Cell‐free protein synthesis system: A new frontier for sustainable biotechnology‐based products

Cell‐free protein synthesis (CFPS) system is an innovative technology with a wide range of potential applications that could challenge current thinking and provide solutions to environmental and health issues. CFPS system has been demonstrated to be a successful way of producing biomolecules in a variety of applications, including the biomedical industry. Although there are still obstacles to overcome, its ease of use, versatility, and capacity for integration with other technologies open the door for it to continue serving as a vital instrument in synthetic biology research and industry. In this review, we mainly focus on the cell‐free based platform for various product productions. Moreover, the challenges in the bio‐therapeutic aspect using cell‐free systems and their future prospective for the improvement and sustainability of the cell free systems.

frequently in high-throughput functional genomics and proteomics.When creating protein arrays like the nucleic acid programmable protein array (NAPPA) or engineering enzymes with display technologies, CFPS system is significant.The major way to link genotype and phenotype (function of expressed protein) is through the cell-free method.In linked transcription and translation system, 2 either mRNA template or DNA template (plasmid DNA or PCR fragments) 3 can produce proteins in a matter of hours (Figure 1).In addition, CFPS systems are essential for the expression of proteins that are poisonous, membrane-bound, viral, and subject to fast intracellular protease-mediated proteolytic destruction (Table 1). 4 In the early days of molecular biology, how amino acids were incorporated into proteins was addressed using the CFPS system. 5In this perspective, many researchers regulated CFPS system that uses the extract of rat liver cells, 6 F I G U R E 1 Animated illustration of cell-free protein synthesis (CFPS) system components required for the reaction.The reaction is set up in an Eppendorf tube containing DNA, amino acids, and buffers mixed along with molecular machinery present in the cellular lysate to initiate transcription and translation for the synthesis of functional proteins.

TA B L E 1
Merits and limitations of the cell-free protein synthesis (CFPS) system.

Merits Limitations
✓ High-throughput protein expression ✓ Poor expression when protein is larger ✓ Simple modification and optimization of protein synthesis conditions ✓ Possible to optimize for small-scale prototype reactions only ✓ Possible to express the difficult-to-express protein ✓ Toxic protein production ✓ Feasible in protein synthesis (usually ∼3-6 h) ✓ Expensive compared to the cell-mediated method ✓ High yields ✓ Yield remains a challenge based on the cell lysate used ✓ Metabolic modification is not required ✓ Enzyme activity may be suppressed or controlled by reaction precursors and by-products ✓ Stable, that is, proteolytic degradation is not common ✓ Selective labeling of several amino acids ✓ An open system allows adding many components to the reaction ✓ Intricate and protracted reagent preparation ✓ Chemical environment manipulation possible ✓ Quick degradation of interest of DNA due to endo-and exonucleases bacteria, 7,8 human and rabbit reticulocytes, 9,10 ascites cells, 11 and wheat germ. 12CFPS systems were initially created as a research tool for understanding the principles of in vitro translation processes. 13Nirenberg and Matthaei first reported on CFPS system applications in 1961, employing an Escherichia coli whole-cell extract to define the first protein codon sequence. 13CFPS system was employed to illuminate the lactose 14 and tryptophan operons in E. coli 15 in the late 1960s and early 1970s.In the present, CFPS systems have replaced traditional cell-based protein expression systems as a reliable and cost-effective substitute. 3CFPS system has an absolute remark in using as a protein synthesis technology that includes pharmaceutical protein production [16][17][18][19] and high output protein libraries for protein evolution and structural genomics. 20,21he advantage of CFPS system over conventional methods (in vivo) is allowing screening without requiring a gene cloning step, making it particularly suited for rapid biological processes or product development pipelines 17,22 (Figure 2).First off, a CFPS system's open design gives experimenters complete control over the circumstances of the reaction.Exogenous elements can F I G U R E 2 Schematic comparison of in vivo protein expression and a cell-free protein synthesis (CFPS) system.The figure illustrates a complete extract preparation for conventional system and CFPS system.be easily added, either at the beginning of the experimentation or else at user-defined time points, including artificial building blocks.Additionally, additives are frequently used to increase protein yield, stability, and folding. 23,24Moreover, CFPS system normally requires 1-2 days for the experimental procedure, including extract preparation, whereas in vivo protein expression might require 1-2 weeks. 1 Because it is the open reaction, the absence of cell wall permits the direct manipulation of the chemical environment, which also helps to regulate the easy sample absorption, concentration optimization, and online reaction monitoring. 25Further, enzyme and substrate addition, customized DNA concentration, waste molecule eradication, cofactor increment, and supplements of non-natural reagents are also practicable. 26he autonomous environment of CFPS system ensures the exceptional flexibility in target selection by allowing the production of difficult-to-express protein. 27,28This adaptability can be additionally employed for synthesizing custom proteins, enabling the creation of tailored medicines 29,17 or proteins with cutting-edge properties like the insertion of non-canonical amino acids 30 for targeted drug delivery. 31The versatility of CFPS system, which just needs a nucleic acid template, and the quick isolation of protein products (few hours after reaction initiation), make it an excellent choice for the cyclical workflow for prototyping synthetic structures. 32Further, directed evolution, 33 a potent method of protein engineering that supports design-build-test-learn, can be fueled by the capacity of CFPS system to replicate biological behaviors. 34ecause proteins vary in complexity, folding, and posttranslational changes, CFPS system conditions must also take these factors into consideration. 35With all of these components combined, CFPS system offers a flexible and simple platform to customize.Additionally, as the system allows for the exclusive targeting of one target protein by metabolic processes, interventions to improve protein synthesis 36 or folding 37 are simply possible.Finally, CFPS system is a desirable platform for the instantaneous synthesis and high-throughput screening of several protein targets because it does not need cell upkeep or time-consuming in vivo manufacturing techniques. 38

