Cell-free System Research Articles

Abstract 407: Development of an integrated multi-modal platform for comprehensive analysis of targeted protein degraders in a drug discovery setting

With growth in the area of targeted protein degradation (TPD), there has been increasing interest in the development of analytical platforms which can facilitate efficient drug discovery processes. We provide a fully developed high throughput analytical platform to study and optimize protein degrader candidates in a comprehensive manner. Our platform consists of multiple orthogonal technologies that generate major mechanistic information and help advance TPD drugs rapidly through the preclinical development stages. Our platform is based on a well-defined degrader development strategy using advanced technologies and automated processes. Major analytical features include: • Binding and binary complex formation: assessment of warheads. Monitoring target protein binding interactions in both cell free and cellular systems, using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and NanoBRET, respectively. • Ternary complex formation evaluation: assessment of degrader-induced protein-protein interactions in both cell free and cell-based systems, using homogeneous time-resolved fluorescence (HTRF) and NanoBRET respectively. • Cellular degradation profiling via a variety of methods, including automated capillary Western blotting, HiBiT and High Content Screening, enabling precise quantification of degradation kinetics and efficacy. • Proteome-wide specificity assessment using mass spectrometry. In our integrated data processing pipeline, we correlate physicochemical properties with degradation efficiency, to support SAR analysis and enhance the rational optimization of degraders. The platform has been proven to work across multiple E3 ligase systems, including CRBN, VHL and other tissue-specific ligases, thus supporting a wide range of TPD programs. Comparative case studies from several client-based programs exemplify the versatility of our platform and its capability to generate comprehensive data for effective decision making. Notably, we achieved high concordance between orthogonal methods. Modular design of the platform enables its segmentation to fit the needs of the clients’ individual specifications whilst retaining standardized work procedures for effective delivery of the project. This comprehensive analytical strategy addresses the challenges of TPD drug discovery and provides the scalability and reliability required for contract research organizations supporting multiple concurrent programs. Citation Format: Magdalena Kieltyka, Maciej Olszewski, Marta Smejda, Kamila Zaremba, Katarzyna Nosek, Magdalena Malik, Katarzyna Szafran. Development of an integrated multi-modal platform for comprehensive analysis of targeted protein degraders in a drug discovery setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 407.

Developing an E. coli-Based Cell-Free Protein Synthesis System for Artificial Spidroin Production and Characterization.

Spider silk spidroins, nature's advanced polymers, have long hampered efficient in vitro production due to their considerable size, repetitive sequences, and aggregation-prone nature. This study harnesses the power of a cell-free protein synthesis (CFPS) system, presenting the first successful in vitro production and detailed characterization of recombinant spider silk major ampullate spidroins (MaSps) utilizing a reformulated and optimizedEscherichia coli based CFPS system. Through systematic optimization, including cell strain engineering via knockout generation, energy sources, crowding agents, and amino acid supplementation, we effectively addressed the specific challenges associated with recombinant spidroin biosynthesis, resulting in high yields of 0.61 mg/mL for MaSp1 (69 kDa) and 0.52 mg/mL for MaSp2 (73 kDa). The synthesized spidroins self-assembled into micelles, facilitating efficient purification compared to in vivo methods, and were further processed into prototype silk fiber products. The functional characterization demonstrated that the purified spidroins maintain essential natural properties, such as phase separation and fiber formation triggered by pH and ions. This tailored CFPS platform also facilitates versatile cosynthesis and serves as an accessible platform for studying the supramolecular coassembly and dynamic interactions among spidroins. This CFPS platform offers a viable alternative to conventional in vivo methods, facilitating innovative approaches for silk protein engineering and biomaterial development in a high-throughput, efficient manner.

