Synthetic Biology Approaches Research Articles

Designing plant flavonoids: harnessing transcriptional regulation and enzyme variation to enhance yield and diversity.

Plant synthetic biology has emerged as a powerful and promising approach to enhance the production of value-added metabolites in plants. Flavonoids, a class of plant secondary metabolites, offer numerous health benefits and have attracted attention for their potential use in plant-based products. However, achieving high yields of specific flavonoids remains challenging due to the complex and diverse metabolic pathways involved in their biosynthesis. In recent years, synthetic biology approaches leveraging transcription factors and enzyme diversity have demonstrated promise in enhancing flavonoid yields and expanding their production repertoire. This review delves into the latest research progress in flavonoid metabolic engineering, encompassing the identification and manipulation of transcription factors and enzymes involved in flavonoid biosynthesis, as well as the deployment of synthetic biology tools for designing metabolic pathways. This review underscores the importance of employing carefully-selected transcription factors to boost plant flavonoid production and harnessing enzyme promiscuity to broaden flavonoid diversity or streamline the biosynthetic steps required for effective metabolic engineering. By harnessing the power of synthetic biology and a deeper understanding of flavonoid biosynthesis, future researchers can potentially transform the landscape of plant-based product development across the food and beverage, pharmaceutical, and cosmetic industries, ultimately benefiting consumers worldwide.

Advances in Metabolic Engineering of Plant Monoterpene Indole Alkaloids.

Monoterpene indole alkaloids (MIAs) encompass a diverse family of over 3000 plant natural products with a wide range of medical applications. Further utilizations of these compounds, however, are hampered due to low levels of abundance in their natural sources, causing difficult isolation and complex multi-steps in uneconomical chemical syntheses. Metabolic engineering of MIA biosynthesis in heterologous hosts is attractive, particularly for increasing the yield of natural products of interest and expanding their chemical diversity. Here, we review recent advances and strategies which have been adopted to engineer microbial and plant systems for the purpose of generating MIAs and discuss the current issues and future developments of manufacturing MIAs by synthetic biology approaches.

A synthetic biology approach to engineering circuits in immune cells.

A synthetic circuit in a biological system involves the designed assembly of genetic elements, biomolecules, or cells to create a defined function. These circuits are central in synthetic biology, enabling the reprogramming of cellular behavior and the engineering of cells with customized responses. In cancer therapeutics, engineering T cells with circuits have the potential to overcome the challenges of current approaches, for example, by allowing specific recognition and killing of cancer cells. Recent advances also facilitate engineering integrated circuits for the controlled release of therapeutic molecules at specified locations, for example, in a solid tumor. In this review, we discuss recent strategies and applications of synthetic receptor circuits aimed at enhancing immune cell functions for cancer immunotherapy. We begin by introducing the concept of circuits in networks at the molecular and cellular scales and provide an analysis of the development and implementation of several synthetic circuits in T cells that have the goal to overcome current challenges in cancer immunotherapy. These include specific targeting of cancer cells, increased T-cell proliferation, and persistence in the tumor microenvironment. By harnessing the power of synthetic biology, and the characteristics of certain circuit architectures, it is now possible to engineer a new generation of immune cells that recognize cancer cells, while minimizing off-target toxicities. We specifically discuss T-cell circuits for antigen density sensing. These circuits allow targeting of solid tumors that share antigens with normal tissues. Additionally, we explore designs for synthetic circuits that could control T-cell differentiation or T-cell fate as well as the concept of synthetic multicellular circuits that leverage cellular communication and division of labor to achieve improved therapeutic efficacy. As our understanding of cell biology expands and novel tools for genome, protein, and cell engineering are developed, we anticipate further innovative approaches to emerge in the design and engineering of circuits in immune cells.

