Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates

Applicability of negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) was examined in trace mixture analyses and structural assignments of some isoprenoid diphosphates. Negative ion FAB-MS spectra using a glycerol matrix of these isoprenoid diphosphates showed predominantly molecular ions (M-H)- together with fragment ions at m/z 177 (H3P2O7)-, 176 (H2P2O7)-, 159 (HP2O6)-, and 79 (PO3)- which were characteristic of the diphosphate ester moiety. The molecular ions did not overlap with peaks arising from any impurities even when crude sample such as butanol extracts from enzymatic reaction mixtures were directly analyzed without any purification. Moreover, collisionally activated dissociation spectra of the molecular ion showed many structurally significant fragment ions which enabled us to elucidate the structures of such irregular alkyl chain moieties as those having a homoisoprenoid skeleton or substituted structures. These studies indicate that negative ion FAB-MS/MS is a simple and useful technique for trace mixture analysis and structure elucidation of isoprenoid diphosphates.     

From flavors and pharmaceuticals to advanced biofuels: Production of isoprenoids in Saccharomyces cerevisiae

Isoprenoids denote the largest group of chemicals in the plant kingdom and are employed for a wide range of applications in the food and pharmaceutical industry. In recent years, isoprenoids have additionally been recognized as suitable replacements for petroleum-derived fuels and could thus promote the transition towards a more sustainable society. To realize the biofuel potential of isoprenoids, a very efficient production system is required. While complex chemical structures as well as the low abundance in nature demonstrate the shortcomings of chemical synthesis and plant extraction, isoprenoids can be produced by genetically engineered microorganisms from renewable carbon sources. In this article, we summarize the development of isoprenoid applications from flavors and pharmaceuticals to advanced biofuels and review the strategies to design microbial cell factories, focusing on Saccharomyces cerevisiae for the production of these compounds. While the high complexity of biosynthetic pathways and the toxicity of certain isoprenoids still denote challenges that need to be addressed, metabolic engineering has enabled large-scale production of several terpenoids and thus, the utilization of these compounds is likely to expand in the future.     

Opportunistic emissions of volatile isoprenoids

Isoprene, monoterpenes and sesquiterpenes are synthesized and emitted by some plant species, but not all plant species have this ability. These volatile, nonessential isoprenoid compounds share the same biochemical precursors as larger essential isoprenoids such as gibberellic acids and carotenoids. They have many protective and ecological functions for the plant species that produce them, but plant species that do not produce these compounds also grow and reproduce successfully. Here, we develop an 'opportunist hypothesis' suggesting that (i) volatile isoprenoid production takes advantage of dimethylallyl diphosphate (DMAPP) and its isomer isopentenyl diphosphate (IPP), which are synthesized primarily to produce essential isoprenoids, and (ii) conditions affecting synthesis of the higher isoprenoids will affect the production and emission of volatile isoprenoids.     

Engineering microbial cell factories for biosynthesis of isoprenoid molecules: beyond lycopene

The isoprenoid superfamily of compounds holds great potential for delivering commercial therapeutics, neutraceuticals and fine chemicals. As such, it has attracted widespread attention and prompted research aimed at metabolic engineering of the pathway for isoprenoid overproduction. The carotenoids in particular, because of their convenient colorimetric screening properties, have facilitated the investigation of new tools for pathway optimization. Because all isoprenoids share common metabolic precursors, genetic platforms resulting from work with carotenoids can be applied to the biosynthesis of other valuable products. In this review we summarize the many tools and methods that have been developed for isoprenoid pathway engineering, and the potential of these technologies for producing other molecules of this family, especially terpenoids.     

Rapid resonance Raman microspectroscopy to probe carbon dioxide fixation by single cells in microbial communities

Photosynthetic microorganisms play crucial roles in aquatic ecosystems and are the major primary producers in global marine ecosystems. The discovery of new bacteria and microalgae that play key roles in CO₂ fixation is hampered by the lack of methods to identify hitherto-unculturable microorganisms. To overcome this problem we studied single microbial cells using stable-isotope probing (SIP) together with resonance Raman (RR) microspectroscopy of carotenoids, the light-absorbing pigments present in most photosynthetic microorganisms. We show that fixation of ¹³CO₂ into carotenoids produces a red shift in single-cell RR (SCRR) spectra and that this SCRR–SIP technique is sufficiently sensitive to detect as little as 10% of ¹³C incorporation. Mass spectrometry (MS) analysis of labelled cellular proteins verifies that the red shift in carotenoid SCRR spectra acts as a reporter of the ¹³C content of single cells. Millisecond Raman imaging of cells in mixed cultures and natural seawater samples was used to identify cells actively fixing CO₂, demonstrating that the SCRR–SIP is a noninvasive method for the rapid and quantitative detection of CO₂ fixation at the single cell level in a microbial community. The SCRR–SIP technique may provide a direct method for screening environmental samples, and could help to reveal the ecophysiology of hitherto-unculturable microorganisms, linking microbial species to their ecological function in the natural environment.     