DEVELOPMENT OF CELL EXTRACT AND SELECTION OF CFPS SYSTEM
A number of things to consider when choosing the best CFPS system method, including the kind of template used, the preferred protein yield, and the intended downstream applications.E. coli extracts made from exponentially developing cells often produce far more protein than extracts made from other sources, like wheat germ and rabbit reticulocytes.Additionally, the price of an E. coli extract is less expensive, and performance varies less from batch to batch.The intricate procedures required to prepare cell extracts in typical methods for CFPS system, however, pose a major obstacle when large quantities of consistent-quality extract are required. 39An effort was made to design extract preparation techniques that did not physically harm cells in order to make them more practical for laboratories with limited equipment.Osmotic shock, freeze-thaw cycles, and lysozyme treatment can all be used to prepare cell extracts. 40ost common type of system to be selected is available as prokaryotic and eukaryotic sources and is dependent on several factors such as the origin of DNA and RNA template, 41,42 protein yield or the requirement of posttranslational modification (PTM) for the protein of interest.In vitro translation systems based on E. coli, wheat germ, rabbit reticulocytes, or insect cell extracts (ICEs) are widely used commercially available systems.Among these, the prokaryotic E. coli system is currently the most widely used protein expression method. 43Because E. coli may be readily fermented in large quantities using inexpensive media and quickly ruptured using high-pressure homogenizers, the preparation for E. coli extraction is straightforward and affordable.Additionally, E. coli-based systems often provide the highest protein yields, and their systems' overall reaction costs are the lowest.A metabolic response in the extract can be triggered by E. coli, which then drives high-level protein synthesis and negates the need for more expensive energy sources like phosphoenolpyruvate.
The most popular eukaryotic extraction methods at the moment are wheat germ extract (WGE), 44 rabbit reticulocyte lysate, 10 and ICE. 45These systems are useful for producing more complicated proteins and have the ability to carry out posttranslational changes that are not possible in E. coli.These eukaryotic systems do, however, typically require more time-consuming extract preparation techniques, which can raise expenses.In batch processes, eukaryotic systems frequently generate less protein than E. coli systems do.A wide range of protein characterization applications, including enzyme engineering employing display technologies and NAPPA, are made possible by the great variety of cell-free protein expression systems.Using modified charged tRNAs or amino acids, the cell-free method also makes it possible to identify individual proteins with fluorescence, biotin, radioactivity, or heavy atoms.
CFPS system could be effective tools for the production of target proteins without using living cells. 46Nevertheless, there are several CFPS system-related issues that still need to be resolved.
Safety: With the permission of the Food and Drug Administration (FDA), the clinical trial using cell-free produced antibody drug from Sutra Biopharma has no usage approach and has been approved. 47Meanwhile, some of the bio-products like biocatalysts and commodity chemicals have also not been in use.Likewise, the global biopharmaceutical company, Ipsen Biopharma, produces products in neuroscience, consumer healthcare, oncology, and rare diseases, for example, botulinum toxin, which is used as a therapeutic for overactivity muscle disorder.However, the workers while manufacturing these products get substantial hazards (unusual for a bio-therapeutic product) due to the extreme toxicity of the products. 48herefore, there are still some unsolved problems in regard to safety.
Optimization: Many independent research studies have been conducted in the past decades, aiming the both competing and complementary technologies using cellfree extract, genetic strains, and different microorganisms (MOs)/cells (including E. coli, yeast, Chinese hamster ovary [CHO], rabbit, wheat germ, HeLa, and recently human blood). 49,50Moreover, there may be some differences within the same strain like components and their activities during their different growth stages, with different growth media.Despite the fact that cell-free biomanufacturing could be conducted in broad ways with design flexibility, such as in microfluidic, 51,52 batch, 19 fedbatch, 53 and semicontinuous/continuous exchange 54,55 operational formats, optimization for each product could be necessary.
Cost: Cell-free protein expression can turn into a costly process because the reagents and equipment are costly or when larger quantities of protein are needed or the proteins showing poor expression.This is normally applied for the more complex cell-free lysates from eukaryotic sources that require the separate purification and recombined. 56The system that uses the fluorescent proteins like green fluorescent protein and mCherry or chromogenic substrate-responsive enzyme such as β-galactosidase, 57,58 which is employed as point-of-care diagnostic, is expensive and difficult due to colorimetric output change in color. 59Moreover, due to the size of the organisms and when they inhabit in the delicate place like lungs of the human body, it becomes more challenging and costly.][63] Instability: Besides expensive, another challenge is instability due to the production of endogenous nuclease.During the reaction, endo-and exonucleases form, which degrade a target DNA rapidly leading to significantly lowering the yield.Further, wheat germ contamination due to several inhibitors during the extraction process also caused the instability in the wheat germ cell-free system as reported elsewhere. 64,65The poly(A) tail at the 3′-end of RNA presents another issue when creating templates for in vitro translation because of long plasmid poly dT/dA sequences unstable during plasmid replication in host cells as reported by Sawasaki et al. 66 Post-translation modifications: Due to their critical significance in numerous cellular functions and biological processes, PTMs are increasingly important to investigate. 67It is challenging to create homogeneously changed proteins for research as they are mostly reversible, compete with one another for the same residues, and have unknown modifying enzymes. 68When expressing proteins, PTMs must be taken into account.They frequently serve a crucial role in the control of proteins on several levels, both in terms of their structure and function. 69CFPS systems have a difficult time dealing with PTMs as they are Overall schematic representation of cell-free protein synthesis system.Preparation of crude cell lysate through pre-lysis and lysis processes (A), the addition of essential supplements, such as amino acids, nucleotides (NTPs), cofactors, energy substrates, salts, and DNA templates for target protein production (B), and the production of various functional proteins and biomaterials through optimized cell-free reactions (C) by supplementing effectors, that is, molecular chaperones, nanodiscs, micelles, protein disulfide isomerase (PDI), and orthogonal translation systems (OTSs).so reliant on the extracted source.For example, prokaryotic sources lack PTMs, which results in a more homogeneous sample but less stability or activity. 70,71Additionally, in a recombinant expression system, there is always a PTM issue because, in the case of prokaryotic expression hosts, it usually expresses eukaryotic proteins in less amount or without PTMs. 72Thus, resulting in the incomplete PTMs when overexpressed and also leads to the oversaturated cellular synthesis machinery 73 resulting in heterogeneous samples.In spite of many obstacles in the PTMs, some genetic code expansion strategies can be applied to study the PTMs.
Even though above challenges have been occurring, however, the solutions to solve these challenges are also being developed in the different fields like medical and chemical industries.Currently, CFPS systems are utilized to develop yields of target protein exceeding 2 g/L of reaction volume, which lasts multiple hours, with costeffective manners. 74Moreover, due to these advances, many new applications have been inspired, such as the production of protein therapeutics, 37 vaccines, 75 and short antimicrobial peptides (AMP) 76 (Figure 3).

