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Jay D Keasling - One of the best experts on this subject based on the ideXlab platform.
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Isoprenoid Drugs, Biofuels, and Chemicals—Artemisinin, Farnesene, and Beyond
Advances in Biochemical Engineering \ Biotechnology, 2015Co-Authors: Kevin W George, Jorge Alonso-gutierrez, Jay D KeaslingAbstract:: Isoprenoids have been identified and used as natural pharmaceuticals, fragrances, solvents, and, more recently, advanced biofuels. Although Isoprenoids are most commonly found in plants, researchers have successfully engineered both the eukaryotic and prokaryotic Isoprenoid biosynthetic pathways to produce these valuable chemicals in microorganisms at high yields. The microbial synthesis of the precursor to artemisinin--an important antimalarial drug produced from the sweet wormwood Artemisia annua--serves as perhaps the most successful example of this approach. Through advances in synthetic biology and metabolic engineering, microbial-derived semisynthetic artemisinin may soon replace plant-derived artemisinin as the primary source of this valuable pharmaceutical. The richness and diversity of Isoprenoid structures also make them ideal candidates for advanced biofuels that may act as "drop-in" replacements for gasoline, diesel, and jet fuel. Indeed, the sesquiterpenes farnesene and bisabolene, monoterpenes pinene and limonene, and hemiterpenes isopentenol and isopentanol have been evaluated as fuels or fuel precursors. As in the artemisinin project, these Isoprenoids have been produced microbially through synthetic biology and metabolic engineering efforts. Here, we provide a brief review of the numerous Isoprenoid compounds that have found use as pharmaceuticals, flavors, commodity chemicals, and, most importantly, advanced biofuels. In each case, we highlight the metabolic engineering strategies that were used to produce these compounds successfully in microbial hosts. In addition, we present a current outlook on microbial Isoprenoid production, with an eye towards the many challenges that must be addressed to achieve higher yields and industrial-scale production.
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Isoprenoid drugs biofuels and chemicals artemisinin farnesene and beyond
Advances in Biochemical Engineering \ Biotechnology, 2015Co-Authors: Jay D Keasling, Kevin W George, Jorge AlonsogutierrezAbstract:: Isoprenoids have been identified and used as natural pharmaceuticals, fragrances, solvents, and, more recently, advanced biofuels. Although Isoprenoids are most commonly found in plants, researchers have successfully engineered both the eukaryotic and prokaryotic Isoprenoid biosynthetic pathways to produce these valuable chemicals in microorganisms at high yields. The microbial synthesis of the precursor to artemisinin--an important antimalarial drug produced from the sweet wormwood Artemisia annua--serves as perhaps the most successful example of this approach. Through advances in synthetic biology and metabolic engineering, microbial-derived semisynthetic artemisinin may soon replace plant-derived artemisinin as the primary source of this valuable pharmaceutical. The richness and diversity of Isoprenoid structures also make them ideal candidates for advanced biofuels that may act as "drop-in" replacements for gasoline, diesel, and jet fuel. Indeed, the sesquiterpenes farnesene and bisabolene, monoterpenes pinene and limonene, and hemiterpenes isopentenol and isopentanol have been evaluated as fuels or fuel precursors. As in the artemisinin project, these Isoprenoids have been produced microbially through synthetic biology and metabolic engineering efforts. Here, we provide a brief review of the numerous Isoprenoid compounds that have found use as pharmaceuticals, flavors, commodity chemicals, and, most importantly, advanced biofuels. In each case, we highlight the metabolic engineering strategies that were used to produce these compounds successfully in microbial hosts. In addition, we present a current outlook on microbial Isoprenoid production, with an eye towards the many challenges that must be addressed to achieve higher yields and industrial-scale production.
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carotenoid based phenotypic screen of the yeast deletion collection reveals new genes with roles in Isoprenoid production
Metabolic Engineering, 2013Co-Authors: Bilge Ozaydin, Helcio Burd, Jay D KeaslingAbstract:Abstract Beside their essential cellular functions, Isoprenoids have value as pharmaceuticals, nutriceuticals, pesticides, and fuel alternatives. Engineering microorganisms for production of Isoprenoids is relatively easy, sustainable, and cost effective in comparison to chemical synthesis or extraction from natural producers. We introduced genes encoding carotenoid biosynthetic enzymes into the haploid yeast deletion collection to identify gene deletions that improved Isoprenoid production. Deletions that showed significant improvement in carotenoid production were further screened for production of bisabolene, an Isoprenoid alternative to petroleum-derived diesel. Combining those deletions with other mevalonate pathway modifications increased production of bisabolene from 40 mg/L to 800 mg/L in shake-flask cultures. In a fermentation process, this engineered strain produced 5.2 g/L of bisabolene.
