DXP Pathway Research Articles

The Identification of β-Ocimene Biosynthetic Pathway Through Mevalonate Acid (MVA) and 1-Deoxy-D-Xylulose 5-Phosphate (DXP) Pathways Using Crude Enzyme Extracts in Indonesian Bay Leaf/Salam Leaf (Syzygium polyanthum).

Salam leaf has a β-ocimene as a key volatile compound that gives a fresh aroma to the food when the salam leaves are involved in the cooking process. As a secondary metabolic product, enzymatic biosynthesis as the early stage of β-ocimene is a factor that needs to be known. Thus, this study was done to identify the mechanism of the two well-known terpenoid biosynthetic pathways, namely Mevalonate Acid (MVA) and 1-Deoxy-D-Xylulose 5-Phosphate (DXP) pathways, in the biosynthesis of β-ocimene in salam leaves. The activity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)-MVA pathway-determining enzyme and 1-deoxy-D-xylulose-5-phosphate synthase (DXS)-DXP pathway-determining enzyme in the crude enzyme and their derivative products of salam leaves were analysed for their changes by differences of substrate ratios and enzyme inhibitors. The results showed that the activity of the HMGR enzyme was lower significantly than the DXS enzyme based on the addition of variations to the substrate ratio. These results were also supported by the enzyme and substrate reaction products, MVA and Isopentenyl diphosphate (IPP) intermediates from the MVA pathway, which were significantly lower when compared to DXP and IPP intermediates from the DXP pathway. As the end product of the reaction, β-ocimene gave a significantly higher value of the DXP pathway than the MVA pathway. Therefore, it can conclude that the mechanism of the biosynthetic pathway of β-ocimene in salam leaves was synthesised via the DXP pathway. The production of β-ocimene could have crosstalk-pathway through the MVA pathway, especially when the DXP pathway was blocked.

Computationally Decoding NudF Residues To Enhance the Yield of the DXP Pathway.

Terpenoids form a large pool of highly diverse organic compounds possessing several economically important properties, including nutritional, aromatic, and pharmacological properties. The 1-deoxy-d-xylulose 5-phosphate (DXP) pathway’s end enzyme, nuclear distribution protein (NudF), interacting with isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), is critical for the synthesis of isoprenol/prenol/downstream compounds. The enzyme is yet to be thoroughly investigated to increase the overall yield of terpenoids in the Bacillus subtilis, which is widely used in industry and is generally regarded as a safe (GRAS) bacterium. The study aims to analyze the evolutionary conservation across the active site for mapping the key residues for mutagenesis studies. The 37-sequence data set, extracted from 103 Bacillus subtilis entries, shows a high phylogenetic divergence, and only six one-motif sequences ASB92783.1, ASB69297.1, ASB56714.1, AOR97677.1, AOL97023.1, and OAZ71765.1 show a monophyly relationship, unlike a complete polyphyly relationship between the other 31 three-motif sequences. Furthermore, only 47 of 179 residues of the representative sequence CUB50584.1 are observed to be significantly conserved. Docking analysis suggests a preferential bias of adenosine diphosphate (ADP)-ribose pyrophosphatase toward IPP, and a nearly threefold energetic difference is observed between IPP and DMAPP. The loops are hereby shown to play a regulatory role in guiding the promiscuity of NudF toward a specific ligand. Computational saturation mutagenesis of the seven hotspot residues identifies two key positions LYS78 and PHE116, orderly encoded within loop1 and loop7, majorly interacting with the ligands DMAPP and IPP, and their mutants K78I/K78L and PHE116D/PHE116E are found to stabilize the overall conformation. Molecular dynamics analysis shows that the IPP complex is significantly more stable than the DMAPP complex, and the NudF structure is very unstable. Besides showing a promiscuous binding of NudF with ligands, the analysis suggests its rate-limiting nature. The study would allow us to customize the metabolic load toward the synthesis of any of the downstream molecules. The findings would pave the way for the development of catalytically improved NudF mutants for the large-scale production of specific terpenoids with significant nutraceutical or commercial value.

Computational and Synthetic Biology Approaches for the Biosynthesis of Antiviral and Anticancer Terpenoids from Bacillus subtilis.

