Diversifying Isoprenoid Platforms via Atypical Carbon Substrates and Non-model Microorganisms

Isoprenoid compounds are biologically ubiquitous, and their characteristic modularity has afforded products ranging from pharmaceuticals to biofuels. Isoprenoid production has been largely successful in Escherichia coli and Saccharomyces cerevisiae with metabolic engineering of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways coupled with the expression of heterologous terpene synthases. Yet conventional microbial chassis pose several major obstacles to successful commercialization including the affordability of sugar substrates at scale, precursor flux limitations, and intermediate feedback-inhibition. Now, recent studies have challenged typical isoprenoid paradigms by expanding the boundaries of terpene biosynthesis and using non-model organisms including those capable of metabolizing atypical C1 substrates. Conversely, investigations of non-model organisms have historically informed optimization in conventional microbes by tuning heterologous gene expression. Here, we review advances in isoprenoid biosynthesis with specific focus on the synergy between model and non-model organisms that may elevate the commercial viability of isoprenoid platforms by addressing the dichotomy between high titer production and inexpensive substrates.

Transcriptomics and Metabolomics Changes Triggered by Inflorescence Removal in Panax notoginseng (Burk.)

The root of Panax notoginseng (Burk.), in which saponins are the major active components, is a famous traditional Chinese medicine used to stop bleeding and to decrease inflammation and heart disease. Inflorescence removal increases the yield and quality of P. notoginseng, but the underlying molecular mechanisms are unknown. Here, the differences between inflorescence-removal treatment and control groups of P. notoginseng were compared using transcriptomics and metabolomics analyses. Illumina sequencing of cDNA libraries prepared from the rhizomes, leaves and roots of the two groups independently identified 6,464, 4,584, and 7,220 differentially expressed genes (DEG), respectively. In total, 345 differentially expressed transcription factors (TFs), including MYB and WRKY family members, were induced by the inflorescence-removal treatment. Additionally, 215 DEGs involved in saponin terpenoid backbone biosynthetic pathways were identified. Most genes involved in the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways were activated by inflorescence removal. The co-expression analysis showed that the low expression levels of flavonoid biosynthesis-related genes (e.g., C4H and F3H) decreased the biosynthesis and accumulation of some flavonoids after inflorescence removal. The results not only provide new insights into the fundamental mechanisms underlying the poorly studied inflorescence-removal process in P. notoginseng and other rhizome crops, but they also represent an important resource for future research on gene functions during inflorescence-removal treatments and the reproductive stage.

Inhibitory Activity of Proanthocyanidins Against Escherichia coli 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is a key enzyme in the methylerythritol phosphate pathway for terpenoid biosynthesis. Furthermore, it is an ideal target for the screening of novel antibiotics because it is present in causative organisms, but absent from humans. To identify more lipophilic DXR inhibitors from natural resources, we tested the DXR inhibitory activity of five proanthocyanidins in this study. The results indicated that all these compounds specifically restrained the activity of DXR, with procyanid B2 exhibiting a relatively low effect against DXR (IC50 ∼ 305 μM) and procyanid C1 displaying moderate activity (IC50 75.1 μM). The other three compounds cinnamtannin A2, cinnamtannin B1, and cinnamtannin D1 (IC50 ∼ 89.3, 105.0, and 97.8 μM, respectively) showed DXR inhibitory effects that were slightly weaker than that of procyanid C1. In addition, based on the initial characterization, the structure–activity relationship of this series of compounds against DXR is discussed.

Sclareol and linalyl acetate are produced by glandular trichomes through the MEP pathway

Sclareol, an antifungal specialized metabolite produced by clary sage, Salvia sclarea, is the starting plant natural molecule used for the hemisynthesis of the perfume ingredient ambroxide. Sclareol is mainly produced in clary sage flower calyces; however, the cellular localization of the sclareol biosynthesis remains unknown. To elucidate the site of sclareol biosynthesis, we analyzed its spatial distribution in the clary sage calyx epidermis using laser desorption/ionization mass spectrometry imaging (LDI–FTICR-MSI) and investigated the expression profile of sclareol biosynthesis genes in isolated glandular trichomes (GTs). We showed that sclareol specifically accumulates in GTs’ gland cells in which sclareol biosynthesis genes are strongly expressed. We next isolated a glabrous beardless mutant and demonstrate that more than 90% of the sclareol is produced by the large capitate GTs. Feeding experiments, using 1-13C-glucose, and specific enzyme inhibitors further revealed that the methylerythritol-phosphate (MEP) biosynthetic pathway is the main source of isopentenyl diphosphate (IPP) precursor used for the biosynthesis of sclareol. Our findings demonstrate that sclareol is an MEP-derived diterpene produced by large capitate GTs in clary sage emphasing the role of GTs as biofactories dedicated to the production of specialized metabolites.

Enhancing Sesquiterpenoid Production from Methane via Synergy of the Methylerythritol Phosphate Pathway and a Short-Cut Route to 1-Deoxy-D-xylulose 5-Phosphate in Methanotrophic Bacteria

Sesquiterpenoids are one of the most diverse classes of isoprenoids which exhibit numerous potentials in industrial biotechnology. The methanotrophs-based methane bioconversion is a promising approach for sustainable production of chemicals and fuels from methane. With intrinsic high carbon flux though the ribulose monophosphate cycle in Methylotuvimicrobium alcaliphilum 20Z, we demonstrated here that employing a short-cut route from ribulose 5-phosphate to 1-deoxy-d-xylulose 5-phosphate (DXP) could enable a more efficient isoprenoid production via the methylerythritol 4-phosphate (MEP) pathway, using α-humulene as a model compound. An additional 2.8-fold increase in α-humulene production yield was achieved by the fusion of the nDXP enzyme and DXP reductase. Additionally, we utilized these engineering strategies for the production of another sesquiterpenoid, α-bisabolene. The synergy of the nDXP and MEP pathways improved the α-bisabolene titer up to 12.24 ± 0.43 mg/gDCW, twofold greater than that of the initial strain. This study expanded the suite of sesquiterpenoids that can be produced from methane and demonstrated the synergistic uses of the nDXP and MEP pathways for improving sesquiterpenoid production in methanotrophic bacteria.

