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12 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Plant terpene specialized metabolism: complex networks or simple linear pathways?

From the perspectives of pathway evolution, discovery and engineering of plant specialized metabolism, the nature of the biosynthetic routes represents a critical aspect. Classical models depict biosynthesis typically from an end-point angle and as linear, for example, connecting central and specialized metabolism. As the number of functionally elucidated routes increased, the enzymatic foundation of complex plant chemistries became increasingly well understood. The perception of linear pathway models has been severely challenged. With a focus on plant terpenoid specialized metabolism, we review here illustrative examples supporting that plants have evolved complex networks driving chemical diversification. The completion of several diterpene, sesquiterpene and monoterpene routes shows complex formation of scaffolds and their subsequent functionalization. These networks show that branch points, including multiple sub-routes, mean that metabolic grids are the rule rather than the exception. This concept presents significant implications for biotechnological production.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

The foxtail millet ( Setaria italica ) terpene synthase gene family

Terpenoid metabolism plays vital roles in stress defense and the environmental adaptation of monocot crops. Here, we describe the identification of the terpene synthase (TPS) gene family of the panicoid food and bioenergy model crop foxtail millet (Setaria italica). The diploid S. italica genome contains 32 TPS genes, 17 of which were biochemically characterized in this study. Unlike other thus far investigated grasses, S. italica contains TPSs producing all three ent-, (+)- and syn-copalyl pyrophosphate stereoisomers that naturally occur as central building blocks in the biosynthesis of distinct monocot diterpenoids. Conversion of these intermediates by the promiscuous TPS SiTPS8 yielded different diterpenoid scaffolds. Additionally, a cytochrome P450 monooxygenase (CYP99A17), which genomically clustered with SiTPS8, catalyzes the C19 hydroxylation of SiTPS8 products to generate the corresponding diterpene alcohols. The presence of syntenic orthologs to about 19% of the S. italica TPSs in related grasses supports a common ancestry of selected pathway branches. Among the identified enzyme products, abietadien-19-ol, syn-pimara-7,15-dien-19-ol and germacrene-D-4-ol were detectable in planta, and gene expression analysis of the biosynthetic TPSs showed distinct and, albeit moderately, inducible expression patterns in response to biotic and abiotic stress. In vitro growth-inhibiting activity of abietadien-19-ol and syn-pimara-7,15-dien-19-ol against Fusarium verticillioides and Fusarium subglutinans may indicate pathogen defensive functions, whereas the low antifungal efficacy of tested sesquiterpenoids supports other bioactivities. Together, these findings expand the known chemical space of monocot terpenoid metabolism to enable further investigations of terpenoid-mediated stress resilience in these agriculturally important species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Improved Biofuel Production through Discovery and Engineering of Terpene Metabolism in Switchgrass

Project Objectives - Of the myriad specialized metabolites that plants deploy to adapt to environmental challenges, terpenes form the largest group. In many major crops, unique terpene blends serve as key stress defenses that directly impact plant fitness and yield. In addition, terpenes, such as bisabolene and pinene, are used for producing renewable biofuels. Essential to advancing a broader use of terpenes for biofuel feedstock engineering is a system-wide knowledge of the diverse biosynthetic machinery and defensive potential of often species-specific terpene blends. The proposed project would merge genome-wide enzyme discovery with comparative –omics, protein structural and plant microbiome studies to define the biosynthesis and stress-defensive functions of the switchgrass (Panicum virgatum) terpene network. These insights would be combined with developing and applying non-transgenic genome editing tools to design plants with desirable terpene blends for higher productivity and biofuel production on marginal lands. As a dedicated lignocellulosic feedstock for U.S. biofuel production with high net energy yield, stress tolerance, and available genome resources, switchgrass is well-suited for devising new avenues for biofuel production. Project Description – The diversity of plant terpene defenses is governed by species-specific families of terpene synthase (TPS) and cytochrome P450 monooxygenase (P450) enzymes. Mining of the switchgrass genome (genotype Alamo) identified ~100 TPS and P450 candidate genes, and combinatorial biochemical analysis of synthesized TPSs and P450s revealed more than a dozen enzymes with common and novel activities. In addition, several identified terpene metabolites and the corresponding transcripts were up-regulated in response to abiotic stressors. These findings demonstrate a unique switchgrass terpene network with probable importance to abiotic stress tolerance, thus providing a large chemical portfolio for optimizing crop resistance, yield, and biofuel composition. Leveraging these preliminary data, we propose to generate a genome-wide map of the switchgrass terpene metabolic network through multi-gene co-expression analyses that allow the efficient cross-validation of TPS and P450 functions. Key enzymes would further be applied to structure-function studies via X-ray protein crystallography, homology modeling and site-directed mutagenesis to gain mechanistic insight into the catalytic specificity of switchgrass terpene metabolism and provide gene and amino acid targets for genome editing. In tandem with terpene pathway discovery, system-wide metabolomics, transcriptomics and proteomics studies in switchgrass accessions of contrasting drought tolerance would define the role of switchgrass terpene metabolism in conferring abiotic stress resilience. Metabolic changes would be assessed in a combined approach of targeted (terpenes) and untargeted metabolite profiling using a high-resolution LC-MS/MS approach, differential gene expression analyses through multiplexed Illumina RNA sequencing, and quantitative analysis of high-priority pathway enzymes using multiple reaction monitoring (MRM). Drawing on these insights, knock-down/out mutants of stress-associated pathway nodes would be generated by optimizing transient virus-induced gene silencing (VIGS) and CRISPR/Cas9 systems under control of the Tobacco Rattle Virus (TRV). The resulting mutant lines would then be analyzed for stress susceptibility and the impact on the root microbiome to define gene functions in planta. Knowledge of terpene pathways, enzyme mechanisms and bioactivities would be applied to enhance switchgrass stress resilience and to tailor-make terpene blends for biofuel production. Here, TRV-enabled CRISPR/Cas9 genome editing, including allele-specific knock-out of redundant genes, engineering of enzyme specificity via structure-guided point mutations, and overexpression of terpene genes relevant to stress-protection or biofuel production, would be used to increase metabolic flux toward desired pathways. Broader Impacts - Integrating the system-wide discovery, mechanistic analysis and non-transgenic genome engineering of the switchgrass terpene network aligns the required steps to unlock the chemical potential of this important metabolite class to generate crops that are more resistant to stress and provide advanced biofuel production in light of rising climate pressures as foreseeable challenges for bioenergy crop cultivation. The proposed project would further offer interdisciplinary student training through active involvement in the project and integration of research concepts and outcomes into newly-developed graduate and undergraduate courses on Plant Biotechnology.