Single‐cell genomics based on Raman sorting reveals novel carotenoid‐containing bacteria in the Red Sea

Cell sorting coupled with single‐cell genomics is a powerful tool to circumvent cultivation of microorganisms and reveal microbial ‘dark matter’. Single‐cell Raman spectra (SCRSs) are label‐free biochemical ‘fingerprints’ of individual cells, which can link the sorted cells to their phenotypic information and ecological functions. We employed a novel Raman‐activated cell ejection (RACE) approach to sort single bacterial cells from a water sample in the Red Sea based on SCRS. Carotenoids are highly diverse pigments and play an important role in phototrophic bacteria, giving strong and distinctive Raman spectra. Here, we showed that individual carotenoid‐containing cells from a Red Sea sample were isolated based on the characteristic SCRS. RACE‐based single‐cell genomics revealed putative novel functional genes related to carotenoid and isoprenoid biosynthesis, as well as previously unknown phototrophic microorganisms including an unculturable Cyanobacteria spp. The potential of Raman sorting coupled to single‐cell genomics has been demonstrated.     

Rapid and robust MALDI-TOF MS techniques for microbial identification: a brief overview of their diverse applications

in mass spectrometry have enabled the investigation of various biological systems by directly analyzing diverse sets of biomolecules (i.e., proteins, lipids, and carbohydrates), thus making a significant impact on the life sciences field. Over the past decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely utilized as a rapid and reliable method for the identification of microorganisms. MALDI-TOF MS has come into widespread use despite its relatively low resolving power (full width at half maximum, FWHM: < 5,000) and its incompatibility with tandem MS analysis, features with which other high-resolution mass spectrometers are equipped. Microbial identification is achieved by searching databases containing mass spectra of peptides and proteins extracted from microorganisms of interest, using scoring algorithms to match analyzed spectra with reference spectra. In this paper, we give a brief overview of the diverse applications of rapid and robust MALDI-TOF MS-based techniques for microbial identification in a variety of fields, such as clinical diagnosis and environmental and food monitoring. We also describe the fundamental principles of MALDI-TOF MS. The general specifications of the two major MS-based microbial identification systems available in the global market (BioTyper® and VITEK® MS Plus) and the distribution of these instruments in Republic of Korea are also discussed. The current review provides an understanding of this emerging microbial identification and classification technology and will help bacteriologists and cell biologists take advantage of this powerful technique.     

Isoprenoids: an evolutionary pool for photoprotection

Plants have evolved several mechanisms for getting rid of excess energy in photosynthetic membranes, some of which involve isoprenoid compounds. In all photosynthetic organisms, the carotenoids beta-carotene and zeaxanthin, and tocopherols serve an important photoprotective role, either by dissipating excess excitation energy as heat or by scavenging reactive oxygen species (ROS) and suppressing lipid peroxidation. Isoprene and some monoterpenes, diterpenes and other carotenoids also occur in some plant lineages. Compelling evidence indicates that these non-ubiquitous isoprenoids might be particularly relevant in adapting plants to adverse climatic conditions by serving as additional and/or alternative protection mechanisms.     