Synthesis and Evaluation of Aquatic Antimicrobial Peptides Derived from Marine Metagenomes Using a High-Throughput Screening Approach

Bacterial diseases cause high mortality and considerable losses in aquaculture. The rapid expansion of intensive aquaculture has further increased the risk of large-scale outbreaks. However, the emergence of drug-resistant bacteria, food safety concerns, and environmental regulations have severely limited the availability of antimicrobial. Compared to traditional antibiotics, antimicrobial peptides (AMPs) offer broad spectrum activity, physicochemical stability, and lower resistance development. However, their low natural yield and high extraction costs along with the time-consuming and expensive nature of traditional drug discovery, pose a challenge. In this study, we applied a machine-learning macro-model to predict AMPs from three macrogenomes in the water column of South American white shrimp aquaculture ponds. The AMP content per megabase in the traditional earthen pond (TC1) was 1.8 times higher than in the biofloc pond (ZA1) and 63% higher than in the elevated pond (ZP11). A total of 1033 potential AMPs were predicted, including 6 anionic linear peptides, 616 cationic linear peptides, and 411 cationic cysteine-containing peptides. After screening based on structural, and physio-chemical properties, we selected 10 candidate peptides. Using a rapid high-throughput cell-free protein expression system, we identified nine peptides with antimicrobial activity against aquatic pathogens. Three were further validated through chemical synthesis. The three antimicrobial peptides (K-5, K-58, K-61) showed some inhibitory effects on all four pathogenic bacteria. The MIC of K-5 against Vibrio alginolyticus was 25 μM, the cell viability of the three peptides was higher than 70% at low concentrations (≤12.5 μM), and the hemolysis rate of K-5 and K-58 was lower than 5% at 200 μM. This study highlights the benefits of machine learning in AMP discovery, demonstrates the potential of cell-free protein synthesis systems for peptide screening, and provides an efficient method for high-throughput AMP identification for aquatic applications.

A tripartite cell-free translation system to study mammalian translation.

Genetic manipulation of cellular systems often leads to the adaptation of gene expression programs, rendering detailed mechanistic insights challenging to isolate and elucidate. The proteome constitutes the ultimate manifestation of gene expression programs with multiple layers of regulation to ensure faithful execution. While current high-throughput techniques to investigate regulation at the level of translation, such as Ribo-Seq and nascent proteomics, can capture nuanced changes in the translational landscape, they suffer from potential confounding factors imposed by adaptation of the cellular states. Cell-free translation systems have been used to elucidate molecular mechanisms for decades, but experimental setups have rigid composition and often rely on non-human model systems and artificially designed mRNA constructs. Here we detail a tripartite cell-free translation system based on the separation of mRNAs, ribosomes and ribosome-depleted cytoplasmic lysate from human cells, allowing for flexible reconstitution of translation reactions, which can be performed in 1-4 days. In this setup, cellular parts such as the cytoplasmic lysate can be kept constant, while ribosome complexes or mRNA can be varied or subjected to treatments or vice versa. We detail how complete mRNA populations can be used as input with subsequent detection of nascent peptides using autoradiography or mass spectrometry. We utilize this protocol to resolve which aspects of the translational machinery are selectively affected by environmental and cellular stress conditions that trigger ribosome stalling and collisions, which have been unresolvable until now.

Establishing a High-Yield Bacillus subtilis-Based Cell-Free Protein Synthesis System for In Vitro Prototyping and Natural Product Biosynthesis.

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.

Channel Aware Power Allocation and Diversity Gain Selection for Mimo Noma System

In recent communications, multiple-input-multiple-output (MIMO), orthogonal frequency division multiplexing (OFDM) and Non-Orthogonal Multiple Access (NOMA), and are major sub-system techniques of 5G wireless communications for optimization of latency, Bit Error Rate (BER) and improvement of throughput. In this paper, the proposed design, manages the resource allocations among the techniques to meet the requirements using NOMA and MIMO. The interactive waterfilling based PA in MIMO and NOMA to improve Quality of Service (QoS) and investigated NOMA cell free massive MIMO system by considering effect of linear and individual channel estimations. The proposed system also optimizes user pairing approach for group users that optimize downlink rate per user so that PA can be acceptable at cost of involvedness. Lastly, the proposed system demonstrates experimental results to different noisy channel to minimize the BER and latency that does not degraded in performance compared to the existing PA. The design is validated under single user, 2, 4, 8 users under different noisy channels. The proposed system also validated for up-link transmission under same channels by interactive waterfilling based PA in MIMO and NOMA. Based on obtained simulation results, BER is optimized by 8%, SNR, throughput and PAPR are optimally obtained by 5.5%, 7% and 6% respectively.