Engineered Escherichia coli platforms for tyrosine-derivative production from phenylalanine using phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system

BackgroundAromatic compounds derived from tyrosine are important and diverse chemicals that have industrial and commercial applications. Although these aromatic compounds can be obtained by extraction from natural producers, their growth is slow, and their content is low. To overcome these problems, many of them have been chemically synthesized from petroleum-based feedstocks. However, because of the environmental burden and depleting availability of feedstock, microbial cell factories are attracting much attention as sustainable and environmentally friendly processes.ResultsTo facilitate development of microbial cell factories for producing tyrosine derivatives, we developed simple and convenient tyrosine-producing Escherichia coli platforms with a bacterial phenylalanine hydroxylase, which converted phenylalanine to tyrosine with tetrahydromonapterin as a cofactor, using a synthetic biology approach. By introducing a tetrahydrobiopterin-regeneration system, the tyrosine titer of the plasmid-based engineered strain was 4.63 g/L in a medium supplemented with 5.00 g/L phenylalanine with a test tube. The strains were successfully used to produce industrially attractive compounds, such as tyrosol with a yield of 1.58 g/L by installing a tyrosol-producing module consisting of genes encoding tyrosine decarboxylase and tyramine oxidase on a plasmid. Gene integration into E. coli chromosomes has an advantage over the use of plasmids because it increases genetic stability without antibiotic feeding to the culture media and enables more flexible pathway engineering by accepting more plasmids with artificial pathway genes. Therefore, we constructed a plasmid-free tyrosine-producing platform by integrating five modules, comprising genes encoding the phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system, into the chromosome. The platform strain could produce 1.04 g/L of 3,4-dihydroxyphenylalanine, a drug medicine, by installing a gene encoding tyrosine hydroxylase and the tetrahydrobiopterin-regeneration system on a plasmid. Moreover, by installing the tyrosol-producing module, tyrosol was produced with a yield of 1.28 g/L.ConclusionsWe developed novel E. coli platforms for producing tyrosine from phenylalanine at multi-gram-per-liter levels in test-tube cultivation. The platforms allowed development and evaluation of microbial cell factories installing various designed tyrosine-derivative biosynthetic pathways at multi-grams-per-liter levels in test tubes.

Application of Synthetic Biology Approaches to High-Yield Production of Mycosporine-like Amino Acids

Ultraviolet (UV) radiation reaching the Earth’s surface is a major societal concern, and therefore, there is a significant consumer demand for cosmetics formulated to mitigate the harmful effects of UV radiation. Synthetic sunscreens being formulated to block UV penetration include inorganic metal oxide particles and organic filters. Lately, organic UV-absorbing compounds are manufactured from non-renewable petrochemicals and, as a result, there is a need to develop a sustainable manufacturing process for efficient, high-level production of a naturally occurring group of UV-absorbing compounds, namely mycosporine-like amino acids (MAAs), for use as a sunscreen additive to skincare products. Currently, the commercial production of MAAs for use in sunscreens is not a viable proposition due to the low yield and the lack of fermentation technology associated with native MAA-producing organisms. This review summarizes the biochemical properties of MAAs, the biosynthetic gene clusters and transcriptional regulations, the associated carbon-flux-driving processes, and the host selection and biosynthetic strategies, with the aim to expand our understanding on engineering suitable cyanobacteria for cost-effective production of natural sunscreens in future practices.

Reprogramming the Cyclization Cascade of epi-Isozizaene Synthase to Generate Alternative Terpene Products.

The class I sesquiterpene cyclase epi-isozizaene synthase from Streptomyces coelicolor (EIZS) catalyzes the transformation of linear farnesyl diphosphate (FPP) into the tricyclic hydrocarbon epi-isozizaene in the biosynthesis of albaflavenone antibiotics. The active site cavity of EIZS is largely framed by four aromatic residues - F95, F96, F198, and W203 - that form a product-shaped contour, serving as a template to chaperone conformations of the flexible substrate and multiple carbocation intermediates leading to epi-isozizaene. Remolding the active site contour by mutagenesis can redirect the cyclization cascade away from epi-isozizaene biosynthesis to generate alternative sesquiterpene products. Here, we present the biochemical and structural characterization of four EIZS mutants in which aromatic residues have been substituted with polar residues (F95S, F96H, F198S, and F198T) to generate alternative cyclization products. Most notably, F95S EIZS generates a mixture of monocyclic sesquiterpene precursors of bisabolane, a D2 diesel fuel substitute. X-ray crystal structures of the characterized mutants reveal subtle changes in the active site contour showing how each aromatic residue influences the chemistry of a different carbocation intermediate in the cyclization cascade. We advance that EIZS may serve as a robust platform for the development of designer cyclases for the generation of high-value sesquiterpene products ranging from pharmaceuticals to biofuels in synthetic biology approaches.

Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus.

Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.

Improvement of Large-Scale Production of Lignocellulosic Bioethanol through Synthetic Biology Approaches: A Comprehensive Review

This review article explores the use of synthetic biology approaches like gene editing and metabolic engineering to create genetically-modified organisms that can produce commercially-valuable products on large-scale, clean environmental pollutants, and serve as efficient biosensors. The Biobrick system includes a registry of available genetic information and a set of standard protocols for assembling and testing them, which finds various significant uses in synthetic biology, such as in the production of "Fourth generation” biofuel. Genetic engineering techniques may be applied to manipulate crops or microbes for more efficient biofuel production. Such synthetic biology techniques also include modification of lignin structure and content, use of bacterial enzymes abundantly expressed in plants, and manipulation of hemicellulose biosynthesis. However, challenges such as high temperature, efficient conversion of all substrate molecules for maximum product yield, efficient pentose fermentation, and tolerance to acetic acid and bioethanol concentration can all impact the yield of the product. This article explores innovative genetic modification approaches designed to decrease lignin levels and enhance crop digestibility in plants, with a specific focus on the model plant Arabidopsis thaliana and herbaceous plants such as Alfalfa. The aim is to manipulate the Monolignol pathway and modify the CSE gene, known for its ability to decrease lignin content. These modifications have been shown to significantly improve saccharification efficiency and increase the yield of lignocellulosic bioethanol. Besides, the role of xylulose-5-phosphate as an intermediate in the non-oxidative pentose phosphate pathway and the oxidative pentose phosphate pathway, and the use of heterologous xylose transporters and modified sugar transporters as potential solutions to improve the biofuel production efficiency is also discussed in this review article.

Pervasive RNA folding is crucial for narnavirus genome maintenance

A synthetic biology approach toward constructing an RNA-based genome expands our understanding of living things and opens avenues for technological advancement. For the precise design of an artificial RNA replicon either from scratch or based on a natural RNA replicon, understanding structure-function relationships of RNA sequences is critical. However, our knowledge remains limited to a few particular structural elements intensively studied so far. Here, we conducted a series of site-directed mutagenesis studies of yeast narnaviruses ScNV20S and ScNV23S, perhaps the simplest natural autonomous RNA replicons, to identify RNA elements required for maintenance and replication. RNA structure disruption corresponding to various portions of the entire narnavirus genome suggests that pervasive RNA folding, in addition to the precise secondary structure of genome termini, is essential for maintenance of the RNA replicon invivo. Computational RNA structure analyses suggest that this scenario likely applies to other "narna-like" viruses. This finding implies selective pressure on these simplest autonomous natural RNA replicons to fold into a unique structure that acquires both thermodynamic and biological stability. We propose the importance of pervasive RNA folding for the design of RNA replicons that could serve as a platform for invivo continuous evolution as well as an interesting model to study the origin of life.

Engineering of increased L-Threonine production in bacteria by combinatorial cloning and machine learning

The goal of this study is to develop a general strategy for bacterial engineering using an integrated synthetic biology and machine learning (ML) approach. This strategy was developed in the context of increasing L-threonine production in Escherichia coli ATCC 21277. A set of 16 genes was initially selected based on metabolic pathway relevance to threonine biosynthesis and used for combinatorial cloning to construct a set of 385 strains to generate training data (i.e., a range of L-threonine titers linked to each of the specific gene combinations). Hybrid (regression/classification) deep learning (DL) models were developed and used to predict additional gene combinations in subsequent rounds of combinatorial cloning for increased L-threonine production based on the training data. As a result, E. coli strains built after just three rounds of iterative combinatorial cloning and model prediction generated higher L-threonine titers (from 2.7 g/L to 8.4 g/L) than those of patented L-threonine strains being used as controls (4–5 g/L). Interesting combinations of genes in L-threonine production included deletions of the tdh, metL, dapA, and dhaM genes as well as overexpression of the pntAB, ppc, and aspC genes. Mechanistic analysis of the metabolic system constraints for the best performing constructs offers ways to improve the models by adjusting weights for specific gene combinations. Graph theory analysis of pairwise gene modifications and corresponding levels of L-threonine production also suggests additional rules that can be incorporated into future ML models.