CFPS system for antibody production
To discover the best candidate for production, a large number of distinct antibody variants must be evaluated in the early stages of antibody creation.Cell-free systems have a wide range of applications, but recently the system has attracted the attention in pharmaceutical companies.Regarding this, cell-based and cell-free expression systems have been compared in the product yield.Jerome et al. compared recombinant human bone morphogenetic protein yield where their team discovered that bone formation was induced in de novo and was synthesized in mammalian cells and extracted as PTM are essential for protein activity. 77Antibodies and monoclonal antibodies, biopharmaceuticals, employed for cancer treatment demonstrate high specificity, long serum half-life, and so on.Generally, antibodies, which are synthesized in CHO cell-based, are expensive and time prolonged. 78However, Martin et al. synthesized antibodies with commercially available CHO extracts for the rapid cell-free expression of monoclonal antibodies. 79Regardless of several studies with CHO extract for therapeutic protein synthesis, the trastuzumab IgG was synthesized in E. coli extract with the yield of nearly 1 g/L. 80Further, Cai et al. illustrated the capability to maintain high-throughput production with the less price improving the productivity. 81Moreover, Xu et al. discovered the antibodies with the cell-free systems that are bispecific "knob into holes." 82These antibodies recognize two targets and are potentially a promising topic in the field for oncology therapies and infectious diseases.Moreover, using eukaryotic cell-free extracts like ICE, glycoproteins also have been successfully synthesized for erythropoietin production. 83Additionally, cell-free platforms have been shown to manufacture a wide variety of different antibodies. 78,3,84However, there are some obstacles to the cell-free production of antibodies due to the high cost of reagents for CFPS system reactions, including T7-RNA polymerase and reagent involving plasmid DNA. 85n spite of fact that, the recycling of these compounds could bring reaction cost down by lowering the total cost by as much as 29%. 85Cell-free platforms will be utilized more frequently to produce antibodies as a result of new developments in the efficiency of CFPS system reactions.