Optimization and Application of the Purified Cell-Free System.

Cell-free protein synthesis (CFPS) is extensively applied in biotechnological research. Unlike the lysate-based CFPS system, the alternative purified cell-free system, protein synthesis using recombinant elements (PURE), has garnered a great attention due to its controllability and flexibility of adjusting individual component for in vitro transcription and translation research. The PURE system has been widely studied in the synthetic biological field, and optimized for its application in prototyping research, non-canonical amino acid incorporation and synthetic cell construction. Herein, the recent optimization strategies for a simple and enhanced PURE system are discussed. In addition, the recent application via the PURE system and the current challenges for the development of a more robust and controllable PURE are highlighted.

Carbon-conserving Bioproduction of Malate in an E. coli-based Cell-Free System.

Carbon-conserving Bioproduction of Malate in an E. coli-based Cell-Free System.

Cell-free synthesis system: An accessible platform from biosensing to biomanufacturing.

Cell-free synthesis system: An accessible platform from biosensing to biomanufacturing.

Cell-Free Reaction System for ATP Regeneration from d-Fructose.

Adenosine triphosphate (ATP)-dependent in vitro bioprocesses, such as cell-free protein synthesis and the production of phosphorylated fine chemicals, are of considerable industrial significance. However, their implementation is mainly hindered by the high cost of ATP. We propose and demonstrate the feasibility of a cell-free ATP regeneration system based on the in situ generation of the high-energy compound acetyl phosphate from low-cost d-fructose and inorganic phosphate substrates. The enzyme cascade chains d-fructose phosphoketolase, d-erythrose isomerase, d-erythrulose phosphoketolase, and glycolaldehyde phosphoketolase activities theoretically enabling production of 3 mol ATP per mol of d-fructose. Through a semirational engineering approach and the screening of nine single-mutation libraries, we optimized the phosphoketolase (PKT) from Bifidobacterium adolescentis, identifying the improved variant Bad.F6Pkt H548N. This mutant exhibited a 5.6-fold increase in d-fructose activity, a 2.2-fold increase in d-erythrulose activity, and a 1.3-fold increase in glycolaldehyde activity compared to the wild-type enzyme. The Bad.F6Pkt H548N mutant was initially implemented in a cell-free reaction system together with an acetate kinase from Geobacillus stearothermophilus and a glycerol kinase from Cellulomonas sp. for the production of glycerol-3 phosphate from ADP and glycerol. We demonstrated the feasibility of ATP regeneration from 25 mM d-fructose with a stoichiometry of 1 mol of ATP per mol of C6 ketose. Subsequently, the reaction system was enhanced by incorporating d-erythrose isomerase activity provided by a l-rhamnose isomerase from Pseudomonas stutzeri. In the complete system, the ATP yield increased to 2.53 mol molfructose-1 with a maximum productivity of 7.2 mM h-1.

Data-driven multi-omics analyses and modelling for bioprocesses

Biomanufacturing has emerged as a crucial driving force for efficient material conversion through engineered cells or cell-free systems. However, the intrinsic spatiotemporal heterogeneity, complexity, and dynamic characteristics of these processes pose significant challenges to systematic understanding, optimization, and regulation. This review summarizes essential methodologies for multi-omics data acquisition and analyses for bioprocesses and outlines modelling approaches based on multi-omics data. Furthermore, we explore practical applications of multi-omics and modelling in fine-tuning process parameters, improving fermentation control, elucidating stress response mechanisms, optimizing nutrient supplementation, and enabling real-time monitoring and adaptive adjustment. The substantial potential offered by integrating multi-omics with computational modelling for precision bioprocessing is also discussed. Finally, we identify current challenges in bioprocess optimization and propose the possible solutions, the implementation of which will significantly deepen understanding and enhance control of complex bioprocesses, ultimately driving the rapid advancement of biomanufacturing.