Protein Expression in Synechococcus PCC 7002: A Quantitative Comparison of Promoters and Integration Sites

Photosynthetic Cyanobacteria can be used as a chassis for different synthetic biology approaches. However, quantitative comparison of tools for engineering, such as those for heterologous gene expression, is often not available. Here, we report the comparative quantification of heterologous protein production in Synechococcussp.PCC 7002 regarding protein expression cassettes and locations of foreign gene integration using sf-GFP as a reporter. We used promoter cpc560 as reference because it was described as a "super strong" promoter. sf-GFP-expression constructs were integrated into neutral sites NS_1, NS_2, NS_3 and the extrachromosomal plasmid pAQ1. The latter induced a sf-GFP level of approximately 10-fold in comparison to a reference promotor expression. Protein-fusion with 6xHis increased sf-GFP as well as expression of sf-GFP fusion with ß subunit of phycocyanin.

In silico Comparative Analysis of Gene and Protein of Plant Lectins

Lectins are a family of proteins that can recognize and bind specific carbohydrates. Plant lectins play various roles in plant defense and can be utilized as insecticidal, antibacterial, antifungal, and antiviral agents. This study compares genes, proteins, and carbohydrate-binding motifs between 15 plant lectins using in silico methods. The lectin genes of Artocarpus hypargyreus Hance, Hordeum vulgare var. Betzes, Triticum aestivum L. cv. Marshall, Galanthus nivalis L., Allium sativum L., Phaseolus vulgaris, Lens culinaris subsp. tomentosus, Robinia pseudoacacia, Glycine max, Cicer arietinum, Pisum sativum, Canavalia ensiformis, Amaranthus caudatus, Amaranthus hypochondriacus, and Musa acuminata subsp. malaccensis were obtained from National Center for Biotechnology Information and Banana Genome Hub. The gene comparison results revealed different characteristics of the 15 plant lectin genes, with A. hypargyreus having the shortest lectin gene and G. max having the longest. Overall, the 15 plant lectin genes have 1–3 exons. Domain predictions revealed the presence of five domains: jacalin, chitin_bind_1, B_lectin, legume lectin, and agglutinin. Furthermore, there were 2 protein sequences from the jacalin domain, 2 protein sequences from the chitin_bind_I domain, 2 protein sequences from the B_lectin domain, and 4 protein sequences from the legume lectin domains that have complete carbohydrate-binding motifs compared to consensus motifs from literature. The data obtained from this study has not been previously reported and can be utilized for future lectin protein production with synthetic biology approaches. This method will allow scientists to obtain plant bioparts for lectin production using a heterologous system, even without plant samples.

Engineering the IL-4/IL-13 axis for targeted immune modulation.

The structurally and functionally related interleukin-4 (IL-4) and IL-13 cytokines play pivotal roles in shaping immune activity. The IL-4/IL-13 axis is best known for its critical role in T helper 2 (Th2) cell-mediated Type 2 inflammation, which protects the host from large multicellular pathogens, such as parasitic helminth worms, and regulates immune responses to allergens. In addition, IL-4 and IL-13 stimulate a wide range of innate and adaptive immune cells, as well as non-hematopoietic cells, to coordinate various functions, including immune regulation, antibody production, and fibrosis. Due to its importance for a broad spectrum of physiological activities, the IL-4/IL-13 network has been targeted through a variety of molecular engineering and synthetic biology approaches to modulate immune behavior and develop novel therapeutics. Here, we review ongoing efforts to manipulate the IL-4/IL-13 axis, including cytokine engineering strategies, formulation of fusion proteins, antagonist development, cell engineering approaches, and biosensor design. We discuss how these strategies have been employed to dissect IL-4 and IL-13 pathways, as well as to discover new immunotherapies targeting allergy, autoimmune diseases, and cancer. Looking ahead, emerging bioengineering tools promise to continue advancing fundamental understanding of IL-4/IL-13 biology and enabling researchers to exploit these insights to develop effective interventions.

Taking Synthetic Biology to the Seas - From Blue Chassis Organisms to Marine Aquaforming.

