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Molecular farming, initially developed to produce therapeutic proteins using genetically modified plants, gained renewed interest during the Ebola and COVID-19 outbreaks and has expanded into functional food ingredients. This article evaluates molecular farming technologies, market potential, and startups, and identifies opportunities in dairy proteins, food enzymes, collagen, and cellular agriculture. Molecular farming, initially developed to produce therapeutic proteins using genetically modified plants, gained renewed interest during the Ebola and COVID-19 outbreaks and has expanded into functional food ingredients. This article evaluates molecular farming technologies, market potential, and startups, and identifies opportunities in dairy proteins, food enzymes, collagen, and cellular agriculture.

Plants offer several unique advantages in the production of recombinant pharmaceuticals for humans and animals. Although numerous recombinant proteins have been expressed in plants, only a small fraction have been successfully put into use. The hugely distinct expression systems between plant and animal cells frequently cause insufficient yield of the recombinant proteins with poor or undesired activity. To overcome the issues that greatly constrain the development of plant-produced pharmaceuticals, great efforts have been made to improve expression systems and develop alternative strategies to increase both the quantity and quality of the recombinant proteins. Recent technological revolutions, such as targeted genome editing, deconstructed vectors, virus-like particles, and humanized glycosylation, have led to great advances in plant molecular farming to meet the industrial manufacturing and clinical application standards. In this review, we discuss the technological advances made in various plant expression platforms, with special focus on the upstream designs and milestone achievements in improving the yield and glycosylation of the plant-produced pharmaceutical proteins.

Plant Molecular Farming (PMF) capitalizes on the unique properties of plants as bioreactors to efficiently produce valuable proteins, pharmaceuticals, and enzymes. This review emphasizes the critical role of transient expression systems, particularly in Nicotiana benthamiana, due to its susceptibility to various pathogens. Viral vector-based transient expression has proven essential during health emergencies like COVID-19, enabling rapid recombinant protein production. The review also evaluates different transient expression platforms and highlights their applications in biopharmaceutical production, education, synthetic biology, and gene editing. Advances in viral vector modification, hydroponics, and Controlled Environment Agriculture (CEA) are presented as transformative innovations enhancing scalability and regulatory compliance. Furthermore, glycoengineering advancements broaden the range of producible biopharmaceuticals, improving global medication access. By exploring these advancements, this review underscores the vast potential of transient expression systems to meet dynamic scientific and market demands, positioning PMF as a vital component in modern biotechnology.

Plant viruses have traditionally been studied as pathogens in the context of understanding the molecular and cellular mechanisms of a particular disease affecting crops. In recent years, viruses have emerged as a new alternative for producing biological nanomaterials and chimeric vaccines. Plant viruses were also used to generate highly efficient expression vectors, revolutionizing plant molecular farming (PMF). Several biological products, including recombinant vaccines, monoclonal antibodies, diagnostic reagents, and other pharmaceutical products produced in plants, have passed their clinical trials and are in their market implementation stage. PMF offers opportunities for fast, adaptive, and low-cost technology to meet ever-growing and critical global health needs. In this review, we summarized the advancements in the virus-like particles-based (VLPs-based) nanotechnologies and the role they played in the production of advanced vaccines, drugs, diagnostic bio-nanomaterials, and other bioactive cargos. We also highlighted various applications and advantages plant-produced vaccines have and their relevance for treating human and animal illnesses. Furthermore, we summarized the plant-based biologics that have passed through clinical trials, the unique challenges they faced, and the challenges they will face to qualify, become available, and succeed on the market.

Production of biologics in plants, or plant molecular pharming, is a promising protein expression technology that is receiving increasing attention from the pharmaceutical industry. Previously, low expression yields of recombinant proteins and the realization that certain post-translational modifications (PTMs) may not occur optimally limited the widespread acceptance of the technology. However, molecular engineering of the plant secretory pathway is now enabling the production of increasingly complex biomolecules using tailored protein-specific approaches to ensure their maturation. These involve the elimination of undesired processing events, and the introduction of heterologous biosynthetic machinery to support the production of specific target proteins. Here, we discuss recent advances in the production of pharmaceutical proteins in plants, which leverage the unique advantages of the technology. Plants are an alternative pharmaceutical manufacturing platform with unique advantages compared with conventional technologies.Engineering the secretory pathway in plants enables the production of biologics that would otherwise accumulate at low levels or in an improperly processed form.Host proteases can be inactivated by co-expressing broad-spectrum protease inhibitors or by manipulating the pH along the secretory pathway.Glycoengineering strategies enable the production of human-like glycoforms that can be tailored to improve their biological activity.Introducing heterologous chaperone machinery improves the production of target proteins where the endogenous machinery does not efficiently mediate folding.Furin processing and tyrosine sulfation can be achieved in planta by introducing the required biosynthetic machinery.

