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.

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

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.

Silk fibroin as an additive for cell-free protein synthesis

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.

CO_(2)-elevated cell-free protein synthesis

Gases are the vital nutrition of all organisms as the precursor of metabolism pathways.As a potential biological process,protein synthesis is inevitably regulated by gas transport and utilization.However,the effect of carbon dioxide(CO_(2))present in many metabolic pathways on protein synthesis has not been studied well.In this work,carbon dioxide combined with oxygen was employed for cell-free protein synthesis(CFPS)in the tube-in-tube reactor with precise control of gas concentration.In this in vitro system,gases could directly affect the protein synthesis process without transmembrane transport.Varied concentrations of carbon dioxide(0-1%)and constant oxygen concentration(21%)were employed for CFPS to assess the effects.The cell-free reactions with 0.3%CO_(2) and 21%O_(2) showed the highest protein yields.The combined effect of CO_(2) and O_(2) also resulted in relatively high protein expression under high oxygen conditions(0.3%CO_(2) and 100%O_(2)).Moreover,metabolomics assays were performed to gain insight into metabolic changes,which showed that CO_(2) slightly improved energy metabolism and redox balance.In particular,the extra supplied CO_(2) activated the decarboxylating reactions and removed toxic metabolites to recover the protein synthesis activity.The exploration of CO_(2) on protein synthesis could provide guiding implications for basic studies and biomanufacturing.

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

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.

Enhanced Poly(ethylene terephthalate)Hydrolase Activity by Protein Engineering

Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate)(PET)at room temperature,and is thus regarded as a potential tool to solve the issue of polyester plastic pollution.Therefore,we explored the interaction between PETase and the substrate(a dimer of the PET monomer ethylene terephthalate,2PET),using a model of PETase and its substrate.In this study,we focused on six key residues around the substrate-binding groove in order to create novel high-efficiency PETase mutants through protein engineering.These PETase mutants were designed and tested.The enzymatic activities of the R61A,L88F,and I179F mutants,which were obtained with a rapid cell-free screening system,exhibited 1.4 fold,2.1 fold,and 2.5 fold increases,respectively,in comparison with wild-type PETase.The I179F mutant showed the highest activity,with the degradation rate of a PET film reaching 22.5 mg perμmol·L^-1 PETase per day.Thus,this study has created enhanced artificial PETase enzymes through the rational protein engineering of key hydrophobic sites,and has further illustrated the potential of biodegradable plastics.

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

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.

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

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.

Cell-free synthesis of stable isotope-labeled internal standards for targeted quantitative proteomics

High-sensitivity mass spectrometry approaches using selected reaction monitoring(SRM)or multiple reaction monitoring(MRM)methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics in specific biological systems.Both high-sensitivity detection of lowabundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal standards.Currently,there are various ways for preparing standards,including chemical peptide synthesis,cellular protein expression,and cell-free protein or peptide synthesis.Cell-free protein synthesis(CFPS)or in vitro translation(IVT)systems in particular provide high-throughput and low-cost preparation methods,and various cell types and reconstituted forms are now commercially available.Herein,we review the use of such systems for precise and reliable protein quantification.

Methods to reduce variability in E. Coli-based cell-free protein expression experiments

Cell-free protein synthesis(CFPS)is an established biotechnology tool that has shown great utility in many applications such as prototyping proteins,building genetic circuits,designing biosensors,and expressing cytotoxic proteins.Although CFPS has been widely deployed,the many,varied methods presented in the literature can be challenging for new users to adopt.From our experience and others who newly enter the field,one of the most frustrating aspects of applying CFPS as a laboratory can be the large levels of variability that are present within experimental replicates.Herein we provide a retrospective summary of CFPS methods that reduce variability significantly.These methods include optimized extract preparation,fully solubilizing the master mix components,and careful mixing of the reaction.These have reduced our coefficient of variation from 97.3%to 1.2%.Moreover,these methods allow complete novices(e.g.semester rotation undergraduate students)to provide data that is comparable to experienced users,thus allowing broader participation in this exciting research area.

ENHANCEMENT OF BRAIN AND SPLEEN LYMPHOCYTE PROTEIN SYNTHESIS ACTIVITIES DURING COLD ADAPTATION IN RATS

In cold surroundings, in both plants and animals (the class of fish, amphibian, mammal, etc.) tissue cells may appear many special anti-freeze proteins or cold adaptation-associated proteins for existence and development and regulate relevant gene expression at transcription and translation levels. With respect to mammals, the regulation and integration

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.

Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort

A renaissance in cell-free protein synthesis(CFPS)is underway,enabled by the acceleration and adoption of synthetic biology methods.CFPS has emerged as a powerful platform technology for synthetic gene network design,biosensing and on-demand biomanufacturing.Whilst primarily of bacterial origin,cell-free extracts derived from a variety of host organisms have been explored,aiming to capitalise on cellular diversity and the advantageous properties associated with those organisms.However,cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields,despite the potential for improved protein folding and posttranslational modifications.Here we describe further development of a Pichia pastoris cell-free platform,a widely used expression host in both academia and the biopharmaceutical industry.Using a minimised Design of Experiments(DOE)approach,we were able to increase the productivity of the system by improving the composition of the complex reaction mixture.This was achieved in a minimal number of experimental runs,within the constraints of the design and without the need for liquid-handling robots.In doing so,we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold,respectively.This study highlights the P.pastoris-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.

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

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.

A linear DNA template-based framework for site-specific unnatural amino acid incorporation

Site-specific incorporation of unnatural amino acids(UNAAs)into proteins using an orthogonal translation system(OTS)has expanded the scope of protein-coding chemistry.The key factor affecting UNAA embedding efficiency is the orthogonality of the OTS.Compared to traditional cell systems,cell-free systems are more convenient to control the reaction process and improve the utilization rate of UNAA.In this study,a linear DNA template-based cell-free unnatural protein synthesis system for rapid high-throughput screening and evolution was proposed.A total of 14 cell extracts were selected for screening out cell extract with high expression level.The result showed that EcAR7ΔAΔSer cell extract was optimal for the cell-free system.In addition,the screening results of four UNAAs,p-propargyloxy-l-phenylalanine(pPaF),p-azyl-phenylalanine(pAzF),p-acetyl-l-phenylalanine(pAcF),and p-benzoyl-l-phenylalanine(pBpF),showed that o-aaRS and o-tRNA of pPaF had good orthogonality.A new pair of corresponding o-aaRS and o-tRNA for pBpF was screened out.These results proved that this method could speed up the screening of optimal OTS components for UNAAs with versatile functions.