Plant Biotechnology最新文献

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.

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.

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.

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.

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-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.

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.

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.

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.

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.