Cell-free protein synthesis enabled rapid prototyping for metabolic engineering and synthetic biology

Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade.However,due to complexity of cellular metabolism,the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering.Recently,cell-free protein synthesis system(CFPS)has been emerging as an enabling alternative to address challenges in biomanufacturing.This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits(biosensors)to speed up design-build-test(DBT)cycles of metabolic engineering and synthetic biology.

Cell-free synthetic biology:Engineering in an open world

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.

Bacterial cell-free expression technology to in vitro systems engineering and optimization

Cell-free expression system is a technology for the synthesis of proteins in vitro.The system is a platform for several bioengineering projects,e.g.cell-free metabolic engineering,evolutionary design of experiments,and synthetic minimal cell construction.Bacterial cell-free protein synthesis system(CFPS)is a robust tool for synthetic biology.The bacteria lysate,the DNA,and the energy module,which are the three optimized sub-systems for in vitro protein synthesis,compose the integrated system.Currently,an optimized E.coli cell-free expression system can produce up to^2.3 mg/mL of a fluorescent reporter protein.Herein,I will describe the features of ATP-regeneration systems for in vitro protein synthesis,and I will present a machine-learning experiment for optimizing the protein yield of E.coli cell-free protein synthesis systems.Moreover,I will introduce experiments on the synthesis of a minimal cell using liposomes as dynamic containers,and E.coli cell-free expression system as biochemical platform for metabolism and gene expression.CFPS can be further integrated with other technologies for novel applications in environmental,medical and material science.

无细胞蛋白表达系统新进展及在生物制药工程中的应用

无细胞蛋白表达体系是一种以细胞抽提物为基础的体外合成蛋白质表达技术,具有遗传背景简单、反应操控简便等特点,已成为研究生物反应系统的重要技术手段。在研究人员的不断努力下,反应体系从原核扩展到真核蛋白质合成体系,而且目标蛋白表达量从毫克级提高到数克级每升,成本不断降低,反应规模可达到百公升级。近年来,无细胞蛋白表达系统在复杂蛋白、毒性蛋白和膜蛋白表达方面的优势逐渐体现,展示了其在生物制药领域的重要应用潜力。总之,无细胞技术已经成为异源蛋白质高效合成和生物制药领域中有巨大潜力的新策略。