Recent advancements in medicinal research have identified several antiviral and anticancer terpenoids that are usually deployed as a source of flavor, fragrances and pharmaceuticals. Under the current COVID-19 pandemic conditions, natural therapeutics with the least side effects are the need of the hour to save the patients, especially, which are pre-affected with other medical complications. Although plants are the major sources of terpenoids; however, for the environmental concerns, the global interest has shifted to the biocatalytic production of molecules from microbial sources. The gram-positive bacterium Bacillus subtilis is a suitable host in this regard due to its GRAS (generally regarded as safe) status, ease in genetic manipulations and wide industrial acceptability. The B. subtilis synthesizes its terpenoid molecules from 1-deoxy-d-xylulose-5-phosphate (DXP) pathway, a common route in almost all microbial strains. Here, we summarize the computational and synthetic biology approaches to improve the production of terpenoid-based therapeutics from B. subtilis by utilizing DXP pathway. We focus on the in-silico approaches for screening the functionally improved enzyme-variants of the two crucial enzymes namely, the DXP synthase (DXS) and Farnesyl Pyrophosphate Synthase (FPPS). The approaches for engineering the active sites are subsequently explained. It will be helpful to construct the functionally improved enzymes for the high-yield production of terpenoid-based anticancer and antiviral metabolites, which would help to reduce the cost and improve the availability of such therapeutics for the humankind.

Manipulation of DXP pathway for andrographolide production in callus cultures of Andrographis paniculata.

Greening of in vitro callus cultures and andrographolide over-accumulation was achieved by manipulating light exposure and media composition, when the biosynthetic cascade was channeled through the DXP pathway. Andrographolide, the primary biologically active compound of Andrographis paniculata, is produced through coordinated action of two pathways, the classical cytosolic mevalonate pathway and the alternative plastidial non-mevalonate pathway (Deoxy-xylulose Phosphate pathway). In vitro callus cultures of A. paniculata are useful sources of production, as well as, manipulation of andrographolide, and the present study was designed to explore the strategy of pathway inhibition for its overproduction. When the cytosolic mevalonate pathway blocker, lovastatin, was applied to callus cultures of A. paniculata, andrographolide production was enhanced in comparison to untreated control. In contrast, treatment of the callus tissue with the DXP-pathway blocker, fosmidomycin, led to depletion in andrographolide production. The present study also showed that silver nitrate, a potent elicitor of andrographolide production in in vitro callus culture, when added in combination with the pathway inhibitors resulted in alterations in andrographolide production. The highest andrographolide production was obtained in callus treated with a combination of silver nitrate and lovastatin, indicating a predominant role of the plastidial DXP pathway in andrographolide biosynthesis. A positive co-relation with chlorophyll content and andrographolide production in in vitro callus cultures (untreated and treated) observed also supported the above assumption. It could be inferred from this study that greening of callus tissue through organellar organization was a potent strategy for enhancing andrographolide accumulation in callus tissues of A. paniculata.

IN-SILICO ANALYSIS OF TERPENOIDS IN SACCHAROMYCES CEREVISIAE

Heterologous production of terpenoids from plants which are of medicinal and industrial interests is getting much attention. For this purpose saccharomyces cerevisiae is the most commonly used host but the yield of terpenoids is much lower. The main aim of this review is to study the terpenoid pathways of saccharomyces cerevisiae, effect of respective host metabolism as well as to study the effect of different carbon sources in silico by means of elementary mode analysis. The production and yield of IPP was main focus point in order to find out the novel metabolic engineering strategy for increasing production of terpenoids. With glucose acting as a substrate, MVA pathway has low potential to produce terpenoids as compared to DXP pathway if we consider formation of precursor. Moreover the carbon source also has impact on yield with nonfermentable source providing more biomass. At last several knock out methodologies were being employed which identified minimal cuts sets for enhanced growth of terpenoids.

Genomic and transcriptional changes in response to pinene tolerance and overproduction in evolved Escherichia coli

α-Pinene is an important monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals and biofuels. Although an evolved strain Escherichia coli YZFP, which had higher tolerance to pinene and titer, has been successfully used to produce high levels of pinene, the pinene titer is much lower than that of hemiterpene (isoprene) and sesquiterpenes (farnesene) to date. Moreover, the overall cellular physiological and metabolic changes caused by higher tolerance to pinene and overproduction of pinene remains unclear. To reveal the mechanism of Escherichia coli YZFP with the higher tolerance to pinene and titer, a comparative genomics and transcriptional level analyses combining with CRISPR activation (CRISPRa) and interference (CRISPRi) were carried out. The results show that the tolerance to pinene and the overproduction of pinene in E. coli may be associated with: 1) the mutations of the DXP pathway genes, the rpoA and some membrane protein genes, and their upregulations of transcription levels; and 2) the mutations of some genes and their downregulation of transcriptional levels. These comparative omics analyses provided some genetic modification strategies to further improve pinene production. Overexpression of the mutated cbpA, tabA, pitA, rpoA, sufBCDS, mutS, ispH, oppF, dusB, dnaK, dxs, dxr and flgFGH genes further improved pinene production. This study also demonstrated that combining comparative omics analysis with CRISPRa and CRISPRi is an efficient technology to quickly find a new metabolic engineering strategy.