Biosynthesis,accumulation and emission of carotenoids, α-tocopherol,plastoquinone, and isoprene in leaves under high photosynthetic irradiance

The localization of isoprenoid lipids in chloroplasts, the accumulation of particular isoprenoids under high irradiance conditions, and channelling of photosynthetically fixed carbon into plastidic thylakoid isoprenoids, volatile isoprenoids, and cytosolic sterols are reviewed. During leaf and chloroplast development in spring plastidic isoprenoid biosynthesis provides primarily thylakoid carotenoids, the phytyl side-chain of chlorophylls and the electron carriers phylloquinone K1, alpha-tocoquinone and alpha-tocopherol, as well as the nona-prenyl side-chain of plastoquinone-9. Under high irradiance, plants develop sun leaves and high light (HL) leaves with sun-type chloroplasts that possess, besides higher photosynthetic CO2 assimilation rates, different quantitative levels of pigments and prenylquinones as compared to shade leaves and low light (LL) leaves. After completion of chloroplast thylakoid synthesis plastidic isoprenoid biosynthesis continues at high irradiance conditions, constantly accumulating alpha-tocopherol (alpha-T) and the reduced form of plastoquinone-9 (PQ-9H2) deposited in the steadily enlarging osmiophilic plastoglobuli, the lipid reservoir of the chloroplast stroma. In sun leaves of beech (Fagus) and in 3-year-old sunlit Ficus leaves the level of alpha-T and PQ-9 can exceed that of chlorophyll b. Most plants respond to HL conditions (sun leaves, leaves suddenly lit by the sun) with a 1.4-2-fold increase of xanthophyll cycle carotenoids (violaxanthin, zeaxanthin, neoxanthin), an enhanced operation of the xanthophyll cycle and an increase of beta-carotene levels. This is documented by significantly lower values for the weight ratio chlorophylls to carotenoids (range: 3.6-4.6) as compared to shade and LL leaves (range: 4.8-7.0). Many plant leaves emit under HL and high temperature conditions at high rates the volatile compounds isoprene (broadleaf trees) or methylbutenol (American ponderosa pines), both of which are formed via the plastidic 1-deoxy-D: -xylulose-phosphate/2-C-methylerythritol 5-phosphate (DOXP/MEP) pathway. Other plants by contrast, accumulate particular mono- and diterpenes. Under adequate photosynthetic conditions the chloroplastidic DOXP/MEP isoprenoid pathway essentially contributes, with its C5 isoprenoid precusors, to cytosolic sterol biosynthesis. The possible cross-talk between the two cellular isoprenoid pathways, the acetate/MVA and the DOXP/MEP pathways, that preferentially proceeds in a plastid-to-cytosol direction, is shortly discussed.     

Related enzymes solve evolutionarily recurrent problems in the metabolism of carotenoids

Chemical transformations of isoprenoids in plants and some bacteria and fungi lead to the production of various conjugated products, including carotenoids. Carotenoids can be cleaved to generate apocarotenoid precursors for signaling molecules such as abscisic and retinoic acids, and for the photosensory pigment retinal. The enzymes that catalyze the various transformations of carotenoids and apocarotenoids are closely related. This evolutionarily distant conservation is unexpected and intriguing. Many aspects of the metabolism of retinoids in vertebrates remain controversial and poorly understood. Because few chemical reactions are possible for this group of compounds, furthering our knowledge of isoprenoid transformation in plants could be beneficial to our understanding of how retinoids and carotenoids are transformed in vertebrates.     

Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase

The methylerythritol 4-phosphate (MEP) pathway synthesizes the precursors for an astonishing diversity of plastid isoprenoids, including the major photosynthetic pigments chlorophylls and carotenoids. Since the identification of the first two enzymes of the pathway, deoxyxylulose 5-phoshate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), they both were proposed as potential control points. Increased DXS activity has been shown to up-regulate the production of plastid isoprenoids in all systems tested, but the relative contribution of DXR to the supply of isoprenoid precursors is less clear. In this work, we have generated transgenic Arabidopsis thaliana plants with altered DXS and DXR enzyme levels, as estimated from their resistance to clomazone and fosmidomycin, respectively. The down-regulation of DXR resulted in variegation, reduced pigmentation and defects in chloroplast development, whereas DXR-overexpressing lines showed an increased accumulation of MEP- derived plastid isoprenoids such as chlorophylls, carotenoids, and taxadiene in transgenic plants engineered to produce this non-native isoprenoid. Changes in DXR levels in transgenic plants did not result in changes in␣DXS gene expression or enzyme accumulation, confirming that the observed effects on plastid isoprenoid levels in DXR-overexpressing lines were not an indirect consequence of altering DXS levels. The results indicate that the biosynthesis of MEP (the first committed intermediate of the pathway) limits the production of downstream isoprenoids in Arabidopsis chloroplasts, supporting a role for DXR in the control of the metabolic flux through the MEP pathway.     

