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

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Synthetic and Systems Biotechnology 2018年第2期3卷 90-96页

作者： Lihong Jiang Jiarun Zhao Jiazhang Lian Zhinan Xu Key Laboratory of Biomass Chemical Engineering of Ministry of Education College of Chemical and Biological EngineeringZhejiang UniversityHangzhou 310027China

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.

关键词： cell-free protein synthesis Metabolic pathway optimization Genetic circuits Metabolic engineering Synthetic biology

Yeast knockout library allows for efficient testing of genomic mutations for cell-free protein synthesis

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Synthetic and Systems Biotechnology 2016年第1期1卷 2-6页

作者： Jennifer *** Lauren G.Clark Alaksh Choudhury C.Eric Hodgman Michael C.Jewett Department of Chemical and Biological Engineering Northwestern University2145 Sheridan RoadEvanstonIL 60208-3120USA Chemistry of Life Processes Institute 2170 Campus DriveEvanstonIL 60208-3120USA Masters in Biotechnology Program Northwestern University2145 Sheridan RoadEvanstonIL 60208-3120USA Robert H.Lurie Comprehensive Cancer Center Northwestern University676 N.St Clair StSuite 1200ChicagoIL 60611-3068USA Simpson Querrey Institute Northwestern University303 E.Superior StSuite 11-131ChicagoIL 60611-2875USA

cell-free protein synthesis(CFPS)systems from crude lysates have benefitted from modifications to their enzyme composition.For example,functionally deleting enzymes in the source strain that are deleterious to CFPS can improve protein synthesis yields.However,making such modifications can take substantial time.As a proof-of-concept to accelerate prototyping capabilities,we assessed the feasibility of using the yeast knockout collection to identify negative effectors in a Saccharomyces cerevisiae CFPS platform.We analyzed extracts made from six deletion strains that targeted the single deletion of potentially negative effectors(e.g.,nucleases).We found a statistically significant increase in luciferase yields upon loss of function of GCN3,PEP4,PPT1,NGL3,and XRN1 with a maximum increase of over 6-fold as compared to the wild type.Our work has implications for yeast CFPS and for rapidly prototyping strains to enable cell-free synthetic biology applications.

关键词： cell-free protein synthesis Saccharomyces cerevisiae Synthetic biology In vitro translation cell-free biology protein expression

Silk fibroin as an additive for cell-free protein synthesis

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Synthetic and Systems Biotechnology 2020年第3期5卷 145-154页

作者： Marilyn S.Lee Chia-Suei Hung Daniel A.Phillips Chelsea C.Buck Maneesh K.Gupta Matthew W.Lux US Army Combat Capabilities Development Command Chemical and Biological Center 8567 Ricketts Point RoadAberdeen Proving GroundMD21010USA US Air Force Research Laboratory 217912th St.B652/R122 Wright-Patterson Air Force BaseOH45433USA US Naval Research Laboratory Center for Bio/Molecular Science and Engineering Bldg.42Room 3034555 Overlook Ave.WashingtonDC 20375UES Inc.4401 Dayton Xenia Rd.BeavercreekOH 45432USA

cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions.Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing,point-of-need manufacturing,and responsive materials.Meanwhile,silk fibroin from the silk worm,Bombyx mori,has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes.In this work,we explore the effects of silk fibroin as an additive in cell-free protein synthesis(CFPS)reactions.Impacts of silk fibroin on CFPS activity and stability after drying,as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed.We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%,which we attribute to macromolecular crowding effects.However,we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes.Further,the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture.Crosslinking attempts did not impact CFPS activity,but did yield localized protein aggregates rather than a hydrogel.We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.

关键词： cell-free systems cell-free protein synthesis CFPS Silk fibroin Preservation

Streamlining the preparation of “endotoxin-free” ClearColi cell extract with autoinduction media for cell-free protein synthesis of the therapeutic protein crisantaspase

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Synthetic and Systems Biotechnology 2019年第4期4卷 220-224页

作者： J.Porter Hunt Emily Long Zhao Mehran Soltani Madison Frei J.Andrew D.Nelson Bradley C.Bundy Department of Chemical Engineering Brigham Young UniversityProvoUTUSA Department of Chemical Engineering Brigham Young University330N Engineering BuildingProvoUT84602USA

An“endotoxin-free”E.coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand.However,preparation of the most complex CFPS reagent–the cell extract–remains time-consuming and labor-intensive because of the relatively slow growth kinetics of the endotoxin-free ClearColiTMBL21(DE3)strain.Here we report a streamlined procedure for preparing E.coli cell extract from ClearColi™using auto-induction media.In this work,the term auto-induction describes cell culture media which eliminates the need for manual induction of protein expression.Culturing Clearcoli™cells in autoinduction media significantly reduces the hands-on time required during extract preparation,and the resulting“endotoxinfree”cell extract maintained the same cell-free protein synthesis capability as extract produced with traditional induction as demonstrated by the high-yield expression of crisantaspase,an FDA approved leukemia therapeutic.It is anticipated that this work will lower the barrier for researchers to enter the field and use this technology as the method to produce endotoxin-free E.coli-based extract for CFPS.