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biosynthesis of plant Isoprenoids perspectives for microbial engineering
Annual Review of Plant Biology, 2009Co-Authors: James P Kirby, Jay D KeaslingAbstract:Isoprenoids are a large and highly diverse group of natural products with many functions in plant primary and secondary metabolism. Isoprenoids are synthesized from common prenyl diphosphate precursors through the action of terpene synthases and terpene-modifying enzymes such as cytochrome P450 monooxygenases. Many Isoprenoids have important applications in areas such as human health and nutrition, and much effort has been directed toward their production in microbial hosts. However, many hurdles must be overcome in the elucidation and functional microbial expression of the genes responsible for biosynthesis of an Isoprenoid of interest. Here, we review investigations into Isoprenoid function and gene discovery in plants as well as the latest advances in Isoprenoid pathway engineering in both plant and microbial hosts.
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metabolic engineering of microorganisms for Isoprenoid production
Natural Product Reports, 2008Co-Authors: James P Kirby, Jay D KeaslingAbstract:Covering: up to the end of 2007 Isoprenoids are ubiquitous in nature and range from essential cell components to unique secondary metabolites. The two Isoprenoid biosynthetic pathways have received much attention from a metabolic engineering standpoint, and significant advances have been made in increasing flux through these pathways. Engineering later steps in Isoprenoid biosynthetic pathways, specifically those related to the functionalization of terpene backbones, is at an earlier stage of development, both in terms of gene discovery and heterologous expression. Here we review recent advances in the metabolic engineering of microbes for Isoprenoid production as well as some novel approaches to gene discovery and expression.
Gregory Stephanopoulos - One of the best experts on this subject based on the ideXlab platform.
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Protein engineering strategies for microbial production of Isoprenoids.
Metabolic Engineering Communications, 2020Co-Authors: Georgios Daletos, Gregory StephanopoulosAbstract:Abstract Isoprenoids comprise one of the most chemically diverse family of natural products with high commercial interest. The structural diversity of Isoprenoids is mainly due to the modular activity of three distinct classes of enzymes, including prenyl diphosphate synthases, terpene synthases, and cytochrome P450s. The heterologous expression of these enzymes in microbial systems is suggested to be a promising sustainable way for the production of Isoprenoids. Several limitations are associated with native enzymes, such as low stability, activity, and expression profiles. To address these challenges, protein engineering has been applied to improve the catalytic activity, selectivity, and substrate turnover of enzymes. In addition, the natural promiscuity and modular fashion of Isoprenoid enzymes render them excellent targets for combinatorial studies and the production of new-to-nature metabolites. In this review, we discuss key individual and multienzyme level strategies for the successful implementation of enzyme engineering towards efficient microbial production of high-value Isoprenoids. Challenges and future directions of protein engineering as a complementary strategy to metabolic engineering are likewise outlined.
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Novel Strategies and Platforms for Industrial Isoprenoid Engineering.
Trends in Biotechnology, 2020Co-Authors: Georgios Daletos, Constantinos Katsimpouras, Gregory StephanopoulosAbstract:Metabolic engineering has emerged as an important tool for reconstructing heterologous Isoprenoid metabolic pathways in microbial hosts. Here, we provide an overview of promising engineering strategies that have proven to be successful for the high-yield production of Isoprenoids. Besides ‘conventional’ approaches, such as the ‘push–pull’ and protein engineering to optimize the Isoprenoid flux and limited catalytic activity of enzymes, we review emerging strategies in the field, including compartmentalization between synthetic consortia members, novel bypass pathways for Isoprenoid synthesis, cell-free systems, and improvement of the lipid content to overcome storage Isoprenoid limitations. Pitfalls, along with lessons learned from the application of these strategies, will be addressed with the hope of guiding future efforts toward cost-effective and sustainable production of Isoprenoids.
