Artificial intelligence for cell-free systems.

Cell-Free Protein Synthesis Containing Disulfide Bonds and Its Application to Protein Engineering

Cell-free synthesis systems can complete the transcription and translation process in vitro to produce complex proteins that are difficult to be expressed in traditional cell-based systems. Such systems also can be used for the assembly of efficient localized multienzyme cascades to synthesize products that are toxic to cells. Cell-free synthesis systems provide a simpler and faster engineering solution than living cells, allowing unprecedented design freedom. This paper reviews the latest progress on the application of cell-free synthesis systems in the field of enzymatic catalysis, including cell-free protein synthesis and cell-free metabolic engineering. In cell-free protein synthesis: complex proteins, toxic proteins, membrane proteins, and artificial proteins containing non-natural amino acids can be easily synthesized by directly controlling the reaction conditions in the cell-free system. In cell-free metabolic engineering, the synthesis of desired products can be made more specific and efficient by designing metabolic pathways and screening biocatalysts based on purified enzymes or crude extracts. Through the combination of cell-free synthesis systems and emerging technologies, such as: synthetic biology, microfluidic control, cofactor regeneration, and artificial scaffolds, we will be able to build increasingly complex biomolecule systems. In the next few years, these technologies are expected to mature and reach industrialization, providing innovative platforms for a wide range of biotechnological applications.

Building variant ribosomes offers opportunities to reveal fundamental principles underlying ribosome biogenesis and to make ribosomes with altered properties. However, cell viability limits mutations that can be made to the ribosome. To address this limitation, the in vitro integrated synthesis, assembly and translation (iSAT) method for ribosome construction from the bottom up was recently developed. Unfortunately, iSAT is complex, costly, and laborious to researchers, partially due to the high cost of reaction buffer containing over 20 components. In this study, we develop iSAT in Escherichiacoli BL21Rosetta2 cell lysates, a commonly used bacterial strain, with a cost-effective poly sugar and nucleotide monophosphate-based metabolic scheme. We achieved a 10-fold increase in protein yield over our base case with an evolutionary design of experiments approach, screening 490 reaction conditions to optimize the reaction buffer. The computationally guided, cell-free, high-throughput technology presented here augments the way we approach multicomponent synthetic biology projects and efforts to repurpose ribosomes.

Chapter 24 - Cell-free synthetic biology as an emerging biotechnology

Development of a robust Escherichia coli-based cell-free protein synthesis application platform.

Cell-free protein synthesis (CFPS) represents a promising technology for efficient protein production targeting especially so called “difficult-to-express” proteins whose synthesis is challenging in conventional in vivo protein production platforms. Chinese hamster ovary (CHO) cells are one of the most prominent and safety approved cell lines for industrial protein production. In this study we demonstrated the ability to produce high yields of various protein types including membrane proteins and single chain variable fragments (scFv) in a continuous exchange cell-free (CECF) system based on CHO cell lysate that contains endogenous microsomal structures. We showed significant improvement of protein yield compared to batch formatted reactions and proved biological activity of synthesized proteins using various analysis technologies. Optimized CECF reaction conditions led to membrane protein yields up to 980 µg/ml, which is the highest protein yield reached in a microsome containing eukaryotic cell-free system presented so far.

Cell-free protein synthesis is emerging as a powerful tool to accelerate the progress of synthetic biology. Notably, cell-free systems that harness extracted synthetic machinery of cells can address many of the issues associated with the complexity and variability of living systems. In particular, cell-free systems can be programmed with various configurations of genetic information, providing great flexibility and accessibility to the field of synthetic biology. Empowered by recent progress, cell-free systems are now evolving into artificial biological systems that can be tailored for various applications, including on-demand biomanufacturing, diagnostics, and new materials design. Here, we review the key developments related to cell-free protein synthesis systems, and discuss the future directions of these promising technologies.

Cell-free systems are emerging as powerful platforms for synthetic biology with widespread applications in both fundamental research, such as artificial cell construction, and practical uses like recombinant protein production. Among these, cell-free protein synthesis (CFPS) plays a crucial role in gene expression for various downstream applications. However, the development of CFPS systems based on certain chassis, such as Bacillus subtilis, still remains limited due to their low in vitro productivity. Here, we report the development of a highly productive CFPS system derived from an engineered B. subtilis 164T7P strain, which contains a genomic integration of the T7 RNA polymerase gene. This modification allows the preparation of cell extracts that inherently contain T7 RNA polymerase, enabling T7 promoter-based transcription without the supplementation of purified T7 RNA polymerase in CFPS reactions. Through systematic optimization of cell extract preparation and key reaction parameters, we achieved the synthesis of 286 ± 16.7 μg/mL of sfGFP in batch reactions, with yields increasing to over 1100 μg/mL in a semicontinuous format that can replenish substrates and remove inhibitory byproducts. We further demonstrated the system's versatility by using it for two synthetic biology applications: prototyping ribosome binding site (RBS) elements and synthesizing pulcherriminic acid─a bioactive cyclodipeptide. The system successfully characterized RBS performance, with in vitro and in vivo rankings correlating with predicted strengths, and expressed two active biosynthetic enzymes (cyclodipeptide synthase─YvmC and cytochrome P450 enzyme─CypX), leading to the production of pulcherriminic acid. Overall, our B. subtilis-based CFPS system offers a robust platform for high-yield protein synthesis, in vitro prototyping of gene regulatory elements, and natural product biosynthesis, highlighting its broad potential for synthetic biology and biotechnology applications.