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae

Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.

Precise precursor rebalancing for isoprenoids production by fine control of gapA expression in Escherichia coli

Biosynthesis of isoprenoids via the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway requires equimolar glyceraldehyde 3-phosphate and pyruvate to divert carbon flux toward the products of interest. Here, we demonstrate that precursor balancing is one of the critical steps for the production of isoprenoids in Escherichia coli. First, the implementation of the synthetic lycopene production pathway as a model system and the amplification of the native DXP pathway were accomplished using synthetic constitutive promoters and redesigned 5′-untranslated regions (5′-UTRs). Next, fine-controlled precursor balancing was investigated by tuning phosphoenolpyruvate synthase (PpsA) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The results showed that tuning-down of gapA improved the specific lycopene content by 45% compared to the overexpression of ppsA. The specific lycopene content in the strains with down-regulated gapA increased by 97% compared to that in the parental strain. Our results indicate that gapA is the best target for precursor balancing to increase biosynthesis of isoprenoids.

Proteomics-based metabolic modeling and characterization of the cellulolytic bacterium Thermobifida fusca.

BackgroundThermobifida fusca is a cellulolytic bacterium with potential to be used as a platform organism for sustainable industrial production of biofuels, pharmaceutical ingredients and other bioprocesses due to its capability of potential to convert plant biomass to value-added chemicals. To best develop T. fusca as a bioprocess organism, it is important to understand its native cellular processes. In the current study, we characterize the metabolic network of T. fusca through reconstruction of a genome-scale metabolic model and proteomics data. The overall goal of this study was to use multiple metabolic models generated by different methods and comparison to experimental data to gain a high-confidence understanding of the T. fusca metabolic network.ResultsWe report the generation of three versions of a metabolic model of Thermobifida fusca sp. XY developed using three different approaches (automated, semi-automated, and proteomics-derived). The model closest to in vivo growth was the proteomics-derived model that consists of 975 reactions involving 1382 metabolites and account for 316 EC numbers (296 genes). The model was optimized for biomass production with the optimal flux of 0.48 doublings per hour when grown on cellobiose with a substrate uptake rate of 0.25 mmole/h. In vivo activity of the DXP pathway for terpenoid biosynthesis was also confirmed using real-time PCR.ConclusionsiTfu296 provides a platform to understand and explore the metabolic capabilities of the actinomycete T. fusca for the potential use in bioprocess industries for the production of biofuel and pharmaceutical ingredients. By comparing different model reconstruction methods, the use of high-throughput proteomics data as a starting point proved to be the most accurate to in vivo growth.

Combinatorial Engineering of 1-Deoxy-D-Xylulose 5-Phosphate Pathway Using Cross-Lapping In Vitro Assembly (CLIVA) Method

The ability to assemble multiple fragments of DNA into a plasmid in a single step is invaluable to studies in metabolic engineering and synthetic biology. Using phosphorothioate chemistry for high efficiency and site specific cleavage of sequences, a novel ligase independent cloning method (cross-lapping in vitro assembly, CLIVA) was systematically and rationally optimized in E. coli. A series of 16 constructs combinatorially expressing genes encoding enzymes in the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway were assembled using multiple DNA modules. A plasmid (21.6 kb) containing 16 pathway genes, was successfully assembled from 7 modules with high efficiency (2.0 x 103 cfu/ µg input DNA) within 2 days. Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene. Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC), an intermediate metabolite in the DXP pathway. The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene. This study demonstrated the utility of CLIVA in the assembly of multiple fragments of DNA into a plasmid which enabled the rapid exploration of biological pathways.