Oleuropein Is a Stimulator of Melanocyte Dendricity: Potential for Treatment of Hypopigmentation

Background/Objectives: Oleuropein (OLP), the key bioactive in olive leaf extracts, has demonstrated various biological benefits. We previously reported on the pro-melanogenic action with increased dendricity of a patented olive leaf extract (Benolea®) that was standardized to 16–24% OLP. In this study, purified OLP was evaluated to identify if it might be the bioactive responsible for the stimulating effects on melanocytes. Moreover, previous studies on OLP have never reported the effects on melanocyte dendricity or melanin export in the medium. Methods: Herein, the effect of OLP on melanogenesis was first evaluated using the B16F10 cell model and validated using the physiological model of normal human melanocytes from Caucasian (lightly pigmented; LP) and Asian (moderately pigmented; MP) skin. The effects of OLP on melanin export in LP and MP cells were indirectly evaluated by dendricity indices. Results: OLP lowered the intracellular melanin content in B16F10 cells by 26.36%, 24.48%, and 27.71% at 100, 150, and 200 µg/mL (all p < 0.01), respectively, with no effect on the intracellular melanin contents of LP or MP cells. OLP treatment did not influence tyrosinase activity in B16F10 cells or MP cells but significantly enhanced the activity in LP cells. The measurement of extracellular melanin showed significantly higher levels for all three cells, although the levels were considerably higher in MP cells, after the adjustment for OLP autoxidation observed in the cell-free system, which caused melanin-like brown coloration. Furthermore, OLP induced morphological alterations of extended dendrites of B16F10 cells that were retained in LP and MP cells. The quantitation of the dendricity of cells treated with OLP at 200 μg/mL revealed that the total dendrite length was increased by 35.24% (p < 0.05) in LP cells and by 58.45% (p < 0.001) in MP cells without any change in the dendrite number. Conclusions: This is the first study to demonstrate the novel finding that OLP possesses a hitherto unreported unique capacity to stimulate melanocyte dendricity, hence establishing the efficacy for use in increasing human pigmentation. Our findings show significance, with a potential application of the compound OLP for addressing human hypopigmentation disorders in clinical settings or for cosmetic uses related to sunless tanning.

Structural insights into lab-coevolved RNA-RBP pairs and applications of synthetic riboswitches in cell-free system.

CS1-LS4 and CS2-LS12 are ultra-high affinity and orthogonal RNA-protein pairs that were identified by PD-SELEX (Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment). To investigate the molecular basis of the lab-coevolved RNA-RBP pairs, we determined the structures of the CS1-LS4 and CS2-LS12 complexes and the LS12 homodimer in an RNA-free state by X-ray crystallography. The structural analyses revealed that the lab-coevolved RNA-RBPs have acquired unique molecular recognition mechanisms, whereas the overall structures of the RNP complexes were similar to the typical kink-turn RNA-L7Ae complex. The orthogonal RNA-RBP pairs were applied to construct high-performance cell-free riboswitches that regulate translation in response to LS4 or LS12. In addition, by using the orthogonal protein-responsive switches, we generated an AND logic gate that outputs staphylococcal γ-hemolysin in cell-free system and carried out hemolysis assay and calcein leakage assay using rabbit red blood cells and artificial cells, respectively.

Effects of Crude Shea Butters and Their Polar Extracts on Singlet Oxygen Quenching and Against Rose Bengal-Induced HaCaT Cell Phototoxicity.

