Synthetic biology, an interdisciplinary field at the intersection of engineering and biology, has garnered considerable attention for its potential applications in plant science. By exploiting engineering principles, synthetic biology enables the redesign and construction of biological systems to manipulate plant traits, metabolic pathways, and responses to environmental stressors. This review explores the evolution and current state of synthetic biology in plants, highlighting key achievements and emerging trends. Synthetic biology offers innovative solutions to longstanding challenges in agriculture and biotechnology for improvement of nutrition and photosynthetic efficiency, useful secondary metabolite production, engineering biosensors, and conferring stress tolerance. Recent advances, such as genome editing technologies, have facilitated precise manipulation of plant genomes, creating new possibilities for crop improvement and sustainable agriculture. Despite its transformative potential, ethical and biosafety considerations underscore the need for responsible deployment of synthetic biology tools in plant research and development. This review provides insights into the burgeoning field of plant synthetic biology, offering a glimpse into its future implications for food security, environmental sustainability, and human health.

Microbial production of valuable plant metabolites is feasible. However, constructing all pathways in a single cell is a formidable challenge, and the extended biosynthetic pathways within cells often result in reduced productivity. To address these challenges, a co-culture system that divides biosynthetic pathways into several host cells and co-cultures has been developed. Various combinations of host cells, along with the optimal conditions for each co-culture, have been documented, leading to the successful production of valuable metabolites. In addition, efficient biosynthesis frequently involves metabolite movement, encompassing substrate uptake, intracellular intermediate transport, and end-product efflux. Recent advances in plant transporters of specialized metabolites have enhanced productivity by harnessing these transporters. This review summarizes the latest findings on co-culture systems and transport engineering and provides insights into the future of valuable metabolite production through the integration of co-culture and transport engineering.

Hydrolyzable tannins (HTs) are a class of polyphenols produced mostly in core eudicot plants. They accumulate in various plant tissues and are considered to function as defense compounds that protect against herbivory, infections, and toxic metals (specifically aluminum ions). Moreover, HTs have industrial and pharmaceutical uses that benefit humans. Elucidating and reconstituting the biosynthesis of HTs is necessary for genetically engineering in planta functions and for efficiently producing HTs for human use. The biosynthesis of HTs is initiated by the formation of gallic acid from the shikimate pathway intermediate 3-dehydroshikimic acid, which is catalyzed by bifunctional dehydroquinate dehydratase/shikimate dehydrogenases (DQD/SDHs). In the second step, UDP glycosyltransferases (UGTs) esterify gallic acid with glucose to form β-glucogallin (1-O-galloyl-β-D-glucose). β-glucogallin is then converted to 1,2,3,4,6-penta-O-galloyl-β-D-glucose through a series of galloylation steps that are catalyzed by galloyltransferases, using β-glucogallin as a galloyl donor. Laccases subsequently catalyze the oxidative coupling between adjacent galloyl groups to form hexahydroxydiphenoyl (HHDP) groups, which are characteristic components of ellagitannins. Furthermore, monomeric ellagitannins can undergo oligomerization via intermolecular oxidative coupling, which is also catalyzed by laccases. To reconstitute the HT biosynthetic pathway in HT-non-accumulating plants, DQD/SDHs and UGTs from Eucalyptus camaldulensis were heterologously co-expressed in Nicotiana benthamiana leaves, which resulted in the production of gallic acid and β-glucogallin. In future studies, this transgenic system will be used to identify genes encoding galloyltransferases and laccases to further elucidate and reconstitute the HT biosynthetic pathway.

Plant biomass is an abundant, renewable resource that offers multiple advantages for the production of green chemicals and recombinant proteins. However, the adoption of plant-based systems by industry is hindered because mammalian and other cell cultures are well-established and better characterized in an industrial setting, and thus it is difficult for plant-based processes to gain a foothold in the marketplace. Therefore, additional benefits of plant-based systems may be essential to tip the balance in favor of sustainable plant-derived products. A crucial factor in biomass valorization is to design mid- to high-value co-products that can be derived cost-effectively from the residual lignocellulose (LC). However, the utility of LC remains limited because LCs are, in general, too recalcitrant for industries to utilize their components (lignin, cellulose, and hemicelluloses). To overcome this issue, in planta engineering to reduce LC recalcitrance has been ongoing in recent decades, with essential input from synthetic biology owing to the complexity of LC pathways and the massive number of genes involved. In this review, we describe recent advances in LC manipulation and eight strategies for redesigning the pathways for lignin and structural glycans to reduce LC recalcitrance while mitigating against the growth penalty associated with yield loss.

