Bernd Markus Lange

A systems biology approach identifies the biochemical mechanisms regulating monoterpenoid essential oil composition in peppermint

The integration of mathematical modeling and experimental testing is emerging as a powerful approach for improving our understanding of the regulation of metabolic pathways. In this study, we report on the development of a kinetic mathematical model that accurately simulates the developmental patterns of monoterpenoid essential oil accumulation in peppermint (Mentha × piperita). This model was then used to evaluate the biochemical processes underlying experimentally determined changes in the monoterpene pathway under low ambient-light intensities, which led to an accumulation of the branchpoint intermediate (+)-pulegone and the side product (+)-menthofuran. Our simulations indicated that the environmentally regulated changes in monoterpene profiles could only be explained when, in addition to effects on biosynthetic enzyme activities, as yet unidentified inhibitory effects of (+)-menthofuran on the branchpoint enzyme pulegone reductase (PR) were assumed. Subsequent in vitro analyses with recombinant protein confirmed that (+)-menthofuran acts as a weak competitive inhibitor of PR (Ki = 300 μM). To evaluate whether the intracellular concentration of (+)-menthofuran was high enough for PR inhibition in vivo, we isolated essential oil-synthesizing secretory cells from peppermint leaves and subjected them to steam distillations. When peppermint plants were grown under low-light conditions, (+)-menthofuran was selectively retained in secretory cells and accumulated to very high levels (up to 20 mM), whereas under regular growth conditions, (+)-menthofuran levels remained very low (<400 μM). These results illustrate the utility of iterative cycles of mathematical modeling and experimental testing to elucidate the mechanisms controlling flux through metabolic pathways.

Improving peppermint essential oil yield and composition by metabolic engineering

Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost.

Open-Access Metabolomics Databases for Natural Product Research: Present Capabilities and Future Potential

Various databases have been developed to aid in assigning structures to spectral peaks observed in metabolomics experiments. In this review article, we discuss the utility of currently available open-access spectral and chemical databases for natural products discovery. We also provide recommendations on how the research community can contribute to further improvements.

The Evolution of Plant Secretory Structures and Emergence of Terpenoid Chemical Diversity

Secretory structures in terrestrial plants appear to have first emerged as intracellular oil bodies in liverworts. In vascular plants, internal secretory structures, such as resin ducts and laticifers, are usually found in conjunction with vascular bundles, whereas subepidermal secretory cavities and epidermal glandular trichomes generally have more complex tissue distribution patterns. The primary function of plant secretory structures is related to defense responses, both constitutive and induced, against herbivores and pathogens. The ability to sequester secondary (or specialized) metabolites and defense proteins in secretory structures was a critical adaptation that shaped plant-herbivore and plant-pathogen interactions. Although this review places particular emphasis on describing the evolution of pathways leading to terpenoids, it also assesses the emergence of other metabolite classes to outline the metabolic capabilities of different plant lineages.

Biosynthesis and Biotechnology of High-Value p-Menthane Monoterpenes, Including Menthol, Carvone, and Limonene

Monoterpenes of the p-menthane group are volatile secondary (or specialized) metabolites found across the plant kingdom. They are dominant constituents of commercially important essential oils obtained from members of the genera Mentha (Lamiaceae), Carum (Apiaceae), Citrus (Rutaceae), and Eucalyptus (Myrtaceae). p-Menthane monoterpenes have also attracted interest as chiral specialty chemicals, and the harvest from natural sources is therefore supplemented by chemical synthesis. More recently, microbial and plant-based platforms for the high-level accumulation of specific target monoterpenes have been developed. In this review chapter, I discuss the properties of the genes and enzymes involved in p-menthane biosynthesis and provide a critical assessment of biotechnological production approaches.

Multiple levels of regulation determine monoterpenoid essential oil compositional variation in the mint family.

The genus Mentha has considerable economic importance as a source of essential oils for the flavor, fragrance, and aromatherapy industries (Lange and Ahkami, 2013). The history of commercial mint cultivars is complex and involves various polyploid hybrids. The most widely grown cultivars are essentially sterile, which has impeded the development of genetic resources. The work presented here aims to shed more light into the factors that determine oil composition, which is important for clonal selection strategies.

