or detergents for functional analysis [9,20] Reliability Most of the reports related to CFPS are currently limited to the research and laboratory level, but progress towards the drug discovery pipeline has been made recently Most reliable and state of the art for protein production and drug discovery purposes, and approved by drug authorities [21] Functional characterization Compared with cell-based systems, standardized biophysical, biochemical assays are limited, but progress has been made recently A wide range of standardized biophysical and biochemical techniques are available for proteins synthesized by cell-based systems [22] Protein yields and downstream applications Yields range from µg/mL (complex proteins) to several mg/mL (cytosolic proteins and few MPs) with more complex proteins Downstream applications are simple and protein can be purified and reconstituted immediately after synthesis Yields can be very high, in the range of mg/mL Downstream applications are possible but need to additionally lyse the cells for MPs [11,21,22] Post-translational modifications (PTMs) PTMs possible (mostly in eukaryotic CF systems with translationally active microsomes) Limited PTMs in prokaryotic and eukaryotic lysates lacking endogenous microsomes. O-Glycosylation not possible All PTMs are possible including O-glycosylation [13] Incorporation of non-standard amino acids Ideal choice for the incorporation of single and multiple noncanonical amino acids Difficult to incorporate non-canonical amino acids due to cell membrane barrier and cytotoxic effects [23,24] Scale of reaction volume Ranging from few µL (chip-based and batch-based in an Eppendorf tube) to 100 L reaction (in a fermenter)…”

“…Completely closed system and difficult to manipulate [22] Automation CF systems can be automated with high throughput screening of multiple templates, starting in an ELISA plate format Generally difficult to automate due to the requirement of larger volumes and aseptic techniques [29] Point of care production of biologics Lyophilized CF lysates are suitable for the production of therapeutic proteins next to the emergency settings Very difficult due to its time-consuming process and requirements of large infrastructure including manufacturing facilities, transport, and cold storage facilities [30,31] Recently, several eukaryotic CF extracts based on Tobacco [35], Leishmania [36], Neurospora [37], yeast cells [38], and human blood cells [39] were characterized and optimized for a limited number of proteins at the laboratory level. There is a growing trend in the development of novel CF platforms for taking advantage of the genetic tools available in the literature and the abundant literature available on the in vivo expression of proteins.…”

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…Envisioning a synthetic cell necessitates defined compartments and the direction of TX‐TL, which are accomplished respectively throughout the operation of lipids, DNA template, and RNA polymerase (Rampioni, Leoniλ, & Stano, 2019). Bottom‐up assembled constructs, despite possessing the potential for biosensing, identification, and responding to the surrounding environment through genetic circuits, are unable of reproduction, as characteristics of living cells (Lu, 2017). Tackling the problem of construction of a self‐duplicating cell model necessitates subunit integration, micro‐fabrication, and cell‐free expression systems to promote the development of minimal cells.…”

Bottom-up synthetic biology: modular design for making artificial platelets

“…With the advent of cell-free systems, studies of protein production and regulation of transcription and translation with specific genetic models have become robust and efficient (16–18). Furthermore, interactions between different molecules (e.g.…”