Summary Nicotiana benthamiana (Australian tobacco) has become a major host for plant‐based recombinant protein production, especially using transient expression. Once a candidate protein has been designed and produced in a suitable variety (e.g. facilitated humanized glycosylation) under reproducible conditions (e.g. in a vertical farm), the product must be extracted from the biomass and purified to meet the application requirements. For example, >95% purity is often expected for biopharmaceuticals. Here, we review options for and challenges of recombinant protein isolation from N. benthamiana biomass taking inspiration from related host systems when appropriate. Specifically, we first introduce typical properties of the biomass to be processed and then discuss options to harvest it and extract the product, for example, using homogenizers. This includes conditioning steps like heat treatment and means to ensure product integrity. Next, we highlight the advantages and limitations of different clarification operations given the high particle loads typically present in N. benthamiana extracts. Afterwards, we look at product purification using both membrane and chromatographic unit operations focusing on the specific aspects of plant molecular farming. We briefly discuss the potentials of modelling and artificial intelligence in downstream process development and conclude with a short outlook on future developments.

Transient expression of recombinant proteins in leaves of Nicotiana benthamiana is routinely employed for both basic research and manufacturing of biopharmaceutical products in plants. Relying on disarmed strains of the bacterial plant pathogen Agrobacterium tumefaciens as a transgene vector, this safe, cost‐effective and easily scalable ‘plant molecular farming’ approach offers a reliable alternative to classical protein expression platforms. Commonly referred to as agroinfiltration, scaled‐up versions of this manufacturing process have now become helpful in the fight against global health issues, such as those rapidly evolving virus strains causing influenza or coronavirus disease 2019. In the past decades, considerable efforts have been deployed to improve the efficacy of Agrobacterium‐mediated expression, including through the development of new binary vectors, the design of strong promoters, and the deployment of approaches to increase levels and stability of transgene mRNAs. By comparison, much less attention has been given to understanding the effects that agroinfiltration unavoidably has on host plants, including the infiltration process itself, the perception of Agrobacterium and the subsequent accumulation of recombinant products throughout the expression phase. Using the upregulation profiles of plant receptor genes during the heterologous expression of virus‐like particles in N. benthamiana leaves, I here describe how some of these host responses interact with each other to form an intricate signalling interplay at the molecular level. I also review host plant's responses to agroinfiltration and highlight strategies that have emerged to improve the efficacy of plant cell biofactories based on the better understanding of this transient expression system.

Summary The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian‐based production systems is increasing. Plant‐based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state‐of‐the‐art mass spectrometry‐based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.

Summary The shared diseases between animals and humans are known as zoonotic diseases and spread infectious diseases among humans. Zoonotic diseases are not only a major burden to livestock industry but also threaten humans accounting for >60% cases of human illness. About 75% of emerging infectious diseases in humans have been reported to originate from zoonotic pathogens. Because antibiotics are frequently used to protect livestock from bacterial diseases, the development of antibiotic‐resistant strains of epidemic and zoonotic pathogens is now a major concern. Live attenuated and killed vaccines are the only option to control these infectious diseases and this approach has been used since 1890. However, major problems with this approach include high cost and injectable vaccines is impractical for >20 billion poultry animals or fish in aquaculture. Plants offer an attractive and affordable platform for vaccines against animal diseases because of their low cost, and they are free of attenuated pathogens and cold chain requirement. Therefore, several plant‐based vaccines against human and animals diseases have been developed recently that undergo clinical and regulatory approval. Plant‐based vaccines serve as ideal booster vaccines that could eliminate multiple boosters of attenuated bacteria or viruses, but requirement of injectable priming with adjuvant is a current limitation. So, new approaches like oral vaccines are needed to overcome this challenge. In this review, we discuss the progress made in plant‐based vaccines against zoonotic or other animal diseases and future challenges in advancing this field.

The ongoing demand for reliable, cost-effective, and scalable diagnostic solutions during the COVID-19 pandemic emphasized the need for innovative production platforms. In this study, we present a plant-based molecular farming (PMF) strategy for the production of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein fused with an Fc region (RBDw-Fc). The RBDw-Fc antigen was transiently expressed in the Nicotiana benthamiana plant, achieving high yields and purity. Its functionality was assessed through antigen–antibody binding assays. The purified antigen was subsequently employed in the development of a rapid diagnostic blot assay capable of screening plasma EDTA samples from pre- and post-vaccinated as well as pre- and post-infected individuals, demonstrating high sensitivity and specificity. Our results show that the RBDw-Fc-based assay is effective for SARS-CoV-2 detection and offers considerable advantages in terms of production speed, scalability, and cost efficiency compared to traditional systems, such as cell-culture-based production. The assay delivers accurate results in just a few minutes, making it particularly suitable for clinical and resource-limited settings. This study highlights the versatility of PMF as a platform for producing high-quality reagents, with promising applications beyond SARS-CoV-2 diagnostics. The RBDw-Fc antigen-based method provides a model for the rapid, economical, and flexible development of screening tools for emerging infectious diseases and future pandemics.