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two step pathway for Isoprenoid synthesis
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Alkiviadis Orfefs Chatzivasileiou, Valerie C A Ward, Steven Edgar, Gregory StephanopoulosAbstract:Isoprenoids comprise a large class of chemicals of significant interest due to their diverse properties. Biological production of Isoprenoids is considered to be the most efficient way for their large-scale production. Isoprenoid biosynthesis has thus far been dependent on pathways inextricably linked to glucose metabolism. These pathways suffer from inherent limitations due to their length, complex regulation, and extensive cofactor requirements. Here, we present a synthetic Isoprenoid pathway that aims to overcome these limitations. This isopentenol utilization pathway (IUP) can produce isopentenyl diphosphate or dimethylallyl diphosphate, the main precursors to Isoprenoid synthesis, through sequential phosphorylation of isopentenol isomers isoprenol or prenol. After identifying suitable enzymes and constructing the pathway, we attempted to probe the limits of the IUP for producing various Isoprenoid downstream products. The IUP flux exceeded the capacity of almost all downstream pathways tested and was competitive with the highest Isoprenoid fluxes reported.
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Engineering Microbes to Synthesize Plant Isoprenoids.
Methods in Enzymology, 2016Co-Authors: Kang Zhou, Steven Edgar, Gregory StephanopoulosAbstract:Abstract Humans constantly look for faster, more economical, and more sustainable ways to produce chemicals that originally harvested from nature. Over the past two decades, substantial progress has been made toward this goal by harnessing enzymes and cells as biocatalysts. For example, enzymes of slow-growing plants can be reconstituted in microbes, which empower them with the ability to produce useful plant metabolic compounds from sugars faster than plants. In this chapter, we provide protocols for producing Isoprenoids – a large group of useful natural products – in microbes. It has been found that expression of genes encoding plant enzymes and selected endogenous genes must be delicately adjusted in microbes, otherwise Isoprenoid production is negatively affected. Therefore, we focus on how to balance gene expression in Escherichia coli and use process engineering to increase its Isoprenoid production. We also introduce our recent work on the use of microbial consortia and provide protocols for using yeast to help E . coli functionalize its Isoprenoid product. Together, the methods and protocols provided here should be useful to researchers who aim to use microbes to synthesize novel Isoprenoids.
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metabolite profiling identified methylerythritol cyclodiphosphate efflux as a limiting step in microbial Isoprenoid production
PLOS ONE, 2012Co-Authors: Kang Zhou, Gregory StephanopoulosAbstract: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.
Michel Rohmer - One of the best experts on this subject based on the ideXlab platform.
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the discovery of a mevalonate independent pathway for Isoprenoid biosynthesis in bacteria algae and higher plants
Natural Product Reports, 1999Co-Authors: Michel RohmerAbstract:1 Isoprenoid biosynthesis 1.1 The mevalonate route to isopentenyl diphosphate 1.2 Isoprenoid biosynthesis in higher plants: some contradictions with the mevalonate pathway 2 The discovery of the mevalonate-independent pathway 2.1 The origin of the discovery: the biosynthesis of bacterial hopanoids 2.2 The origin of the carbon atoms of isoprenic units in the mevalonate-independent pathway 2.3 d-Glyceraldehyde 3-phosphate and pyruvate as the first precursors of isopentenyl diphosphate 3 Towards the identification of intermediates and enzymes of the new pathway 3.1 1-Deoxy-d-xylulose 5-phosphate and 1-deoxy-d-xylulose 5-phosphate synthase 3.2 2-C-Methyl-d-erythritol 4-phosphate and 1-deoxy-d-xylulose 5-phosphate reducto-isomerase 4 The distribution of the glyceraldehyde 3-phosphate/pyruvate pathway amongst prokaryotes 5 The distribution of the GAP/pyruvate pathway amongst phototrophic eukaryotes 5.1 Essential plant chloroplast Isoprenoids and sterols from green algae 5.2 Isoprenoids from secondary metabolism 5.3 Intermediate exchanges between the mevalonate and the GAP/pyruvate pathways in plants 6 Conclusion 7 Acknowledgments 8 References
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distribution of the mevalonate and glyceraldehyde phosphate pyruvate pathways for Isoprenoid biosynthesis in unicellular algae and the cyanobacterium synechocystis pcc 6714
Biochemical Journal, 1998Co-Authors: Andrea Disch, Jorg Schwender, Hartmut K Lichtenthaler, Christian Muller, Michel RohmerAbstract:Isopentenyl diphosphate, the universal Isoprenoid precursor, can be produced by two different biosynthetic routes: either via the acetate/mevalonate (MVA) pathway, or via the more recently identified MVA-independent glyceraldehyde phosphate/pyruvate pathway. These two pathways are easily differentiated by incorporation of [1- 13 C]glucose and analysis of the resulting labelling patterns found in the Isoprenoids. This method was successfully applied to several unicellular algae raised under heterotrophic growth conditions and allowed for the identification of the pathways that were utilized for Isoprenoid biosynthesis. All Isoprenoids examined (sterols, phytol, carotenoids) of the green algae Chlorella fusca and Chlamydomonas reinhardtii were synthesized via the GAP/pyruvate pathway, as in another previously investigated green alga, Scenedesmus obliquus , which was also shown in this study to synthesize ubiquinone by the same MVA-independent route. In the red alga Cyanidium caldarium and in the Chrysophyte Ochromonas danica a clear dichotomy was observed: as in higher plants, sterols were formed via the MVA route, whereas chloroplast Isoprenoids (phytol in Cy. caldarium and O. danica and β-carotene in O. danica ) were synthesized via the GAP/pyruvate route. In contrast, the Euglenophyte Euglena gracilis synthesized ergosterol, as well as phytol, via the acetate/MVA route. Similar feeding experiments were performed with the cyanobacterium Synechocystis PCC 6714 using [1- 13 C]- and [6- 13 C]-glucose. The two Isoprenoids examined, phytol and β-carotene, were shown to have the typical labelling pattern derived from the GAP/pyruvate route.