Patterns of Metabolite Changes Identified from Large-Scale Gene Perturbations in Arabidopsis Using a Genome-Scale Metabolic Network

Global patterns of metabolic responses upon single gene perturbations are specific to gene functions, but they are coordinated with characteristics of the perturbed genes. Metabolomics enables quantitative evaluation of metabolic changes caused by genetic or environmental perturbations. However, little is known about how perturbing a single gene changes the metabolic system as a whole and which network and functional properties are involved in this response. To answer this question, we investigated the metabolite profiles from 136 mutants with single gene perturbations of functionally diverse Arabidopsis (Arabidopsis thaliana) genes. Fewer than 10 metabolites were changed significantly relative to the wild type in most of the mutants, indicating that the metabolic network was robust to perturbations of single metabolic genes. These changed metabolites were closer to each other in a genome-scale metabolic network than expected by chance, supporting the notion that the genetic perturbations changed the network more locally than globally. Surprisingly, the changed metabolites were close to the perturbed reactions in only 30% of the mutants of the well-characterized genes. To determine the factors that contributed to the distance between the observed metabolic changes and the perturbation site in the network, we examined nine network and functional properties of the perturbed genes. Only the isozyme number affected the distance between the perturbed reactions and changed metabolites. This study revealed patterns of metabolic changes from large-scale gene perturbations and relationships between characteristics of the perturbed genes and metabolic changes.

Comprehensive Assessment of Transcriptional Regulation Facilitates Metabolic Engineering of Isoprenoid Accumulation in Arabidopsis

A population of Arabidopsis lines with modulated levels of expression of each gene involved in isoprenoid precursor pathways was used to develop engineering strategies for increasing sterols. In plants, two spatially separated pathways provide the precursors for isoprenoid biosynthesis. We generated transgenic Arabidopsis (Arabidopsis thaliana) lines with modulated levels of expression of each individual gene involved in the cytosolic/peroxisomal mevalonate and plastidial methylerythritol phosphate pathways. By assessing the correlation of transgene expression levels with isoprenoid marker metabolites (gene-to-metabolite correlation), we determined the relative importance of transcriptional control at each individual step of isoprenoid precursor biosynthesis. The accumulation patterns of metabolic intermediates (metabolite-to-gene correlation) were then used to infer flux bottlenecks in the sterol pathway. The extent of metabolic cross talk, the exchange of isoprenoid intermediates between compartmentalized pathways, was assessed by a combination of gene-to-metabolite and metabolite-to-metabolite correlation analyses. This strategy allowed the selection of genes to be modulated by metabolic engineering, and we demonstrate that the overexpression of predictable combinations of genes can be used to significantly enhance flux toward specific end products of the sterol pathway. Transgenic plants accumulating increased amounts of sterols are characterized by significantly elevated biomass, which can be a desirable trait in crop and biofuel plants.

Validation of a microscale extraction and high-throughput UHPLC-QTOF-MS analysis method for huperzine A in Huperzia.

Herein we report on an improved method for the microscale extraction of huperzine A (HupA), an acetylcholinesterase-inhibiting alkaloid, from as little as 3 mg of tissue homogenate from the clubmoss Huperzia squarrosa (G. Forst.) Trevis with 99.95% recovery. We also validated a novel UHPLC-QTOF-MS method for the high-throughput analysis of H. squarrosa extracts in only 6 min, which, in combination with the very low limit of detection (20 pg on column) and the wide linear range for quantification (20-10,000 pg on column), allow for a highly efficient screening of extracts containing varying amounts of HupA. Utilization of this methodology has the potential to conserve valuable plant resources.

Online resources for gene discovery and biochemical research with aromatic and medicinal plants

The elucidation of pathways leading to the signature metabolites accumulated in aromatic and medicinal plants has traditionally been fairly slow in comparison to progress made with much simpler microbes, where gene clustering for natural product biosynthesis is very common. However, recent advances in next-generation sequencing and innovative new approaches to gene discovery have narrowed this gap, and the elucidation of even complex plant pathways has now become significantly faster. A wealth of sequence data has been generated with aromatic and medicinal plants, and it has become increasingly difficult for researchers to stay abreast of new developments. In this review article, I will summarize the functionality of currently available genomics databases and will comment on future needs to maximize their utility.