Phenylethanoid glycosides (PhGs), with a C₆-C₂ glucoside unit as the basic skeleton, are specialized (secondary) metabolites found in several medicinal plants. As PhGs exhibit various pharmacological activities, they are expected to be used as lead compounds in drug discovery. However, mass-production systems have not yet been established even for acteoside, a typical PhG that is widely distributed in nature (more than 150 species). This review focuses on recent studies on the accumulation and distribution of PhGs in plants, biosynthetic pathways of PhGs, and the bioproduction of PhGs.

Plant-made pharmaceuticals (PMP) have great potential in terms of production costs, scalability, safety, environmental protection, and consumer acceptability. The first PMP were antibodies and antigens produced in stably transformed transgenic plants in the around 90s. Even though the effort using stable transgenic plants is still going on, the mainstream of PMP production has shifted to transient expression in Nicotiana benthamiana. This system involves the expression vectors by Agrobacterium, and its efficiency has been improved by the development of new vector systems and host engineering. The COVID-19 outbreak accelerated this trend through efforts to produce vaccines in plants. Transient expression systems have been improved and diversified by the development of plant virus vectors, which can be classified as full and deconstructed vectors. Full virus vectors spread systemically, allowing for protein production in the entire plant. Compared with conventional agroinfiltration vectors, excellent virus vectors result in higher protein production. Engineering of host plants has included knocking out gene-silencing systems to increase protein production, and the introduction of glycan modification enzymes so that plant-made proteins more resemble animal-made proteins. Hydroponic cultivation systems in plant factories and environmental controls have contributed to efficient protein production in plants. Considering their advantages and small environmental impact, PMP should be more widely adopted for pharmaceuticals’ production. However, the initial investment and running costs of plant factories are higher than open filed cultivation. The next objectives are to develop next-generation low-cost plant factories that use renewable energy and recycle materials based on the idea of circular economy.

To efficiently produce useful compounds using biological cells, it is essential to optimally design all metabolic reactions and pathways, including not only the flow of carbon within the cell but also the production and consumption of energy and the balance of oxidation-reduction. Computational scientific methods are effective for the rational design of metabolic pathways and the optimization of metabolic fluxes. Based on this blueprint, it is crucial to accurately construct the cell, test and analyze whether it conforms to the design, and learn from the results to redesign the system in an effective cycle. This review introduces essential metabolic design techniques in synthetic biology and discusses the potential of using plant cells or plant genes effectively in synthetic biology for the production of useful compounds.

Structurally diverse benzylisoquinoline alkaloids (BIAs) are found in specific plant families, some of which are desirable for their efficient production because of their strong biological activities. Corydalis plants (e.g., Corydalis yanhusuo) of the family Papaveraceae also produce various BIAs; thus, they have been used in traditional Chinese medicine. Because metabolic engineering and synthetic biology using microorganisms are promising technologies for the effective production of useful metabolites, elucidation of the biosynthetic pathway of each BIA is indispensable. Although several enzyme genes involved in the biosynthesis of Corydalis BIAs have recently been isolated, many remain unknown, such as the protoberberine alkaloid C-methyltransferase involved in the biosynthesis of corydaline, one of the main BIAs found in the tubers of Corydalis plants. In this study, we performed transcriptome analysis combined with metabolite profiling of different tissues of Corydalis solida. Based on the high accumulation of several BIAs, including protopine, allocryptopine, and corydaline, genes encoding putative biosynthetic enzymes, including cytochrome P450, methyltransferase, and oxidase proteins, that were highly expressed in the tubers were screened. Two OMT genes, CsOMT1 and CsOMT2, were highly expressed in the tuber, and further characterization using crude enzyme preparations demonstrated that CsOMT1 showed 7-O-methylation activity against reticuline, whereas CsOMT2 catalyzed 9-O-methylation of scoulerine, followed by 2-O-methylation of tetrahydrocolumbamine. Our findings provide valuable information for the isolation of novel biosynthetic enzyme genes in Corydalis species.