关键词： Auto-induction Autoinduction cell-free protein synthesis CFPS Endotoxin-free Crisantaspase Clearcoli

Toward efficient multiple-site incorporation of unnatural amino acids using cell-free translation system

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Synthetic and Systems Biotechnology 2022年第1期7卷 522-532页

作者： Jiaqi Hou Xinjie Chen Nan Jiang Yanan Wang Yi Cui Lianju Ma Ying Lin Yuan Lu College of Life Sciences Shenyang Normal UniversityShenyang110034China Key Laboratory of Industrial Biocatalysis Ministry of EducationDepartment of Chemical EngineeringTsinghua UniversityBeijing100084China Guangdong Key Laboratory of Fermentation and Enzyme Engineering School of Biology and Biological EngineeringSouth China University of TechnologyGuangzhou510006China

Amber suppression has been widely used to incorporate unnatural amino acids(UNAAs)with unique structures or functional side-chain groups into specific sites of the target protein,which expands the scope of protein-coding chemistry.However,this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein,which limits the development of unnatural proteins.To address this challenge,the suppression method using multiple termination codons(TAG,TAA or TGA)was proposed,and cell-free unnatural protein synthesis(CFUPS)system was employed.By the analysis of incorporating 3 different UNAAs(p-propargyloxy-L-phenylalanine,p-azyl-phenylalanine and L-4-Iodophenylalanine)and mass spectrometry,the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation.The CFUPS conditions were further optimized for better UNAA incorporation efficiency,including the orthogonal translation system(OTS)components,magnesium ions,and the redox environment.This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs,thereby synthesizing unnatural proteins with novel physicochemical functions.

关键词： cell-free protein synthesis Orthogonal translation system Unnatural protein Unnatural amino acid Terminal codon suppression

cell-free expression of NO synthase and P450 enzyme for the biosynthesis of an unnatural amino acid L-4-nitrotryptophan

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Synthetic and Systems Biotechnology 2022年第2期7卷 775-783页

作者： Xintong Tian Wan-Qiu Liu Huiling Xu Xiangyang Ji Yushi Liu Jian Li School of Physical Science and Technology ShanghaiTech UniversityShanghai201210China

cell-free system has emerged as a powerful platform with a wide range of in vitro applications and recently has contributed to express metabolic pathways for biosynthesis.Here we report in vitro construction of a native biosynthetic pathway for L-4-nitrotryptophan(L-4-nitro-Trp)synthesis using an Escherichia coli-based cell-free protein synthesis(CFPS)system.Naturally,a nitric oxide(NO)synthase(TxtD)and a cytochrome P450 enzyme(TxtE)are responsible for synthesizing L-4-nitro-Trp,which serves as one substrate for the biosynthesis of a nonribosomal peptide herbicide thaxtomin A.Recombinant coexpression of TxtD and TxtE in a heterologous host like E.coli for L-4-nitro-Trp production has not been achieved so far due to the poor or insoluble expression of TxtD.Using CFPS,TxtD and TxtE were successfully expressed in vitro,enabling the formation of L-4-nitro-Trp.After optimization,the cell-free system was able to synthesize approximately 360μM L-4-nitro-Trp within 16 h.Overall,this work expands the application scope of CFPS for study and synthesis of nitro-containing compounds,which are important building blocks widely used in pharmaceuticals,agrochemicals,and industrial chemicals.

关键词： cell-free protein synthesis NO synthase P450 enzyme L-4-nitrotryptophan In vitro biosynthesis Synthetic biology

cell-free synthetic biology for in vitro biosynthesis of pharmaceutical natural products

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Synthetic and Systems Biotechnology 2018年第2期3卷 83-89页

作者： Jian Li Lingkai Zhang Wanqiu Liu School of Physical Science and Technology ShanghaiTech UniversityShanghai 201210China

Natural products with significant biological activities continuously act as rich sources for drug discovery and development.To harness the potential of these valuable compounds,robust methods need to be developed for their rapid and sustainable production.cell-free biosynthesis of pharmaceutical natural products by in vitro reconstruction of the entire biosynthetic pathways represents one such solution.In this review,we focus on in vitro biosynthesis of two important classes of natural products,polyketides(PKs)and nonribosomal peptides(NRPs).First,we summarize purified enzyme-based systems for the biosynthesis of PKs,NRPs,and PK/NRP hybrids.Then,we introduce the cell-free protein synthesis(CFPS)-based technology for natural product production.With that,we discuss challenges and opportunities of cell-free synthetic biology for in vitro biosynthesis of natural products.

关键词： cell-free synthetic biology Purified enzymes cell-free protein synthesis Polyketides Nonribosomal peptides In vitro biosynthesis Natural products

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

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Synthetic and Systems Biotechnology 2017年第2期2卷 97-104页

作者： Filippo Caschera Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris(ESPCI Paris) CNRS UMR 708310 Rue Vauquelin75231 Paris Cedex 05France

cell-free expression system is a technology for the synthesis of proteins in vitro.The system is a platform for several bioengineering projects,e.***.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.

关键词： cell-free protein synthesis Engineering Optimization Polysaccharide ATP-regeneration Machine-learning Synthetic minimal cell

cell-free synthetic biology:Engineering in an open world

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Synthetic and Systems Biotechnology 2017年第1期2卷 23-27页

作者： Yuan Lu Key Lab of Industrial Biocatalysis Ministry of EducationDepartment of Chemical EngineeringTsinghua UniversityBeijing 100084China Institute of Biochemical Engineering Department of Chemical EngineeringTsinghua UniversityBeijing 100084China

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.

关键词： cell-free synthetic biology cell-free protein synthesis protein engineering Metabolic engineering Artificial cell Unnatural amino acids