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1.  A modular approach for facile biosynthesis of labdane-related diterpenes 
Labdane-related diterpenoids are a large group of over 5,000 natural products whose biosynthesis typically proceeds through a labdadienyl/copalyl diphosphate (CPP) intermediate to a further cyclized and/or rearranged hydrocarbon diterpene en route to more elaborated compounds. Here we report a modular approach for facile biosynthesis of labdane-related diterpenes wherein base pGGxC vectors capable of introducing bacterial production of any one of the three common stereoisomers of CPP can be co-introduced with diterpene synthases that convert these CPP intermediates to specific diterpene hydrocarbon skeletal structures. The utility of this approach is demonstrated by individually engineering E. coli to produce any one of eight different diterpene skeletal structures, which collectively serve as precursors to literally thousands of distinct natural products.
doi:10.1021/ja071158n
PMCID: PMC2518946  PMID: 17480080
2.  Biosynthesis of β-carotene in engineered E. coli using the MEP and MVA pathways 
Microbial Cell Factories  2014;13(1):160.
Background
β-carotene is a carotenoid compound that has been widely used not only in the industrial production of pharmaceuticals but also as nutraceuticals, animal feed additives, functional cosmetics, and food colorants. Currently, more than 90% of commercial β-carotene is produced by chemical synthesis. Due to the growing public concern over food safety, the use of chemically synthesized β-carotene as food additives or functional cosmetic agents has been severely controlled in recent years. This has reignited the enthusiasm for seeking natural β-carotene in large-scale fermentative production by microorganisms.
Results
To increase β-carotene production by improving the isopentenyl pyrophosphate (IPP) and geranyl diphospate (GPP) concentration in the cell, the optimized MEP (methylerythritol 4-phosphate) pathway containing 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and isopentenyl pyrophosphate isomerase (FNI) from Bacillus subtilis, geranyl diphosphate synthase (GPPS2) from Abies grandis have been co-expressed in an engineered E. coli strain. To further enhance the production of β-carotene, the hybrid MVA (mevalonate) pathway has been introduced into an engineered E. coli strain, co-expressed with the optimized MEP pathway and GPPS2. The final genetically modified strain, YJM49, can accumulate 122.4±6.2 mg/L β-carotene in flask culture, approximately 113-fold and 1.7 times greater than strain YJM39, which carries the native MEP pathway, and YJM45, which harbors the MVA pathway and the native MEP pathway, respectively. Subsequently, the fermentation process was optimized to enhance β-carotene production with a maximum titer of 256.8±10.4 mg/L. Finally, the fed-batch fermentation of β-carotene was evaluated using the optimized culture conditions. After induction for 56 h, the final engineered strain YJM49 accumulated 3.2 g/L β-carotene with a volumetric productivity of 0.37 mg/(L · h · OD600) in aerobic fed-batch fermentation, and the conversion efficiency of glycerol to β-carotene (gram to gram) reached 2.76%.
Conclusions
In this paper, by using metabolic engineering techniques, the more efficient biosynthetic pathway of β-carotene was successfully assembled in E. coli BL21(DE3) with the optimized MEP (methylerythritol 4-phosphate) pathway, the gene for GPPS2 from Abies grandis, the hybrid MVA (mevalonate) pathway and β-carotene synthesis genes from Erwinia herbicola.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-014-0160-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12934-014-0160-x
PMCID: PMC4239400  PMID: 25403509
β-carotene; MEP pathway; MVA pathway; E. coli
3.  MEP pathway-mediated isopentenol production in metabolically engineered Escherichia coli 
Microbial Cell Factories  2014;13(1):135.
Background
Isopentenols, such as prenol and isoprenol, are promising advanced biofuels because of their higher energy densities and better combustion efficiencies compared with ethanol. Microbial production of isopentenols has been developed recently via metabolically engineered E. coli. However, current yields remain low and the underlying pathways require systematic optimization.