Synthetic biological circuits are the foundation for the ultimate goals of controlling cells and building artificial cells from the ground up. To get closer to these goals in a more efficient way, we utilize a cell-free transcription-translation system to help perfect biological circuits for the simplicity, freedom, and convenience that the system offers. In this thesis, we demonstrate three distinct aspects of biological circuits in a cell-free transcription-translation system: circuit dynamics, phosphorylation, and membrane proteins. We start with a simple feedforward circuit, which shows dynamic responses to the input. We first prototype the feedforward circuit in the cell-free system with the aid of mathematical modeling. Then, based on the knowledge learned from prototyping, we successfully implement the circuit in cells. Not only do we show that a circuit with dynamics can be prototyped in the cell- free system, but we also test a more complicated circuit involving a phosphorylation cycle. The phosphorylation-based insulator circuit is prototyped and then a model created for the circuit is shown to be identifiable in the cell-free system. To further expand the capability of the cell-free system, we demonstrate that biologically active membrane proteins can be generated in the cell-free system with engineering, suggesting that even biological circuits requiring membrane proteins can be prototyped in the system. These results help advance our knowledge of both biological circuits and the cell-free transcription-translation system, and bring us one step closer to our ultimate goals of implementing control theory in synthetic biology.

Role of disulfide bond isomerase DsbC, calcium ions, and hemin in cell-free protein synthesis of active manganese peroxidase isolated from Phanerochaete chrysosporium

During the early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owing to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from "traditional" E. coli, rabbit reticulocyte lysate, and wheat germ extracts, to recent insect and human cell extracts, to defined systems reconstituted from purified recombinant components. Although each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high-throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This unit provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology.

The bottom-up synthesis of a minimal biological cell is achieved by integrating and connecting three fundamental modules: metabolism, information and self-organization (Noireaux et al., 2011). The execution and connection of these three parts into cell-sized liposomes should generate a system capable of self-reproduction and ultimately evolution. Each of these molecular sub systems is the result of a forward engineering process where bio-parts, e.g. sugars, proteins, phospholipids and nucleotides are interlocked in a functional way (Mann 2008). The creation of a minimal biological cell is certainly one of the most challenging goals of the synthetic biology community (Porcar et al., 2011). The objective of this research area is to elucidate the fundamental design principles found in biology and to understand cellular functions by applying a reductionist approach (Elowitz, 2010). This work also promotes the development of new technologies based on life’s principles (Bedau et al. 2010). The functionalities of biological cells are dependent on the activity of molecular machineries (enzymes). However, such machineries require energy for their physiological function. Therefore, in order to attempt the construction of wet artificial life is essential to reconstitute an in vitro metabolic network that supports the cellular energetic. In biological systems, the energy requirements are stored in the high-energy molecule adenosine triphosphate (ATP). The conversion from ATP to adenosine diphosphate (ADP) is crucial for energy generation. In particular, the hydrolysis of one chemical bond releases inorganic phosphate (iP) and liberates energy used for life processes. Noireaux’s lab has recently developed a unique cell-free transcription-translation (TX-TL) system for synthetic biology applications, as for instance, the possibility of executing DNA programs made of up to ≈ 60 genes (Shin and Noireaux, 2012a, 2012b). In a cell-free system, the rate of protein synthesis depends exponentially on the adenylate energy charge (Atkinson 1968; Matveev et al., 1996). Protein degradation by AAA+ proteases is also highly dependent on the pool of chemical energy available. Therefore, efficient long-lived ATP regeneration and byproducts recycling is at the heart of a minimal cell construction by allowing the execution of larger and larger DNA programs with interesting dynamical behaviors. We will present our recent efforts to design an in vitro metabolism for efficient protein synthesis. This new metabolic scheme relies on the catabolism of polysugars molecules as energetic resources, and it only exploits the endogenous enzymes present in the cellular extract. Cell-free protein synthesis is improved by addition of maltose or maltodextrin in the reaction mixture. The initial phosphorylation of maltose or maltodextrin produces either glucose or glucose-6-phosphate, which are intermediates of the glycolysis. In turn, this allows for higher level of sustained ATP concentration through recycling of iP, the byproduct of the transcription/translation processes. We will present biochemical experiments that quantitatively measure several system’s parameters: concentration of synthesized protein (a reporter gene eGFP), level of ATP and inorganic phosphate, as well as pH fluctuations during in vitro protein synthesis. Recently, we reported the highest protein yield ever achieved with an E. coli cell-free expression system with this new metabolism (Caschera and Noireaux 2013). Therefore, compared to others cell-free expression systems used to design a minimal cell (Ichihashi et al. 2010), it represents a more powerful solution in term of adenylate energy charge. We are now using this system to develop a minimal cell. One of the current bottlenecks in this research area is the encapsulation of the cell-free TX-TL reaction into cell-sized vesicles of complex phospholipid composition. Further optimization of the reaction mixture for cell-free protein synthesis, as well as its integration into liposome with an active membrane (Noireaux and Libchaber 2004), could be accelerated exploiting a machine learning approach coupled to robotic workstation for liquid handling (Caschera et al. 2010, 2011).