Biosynthesis of Natural Products

Natural products are in the form of primary and secondary metabolites and are isolated chemical compounds or substances from living organisms. Terpenes, Phenolic compounds, and Nitrogen-containing compounds are secondary metabolites. The biosyntheses of secondary metabolites are derived from primary metabolism pathways, which consist of a tricarboxylic acid cycle (TCA), methylerythritol phosphate pathway (MEP), mevalonic and shikimic acid pathway. This chapter provides an overview of the diversity of secondary metabolites in plants, their multiple biological functions, and multi-faceted cultural history.

Biosynthesis Pathways of Vitamin E and Its Derivatives in Plants

Naturally occurring vitamin E, comprised of four forms each of tocopherols and tocotrienols, are synthesized solely by photosynthetic organisms and function primarily as antioxidants. The structural motifs of the vitamin E family and specifically the chroman moiety, are amenable to various modifications in order to improve their bioactivities towards numerous therapeutic targets. Tocopherols are lipophilic antioxidants and together with tocotrienols belong to the vitamin-E family. These lipid-soluble compounds are potent antioxidants that protect polyunsaturated fatty acids from lipid peroxidation. Biosynthetic pathways of plants producing a diverse array of natural products that are important for plant function, agriculture, and human nutrition. Edible plant-derived products, notably seed oils, are the main sources of vitamin E in the human diet. The biosynthesis of tocopherols takes place mainly in plastids of higher plants from precursors derived from two metabolic pathways: homogentisic acid, an intermediate of degradation of aromatic amino acids, and phytyldiphosphate, which arises from methylerythritol phosphate pathway. Tocopherols and tocotrienols play an important roles in the oxidative stability of vegetable oils and in the nutritional quality of crop plants for human and livestock diets. Here, we review major biosynthetic pathways, including common precursors and competitive pathways of the vitamin E and its derivatives in plants.

Molecular Cloning and Functional Analysis of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase from Santalum album

Sandalwood (Santalum album L.) heartwood-derived essential oil contains a high content of sesquiterpenoids that are economically highly valued and widely used in the fragrance industry. Sesquiterpenoids are biosynthesized via the mevalonate acid and methylerythritol phosphate (MEP) pathways, which are also the sources of precursors for photosynthetic pigments. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is a secondary rate-limiting enzyme in the MEP pathway. In this paper, the 1416-bp open reading frame of SaDXR and its 897-bp promoter region, which contains putative conserved cis-elements involved in stress responsiveness (HSE and TC-rich repeats), hormone signaling (abscisic acid, gibberellin and salicylic acid) and light responsiveness, were cloned from 7-year-old S. album trees. A bioinformatics analysis suggested that SaDXR encodes a functional and conserved DXR protein. SaDXR was widely expressed in multiple tissues, including roots, twigs, stem sapwood, leaves, flowers, fruit and stem heartwood, displaying significantly higher levels in tissues with photosynthetic pigments, like twigs, leaves and flowers. SaDXR mRNA expression increased in etiolated seedlings exposed to light, and the content of chlorophylls and carotenoids was enhanced in all 35S::SaDXR transgenic Arabidopsis thaliana lines, consistent with the SaDXR expression level. SaDXR was also stimulated by MeJA and H2O2 in seedling roots. α-Santalol content decreased in response to fosmidomycin, a DXR inhibitor. These results suggest that SaDXR plays an important role in the biosynthesis of photosynthetic pigments, shifting the flux to sandalwood-specific sesquiterpenoids.

The eukaryotic MEP-pathway genes are evolutionarily conserved and originated from Chlaymidia and cyanobacteria

Background Isoprenoids are the most ancient and essential class of metabolites produced in all organisms, either via mevalonate (MVA)-and/or methylerythritol phosphate (MEP)-pathways. The MEP-pathway is present in all plastid-bearing organisms and most eubacteria. However, no comprehensive study reveals the origination and evolutionary characteristics of MEP-pathway genes in eukaryotes. Results Here, detailed bioinformatics analyses of the MEP-pathway provide an in-depth understanding the evolutionary history of this indispensable biochemical route, and offer a basis for the co-existence of the cytosolic MVA- and plastidial MEP-pathway in plants given the established exchange of the end products between the two isoprenoid-biosynthesis pathways. Here, phylogenetic analyses establish the contributions of both cyanobacteria and Chlamydiae sequences to the plant’s MEP-pathway genes. Moreover, Phylogenetic and inter-species syntenic block analyses demonstrate that six of the seven MEP-pathway genes have predominantly remained as single-copy in land plants in spite of multiple whole-genome duplication events (WGDs). Substitution rate and domain studies display the evolutionary conservation of these genes, reinforced by their high expression levels. Distinct phenotypic variation among plants with reduced expression levels of individual MEP-pathway genes confirm the indispensable function of each nuclear-encoded plastid-targeted MEP-pathway enzyme in plant growth and development. Conclusion Collectively, these findings reveal the polyphyletic origin and restrict conservation of MEP-pathway genes, and reinforce the potential function of the individual enzymes beyond production of the isoprenoids intermediates.