Shea butter (SB) is a raw material fat obtained from Vitellaria paradoxa C.F. Gaertn kernels. We investigated the direct and indirect protective effects of 10 traditional and industrial SBs and their polar extracts on cell-free systems using ABTS and DPPH radical scavenging assays as well as on singlet oxygen (1O2) produced by Rose Bengal (RB) photosensitization. Their effects against RB-induced HaCaT cell phototoxicity were also explored. A spectrophotometric assay and HPLC were performed to quantify and identify phenolic content, which was between 14.16 and 82.99 ppm pyrogallol equivalent. These variations could be due to the SB origin and extraction process. These polar fractions exhibited moderate DPPH and strong ABTS radical-scavenging activity. By applying the UV-visible technique, we demonstrated that SBs and their phenolic compounds behave as 1O2 quenchers in a dose-dependent manner. Moreover, using a UVR-like model after the irradiation of RB, both polar extracts and crude SB exhibited photoprotective effects, highlighting the indirect protective action. In acellular and cellular models, SB and its polar extracts can act as a free radical scavenger against reactive oxygen species and 1O2 quenchers. Due to the maximum absorbance of SB at 280 nm and the antioxidant effect of 1O2 quenching, SB polar extracts exhibit photoprotective properties.

Design Principles for Polymerase Strand Recycling Circuits.

Cell-free biosensing systems are being engineered as versatile and programmable diagnostic technologies. A core component of cell-free biosensors are programmable molecular circuits that improve biosensor speed, sensitivity and specificity by performing molecular computations such as logic evaluation and signal amplification. In previous work, we developed one such circuit system called Polymerase Strand Recycling (PSR) which amplifies cell-free molecular circuits by using T7 RNA polymerase off-target transcription to recycle nucleic acid inputs. We showed that PSR circuits can be configured to detect RNA target inputs as well as be interfaced with allosteric transcription factor-based biosensors to amplify signal and enhance sensitivity. Here we expand the development of PSR circuit design principles to generalize the platform for detecting a diverse set of model microRNA inputs. We show that PSR circuit function can be enhanced through engineering T7 RNAP, and present troubleshooting strategies to optimize PSR circuit performance.

Live-cell synthesis of biocompatible quantum dots.

Quantum dots (QDs) exhibit fluorescence properties with promising prospects for biomedical applications; however, the QDs synthesized in organic solvents shows poor biocompatibility, limiting their use in biological systems. We developed an approach for synthesizing QDs in live cells by coupling a series of intracellular metabolic pathways in a precise spatial and temporal sequence. We have validated this approach in yeast (Saccharomyces cerevisiae), Staphylococcus aureus, Michigan Cancer Foundation-7 (MCF-7) and Madin-Darby canine kidney (MDCK) cells. The intracellularly synthesized QDs are inherently stable and biocompatible, making them suitable for the direct in situ labeling of cells and cell-derived vesicles. Here, we provide an optimized workflow for the live-cell synthesis of QDs by using S. cerevisiae, S. aureus or MCF-7 cells. In addition, we detail a cell-free aqueous synthetic system (quasi-biosynthesis) containing enzymes, electrolytes, peptides and coenzymes, which closely mimics the intracellular synthetic conditions used in our cell culture system. In this solution, we synthesize biocompatible ultrasmall QDs that are easier to purify and characterize than those synthesized in cells. The live-cell-synthesized QDs can be used for bioimaging and microvesicle detection, whereas the quasi-biosynthesized QDs are suitable for applications such as biodetection, biolabeling and real-time imaging. The procedure can be completed in 3-4 d for live-cell QD synthesis and 2 h for the quasi-biosynthesis of QDs. The procedure is suitable for users with expertise in chemistry, biology, materials science and synthetic biology. This approach encourages interested researchers to engage in the field of QDs and develop further biomedical applications.

A Rapid and Modular Nanobody Assay for Plug-and-Play Antigen Detection.