In silico profiling of Escherichia coli and Saccharomyces cerevisiae as terpenoid factories

BackgroundHeterologous microbial production of rare plant terpenoids of medicinal or industrial interest is attracting more and more attention but terpenoid yields are still low. Escherichia coli and Saccharomyces cerevisiae are the most widely used heterologous hosts; a direct comparison of both hosts based on experimental data is difficult though. Hence, the terpenoid pathways of E. coli (via 1-deoxy-D-xylulose 5-phosphate, DXP) and S. cerevisiae (via mevalonate, MVA), the impact of the respective hosts metabolism as well as the impact of different carbon sources were compared in silico by means of elementary mode analysis. The focus was set on the yield of isopentenyl diphosphate (IPP), the general terpenoid precursor, to identify new metabolic engineering strategies for an enhanced terpenoid yield.ResultsStarting from the respective precursor metabolites of the terpenoid pathways (pyruvate and glyceraldehyde-3-phosphate for the DXP pathway and acetyl-CoA for the MVA pathway) and considering only carbon stoichiometry, the two terpenoid pathways are identical with respect to carbon yield. However, with glucose as substrate, the MVA pathway has a lower potential to supply terpenoids in high yields than the DXP pathway if the formation of the required precursors is taken into account, due to the carbon loss in the formation of acetyl-CoA. This maximum yield is further reduced in both hosts when the required energy and reduction equivalents are considered. Moreover, the choice of carbon source (glucose, xylose, ethanol or glycerol) has an effect on terpenoid yield with non-fermentable carbon sources being more promising. Both hosts have deficiencies in energy and redox equivalents for high yield terpenoid production leading to new overexpression strategies (heterologous enzymes/pathways) for an enhanced terpenoid yield. Finally, several knockout strategies are identified using constrained minimal cut sets enforcing a coupling of growth to a terpenoid yield which is higher than any yield published in scientific literature so far.ConclusionsThis study provides for the first time a comprehensive and detailed in silico comparison of the most prominent heterologous hosts E. coli and S. cerevisiae as terpenoid factories giving an overview on several promising metabolic engineering strategies paving the way for an enhanced terpenoid yield.

Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production

Isoprenoids are natural products that are all derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These precursors are synthesized either by the mevalonate (MVA) pathway or the 1-Deoxy-D-Xylulose 5-Phosphate (DXP) pathway. Metabolic engineering of microbes has enabled overproduction of various isoprenoid products from the DXP pathway including lycopene, artemisinic acid, taxadiene and levopimaradiene. To date, there is no method to accurately measure all the DXP metabolic intermediates simultaneously so as to enable the identification of potential flux limiting steps. In this study, a solid phase extraction coupled with ultra performance liquid chromatography mass spectrometry (SPE UPLC-MS) method was developed. This method was used to measure the DXP intermediates in genetically engineered E. coli. Unexpectedly, methylerythritol cyclodiphosphate (MEC) was found to efflux when certain enzymes of the pathway were over-expressed, demonstrating the existence of a novel competing pathway branch in the DXP metabolism. Guided by these findings, ispG was overexpressed and was found to effectively reduce the efflux of MEC inside the cells, resulting in a significant increase in downstream isoprenoid production. This study demonstrated the necessity to quantify metabolites enabling the identification of a hitherto unrecognized pathway and provided useful insights into rational design in metabolic engineering.

Biosynthesis of artemisinin – revisited

Artemisinin is a well-known antimalarial drug isolated from the Artemisia annua plant. The biosynthesis of this well-known molecule has been reinvestigated by using [1-13C]acetate, [2-13C]acetate, and [1,6-13C2]glucose. The 13C peak enrichment in artemisinin was observed in six and nine carbon atoms from [1-13C]acetate and [2-13C]acetate, respectively. The 13C NMR spectra of 13C-enriched artemisinin suggested that the mevalonic acid (MVA) pathway is the predominant route to biosynthesis of this sesquiterpene. On the other hand, the peak enrichment of five carbons of 13C-artemisinin including carbon atoms originating from methyls of dimethylallyl group of geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) was observed from [1,6-13C2]glucose. This suggested that GPP which is supposed to be biosynthesized in plastids travels from plastids to cytosol through the plastidial wall and combines with isopentenyl pyrophosphate (IPP) to form the (E,E)-FPP which finally cyclizes and oxidizes to artemisinin. In this way the DXP pathway also contributes to the biosynthesis of this sesquiterpene.