Strategies of isoprenoids production in engineered bacteria

Isoprenoids are the largest family of natural products, over 40 000 compounds have been described, which have been widely used in various fields. Currently, the isoprenoid products are mainly produced by natural extraction or chemical synthesis, however, limited yield and high cost is far behind the increasing need. Most bacteria synthesize the precursors of isoprenoids through the methylerythritol 4‐phosphate pathway, microbial synthesis of isoprenoids by fermentation becomes more attractive mainly in terms of environmental concern and renewable resources. In this review, the strategies of isoprenoid production in bacteria by synthetic biology are discussed. Introducing foreign genes associated with desired products made it possible to produce isoprenoids in bacteria. Furthermore, the yield of isoprenoids is increased by the strategies of overexpression of native or foreign genes, introducing heterologous mevalonate pathway, balancing of the precursors and inactivating the competing pathway, these methods were used separately or simultaneously.     

Supply of precursors for carotenoid biosynthesis in plants

Carotenoids are isoprenoids of industrial and nutritional interest produced by all photosynthetic organisms, including plants. Too often, the metabolic engineering of plant carotenogenesis has been obstructed by our limited knowledge on how the endogenous pathway interacts with other related metabolic pathways, particularly with those involved in the production of isoprenoid precursors. However, recent discoveries are providing new insights into this field. All isoprenoids derive from prenyl diphosphate precursors. In the case of carotenoids, these precursors are produced predominantly by the methylerythritol 4-phosphate (MEP) pathway in plants. This review focuses on the progress in our understanding of how manipulation of the MEP pathway impacts carotenoid biosynthesis and on the discoveries underlining the central importance of coordinating the supply of MEP-derived precursors with the biosynthesis of carotenoids and other derived isoprenoids.     

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

Isoprenoids are the most numerous and structurally diverse family of natural products. Terpenoids, a class of isoprenoids often isolated from plants, are used as commercial flavor and fragrance compounds and antimalarial or anticancer drugs. Because plant tissue extractions typically yield low terpenoid concentrations, we sought an alternative method to produce high-value terpenoid compounds, such as the antimalarial drug artemisinin, in a microbial host. We engineered the expression of a synthetic amorpha-4,11-diene synthase gene and the mevalonate isoprenoid pathway from Saccharomyces cerevisiae in Escherichia coli. Concentrations of amorphadiene, the sesquiterpene olefin precursor to artemisinin, reached 24 microg caryophyllene equivalent/ml. Because isopentenyl and dimethylallyl pyrophosphates are the universal precursors to all isoprenoids, the strains developed in this study can serve as platform hosts for the production of any terpenoid compound for which a terpene synthase gene is available.     

Genome organization in Arabidopsis thaliana: a survey for genes involved in isoprenoid and chlorophyll metabolism

The isoprenoid biosynthetic pathway provides intermediates for the synthesis of a multitude of natural products which serve numerous biochemical functions in plants: sterols (isoprenoids with a C30 backbone) are essential components of membranes; carotenoids (C40) and chlorophylls (which contain a C20 isoprenoid side-chain) act as photosynthetic pigments; plastoquinone, phylloquinone and ubiquinone (all of which contain long isoprenoid side-chains) participate in electron transport chains; gibberellins (C20), brassinosteroids (C30) and abscisic acid (C15) are phytohormones derived from isoprenoid intermediates; prenylation of proteins (with C15 or C20 isoprenoid moieties) may mediate subcellular targeting and regulation of activity; and several monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20) have been demonstrated to be involved in plant defense. Here we present a comprehensive analysis of genes coding for enzymes involved in the metabolism of isoprenoid-derived compounds in Arabidopsis thaliana. By combining homology and sequence motif searches with knowledge regarding the phylogenetic distribution of pathways of isoprenoid metabolism across species, candidate genes for these pathways in A. thaliana were obtained. A detailed analysis of the vicinity of chromosome loci for genes of isoprenoid metabolism in A. thaliana provided evidence for the clustering of genes involved in common pathways. Multiple sequence alignments were used to estimate the number of genes in gene families and sequence relationship trees were utilized to classify their individual members. The integration of all these datasets allows the generation of a knowledge-based metabolic map of isoprenoid metabolic pathways in A. thaliana and provides a substantial improvement of the currently available gene annotation.     