Triterpenoids, a group of specialized plant metabolites with substantial structural diversity, are promising for healthcare applications. Ursolic acid, a pentacyclic triterpenoid with therapeutic potential, is also important as a precursor of corosolic acid, which is known as a “phyto-insulin” for its insulin-like properties. Ursolic acid is synthesized from a linear 30-carbon precursor 2,3-oxidosqualene via cyclization to produce triterpene scaffold α-amyrin, followed by a series of oxidation steps at the C-28 position mediated by cytochrome P450 monooxygenases (CYPs) in the CYP716A subfamily. The Tsukuba system was developed for the high-level transient expression of foreign proteins in plant cells based on the use of a binary vector equipped with geminiviral replication system and a double terminator. In this study, we used the Tsukuba system to produce ursolic acid in Nicotiana benthamiana leaves via transient pathway reconstruction. We used an oxidosqualene cyclase identified from the medicinal legume Bauhinia forficata, exhibiting a preponderant α-amyrin-producing activity. Wild-type Medicago truncatula CYP716A12 and its mutants were assessed in terms of ursolic acid production. We improved the performance of MtCYP716A12 by co-expressing it with the appropriate cytochrome P450 reductase (CPR) isozyme as an electron-transfer partner and tested different Agrobacterium infiltration ratios to optimize the CPR : CYP ratio to maximize ursolic acid production. We also achieved high yield of corosolic acid by co-expressing Avicennia marina CYP716C53 with ursolic acid biosynthetic enzymes. Moreover, engineering of AmCYP716C53 significantly improved corosolic acid yield, resulting in a yield exceeding the content found in banaba leaves, a well-known rich source of corosolic acid.

Potatoes produce steroidal glycoalkaloids (SGAs), toxic secondary metabolites associated with food poisoning. SGAs are synthesized by multiple biosynthetic enzymes. Knockdown of the CYP88B1 gene, also known as PGA3 or GAME4, is predicted to reduce toxic SGAs and accumulate steroidal saponins. These saponins not only serve as a source of steroidal drugs but are also anticipated to confer disease resistance to potatoes. In this study, we employed transcription activator-like effector nucleases (TALENs) for genome editing to disrupt CYP88B1. We introduced the TALEN expression vector via Agrobacterium-mediated transformation into seven potato lines. In six of these lines, disruption of the CYP88B1 gene was confirmed. Liquid chromatography-mass spectrometry analysis revealed that SGAs were reduced to undetectable levels, corroborating the accumulation of steroidal saponins observed in previous knockdown studies. Our findings demonstrate the feasibility of generating low-toxicity potato lines through CYP88B1 gene disruption using genome editing techniques.

Sweet potatoes (Ipomoea batatas) produce glucose esters from cinnamates during stress responses. We isolated a glucosyltransferase, IbGT1 (UGT84A20), from sweet potato and characterized its recombinant enzyme heterologously expressed in Escherichia coli. The recombinant IbGT1 enzyme reacted with cinnamates, especially p-coumaric acid, ferulic acid, and sinapic acid, and showed activity against benzoates with an optimum pH of 5.0–5.5. The enzyme also reacted with flavonoids under alkaline conditions (pH 8.0), with the main product being 7-O-glucosides. Bioconversion using E. coli expressing IbGT1 (Ec-IbGT1) as a whole cell biocatalyst converted 200 µM cinnamates such as p-coumaric acid and benzoates such as p-hydroxybenzoic acid into their glucose esters mostly within 3–6 h. The conversion rates for caffeic acid and sinapic acid were much lower than those for the other substrates tested, which took more than 6 h, despite the enzymatic activities of IbGT1 against these compounds being comparable to the others. Scaled-up production combined with repeated administration of sinapic acid, trans-cinnamic acid and p-hydroxybenzoic acid at a concentration of 200 µM yielded their glucose esters with conversion rates of 74–95% in 20–30 h. These results suggest that the Ec-IbGT1 system efficiently produces these glucose esters.

Plants produce structurally diverse triterpenes (triterpenoids and steroids). Their biosynthesis occurs from a common precursor, namely 2,3-oxidosqualene, followed by cyclization catalyzed by oxidosqualene cyclases (OSCs) to yield various triterpene skeletons. Steroids, which are biosynthesized from cycloartenol or lanosterol, are essential primary metabolites in most plant species, along with lineage-specific steroids, such as steroidal glycoalkaloids found in the Solanum species. Other diverse triterpene skeletons are converted into triterpenoids, often classified as specialized compounds that are biosynthesized only in a limited number of plant species with tissue- or cell-type-specific accumulation in plants. Recent studies have identified various tailoring enzymes involved in the structural diversification of triterpenes as well as transcription factors that regulate the expression of these enzymes. However, the coverage of these proteins is scarce in publicly available databases for curated proteins or enzymes, which complicates the functional annotation of newly assembled genomes or transcriptome sequences. Here, we created the Triterpene RDF, a manually curated database of enzymes and transcription factors involved in plant triterpene biosynthesis. The database (https://github.com/ktamura2021/triterpene_rdf/) contains 532 proteins, with links to the UniProt Knowledgebase or NCBI protein database, and it enables direct download of a set of protein sequences filtered by protein type or taxonomy. Triterpene RDF will enhance the functional annotation of enzymes and regulatory elements for triterpene biosynthesis, in a current expansion of availability of genomic information on various plant species.