09 BIOMASS FUELS↗
DOE OSTI2025

A multifunctional sesquiterpene synthase integrates with cytochrome P450s to reinforce the terpenoid defense network in maize

Terpenoids, the largest and most structurally diverse class of plant natural products, play essential roles in maize defense and ecological interactions. In this study, we identified and functionally characterized a sesquiterpenoid-based defense pathway in maize centered on α-santalenoic acid, a pathogen-inducible sesquiterpenoid antibiotic. Using a combination of metabolite-based genome-wide association studies (mGWAS), linkage mapping, and heterologous expression assays, we identified ZmTPS9 as a multiproduct terpene synthase that primarily produces α-santalene and β-bisabolene. Sequence analysis and site-directed mutagenesis revealed that threonine at position 413 is critical for enzyme activity, with its deletion resulting in a complete loss of enzyme activity. The sesquiterpene hydrocarbons produced by ZmTPS9 are further oxidized by three cytochrome P450 monooxygenases, ZmCYP71Z16, ZmCYP71Z18, and ZmCYP71Z19, to yield antimicrobial metabolites including α-santalenoic acid, zealexin D1 (ZD1), and zealexin D2 (ZD2). Together, these findings demonstrate a convergent biosynthetic strategy in maize, where multiproduct terpene synthases and promiscuous P450s collaboratively generate a flexible and robust terpenoid defense network.

a-santalenoic acid↗
DOE OSTI2023

Maize Terpene Synthase 8 (ZmTPS8) Contributes to a Complex Blend of Fungal-Elicited Antibiotics

In maize (Zea mays), fungal-elicited immune responses include the accumulation of terpene synthase (TPS) and cytochrome P450 monooxygenases (CYP) enzymes resulting in complex antibiotic arrays of sesquiterpenoids and diterpenoids, including α/β-selinene derivatives, zealexins, kauralexins and dolabralexins. To uncover additional antibiotic families, we conducted metabolic profiling of elicited stem tissues in mapping populations, which included B73 × M162W recombinant inbred lines and the Goodman diversity panel. Five candidate sesquiterpenoids associated with a chromosome 1 locus spanning the location of ZmTPS27 and ZmTPS8. Heterologous enzyme co-expression studies of ZmTPS27 in Nicotiana benthamiana resulted in geraniol production while ZmTPS8 yielded α-copaene, δ-cadinene and sesquiterpene alcohols consistent with epi-cubebol, cubebol, copan-3-ol and copaborneol matching the association mapping efforts. ZmTPS8 is an established multiproduct α-copaene synthase; however, ZmTPS8-derived sesquiterpene alcohols are rarely encountered in maize tissues. A genome wide association study further linked an unknown sesquiterpene acid to ZmTPS8 and combined ZmTPS8-ZmCYP71Z19 heterologous enzyme co-expression studies yielded the same product. To consider defensive roles for ZmTPS8, in vitro bioassays with cubebol demonstrated significant antifungal activity against both Fusarium graminearum and Aspergillus parasiticus. As a genetically variable biochemical trait, ZmTPS8 contributes to the cocktail of terpenoid antibiotics present following complex interactions between wounding and fungal elicitation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Terpenes modulate bacterial and fungal growth and sorghum rhizobiome communities