CFPS system for antimicrobial peptide production
One of the most effective types of innate chemical defenses used by eukaryotic cells against bacteria may be natural AMPs.Numerous AMPs were extracted from natural sources, and thousands of synthetic versions with allencompassing antibacterial properties were also found.Many of these peptides showed bactericidal, antiviral, and anticancer properties. 86As AMPs are generally cationic and amphipathic in nature, their interaction with the lipid cell membranes can lead to the disruption of lipid membranes. 87The vast range of actions of AMP mechanisms, which include disrupting cell membrane stability and inhibiting molecular processes like protein synthesis and enzyme activity, depend largely on the organism from which they are formed. 88,89Only a few AMPs may be created in this manner due to the bacteriolytic nature of AMPs, which frequently contradicts the hosts' culture requirements. 90 potentially effective alternative for the creation of AMPs is CFPS system.91 It does not suffer from the toxicity of the freshly created AMPs 91 and enables for the assimilation of noncanonical amino acids.92 It can accept linear DNA templates produced by polymerase chain reaction 93 and does not require the addition of signal peptides or leader sequences, nor does it require specialized transporters for the secretion of mature peptides.94 In addition, it does not necessitate difficult cloning procedures. 95All kinds of living organisms contain AMPs, which are small polypeptide molecules with an average amino acid composition of between 12 and 50.96 In mammals, epithelial cells and phagocytes both ribosomally generate these compounds, which are formed as secondary metabolites and are a component of innate immunity (cells of the immune system). Thse peptides are present in tissues and mucous membranes, the latter of which are home to a wide variety of commensal and pathogenic microbes.Some of these peptides have a broad range of antimicrobial activity, suppressing or killing a variety of MOs, including viruses, protozoans, and Gram-positive or -negative bacteria, and fungus.][99][100][101]

