Plant Metabolic Engineering Research Articles

Evolution of aromatic amino acid metabolism in plants: a key driving force behind plant chemical diversity in aromatic natural products.

A diverse array of plant aromatic compounds contributes to the tremendous chemical diversity in the plant kingdom that cannot be seen in microbes or animals. Such chemodiversity of aromatic natural products has emerged, occasionally in a lineage-specific manner, to adopt to challenging environmental niches, as various aromatic specialized metabolites play indispensable roles in plant development and stress responses (e.g. lignin, phytohormones, pigments and defence compounds). These aromatic natural products are synthesized from aromatic amino acids (AAAs), l-tyrosine, l-phenylalanine and l-tryptophan. While amino acid metabolism is generally assumed to be conserved between animals, microbes and plants, recent phylogenomic, biochemical and metabolomic studies have revealed the diversity of the AAA metabolism that supports efficient carbon allocation to downstream biosynthetic pathways of AAA-derived metabolites in plants. This review showcases the intra- and inter-kingdom diversification and origin of committed enzymes involved in plant AAA biosynthesis and catabolism and their potential application as genetic tools for plant metabolic engineering. I also discuss evolutionary trends in the diversification of plant AAA metabolism that expands the chemical diversity of AAA-derived aromatic natural products in plants. This article is part of the theme issue 'The evolution of plant metabolism'.

Evolutionary trajectory of transcription factors and selection of targets for metabolic engineering.

Transcription factors (TFs) provide potentially powerful tools for plant metabolic engineering as they often control multiple genes in a metabolic pathway. However, selecting the best TF for a particular pathway has been challenging, and the selection often relies significantly on phylogenetic relationships. Here, we offer examples where evolutionary relationships have facilitated the selection of the suitable TFs, alongside situations where such relationships are misleading from the perspective of metabolic engineering. We argue that the evolutionary trajectory of a particular TF might be a better indicator than protein sequence homology alone in helping decide the best targets for plant metabolic engineering efforts. This article is part of the theme issue 'The evolution of plant metabolism'.

Strategies, Achievements, and Potential Challenges of Plant and Microbial Chassis in the Biosynthesis of Plant Secondary Metabolites.

Diverse secondary metabolites in plants, with their rich biological activities, have long been important sources for human medicine, food additives, pesticides, etc. However, the large-scale cultivation of host plants consumes land resources and is susceptible to pest and disease problems. Additionally, the multi-step and demanding nature of chemical synthesis adds to production costs, limiting their widespread application. In vitro cultivation and the metabolic engineering of plants have significantly enhanced the synthesis of secondary metabolites with successful industrial production cases. As synthetic biology advances, more research is focusing on heterologous synthesis using microorganisms. This review provides a comprehensive comparison between these two chassis, evaluating their performance in the synthesis of various types of secondary metabolites from the perspectives of yield and strategies. It also discusses the challenges they face and offers insights into future efforts and directions.

Isotopically nonstationary metabolic flux analysis of plants: recent progress and future opportunities.

Metabolic flux analysis (MFA) is a valuable tool for quantifying cellular phenotypes and to guide plant metabolic engineering. By introducing stable isotopic tracers and employing mathematical models, MFA can quantify the rates of metabolic reactions through biochemical pathways. Recent applications of isotopically nonstationary MFA (INST-MFA) to plants have elucidated nonintuitive metabolism in leaves under optimal and stress conditions, described coupled fluxes for fast-growing algae, and produced a synergistic multi-organ flux map that is a first in MFA for any biological system. These insights could not be elucidated through other approaches and show the potential of INST-MFA to correct an oversimplified understanding of plant metabolism.

Plant-Produced Viral Nanoparticles as a Functionalized Catalytic Support for Metabolic Engineering.

Substrate channeling could be very useful for plant metabolic engineering; hence, we propose that functionalized supramolecular self-assembly scaffolds can act as enzymatic hubs able to perform reactions in close contiguity. Virus nanoparticles (VNPs) offer an opportunity in this context, and we present a functionalization strategy to display different enzymes on the outer surface of three different VNPs produced in plants. Tomato bushy stunt virus (TBSV) and Potato virus X (PVX) plant viruses were functionalized by the genetic fusion of the E-coil peptide coding sequence to their respective coat proteins genes, while the enzyme lichenase was tagged with the K-coil peptide. Immobilized E-coil VNPs were able to interact in vitro with the plant-produced functionalized lichenase, and catalysis was demonstrated by employing a lichenase assay. To prove this concept in planta, the Hepatitis B core (HBc) virus-like particles (VLPs) were similarly functionalized by genetic fusion with the E-coil sequence, while acyl-activating enzyme 1, olivetolic acid synthase, and olivetolic acid cyclase enzymes were tagged with the K-coil. The transient co-expression of the K-coil-enzymes together with E-coil-VLPs allowed the establishment of the heterologous cannabinoid precursor biosynthetic pathway. Noteworthy, a significantly higher yield of olivetolic acid glucoside was achieved when the scaffold E-coil-VLPs were employed.

Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

AI to enable plant cell metabolic engineering.

Plant metabolic engineering must take into consideration the heterogeneous cell types that play a role in metabolite production; cells do not participate equally. We posit that artificial intelligence (AI) developed for biomedical purposes can be applied to plant cell characterization to accelerate the development of metabolic engineering strategies in plants.

Brewing wellness: sugar-based synthesis of anti-obesity drug celastrol

Addressing the burgeoning global obesity crisis, Zhao et al. recently presented innovative developments in celastrol synthesis, a potent anti-obesity agent. They unified plant biochemistry, metabolic engineering, and chemistry to map an effective celastrol biosynthetic path in yeast. The study emphasizes new opportunities for the commercial production of celastrol, revealing future potential for specialized metabolite production.

Functional characterization of CYP81C16 involved in the tanshinone biosynthetic pathway in Salvia miltiorrhiza

Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.

Elucidating the impact of in vitro cultivation on Nicotiana tabacum metabolism through combined in silico modeling and multiomics analysis.

The systematical characterization and understanding of the metabolic behaviors are the basis of the efficient plant metabolic engineering and synthetic biology. Genome-scale metabolic networks (GSMNs) are indispensable tools for the comprehensive characterization of overall metabolic profile. Here we first constructed a GSMN of tobacco, which is one of the most widely used plant chassis, and then combined the tobacco GSMN and multiomics analysis to systematically elucidate the impact of in-vitro cultivation on the tobacco metabolic network. In-vitro cultivation is a widely used technique for plant cultivation, not only in the field of basic research but also for the rapid propagation of valuable horticultural and pharmaceutical plants. However, the systemic effects of in-vitro cultivation on overall plant metabolism could easily be overlooked and are still poorly understood. We found that in-vitro tobacco showed slower growth, less biomass and suppressed photosynthesis than soil-grown tobacco. Many changes of metabolites and metabolic pathways between in-vitro and soil-grown tobacco plants were identified, which notably revealed a significant increase of the amino acids content under in-vitro condition. The in silico investigation showed that in-vitro tobacco downregulated photosynthesis and primary carbon metabolism, while significantly upregulated the GS/GOGAT cycle, as well as producing more energy and less NADH/NADPH to acclimate in-vitro growth demands. Altogether, the combination of experimental and in silico analyses offers an unprecedented view of tobacco metabolism, with valuable insights into the impact of in-vitro cultivation, enabling more efficient utilization of in-vitro techniques for plant propagation and metabolic engineering.

Plastid engineering using episomal DNA.

Novel episomal systems have the potential to accelerate plastid genetic engineering for application in plant synthetic biology. Plastids represent valuable subcellular compartments for genetic engineering of plants with intrinsic advantages to engineering the nucleus. The ability to perform site-specific transgene integration by homologous recombination (HR), coordination of transgene expression in operons, and high production of heterologous proteins, all make plastids an attractive target for synthetic biology. Typically, plastid engineering is performed by homologous recombination; however, episomal-replicating vectors have the potential to accelerate the design/build/test cycles for plastid engineering. By accelerating the timeline from design to validation, it will be possible to generate translational breakthroughs in fields ranging from agriculture to biopharmaceuticals. Episomal-based plastid engineering will allow precise single step metabolic engineering in plants enabling the installation of complex synthetic circuits with the ambitious goal of reaching similar efficiency and flexibility of to the state-of-the-art genetic engineering of prokaryotic systems. The prospect to design novel episomal systems for production of transplastomic marker-free plants will also improve biosafety for eventual release in agriculture.

Structure-driven protein engineering for production of valuable natural products.

Proteins are the most frequently used biocatalysts, and their structures determine their functions. Modifying the functions of proteins on the basis of their structures lies at the heart of protein engineering, opening a new horizon for metabolic engineering by efficiently generating stable enzymes. Many attempts at classical metabolic engineering have focused on improving specific metabolic fluxes and producing more valuable natural products by increasing gene expression levels and enzyme concentrations. However, most naturally occurring enzymes show limitations, and such limitations have hindered practical applications. Here we review recent advances in protein engineering in synthetic biology, chemoenzymatic synthesis, and plant metabolic engineering and describe opportunities for designing and constructing novel enzymes or proteins with desirable properties to obtain more active natural products.