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biosynthesis of Isoprenoids in higher plant chloroplasts proceeds via a mevalonate independent pathway
FEBS Letters, 1997Co-Authors: Hartmut K Lichtenthaler, Jorg Schwender, Andrea Disch, Michel RohmerAbstract:Isopentenyl diphosphate (IPP) is the biological C5 precursor of Isoprenoids. By labeling experiments using [1-13C]glucose, higher plants were shown to possess two distinct biosynthetic routes for IPP biosynthesis: while the cytoplasmic sterols were formed via the acetate/mevalonate pathway, the chloroplast-bound Isoprenoids (β-carotene, lutein, prenyl chains of chlorophylls and plastoquinone-9) were synthesized via a novel IPP biosynthesis pathway (glyceraldehyde phosphate/pyruvate pathway) which was first found in eubacteria and a green alga. The dichotomy in Isoprenoid biosynthesis in higher plants allows a reasonable interpretation of previous odd and inconclusive results concerning the biosynthesis of chloroplast Isoprenoids, which so far had mainly been interpreted in the frame of models using compartmentation of the mevalonate pathway.
Wilhelm Gruissem - One of the best experts on this subject based on the ideXlab platform.
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network analysis of the mva and mep pathways for Isoprenoid synthesis
Annual Review of Plant Biology, 2013Co-Authors: Eva Vranova, Diana Coman, Wilhelm GruissemAbstract:Isoprenoid biosynthesis is essential for all living organisms, and Isoprenoids are also of industrial and agricultural interest. All Isoprenoids are derived from prenyl diphosphate (prenyl-PP) precursors. Unlike Isoprenoid biosynthesis in other living organisms, prenyl-PP, as the precursor of all Isoprenoids in plants, is synthesized by two independent pathways: the mevalonate (MVA) pathway in the cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. This review focuses on progress in our understanding of how the precursors for Isoprenoid biosynthesis are synthesized in the two subcellular compartments, how the underlying pathway gene networks are organized and regulated, and how network perturbations impact each pathway and plant development. Because of the wealth of data on Isoprenoid biosynthesis, we emphasize research in Arabidopsis thaliana and compare the synthesis of Isoprenoid precursor molecules in this model plant with their synthesis in other prokaryotic and eukaryotic organisms.
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structure and dynamics of the Isoprenoid pathway network
Molecular Plant, 2012Co-Authors: Eva Vranova, Diana Coman, Wilhelm GruissemAbstract:Isoprenoids are functionally and structurally the most diverse group of plant metabolites reported to date. They can function as primary metabolites, participating in essential plant cellular processes, and as secondary metabolites, of which many have substantial commercial, pharmacological, and agricultural value. Isoprenoid end products participate in plants in a wide range of physiological processes acting in them both synergistically, such as chlorophyll and carotenoids during photosynthesis, or antagonistically, such as gibberellic acid and abscisic acid during seed germination. It is therefore expected that fluxes via Isoprenoid metabolic network are tightly controlled both temporally and spatially, and that this control occurs at different levels of regulation and in an orchestrated manner over the entire Isoprenoid metabolic network. In this review, we summarize our current knowledge of the topology of the plant Isoprenoid pathway network and its regulation at the gene expression level following diverse stimuli. We conclude by discussing agronomical and biotechnological applications emerging from the plant Isoprenoid metabolism and provide an outlook on future directions in the systems analysis of the plant Isoprenoid pathway network.