In silico analysis and experimental improvement of taxadiene heterologous biosynthesis in Escherichia coli

The biosynthesis of terpenoids in heterologous hosts has become increasingly popular. Isopentenyl diphosphate (IPP) is the central precursor of all isoprenoids, and the synthesis can proceed via two separate pathways in different organisms: The 1-deoxylulose 5-phosphate (DXP) pathway and the mevalonate (MVA) pathway. In this study, an in silico comparison was made between the maximum theoretical IPP yields and the thermodynamic properties of the DXP and MVA pathways using different hosts and carbon sources. We found that Escherichia coli and its DXP pathway have the most potential for IPP production. Consequently, codon usage redesign, and combinations of chromosomal engineering and various strains were considered for optimizing taxadiene biosynthesis through the endogenic DXP pathway. A high production strain yielding 876 ± 60 mg/L taxadiene, with an overall volumetric productivity of 8.9 mg/(L × h), was successfully obtained by combining the chromosomal engineered upstream DXP pathway and the downstream taxadiene biosynthesis pathway. This is the highest yield thus far reported for taxadiene production in a heterologous host. These results indicate that genetic manipulation of the DXP pathway has great potential to be used for production of terpenoids, and that chromosomal engineering is a powerful tool for heterologous biosynthesis of natural products.

A genomics resource for investigating regulation of essential oil production in Lavandula angustifolia

We are developing Lavandula angustifolia (lavender) as a model system for investigating molecular regulation of essential oil (a mixture of mono- and sesquiterpenes) production in plants. As an initial step toward building the necessary 'genomics toolbox' for this species, we constructed two cDNA libraries from lavender leaves and flowers, and obtained sequence information for 14,213 high-quality expressed sequence tags (ESTs). Based on homology to sequences present in GenBank, our EST collection contains orthologs for genes involved in the 1-deoxy-D: -xylulose-5-phosphate (DXP) and the mevalonic acid (MVA) pathways of terpenoid biosynthesis, and for known terpene synthases and prenyl transferases. To gain insight into the regulation of terpene metabolism in lavender flowers, we evaluated the transcriptional activity of the genes encoding for 1-deoxy-D: -xylulose-5-phosphate synthase (DXS) and HMG-CoA reductase (HMGR), which represent regulatory steps of the DXP and MVA pathways, respectively, in glandular trichomes (oil glands) by real-time PCR. While HMGR transcripts were barely detectable, DXS was heavily expressed in this tissue, indicating that essential oil constituents are predominantly produced through the DXP pathway in lavender glandular trichomes. As anticipated, the linalool synthase (LinS)-the gene responsible for the production of linalool, a major constituent of lavender essential oil-was also strongly expressed in glands. Surprisingly, the most abundant transcript in floral glandular trichomes corresponded to a sesquiterpene synthase (cadinene synthase, CadS), although sesquiterpenes are minor constituents of lavender essential oils. This result, coupled to the weak activity of the MVA pathway (the main route for sesquiterpene production) in trichomes, indicates that precursor supply may represent a bottleneck in the biosynthesis of sesquiterpenes in lavender flowers.

Carotenoids of Gemmatimonas aurantiaca (Gemmatimonadetes): identification of a novel carotenoid, deoxyoscillol 2-rhamnoside, and proposed biosynthetic pathway of oscillol 2,2′-dirhamnoside

Gemmatimonas aurantiaca strain T-27(T) is an orange-coloured, Gram-negative, facultatively aerobic, polyphosphate-accumulating bacterium belonging to a recently proposed phylum, Gemmatimonadetes. We purified its pigments and identified them as carotenoids and their glycoside derivatives using spectral data. The major carotenoid was (2S,2' S)-oscillol 2,2'-di-(alpha-l-rhamnoside), and the minor carotenoids were (2S)-deoxyoscillol 2-( alpha-l-rhamnoside) and didemethylspirilloxanthin. Deoxyoscillol 2-rhamnoside is a novel carotenoid. Oscillol 2,2'-diglycosides have hitherto only been reported in a limited number of cyanobacteria, and this is believed to be the first finding of such carotenoids in another bacterial phylum. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence, we propose a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. We propose the involvement of geranylgeranyl pyrophosphate synthase (CrtE), phytoene synthase (CrtB) and phytoene desaturase (CrtI) for lycopene synthesis; and of carotenoid 1,2-hydratase (CruF) and carotenoid 2-O-rhamnosyltransferase (CruG) for oscillol 2,2'-dirhamnoside synthesis. Further, isopentenyl pyrophosphate could be synthesized by a non-mevalonate pathway (DXP pathway).