Rapid and portable antigen detection is essential for managing infectious diseases and responding to toxic exposures, yet current methods face significant limitations. Highly sensitive platforms like the Enzyme-Linked Immunosorbent Assay (ELISA) are time- and cost-prohibitive for point-of-need detection, while portable options like lateral flow assays (LFAs) require systemic overhauls for new targets. Furthermore, the complex infrastructure, high production costs, and extended timelines for assay development constrain manufacturing of traditional diagnostic platforms in low-resource settings. To address these challenges, we describe the Rapid and Modular Nanobody Assay (RAMONA) as a versatile antigen detection platform that leverages nanobody-coiled coil fusion proteins for modular integration with downstream readout methods. RAMONA merges the portability of LFAs with the benefits of nanobodies, such as their smaller size, improved solubility, and compatibility with cell-free protein synthesis systems, enabling on-demand biomanufacturing and rapid adaptation for diverse targets. We demonstrate assay generalizability through the detection of three distinct protein targets, robustness across various temperatures and incubation periods, and compatibility with saliva samples and cell-free synthesis. Detection occurs in under 30 minutes, with results strongly and positively correlating to ELISA data while requiring minimal resources. Moreover, RAMONA supports multiplexed detection of three antigens simultaneously using orthogonal capture probes. By overcoming several limitations of traditional immunoassays, RAMONA represents a significant advancement in rapid, adaptable, and field-deployable antigen detection technologies.

In vitro differentiation of common lymphoid progenitor cells into B cell using stromal cell free culture system.

The OP9 culture system is an important in vitro model for B cell development. However, the complex nature of operations and the intrinsic variability of stromal cell functionality, which can be influenced by factors such as radiation exposure or contamination, pose considerable challenges to their wider application. Currently, there exists a paucity of studies documenting in vitro B cell differentiation culture systems that exclude stromal cells, and the experimental methodologies available for reference remain limited. This report elucidates a robust stromal cell-free culture system. Specifically, bovine serum albumin (BSA) or fetal bovine serum (FBS), in conjunction with interleukin-7 (IL-7), Flt3 ligand (Flt3L), and stem cell factor (SCF), were incorporated into X-VIVO15 medium. This system proficiently facilitates the directed differentiation of common lymphoid progenitor cells (CLP), defined as lineage-CD127 + CD117lowsca-1lowCD135+, into B lymphocytes in vitro, achieving an amplification factor of up to one hundredfold.We examined the roles of IL-7, Flt3L and SCF in differentiation of B cells from CLP in this culture system. Our findings indicate that IL-7 is a pivotal cytokine essential for B cell differentiation in vitro, demonstrating a notable synergistic impact when combined with SCF and FLT3L. Moreover, this system is capable of supporting the differentiation of hematopoietic stem cells (HSCs) and lymphoid-primed multipotent progenitor cells (LMPPs) into B cells in vitro. The findings substantiated the efficacy of the culture system in investigating the in vitro differentiation of bone marrow-derived progenitor cells into B cells and elucidated the specific roles of BSA, FBS and three cytokines (IL-7, FLT3L and SCF) in promoting efficient B lineage differentiation.

Cell-free systems: A synthetic biology tool for rapid prototyping in metabolic engineering.

Cell-free systems: A synthetic biology tool for rapid prototyping in metabolic engineering.

Navigating the challenges of engineering composite specialized metabolite pathways in plants.

Plants are a valuable source of diverse specialized metabolites with numerous applications. However, these compounds are often produced in limited quantities, particularly under unfavorable ecological conditions. To achieve sufficient levels of target metabolites, alternative strategies such as pathway engineering in heterologous systems like microbes (e.g., bacteria and fungi) or cell-free systems can be employed. Another approach is plant engineering, which aims to either enhance the native production in the original plant or reconstruct the target pathway in a model plant system. Although increasing metabolite production in the native plant is a promising strategy, these source plants are often exotic and pose significant challenges for genetic manipulation. Effective pathway engineering requires comprehensive prior knowledge of the genes and enzymes involved, as well as the precursor, intermediate, branching, and final metabolites. Thus, a thorough elucidation of the biosynthetic pathway is closely linked to successful metabolic engineering in host or model systems. In this review, we highlight recent advances in strategies for biosynthetic pathway elucidation and metabolic engineering. We focus on efforts to engineer complex, multi-step pathways that require the expression of at least eight genes for transient and three genes for stable transformation. Reports on the engineering of complex pathways in stably transformed plants remain relatively scarce. We discuss the major hurdles in pathway elucidation and strategies for overcoming them, followed by an overview of achievements, challenges, and solutions in pathway reconstitution through metabolic engineering. Recent advances including computer-based predictions offer valuable platforms for the sustainable production of specialized metabolites in plants.