Oceans cover 71% of Earth's surface and are home to hundreds of thousands of species, many of which are microbial. Knowledge about marine microbes has strongly increased in the past decades due to global sampling expeditions and hundreds of detailed studies on marine microbial ecology, physiology, and biogeochemistry. However, the translation of this knowledge into biotechnological applications or synthetic biology approaches using marine microbes has been limited so far. This review highlights key examples of marine bacteria in synthetic biology and metabolic engineering, and outlines possible future work based on the emerging marine chassis organisms Vibrio natriegens and Halomonas bluephagenesis. Furthermore, the valorization of algal polysaccharides by genetically enhanced microbes is presented as an example for the opportunities and challenges associated with blue biotechnology. Finally, new roles for marine synthetic biology in tackling pressing global challenges, including climate change and marine pollution, are discussed.

Metabolic Engineering and Synthetic Biology Approaches for the Heterologous Production of Aromatic Polyketides.

Polyketides are a diverse set of natural products with versatile applications as pharmaceuticals, nutraceuticals, and cosmetics, to name a few. Of several types of polyketides, aromatic polyketides comprising type II and III polyketides contain many chemicals important for human health such as antibiotics and anticancer agents. Most aromatic polyketides are produced from soil bacteria or plants, which are difficult to engineer and grow slowly in industrial settings. To this end, metabolic engineering and synthetic biology have been employed to efficiently engineer heterologous model microorganisms for enhanced production of important aromatic polyketides. In this review, we discuss the recent advancement in metabolic engineering and synthetic biology strategies for the production of type II and type III polyketides in model microorganisms. Future challenges and prospects of aromatic polyketide biosynthesis by synthetic biology and enzyme engineering approaches are also discussed.

Genetic and Environmental Factors Influencing the Production of Select Fungal Colorants: Challenges and Opportunities in Industrial Applications.

Natural colorants, mostly of plant and fungal origins, offer advantages over chemically synthetic colorants in terms of alleviating environmental pollution and promoting human health. The market value of natural colorants has been increasing significantly across the globe. Due to the ease of artificially culturing most fungi in the laboratory and in industrial settings, fungi have emerged as the organisms of choice for producing many natural colorants. Indeed, there is a wide variety of colorful fungi and a diversity in the structure and bioactivity of fungal colorants. Such broad diversities have spurred significant research efforts in fungi to search for natural alternatives to synthetic colorants. Here, we review recent research on the genetic and environmental factors influencing the production of three major types of natural fungal colorants: carotenoids, melanins, and polyketide-derived colorants. We highlight how molecular genetic studies and environmental condition manipulations are helping to overcome some of the challenges associated with value-added and large-scale productions of these colorants. We finish by discussing potential future trends, including synthetic biology approaches, in the commercial production of fungal colorants.

Adaptive diversity in structure and function of C4 photosynthetic components.

Many tropical and subtropical plant lineages have independently evolved C4 photosynthesis. The convergent evolution of this complex functional trait from different ancestors is reflected in variations in the structural and biochemical characteristics of C4 components such as enzymes and cellular specializations. The mechanism of C4 carbon concentration mostly involves coordinated function of mesophyll and bundle sheath cells. Important adaptations of the C4 syndrome include increased vein density and the development of photosynthetic bundle sheath cells with low gas conductance. In addition, the enzymes and transporters of the C4 pathway evolved via the co-option of multiple genes, each derived from a specific lineage of isoforms present in nonC4-ancestors. In particular, the adaptation of C4 enzymes resulted in a variety of structural and biochemical modifications, generally leading to increased catalytic efficiency and regulation by metabolites and post-translational modifications. Differences in these adaptations are particularly evident in the C4-acid decarboxylation step, which can be catalyzed by three decarboxylases that define the C4 subtypes. Associated with the biochemical subtypes, there are also differences in the extend of grana staking and localization of bundle sheath cells chloroplasts. The presence of a suberin layer and symplastic connections also likely vary among the different C4-subtypes. This review examines the current understanding of the diversity of structural and functional changes in key components of the C4 carbon concentration mechanism. This knowledge is necessary not only to identify divergent solutions for convergent optimization of C4 components in different C4 lineages, but also to guide their creation for rational synthetic biology approaches.

Improvements in Bioremediation Agents and Their Modified Strains in Mediating Environmental Pollution.