Callus cultures are fundamental for plant propagation, genetic transformation, and emerging biotechnological applications that use cellular factories to produce high-value metabolites like plant-based drugs. These applications exploit the diverse metabolic capabilities of various plant species. However, optimizing culture conditions for specific applications necessitates a deep understanding of the transcriptome, metabolome, and phytohormone profiles of different species. Comprehensive comparative studies of callus characteristics across species are limited. Here, we analyzed the transcriptome, metabolome, and phytohormone profiles of callus cultures from tobacco (Nicotiana tabacum), rice (Oryza sativa), and two bamboo species (Phyllostachys nigra and P. bambusoides). Multivariate analyses of metabolome data revealed similar metabolic trends in these diverse callus cultures and identified metabolites that differ between species. Hormone profiling showed distinct species-specific patterns and notable cytokinin diversity, even between the bamboo species. Moreover, a comparative analysis of 8,256 pairs of syntenic genes between rice and bamboo revealed that 84.7% of these orthologs showed differential expression, indicating significant transcriptomic diversity despite phylogenomic relatedness. Transcriptional regulation of developing organs often involves conserved gene expression patterns across species; however, our findings suggest that callus formation may relax evolutionary constraints on these regulatory programs. These results illustrate the molecular diversity in callus cultures from multiple plant species, emphasizing the need to map this variability comprehensively to fully exploit the biotechnological potential of plant callus cultures.

Lithospermum erythrorhizon (Boraginaceae) produces shikonin/alkannin, an enantiomeric pair of red naphthoquinone pigments with diverse biological activities. For the industrial production of shikonin/alkannin derivatives, a cell suspension culture system of L. erythrorhizon has been established. To produce shikonin/alkannin derivatives more efficiently in cultured cells, it is essential to understand the shikonin/alkannin biosynthetic pathway, which has not been fully elucidated. A previous study suggested that a conversion of (Z)- to (E)-3″-hydroxygeranylhydroquinone (3″-OH-GHQ) is a branching point of the shikonin/alkannin biosynthetic pathway and the shikonofuran biosynthetic pathway in L. erythrorhizon cell cultures. However, it is not clear whether (E)-3″-OH-GHQ is an intermediate of both pathways. This study performed a feeding assay with three deuterium-labeled compounds including (E)-3″-OH-GHQ and its (Z)-isomer, and showed that (E)-3″-OH-GHQ was not involved in the shikonin/alkannin and shikonofuran biosynthetic pathways.

For the optimal production of specialized (secondary) metabolites in plant hosts, a comprehensive understanding of their regulatory mechanisms is imperative. Bioactive C-28-oxidized triterpenes, such as oleanolic, ursolic, and betulinic acids, are metabolites ubiquitously found across the plant kingdom; however the precise regulatory mechanisms governing their biosynthesis remain elusive. Previously, we demonstrated that the clade Ia bHLH transcription factor, LjbHLH50, plays a pivotal role in the upregulation of betulinic acid biosynthesis in Lotus japonicus. However, inconsistent outcomes have been observed in transient effector-reporter assays, which are commonly employed in transcription factor studies. Thus, in the present study, we sought to further characterize LjbHLH50 by examining the ectopic expression of BpbHLH9, a homolog of LjbHLH50 in Betula platyphylla, in L. japonicus hairy roots. Remarkably, BpbHLH9 expression elicited metabolic and transcriptomic alterations almost similar to those induced by LjbHLH50 overexpression, highlighting the conserved function of clade Ia bHLHs. Through RNA-sequencing analysis, we found that LjbHLH50 was upregulated by ectopic BpbHLH9 expression, implying the existence of a self-activating loop in clade Ia bHLHs that facilitates enhanced betulinic acid biosynthesis. Notably, among the clade Ia bHLHs homologous to BpbHLH9, LjbHLH50 and two LjbHLH50 paralogs were upregulated upon BpbHLH9 induction, underscoring the central role of these clade Ia bHLHs in betulinic acid biosynthesis regulatory networks in L. japonicus hairy roots.