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AtIPD: A Curated Database of Arabidopsis Isoprenoid Pathway Models and Genes for Isoprenoid Network Analysis
Plant Physiology, 2011Co-Authors: Eva Vranova, Matthias Hirsch-hoffmann, Wilhelm GruissemAbstract:Isoprenoid biosynthesis is one of the essential metabolic pathways in plants and other organisms. Despite the importance of Isoprenoids for plant functions, not much is known about the regulation of Isoprenoid synthesis. Quantitative technologies and systems approaches are now increasingly used to investigate the regulation of metabolic pathways and networks. Prerequisite for systems approaches is the knowledge of network elements and topologies. Information that can be extracted from the public metabolic pathway databases such as AraCyc and KEGG is often not sufficiently comprehensive and current. Therefore we have built a database of manually curated Isoprenoid pathway models and genes, the Arabidopsis thaliana Isoprenoid Pathway Database (AtIPD; http://www.atipd.ethz.ch). The database was compiled using information on pathways and pathway genes from BioPathAt (Lange and Ghassemian, 2003, 2005), KEGG (http://www.genome.jp/kegg), AraCyc (http://www.arabidopsis.org/biocyc), SUBA (http://suba.plantenergy.uwa.edu.au), and from the literature. AtIPD can be searched or browsed to extract data and external links related to Isoprenoid pathway models, enzyme activities or subcellular enzyme localizations. To display quantitative gene-related data on curated pathway models, we created image annotation and mapping files for integrated use with the MapMan tool (http://mapman.gabipd.org/web/guest/mapman). Additionally, we built SBML XML files of the Isoprenoid pathway images using the Cell DesignerTM tool (http://www.celldesigner.org). Users can download all image and annotation files for customization, e.g., adding pathway structural and regulatory network elements or modifying pathway images to visualize other quantitative protein or metabolite data. AtIPD therefore represents a valuable resource for Isoprenoid network analysis.
Norihiko Misawa - One of the best experts on this subject based on the ideXlab platform.
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Novel approaches and achievements in biosynthesis of functional Isoprenoids in Escherichia coli
Applied Microbiology and Biotechnology, 2009Co-Authors: Hisashi Harada, Norihiko MisawaAbstract:Isoprenoids, also referred to as terpenes, are the most diverse class of natural products appearing in a variety of natural sources, specifically in higher plants, and have a wide range of biological functions. This review describes novel or recent approaches and achievements in pathway engineering of Escherichia coli towards efficient biosynthesis of functional Isoprenoids, specifically carotenoids and sesquiterpene, following description of “regularity and simplicity” in the biosynthesis of Isoprenoid basic structures. The introduction of heterologous mevalonate pathway-based genes into E . coli has been shown to improve the productivity of carotenoids or sesquiterpenes that are synthesized from farnesyl diphosphate. This achievement also enables relevant researchers to efficiently analyze an isolated gene candidate for a terpene synthase (terpene cyclase).
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expression of an exogenous isopentenyl diphosphate isomerase gene enhances Isoprenoid biosynthesis in escherichia coli
Biochemical Journal, 1997Co-Authors: Susumu Kajiwara, Paul D Fraser, Keiji Kondo, Norihiko MisawaAbstract:Escherichia coli expressing the Erwinia carotenoid biosynthesis genes, crtE, crtB, crtI and crtY, form yellow-coloured colonies due to the presence of beta-carotene. This host was used as a visible marker for evaluating regulatory systems operating in Isoprenoid biosynthesis of E. coli. cDNAs enhancing carotenoid levels were isolated from the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. Nucleotide sequence analysis indicated that they coded for proteins similar to isopentenyl diphosphate (IPP) isomerase of the yeast Saccharomyces cerevisiae. Determination of enzymic activity confirmed the identity of the gene products as IPP isomerases. The corresponding gene was isolated from the genomic library of S. cerevisiae based on its nucleotide sequence, and was confirmed to have the same effect as the above two IPP isomerase genes when introduced into the E. coli transformant accumulating beta-carotene. In the three E. coli strains carrying the individual exogenous IPP isomerase genes, the increases in carotenoid levels are comparable to the increases in IPP isomerase enzyme activity with reference to control strains possessing the endogenous gene alone. These results imply that IPP isomerase forms an influential step in Isoprenoid biosynthesis of the prokaryote E. coli, with potential for the efficient production of industrially useful Isoprenoids by metabolic engineering.