Flavonoid Production: Current Trends in Plant Metabolic Engineering and De Novo Microbial Production.

Flavonoids are secondary metabolites that represent a heterogeneous family of plant polyphenolic compounds. Recent research has determined that the health benefits of fruits and vegetables, as well as the therapeutic potential of medicinal plants, are based on the presence of various bioactive natural products, including a high proportion of flavonoids. With current trends in plant metabolite research, flavonoids have become the center of attention due to their significant bioactivity associated with anti-cancer, antioxidant, anti-inflammatory, and anti-microbial activities. However, the use of traditional approaches, widely associated with the production of flavonoids, including plant extraction and chemical synthesis, has not been able to establish a scalable route for large-scale production on an industrial level. The renovation of biosynthetic pathways in plants and industrially significant microbes using advanced genetic engineering tools offers substantial promise for the exploration and scalable production of flavonoids. Recently, the co-culture engineering approach has emerged to prevail over the constraints and limitations of the conventional monoculture approach by harnessing the power of two or more strains of engineered microbes to reconstruct the target biosynthetic pathway. In this review, current perspectives on the biosynthesis and metabolic engineering of flavonoids in plants have been summarized. Special emphasis is placed on the most recent developments in the microbial production of major classes of flavonoids. Finally, we describe the recent achievements in genetic engineering for the combinatorial biosynthesis of flavonoids by reconstructing synthesis pathways in microorganisms via a co-culture strategy to obtain high amounts of specific bioactive compounds.

Plant Metabolic Engineering by Multigene Stacking: Synthesis of Diverse Mogrosides.

Mogrosides are a group of health-promoting natural products that extracted from Siraitia grosvenorii fruit (Luo-han-guo or monk fruit), which exhibited a promising practical application in natural sweeteners and pharmaceutical development. However, the production of mogrosides is inadequate to meet the need worldwide, and uneconomical synthetic chemistry methods are not generally recommended for structural complexity. To address this issue, an in-fusion based gene stacking strategy (IGS) for multigene stacking has been developed to assemble 6 mogrosides synthase genes in pCAMBIA1300. Metabolic engineering of Nicotiana benthamiana and Arabidopsis thaliana to produce mogrosides from 2,3-oxidosqualene was carried out. Moreover, a validated HPLC-MS/MS method was used for the quantitative analysis of mogrosides in transgenic plants. Herein, engineered Arabidopsis thaliana produced siamenoside I ranging from 29.65 to 1036.96 ng/g FW, and the content of mogroside III at 202.75 ng/g FW, respectively. The production of mogroside III was from 148.30 to 252.73 ng/g FW, and mogroside II-E with concentration between 339.27 and 5663.55 ng/g FW in the engineered tobacco, respectively. This study provides information potentially applicable to develop a powerful and green toolkit for the production of mogrosides.

Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements.

While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into Nicotiana benthamiana leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.

Engineering Catharanthus roseus monoterpenoid indole alkaloid pathway in yeast.

Catharanthus roseus (Madagascar periwinkle), a medicinal plant possessing high pharmacological attributes, is widely recognized for the biosynthesis of anticancer monoterpenoid indole alkaloids (MIAs) - vinblastine and vincristine. The plant is known to biosynthesize more than 130 different bioactive MIAs, highly acclaimed in traditional and modern medicinal therapies. The MIA biosynthesis is strictly regulated at developmental and spatial-temporal stages and requires a well-defined cellular and sub-cellular compartmentation for completion of the entire MIAs biosynthesis. However, due to their cytotoxic nature, the production of vinblastine and vincristine occurs in low concentrations in planta and the absence of chemical synthesis alternatives projects a huge gap in demand and supply, leading to high market price. With research investigations spanning more than four decades, plant tissue culture and metabolic engineering (ME)-based studies were attempted to explore, understand, explain, improve and enhance the MIA biosynthesis using homologous and heterologous systems. Presently, metabolic engineering and synthetic biology are the two powerful tools that are contributing majorly in elucidating MIA biosynthesis. This review concentrates mainly on the efforts made through metabolic engineering of MIAs in heterologous microbial factories. KEY POINTS: • Yeast engineering provides alternative production source of phytomolecules • Yeast engineering also helps to discover missing plant pathway enzymes and genes.

Identification and Characterization of Two Regiospecific Tricetin UDP-Dependent Glycosyltransferases from Pomegranate (Punica granatum L.).