The Fourth Industrial Revolution (Industry 4.0) envisions smart factories where cyber-physical systems (CPS), Industrial Internet of Things (IIoT), and advanced analytics converge to enable autonomous, data-driven manufacturing. Central to this vision is the synergistic integration of Artificial Intelligence (AI) and Machine Learning (ML), which enhances decision-making, automation, and adaptability. AI/ML techniques—including deep learning (DL), reinforcement learning (RL), computer vision, and predictive analytics—interoperate with digital twins, edge/cloud computing, and IIoT networks to enable real-time process optimization, self-diagnosing systems, and intelligent robotics (Lee et al., 2018). This paradigm shifts leverages AI’s strengths (e.g., symbolic reasoning and optimization) alongside ML’s data-driven pattern recognition, creating a unified framework that transcends traditional siloed approaches. Recent advances highlight how AI/ML-driven industrial analytics improve anomaly detection, prescriptive maintenance, and adaptive control, while autonomous RL agents optimize production workflows (McKinsey Digital, 2021). Key technologies such as physics-informed digital twins and edge AI exemplify this synergy: AI enhances twin-based simulations for ML training, while generative models (e.g., GANs) refine digital twin fidelity. Conversely, ML-driven sensor fusion bridges gaps between physical and virtual systems, enabling closed-loop intelligence. This paper systematically reviews these developments through five lenses: (1) the evolution of Industry 4.0 and its AI/ML foundations; (2) literature synthesis of prior integration frameworks; (3) emerging architectures for AI/ML in smart manufacturing; (4) high-impact applications (e.g., vision-based quality inspection, collaborative robotics, self-healing supply chains); and (5) enabling technologies (e.g., AR/VR interfaces, 5G-edge AI, blockchain-secured CPS). We also analyze critical barriers, including data silos, real-time ML deployment challenges, adversarial AI risks, and ethical workforce transitions (WEF, 2023). Finally, we propose future trajectories, such as cognitive digital twins, AI-for-sustainability, and neuromorphic computing for low-latency control. Our findings underscore that convergent AI+ML systems—not standalone tools—are pivotal to realizing Industry 4.0’s full potential.

Polysaccharide nanogels have been demonstrated to aid the refolding processes of chemically or thermally denatured proteins, a function that is similar to that of natural molecular chaperones. In this study, we examined the possibilities of using the nanogel chaperone system to mediate protein folding in a cell-free (in vitro) protein synthesis system containing transcription/translation factors. High-performance liquid chromatography showed that a polysaccharide nanogel comprising cholesteryl group-bearing pullulan (CHP) trapped unfolded or partially folded green fluorescent protein (GFP) expressed in the cell-free system. The protein release and refolding processes, which are induced by ATP in natural molecular chaperone systems, were also simulated by methyl-β-cyclodextrin (M-β-CD). The CHP nanogels dissociate on complexation with M-β-CD to yield dissociated CHP. Thus, the dissociation of the CHP nanogel–protein complex subsequently allows for the release and folding of GFP. The folding kinetics in the presence of the CHP nanogel and M-β-CD was comparable to that of spontaneous folding in the absence of CHP/M-β-CD, indicating that the CHP nanogels did not affect protein synthesis in the cell-free system, providing correctly folded active proteins. The molecular chaperone function of polysaccharide nanogel was demonstrated for the folding of newly synthesized green fluorescent protein (GFP) in a cell-free protein synthesis system. Nanogel comprising cholesteryl group-bearing pullulan (CHP) trapped unfolded or partially folded GFP expressed in the cell-free system. The structure of the CHP nanogel was disrupted by the addition of cyclodextrins, and the protein complexed with the nanogel was released and folded into the mature form.

Due to recent advances in genome sequencing, there has been a dramatic increase in the quantity of genetic information, which has lead to an even greater demand for a faster, more parallel expression system. Therefore, interest in cell-free protein synthesis, as an alternative method for high-throughput gene expression, has been revived. In contrast toin vivo gene expression methods, cell-free protein synthesis provides a completely open system for direct access to the reaction conditions. We have developed an efficient cell-free protein synthesis system by optimizing the energy source and S30 extract. Under the optimized conditions, approximately 650 μg/mL of protein was produced after 2 h of incubation, with the developed system further modified for the efficient expression of PCR-amplified DNA. When the concentrations of DNA, magnesium, and amino acids were optimized for the production of PCR-based cell-free protein synthesis, the protein yield was comparable to that from the plasmid template.