Environmental pollution has been a significant concern around the globe as the release of toxic pollutants is associated with carcinogenic, mutagenic, and teratogenic impacts on living organisms. Since microorganisms have the natural potential to degrade toxic metabolites into nontoxic forms, an eco-friendly approach known as bioremediation has been used to tackle toxic-induced pollution. Bioremediation has three fundamental levels, i.e., natural attenuation, bio-augmentation, and biostimulation in which the synthetic biology approach has been lately utilized to enhance the conventional bioremediation techniques. Recently, a more advanced approach of programmable nucleases such as zinc finger nucleases, tale-like effector nucleases, and clustered regularly interspaced short palindromic repeats Cas is being employed to engineer several bacterial, fungal, and algal strains for targeted mutagenesis by knocking in and out specific genes which are involved in reconstructing the metabolic pathways of native microbes. These genetically engineered microorganisms possess heavy metal resistance, greater substrate range, enhanced enzymatic activity, and binding affinity which accelerate the biodegradation of toxic pollutants to environmentally safe levels. This review provides a comprehensive understanding of how we can correlate the novel genetics-based approaches employed to produce genetically engineered microorganisms to enhance the biodegradation of hazardous pollutants, hence, developing a clean and sustainable ecosystem.

Integration of multiple stress signals in plants using synthetic Boolean logic gates.

As photosynthetic organisms, plants have a potential role in the sustainable production of high-value products such as medicines, biofuels, and chemical feedstocks. With effective engineering using synthetic biology approaches, plant-based platforms could conceivably be designed to minimize the costs and waste of production for materials that would otherwise be uneconomical. Additionally, modern agricultural crops could be engineered to be more productive, resilient, or restorative in different or rapidly changing environments and climates. Information-processing genetic devices and circuits containing multiple interacting parts that behave predictably must be developed to achieve these complex goals. A genetic Boolean AND logic gate is a device that computes the presence or absence of 2 inputs (signals and stimuli) and produces an output (response) only when both inputs are present. We optimized individual genetic components and used synthetic protein heterodimerizing domains to rationally assemble genetic AND logic gates that integrate 2 hormonal inputs in transgenic Arabidopsis thaliana plants. These AND gates produce an output only in the presence of both abscisic acid and auxin but not when either or neither hormone is present. The AND logic gate can also integrate signals resulting from 2 plant stresses, cold temperature and bacterial infection, to produce a response. The design principles used here are generalizable, and, therefore, multiple orthogonal AND gates could be assembled and rationally layered to process complex genetic information in plants. These layered logic gates may be used in genetic circuits to probe fundamental questions in plant biology, such as hormonal crosstalk, in addition to plant engineering for bioproduction.

Integrative analysis of metabolite and transcriptome reveals biosynthetic pathway and candidate genes for eupatilin and jaceosidin biosynthesis in Artemisia argyi.

Artemisia argyi (A. argyi) is a medicinal plant belonging to the Asteraceae family and Artemisia genus. Flavonoids abundant in A. argyi are associated with anti-inflammatory, anticancer, and antioxidative effects. Eupatilin and jaceosidin are representative polymethoxy flavonoids with medicinal properties significant enough to warrant the development of drugs using their components. However, the biosynthetic pathways and related genes of these compounds have not been fully explored in A. argyi. This study comprehensively analyzed the transcriptome data and flavonoids contents from four different tissues of A. argyi (young leaves, old leaves, trichomes collected from stems, and stems without trichomes) for the first time. We obtained 41,398 unigenes through the de-novo assembly of transcriptome data and mined promising candidate genes involved in the biosynthesis of eupatilin and jaceosidin using differentially expressed genes, hierarchical clustering, phylogenetic tree, and weighted gene co-expression analysis. Our analysis led to the identification of a total of 7,265 DEGs, among which 153 genes were annotated as flavonoid-related genes. In particular, we were able to identify eight putative flavone-6-hydroxylase (F6H) genes, which were responsible for providing a methyl group acceptor into flavone basic skeleton. Furthermore, five O-methyltransferases (OMTs) gene were identified, which were required for the site-specific O-methylation during the biosynthesis of eupatilin and jaceosidin. Although further validation would be necessary, our findings pave the way for the modification and mass-production of pharmacologically important polymethoxy flavonoids through genetic engineering and synthetic biological approaches.