Combining Genotype Improvement and Statistical Media Optimization for Isoprenoid Production in E. coli

Isoprenoids are a large and diverse class of compounds that includes many high value natural products and are thus in great demand. To meet the increasing demand for isoprenoid compounds, metabolic engineering of microbes has been used to produce isoprenoids in an economical and sustainable manner. To achieve high isoprenoid yields using this technology, the availability of metabolic precursors feeding the deoxyxylulose phosphate (DXP) pathway, responsible for isoprenoid biosynthesis, has to be optimized. In this study, phosphoenolpyruvate, a vital DXP pathway precursor, was enriched by deleting the genes encoding the carbohydrate phosphotransferase system (PTS) in E. coli. Production of lycopene (a C40 isoprenoid) was maximized by optimizing growth medium and culture conditions. In optimized conditions, the lycopene yield from PTS mutant was seven fold higher than that obtained from the wild type strain. This resulted in the highest reported specific yield of lycopene produced from the DXP pathway in E. coli to date (20,000 µg/g dry cell weight). Both the copy number of the plasmid encoding the lycopene biosynthetic genes and the expression were found to be increased in the optimized media. Deletion of PTS together with a similar optimization strategy was also successful in enhancing the production of amorpha-1,4-diene, a distinct C15 isoprenoid, suggesting that the approaches developed herein can be generally applied to optimize production of other isoprenoids.     

Electricity generation by microorganisms in the sediment-water interface of an extreme acidic microcosm

The attachment of microorganisms to electrodes is of great interest for electricity generation in microbial fuel cells (MFC) or other applications in bioelectrochemical systems (BES). In this work, a microcosm of the acidic ecosystem of Río Tinto was built and graphite electrodes were introduced at different points. This allowed the study of electricity generation in the sediment/water interface and the involvement of acidophilic microorganisms as biocatalysts of the anodic and cathodic reactions in a fuel-cell configuration. Current densities and power outputs of up to 3.5 A/m2 and 0.3 W/m2, respectively, were measured at pH 3. Microbial analyses of the electrode surfaces showed that Acidiphilium spp., which uses organic compounds as electron donors, were the predominant biocatalysts of the anodic reactions, whereas the aerobic iron oxidizers Acidithiobacillus ferrooxidans and Leptospirillum spp. were detected mainly on the cathode surface.     

In silico fragmentation for computer assisted identification of metabolite mass spectra

Background  

Mass spectrometry has become the analytical method of choice in metabolomics research. The identification of unknown compounds is the main bottleneck. In addition to the precursor mass, tandem MS spectra carry informative fragment peaks, but the coverage of spectral libraries of measured reference compounds are far from covering the complete chemical space. Compound libraries such as PubChem or KEGG describe a larger number of compounds, which can be used to compare their in silico fragmentation with spectra of unknown metabolites.     

Pathway engineering for functional isoprenoids

Pathway engineering is to engineer biosynthetic pathways for compounds of interests in heterologous organisms such as microbes and higher plants, which has also been one of the most important fields in metabolic engineering and synthetic biology. This review focuses on pathway engineering researches for the production of functional isoprenoids containing monoterpenes, sesquiterpenes, diterpenes, and triterpenes as well as carotenoids and for the elucidation of relevant biosynthesis genes and enzymes, which have been performed in the last two years. As microbial hosts, Escherichia coli and Saccharomyces cerevisiae have often been employed, since they, specifically the former, are fully amenable to genetic manipulations with extensive molecular resources. Various crops have also been used as the hosts for engineering pathways of functional isoprenoids of the plant origin, particularly carotenoids.     

Evidence of isoprenoid precursor toxicity in Bacillus subtilis

The mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways for isoprenoid biosynthesis both culminate in the production of the two-five carbon prenyl diphosphates: dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). These are the building blocks for higher isoprenoids, including many that have industrial and pharmaceutical applications. With growing interest in producing commercial isoprenoids through microbial engineering, reports have appeared of toxicity associated with the accumulation of prenyl diphosphates in Escherichia coli expressing a heterologous MVA pathway. Here we explored whether similar prenyl diphosphate toxicity, related to MEP pathway flux, could also be observed in the bacterium Bacillus subtilis. After genetic and metabolic manipulations of the endogenous MEP pathway in B. subtilis, measurements of cell growth, MEP pathway flux, and DMAPP contents suggested cytotoxicity related to prenyl diphosphate accumulation. These results have implications as to understanding the factors impacting isoprenoid biosynthesis in microbial systems.