ABSTRACT Terpenes are among the oldest and largest class of plant-specialized bioproducts that are known to affect plant development, adaptation, and biological interactions. While their biosynthesis, evolution, and function in aboveground interactions with insects and individual microbial species are well studied, how different terpenes impact plant microbiomes belowground is much less understood. Here we designed an experiment to assess how belowground exogenous applications of monoterpenes (1,8-cineole and linalool) and a sesquiterpene (nerolidol) delivered through an artificial root system impacted its belowground bacterial and fungal microbiome. We found that the terpene applications had significant and variable impacts on bacterial and fungal communities, depending on terpene class and concentration; however, these impacts were localized to the artificial root system and the fungal rhizosphere. We complemented this experiment with pure culture bioassays on responsive bacteria and fungi isolated from the sorghum rhizobiome. Overall, higher concentrations (200 µM) of nerolidol were inhibitory to Ferrovibrium and tested Firmicutes. While fungal isolates of Penicillium and Periconia were also more inhibited by higher concentrations (200 µM) of nerolidol, Clonostachys was enhanced at this higher level and together with Humicola was inhibited by the lower concentration tested (100 µM). On the other hand, 1,8-cineole had an inhibitory effect on Orbilia at both tested concentrations but had a promotive effect at 100 µM on Penicillium and Periconia . Similarly, linalool at 100 µM had significant growth promotion in Mortierella , but an inhibitory effect for Orbilia . Together, these results highlight the variable direct effects of terpenes on single microbial isolates and demonstrate the complexity of microbe-terpene interactions in the rhizobiome. Importance Terpenes represent one of the largest and oldest classes of plant-specialized metabolism, but their role in the belowground microbiome is poorly understood. Here, we used a “rhizobox” mesocosm experimental set-up to supply different concentrations and classes of terpenes into the soil compartment with growing sorghum for 1 month to assess how these terpenes affect sorghum bacterial and fungal rhizobiome communities. Changes in bacterial and fungal communities between treatments belowground were characterized, followed by bioassays screening on bacterial and fungal isolates from the sorghum rhizosphere against terpenes to validate direct microbial responses. We found that microbial growth stimulatory and inhibitory effects were localized, terpene specific, dose dependent, and transient in time. This work paves the way for engineering terpene metabolisms in plant microbiomes for improved sustainable agriculture and bioenergy crop production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Genome-scale modelling of the primary-specialized metabolism interface

Environmental challenges and development require plants to reallocate resources between primary and specialized metabolites to survive. Genome-scale metabolic models, which map carbon flux through metabolic pathways, are a valuable tool in the study of tradeoffs that arise at this interface. Due to annotation gaps, models that characterize all the enzymatic steps in individual specialized pathways and their linkages to each other and to central carbon metabolism are difficult to construct. Recent studies have successfully curated subsystems of specialized metabolism and characterized the interfaces where flux is diverted to the precursors of glucosinolates, terpenes, and anthocyanins. Although advances in metabolite profiling can help to constrain models at this interface, quantitative analysis remains challenging because of the different timescales on which specialized metabolites from constitutive and reactive pathways accumulate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Hyaloscypha finlandica Metabolome Repository

This repository provides the curated data tables, manuscript figure and table exports, dependency records, and workflow scripts supporting an integrated comparative genomics and untargeted LC-MS/MS metabolomics analysis of Hyaloscypha finlandica strain PMI 746, a root-associated dark septate endophyte of poplar. The repository includes genome-mining summaries from antiSMASH, FunBGCeX, BGC-Prophet, and BiG-SCAPE; processed metabolomics inputs; metabolite annotation evidence; statistical outputs; and publication-facing figures and tables. Raw LC-MS/MS spectra, full genome/protein downloads, and large generated tool outputs are referenced through public archive/accession records and are not stored in Git.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