CFPS system for onconase production
Onconase, a highly cytotoxic which belongs to RNase superfamily, 102 is a monomer secreted in oocytes and early embryos of Rana pipiens that shares 30% sequence identity with the prototype RNase A. [103][104][105] Onconase avoids the interaction with cytosolic ribonuclease inhibitor [106][107][108] to confer a high cytotoxic activity against several human cancer cell types like glioma cells 109 and lymphoma B cells. 110s a treatment for unresectable malignant mesothelioma and non-small cell lung cancer, respectively, Ardelt et al., tried onconase in Phase II and Phase III clinical trials. 111ue to many promising applications, such as anticancer treatment, immune suppression, and vaccines, biotherapeutics is now gaining more interest.So, regarding this, utilizing CFPS system is one possible method for streamlining the expression and screening of such therapeutics.Therefore, as a result of their flexible and open reaction conditions, cell-free systems are an appealing alternative to in vivo manufacturing.Amin et al. showed how the difficult-to-express cytotoxic protein onconase could be expressed and directly screened in a CFPS system for the therapeutic treatment of cancer.Further, onconase expressed using cell-free system is soluble and active form with the yield increased to 56 times as well as with greater than 95% solubility, which allows downstream characterization without the need for purification. 53Moreover, due to its ability to resist inhibition by RNase A inhibitor in the presence of salt concentration, 112 it provides beneficial anticancer properties allowing the RNase to function after the endocytosis despite the potential presence of RNase inhibitor. 113,114

CFPS system for vaccine production
Not only in the field of production of different enzymatic biocatalysts or biochemical products but also cell-free system could be applied for the synthesis of conjugate vaccines for the treatment of various diseases.Due to the growing threat posed by antimicrobial-resistant bacteria, which some have suggested could be the source of the next pandemic.However, the conjugate vaccines comprise either pathogen-specific capsular or O-antigen polysaccharides that are linked to an immune-stimulatory protein carrier and are directed at the prevention of bacterial infections. 115,116Although conjugate vaccines have been shown to be safe and effective, global childhood vaccination rates for these vaccines are still as low as 30%, and the majority of the remaining disease burden is caused by insufficient access to or inadequate immunization coverage. 117he researchers created a new manufacturing platform dubbed in vitro conjugate vaccine expression (iVAX) that makes use of cell-free biology, a technique in which researchers take off a cell's outer wall (or membrane) and reuse its internal machinery.Feldman et al. reported that polysaccharide-protein bio-conjugates could be made in E. coli utilizing protein-glycan coupling technology (PGCT). 118Another example is that Stark et al. also developed iVAX cell-free platform for portable, on-demand bio-conjugate vaccine production in Francisella tularensis strain, which is detoxified to ensure the safety of conjugated vaccine products using freeze-dried. 119Further, they also reported that when compared to a bio-conjugate made from living E. coli cells using PGCT, iVAX-derived bioconjugates are much more successful at eliciting FtLPSspecific IgGs.This is due to when compared to their PGCT-derived counterparts, bio-conjugates with higher molecular weight and more carbohydrate loading are decorated with extensive glycosylation, which can only be seen in vitro. 120

CFPS system for insulin-like growth factor I production
Neurotrophic factors (NF), which boost the development of the central nervous system, play a critical role in neuronal growth, survival, and migration.Due to this, NF has gained attention in recent research targeting the possible treatment for several neurodevelopmental and other central nervous system disorders.Among such factors, insulin-like growth factor-1 (IGF-1), the polypeptide of 70 amino acids, 121 belongs to a superfamily of insulin-like hormones and is mainly produced in adults by the liver in response to growth hormone.As IGF-1 shows permeating character and complexity in the mechanism of molecular expression, regulation, and function, it has gained interest recently in treating neurodevelopmental disorders. 122o date, several expression systems, such as E. coli, 123 yeast, 124 cell-free systems, 125 transgenic plants, 126 and rabbits, 127 have been applied to produce IGF-1. 123Among these expression systems, recombinant E. coli has been more beneficial rather than other hosts of easy handling and culture, and high yields make it the most popular host to produce IGF-1. 128Ranjbari et al. (2015) expressed 0.694 g/L IGF-1 internally as an inclusion body that highly burdened the successive purification.Taking this into account, one of the recombinant E. coli cell-free system was introduced to biosynthesized soluble IGF-1 with the production titer of 400 mg/L. 125