Tricetin (5,7,3′,4′,5′-pentahydroxyflavone) is a dietary flavone from flowers of Myrtales plants with demonstrated functions in promoting human health. By contrast, the bioactivity of its glucosylated derivative tricetin 4′-O-glucoside has not been extensively explored. We conducted metabolite profiling analysis of pomegranate (a Myrtales plant) floral tissues and revealed that tricetin and tricetin 4′-O-glucoside accumulate in anthers, but not petals. In addition, the comparative analysis of anther and petal transcriptomes identified 10 UGTs that are more highly expressed in anthers than petals. Of the 10 UGTs, PgUGT76Z1 and PgUGT73AL1 glucosylated specifically at the 4′-O position of tricetin to form tricetin 4′-O-glucoside. The phylogenetic analysis indicated that PgUGT76Z1 and PgUGT73AL1 belong to different plant UGT groups, suggesting a convergent evolution of these tricetin UGTs. Overall, identification and characterization of PgUGT76Z1 and PgUGT73AL1 not only provides evolutionary insights into tricetin glucosylation, but also offers an opportunity to produce tricetin 4′-O-glucoside in large quantities through microbial biotransformation or plant metabolic engineering, thus facilitating the investigation of tricetin 4′-O-glucoside bioactivities.

An Efficient Modular Gateway Recombinase-Based Gene Stacking System for Generating Multi-Trait Transgenic Plants.

Transgenic technology can transfer favorable traits regardless of reproductive isolation and is an important method in plant synthetic biology and genetic improvement. Complex metabolic pathway modification and pyramiding breeding strategies often require the introduction of multiple genes at once, but the current vector assembly systems for constructing multigene expression cassettes are not completely satisfactory. In this study, a new in vitro gene stacking system, GuanNan Stacking (GNS), was developed. Through the introduction of Type IIS restriction enzyme-mediated Golden Gate cloning, GNS allows the modular, standardized assembly of target gene expression cassettes. Because of the introduction of Gateway recombination, GNS facilitates the cloning of superlarge transgene expression cassettes, allows multiple expression cassettes to be efficiently assembled in a binary vector simultaneously, and is compatible with the Cre enzyme-mediated marker deletion mechanism. The linked dual positive-negative marker selection strategy ensures the efficient acquisition of target recombinant plasmids without prokaryotic selection markers in the T-DNA region. The host-independent negative selection marker combined with the TAC backbone ensures the cloning and transfer of large T-DNAs (>100 kb). Using the GNS system, we constructed a binary vector containing five foreign gene expression cassettes and obtained transgenic rice carrying the target traits, proving that the method developed in this research is a powerful tool for plant metabolic engineering and compound trait transgenic breeding.

The production of wax esters in transgenic plants: towards a sustainable source of bio-lubricants.

Wax esters are high-value compounds used as feedstocks for the production of lubricants, pharmaceuticals, and cosmetics. Currently, they are produced mostly from fossil reserves using chemical synthesis, but this cannot meet increasing demand and has a negative environmental impact. Natural wax esters are also obtained from Simmondsia chinensis (jojoba) but comparably in very low amounts and expensively. Therefore, metabolic engineering of plants, especially of the seed storage lipid metabolism of oil crops, represents an attractive strategy for renewable, sustainable, and environmentally friendly production of wax esters tailored to industrial applications. Utilization of wax ester-synthesizing enzymes with defined specificities and modulation of the acyl-CoA pools by various genetic engineering approaches can lead to obtaining wax esters with desired compositions and properties. However, obtaining high amounts of wax esters is still challenging due to their negative impact on seed germination and yield. In this review, we describe recent progress in establishing non-food-plant platforms for wax ester production and discuss their advantages and limitations as well as future prospects.

Anti-Inflammatory and Immunoregulatory Action of Sesquiterpene Lactones.

Sesquiterpene lactones (SL), characterized by their high prevalence in the Asteraceae family, are one of the major groups of secondary metabolites found in plants. Researchers from distinct research fields, including pharmacology, medicine, and agriculture, are interested in their biological potential. With new SL discovered in the last years, new biological activities have been tested, different action mechanisms (synergistic and/or antagonistic effects), as well as molecular structure–activity relationships described. The review identifies the main sesquiterpene lactones with interconnections between immune responses and anti-inflammatory actions, within different cellular models as well in in vivo studies. Bioaccessibility and bioavailability, as well as molecular structure–activity relationships are addressed. Additionally, plant metabolic engineering, and the impact of sesquiterpene lactone extraction methodologies are presented, with the perspective of biological activity enhancement. Sesquiterpene lactones derivatives are also addressed. This review summarizes the current knowledge regarding the therapeutic potential of sesquiterpene lactones within immune and inflammatory activities, highlighting trends and opportunities for their pharmaceutical/clinical use.