Programming cells by multiplex genome engineering and accelerated evolution

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.

A Novel Pathway for Sesquiterpene Biosynthesis fromZ,Z-Farnesyl Pyrophosphate in the Wild TomatoSolanum habrochaites

In the wild tomato Solanum habrochaites, the Sst2 locus on chromosome 8 is responsible for the biosynthesis of several class II sesquiterpene olefins by glandular trichomes. Analysis of a trichome-specific EST collection from S. habrochaites revealed two candidate genes for the synthesis of Sst2-associated sesquiterpenes. zFPS encodes a protein with homology to Z-isoprenyl pyrophosphate synthases and SBS (for Santalene and Bergamotene Synthase) encodes a terpene synthase with homology to kaurene synthases. Both genes were found to cosegregate with the Sst2 locus. Recombinant zFPS protein catalyzed the synthesis of Z,Z-FPP from isopentenylpyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP), while coincubation of zFPS and SBS with the same substrates yielded a mixture of olefins identical to the Sst2-associated sesquiterpenes, including (+)-alpha-santalene, (+)-endo-beta-bergamotene, and (-)-endo-alpha-bergamotene. In addition, headspace analysis of tobacco (Nicotiana sylvestris) plants expressing zFPS and SBS in glandular trichomes afforded the same mix of sesquiterpenes. Each of these proteins contains a putative plastid targeting sequence that mediates transport of a fused green fluorescent protein to the chloroplasts, suggesting that the biosynthesis of these sesquiterpenes uses IPP and DMAPP from the plastidic DXP pathway. These results provide novel insights into sesquiterpene biosynthesis and have general implications concerning sesquiterpene engineering in plants.

Quantitative 2H NMR spectroscopy

The selectivity of deuterium distribution between the nonequivalent positions in 3-carene (1), 4-α-acetyl-2-carene (2), and 4-(1-hydroxyethyl)-2-carene (3) has been measured by 2H-{1H} NMR spectroscopy at the natural abundance of deuterium. These “H/D-isotope portraits” were shown to be typical of terpenes and terpenoids produced in plants via the biosynthetic DXP pathway. The mechanism of acylation of 1 was studied by the density functional theory method (PBE functional, TZ2p basis set). The six-membered ring in compound 1 is planar. However, the endo attack of electrophiles on this ring is more favorable both kinetically and thermodynamically. It was shown both experimentally and theoretically that the elimination of a hydrogen atom in the second reaction step proceeds stereoselectively at the C(2) atom from the anti position with respect to the three-membered ring and occurs with pronounced nucleophilic assistance from the carbonyl group.

Mechanistic Studies of IspH in the Deoxyxylulose Phosphate Pathway: Heterolytic C−O Bond Cleavage at C4 Position

Isoprenoids are one of the largest and most structurally diverse groups of metabolites in nature. their biosyntheses require two precursors, isopentenyl diphosphate (ipp) and its isomer, dimethylallyl diphosphate (dmapp). there are two different pathways for the synthesis of ipp and dmapp: the deoxyxylulose phosphate (dxp) pathway and the mevalonic acid (mva) pathway. more importantly, these two pathways have a well-defined distribution among different kingdoms. most pathogenic bacteria and protozoan parasites utilize the dxp pathway, while animals synthesize their isoprenoid precursors from acety-coa via the mva pathway. plants have both dxp and mva pathways. thus, mechanistic studies on the dxp pathway enzymes may lead to the development of mechanism-based inhibitors as herbicides, broad-spectrum antibiotics, and antimalaria drugs. isph in the dxp pathway catalyzes the reductive dehydration of (e)-4-hydroxy-3-metho-2-butenyl diphosphate (hmbpp) to form ipp and dmapp, the last step in the dxp pathway. recent epr studies reported in literature suggest that isph is a unique iron-site-containing [4fe-4s] protein. in this study, we studied the isph-catalyzed reductive dehydration mechanism using two substrate analogues. our data reported herein provide evidence to not only support the integrity of the c1 position c-o bond during reaction, they also suggest a heterolytic c-o bond cleavage at the c4 position for isph-catatyzed reductive dehydration reaction. our kinetic studies also suggest that the c4 hydroxyl group is involved in substrate binding. because the isph-catalyzed reductive dehydration reaction does not fall into the two known classes of unique iron-site-containing [4fe-4s] proteins, aconitase-type and radical sam-type enzymes, isph may represent a new class of iron-sulfur-containing proteins.