CFPS system for crisantaspase production
In the therapeutic world, CFPS system has become the main targeting research field to produce antibodies.The type of CFPS system may have an impact on the final product, but it is an essential platform that can quickly and affordably synthesis therapeutically significant compounds.In this situation, two E. coli CFPS system platforms were created, one based on cell extract and the other being a general cell-free platform.The generic platform produced therapeutic proteins, antibody fragments, and vaccines with greater expression, with titers of 0.71, 0.23, and 0.3 mg/mL, respectively. 129o prevent the buildup of E. coli endotoxin in the finished product, any therapeutic protein made from E. coli requires time-consuming and expensive purification procedures.CFPS system produced from ClearColi cells crude extract can serve as the starting point for the synthesis of cell-free, endotoxin-free targeted therapies.As opposed to other E. coli strains, ClearColi cells lack the endotoxin that is often present in those cells.However, because of their slower proliferation as well as osmolarity tolerance, the technique for extract preparation is subtly different. 130A study exemplified the crisantaspase production from ClearColi using cell lysate comprising reduced E. coli endotoxin removing the cost and extensive phases for its purification.Nearly 1 mg/mL titer of crisantaspase was achieved comparable to that produced by the E. coli BL21 strain extract.Likewise, Porter et al. ( 2020) utilized the open CFPS system environment with ASP sup-plementary showing a 72% improvement in crisantaspase achievement. 131Additionally, E. coli-based CFPS system exhibited the consistent production of a sizeable amount of the therapeutic protein crisantaspase that is clinically meaningful.As crisantaspase is a therapeutic protein, it can be used in cancer therapy that is approved by the FDA. 130

CFPS system for streptokinase production
An extracellular excreted protein is used clinically as an intravenous thrombolytic agent for the treatment of acute myocardial infraction.As it is a non-fibrin-specific cytoplasmic enzyme, it acts to break down fibrin by stimulating the circulatory plasminogen. 132FPS system has evolved as a dynamic technology platform that can fulfill the increasing demand for the simple and effectual production of protein.For instance, new developments have enabled the factory-scale biosynthesis of granulocyte-macrophage colony-initiating agents at a low cost. 19In this prospective (Tran et al., 2018), 0.5 mg/mL streptokinase has been produced through CFPS using HeLa and CHO cell lysates.Similarly, Kawasaki et al.  (2003) reported around 0.013 mg/mL of single-chain antibody against Salmonella O-antigen with the help of CFPS system with WGE. 133

CFPS system for botulinum toxin production
Botulinum neurotoxins, being the most poisonous substances, can kill more than 1 million people. 134Historically, botulinum was considered a bioweapon until it became the first licensed in the United States for human disease treatment consisting of cervical torticollis, strabismus, and blepharospasm associated with dystonia treatment. 135,136ue to higher productivity than cellular production systems, CFPS system has been considered the potential tool that can be efficiently directed toward recombinant protein production. 137,138Moreover, CFPS system can be used to produce personalized therapeutic vaccines, including heavy-chain proteins for botulinum toxins. 139everal types of subunit vaccines have been produced with the use of an E. coli-based CFPS system.Following this, with the use of the CFPS system, botulinum was synthesized with a yield of 1 g/L as reported by Zichel et al. 139 Further, with the use of optimized reaction conditions in the presence of redox buffer, disulfide bond C, and chaperon, the yield of approximately 30 μg of botulinum neurotoxin serotype B (BoNT/B) was reported. 140