ShadeLab/PAPER_Howe_2023_switchgrass_MetaT

The raw data (metagenomes and metatranscriptomes) for this study are available in the Joint Genomes Institute Genome Portal (https://genome.jgi.doe.gov/portal/ Project ID 503249) with projects designated by year and product type. The MAG genomes analyzed in this paper are available on NCBI, as bioproject PRJNA800073. Plants and microorganisms form beneficial associations. Understanding plant-microbe interactions will inform microbiome management to enhance crop productivity and resilience to stress. Here, we apply a genome-centric approach to identify ecologically important leaf microbiome members on field-grown switchgrass and miscanthus and to quantify their activities for switchgrass over two growing seasons. We integrate metagenome and metatranscriptome sequencing from 192 leaf samples collected over representative time points in crop phenology. We curated 40 medium- and high-quality metagenome-assembled-genomes (MAGs) and focused analysis on seasonal transcript recruitment to them. Classes represented by these focal MAGs (Actinomycetia, Alpha- and Gamma- Proteobacteria, and Bacteroidota) were active and had increases in transcripts for short-chain dehydrogenase, molybdopterin oxidoreductase, and polyketide cyclase in the late season. The majority of MAGs had activated stress-associated pathways, including trehalose metabolism, indole acetic acid degradation, betaine biosynthesis, and reactive oxygen species degradation, suggesting direct engagement with the host environment. We also detected seasonally activated biosynthetic pathways for terpenes (carotenoids and isoprenoids) and for various non-ribosomal peptide pathways that were poorly annotated. Overall, this study overcame laboratory and bioinformatic challenges associated with field-based leaf metatranscriptome analysis to inform both general and likely specialized activities of these phyllosphere populations. These activities collectively support that leaf-associated bacterial populations are seasonally dynamic, responsive to host cues, and interactively engage in feedback with the plant. This analysis represented quality filtering of metagenomes and metatranscriptomes (data-preparation folder), metagenome assemblies (metagenome-assembly folder) and metagenome-assembled genome binning, curation, refinement, annotation (mag-evaluation folder). Abundances of sequencing libraries were calculated based on reads mapped (mapping folder). Additionallly, analysis of our annotated results are also included (analysis folder).

Howe, Adina↗
DOE OSTI2023

Uncovering a miltiradiene biosynthetic gene cluster in the Lamiaceae reveals a dynamic evolutionary trajectory

The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris, Plectranthus barbatus, and Leonotis leonurus. We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Engineering Terpene Production Pathways in Methylobacterium extorquens AM1

Terpenes are diverse specialized metabolites naturally found within plants and have important roles in inter-species communication, adaptation and interaction with the environment. Their industrial applications span a broad range, including fragrances, flavors, cosmetics, natural colorants to agrochemicals and therapeutics, yet formal chemical synthesis is economically challenging due to structural complexities. Engineering terpene biosynthesis could represent an alternative in microbial biotechnological workhorses, such as Saccharomyces cerevisiae or Escherichi coli, utilizing sugars or complex media as feedstocks. Host species that metabolize renewable and affordable carbon sources may offer unique sustainable biotechnological alternatives. Methylotrophs are bacteria with the capacity to utilize one-carbon feedstocks, such as methanol or formate. They colonize the phyllosphere (above-ground area) of plants, and many accumulate abundant carotenoid pigments. Methylotrophs have the capacity to take up and use a subset of the rare earth elements known as lanthanides. These metals can enhance one-carbon (methylotrophic) metabolism. Here, we investigated whether manipulating the metabolism enables and enhances terpene production. A carotenoid-deficient mutant potentially liberates carbon, which may contribute to bioproduct accumulation. To test this hypothesis, terpene-producing bacterial strains regulated by two distinct promoters were generated. Wildtype Methylobacterium extorquens, ∆Meta1_3665, a methylotrophic mutant lacking the carotenoid pathway, and an E. coli strain were transformed with an exogenous terpene pathway and grown both in the presence and absence of lanthanides. The extraction, and the comparison of analytical profiles, provided evidence that engineered cultured M. extorquens under control of a native, inducible methylotrophic promoter can yield the sesquiterpene patchoulol when supplemented with lanthanide. In contrast, using a moderate-strength constitutive promoter failed to give production. We demonstrated colonization of the phyllosphere with the engineered strains, supporting the future engineering of selected species of the plant microbiome and with promising implications for the synthetic biology of small molecules.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Imaging plant metabolism in situ

Abstract Mass spectrometry imaging (MSI) has emerged as an invaluable analytical technique for investigating the spatial distribution of molecules within biological systems. In the realm of plant science, MSI is increasingly employed to explore metabolic processes across a wide array of plant tissues, including those in leaves, fruits, stems, roots, and seeds, spanning various plant systems such as model species, staple and energy crops, and medicinal plants. By generating spatial maps of metabolites, MSI has elucidated the distribution patterns of diverse metabolites and phytochemicals, encompassing lipids, carbohydrates, amino acids, organic acids, phenolics, terpenes, alkaloids, vitamins, pigments, and others, thereby providing insights into their metabolic pathways and functional roles. In this review, we present recent MSI studies that demonstrate the advances made in visualizing the plant spatial metabolome. Moreover, we emphasize the technical progress that enhances the identification and interpretation of spatial metabolite maps. Within a mere decade since the inception of plant MSI studies, this robust technology is poised to continue as a vital tool for tackling complex challenges in plant metabolism.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