CONCLUSION
The CFPS system is a dependable and sturdy system and has gone through several repetitive improvements.The CFPS system has advantages over chemical synthesis and cell-based expression.Despite these challenges, cell-free systems are considered next generation platform for a wide range of applications in biopharmaceuticals.As a result, a large number of proteins have previously been produced in different kinds of cell extracts, both on a large scale and in high-yield synthesis.The CFPS system is becoming more and more appropriate for industrial usage due to advancements in cost reduction and reaction time.
As next-generation DNA sequencing technology advances quickly, more proteins can now be produced synthetically rather than being extracted from their natural sources.
Because of this, they could be produced synthetically instead of using animals and plants, which would make it more cost-effective, sustainable, and environmentally beneficial.
The platform is perfect for developing new genetic and metabolic circuits due to the benefits of CFPS systems in reaction condition tolerance and customization.As cell-free synthesis systems are used increasingly, both experimental and commercial procedures will become more effective.Due to these benefits, the CFPS system can also be utilized for drug discovery, microarray analysis, to synthesize natural products for pharmaceutical biomaterials applications, to identify CRISPR enzymes, to synthesize PETase, and other purposes.Decentralization of manufacturing plants is an additional prospect that the CFPS system can present.Due to the fact that the cell is no longer alive, there is no genetically modified organism that would prevent the DNA from being sent separately from the extract.The CFPS system is an innovative technology with a wide range of potential applications that could challenge current thinking and provide solutions to environmental and health issues.Cell-free platforms are not flawless, extract-based systems still experience inefficient side reactions during energy metabolism, and enzyme-based systems still experience cost-prohibitive scaling issues; however, only a decade ago, many of these platforms' current capabilities were thought to be impractical, and the systems are developing quickly.

FUTURE PERSPECTIVE AND CHALLENGES
With the advent of various cell extracts and methods that encourage the creation of complex proteins, including those with various PTM needs and functionality, CFPS sys-tem possibilities are fast expanding.It is simple to envision this technology being the main protein synthesis platform in the future due to its many benefits.Although CFPS systems have great potential, there are still a number of issues that must be resolved before they are widely used in nonacademic settings.Complex PTM integration and resource regeneration are two of the most urgent.Many proteins, some of which may have therapeutic potential, depend on PTMs for appropriate folding and operation (especially in eukaryotes).In order to supply specific PTMs, some researchers have created enriched prokaryotic CFPS systems. 141,142tilizing mammalian CFPS systems would be advantageous from a therapeutic standpoint because of the extract's lower immunogenicity, even though improvements in the complexity of products from prokaryotic systems are undoubtedly important.Despite some improvements in the creation of internal mammalian extracts, 143,144 commercial products are still quite expensive and are only offered as separate transcription or translation kits at the moment.In order to make standardized practices and goods more accessible, further work is still required.The issue of depleting resources must also be resolved in order to create synergies between CFPS system and other synthetic biology technologies.Large-scale batch cultures 22 and integrative systems with microfluidic platforms, 145 which allow for the exchange of nutrient solutions, have made significant headway, but there is still work to be done on a smaller scale, especially when considering the creation of synthetic cells.To be fully independent, they need to be able to evacuate or recycle waste materials and gather new macromolecules for protein synthesis from the environment.Although reports of synthetic cells with the ability to carry out such molecular exchanges have been made, 146 the cells in this case were evaluated in an in vitro solution with sufficient nutrition rather than in a physiologically realistic environment.Therefore, more work needs to be done before artificial cells are considered fully self-sustaining in vivo.However, if cells were utilized as therapeutic agents, they might not need to be self-sustaining as they might not need to be active for long periods of time.
The creation of enzymes using genetically encoded nonnatural amino acids is another burgeoning future trend that Zhou and Roelfes et al. put into the practice by inserting a non-natural amino acid as the catalytic site of the transcription factor Lactococcal multidrug resistance Regulator and with a complex containing copper.Overall, CFPS system has been demonstrated to be a successful way of producing biomolecules in a variety of applications, including the biomedical industry.Although there are still obstacles to overcome, its ease of use, versatility, and capacity for integration with other technologies open the door for it to continue serving as a vital instrument in synthetic biology research and industry.
Due to the absence of living cells, most of the bioengineers and scientists had ignored or underappreciated the potential of cell-free biosynthesis for the manufacturing of biopharmaceutical products, but far from these, it could be game-changing in the technological fields, which can bring the evolution in the research of science.Moreover, because of the unspecified and complex composition of CFPS system extracts, the activity still is challenging.A good example is side reactions or inhibition by extract components may influence the activity of the synthesized target protein and for the target protein isolation could be done by purification by removing the other components or by immobilizing of target protein.

A C K N O W L E D G M E N T S
This research was carried out with the support of the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5362322).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
No conflicts of interest are declared.