Results
In this study, we targeted the E. coli native 2-methyl-(D)-erythritol-4-phosphate (MEP) pathway and its upstream glycolysis pathway for the optimization of isopentenol production. Two codon optimized genes, nudF and yhfR from Bacillus subtilis, were synthesized and expressed in E. coli W3110 to confer the isopentenol production of the strain. Two key enzymes (IspG and Dxs) were then overexpressed to optimize the E. coli native MEP pathway, which led to a significant increase (3.3-fold) in isopentenol production. Subsequently, the glycolysis pathway was tuned to enhance the precursor and NADPH supplies for the MEP pathway by activating the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (ED), which resulted in additional 1.9 folds of increase in isopentenol production. A 5 L-scale batch cultivation experiment was finally implemented, showing a total of 61.9 mg L−1 isopentenol production from 20 g L−1 of glucose.
Conclusion
The isopentenol production was successfully increased through multi-step optimization of the MEP and its upstream glycolysis pathways. It demonstrated that the total fluxes and their balance of the precursors of the MEP pathway are of critical importance in isopentenol production. In the future, an elucidation of the contribution of PPP and ED to MEP is needed for further optimization of isopentenol production.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-014-0135-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12934-014-0135-y
PMCID: PMC4172795  PMID: 25212876
Isopentenol; MEP pathway; Entner-Doudoroff pathway; Pentose phosphate pathway (PPP); Escherichia coli; Metabolic engineering
4.  Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of astaxanthin 
Background
The xanthophyll astaxanthin is a high-value compound with applications in the nutraceutical, cosmetic, food, and animal feed industries. Besides chemical synthesis and extraction from naturally producing organisms like Haematococcus pluvialis, heterologous biosynthesis in non-carotenogenic microorganisms like Escherichia coli, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of E. coli strains have led to a significant increase in the productivity of carotenoids like lycopene or β-carotene by increasing the metabolic flux towards the isoprenoid precursors. For the heterologous biosynthesis of astaxanthin in E. coli, however, the conversion of β-carotene to astaxanthin is obviously the most critical step towards an efficient biosynthesis of astaxanthin.
Results
Here we report the construction of the first plasmid-free E. coli strain that produces astaxanthin as the sole carotenoid compound with a yield of 1.4 mg/g cdw (E. coli BW-ASTA). This engineered E. coli strain harbors xanthophyll biosynthetic genes from Pantoea ananatis and Nostoc punctiforme as individual expression cassettes on the chromosome and is based on a β-carotene-producing strain (E. coli BW-CARO) recently developed in our lab. E. coli BW-CARO has an enhanced biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) and produces β-carotene in a concentration of 6.2 mg/g cdw. The expression of crtEBIY along with the β-carotene-ketolase gene crtW148 (NpF4798) and the β-carotene-hydroxylase gene (crtZ) under controlled expression conditions in E. coli BW-ASTA directed the pathway exclusively towards the desired product astaxanthin (1.4 mg/g cdw).
Conclusions
By using the λ-Red recombineering technique, genes encoding for the astaxanthin biosynthesis pathway were stably integrated into the chromosome of E. coli. The expression levels of chromosomal integrated recombinant biosynthetic genes were varied and adjusted to improve the ratios of carotenoids produced by this E. coli strain. The strategy presented, which combines chromosomal integration of biosynthetic genes with the possibility of adjusting expression by using different promoters, might be useful as a general approach for the construction of stable heterologous production strains synthesizing natural products. This is the case especially for heterologous pathways where excessive protein overexpression is a hindrance.
doi:10.1186/1475-2859-10-29
PMCID: PMC3111352  PMID: 21521516
astaxanthin; chromosomal integration; E. coli
5.  High-level diterpene production by transient expression in Nicotiana benthamiana 
Plant Methods  2013;9:46.
Background
Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.
Results
Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2.
Conclusion
With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.
doi:10.1186/1746-4811-9-46
PMCID: PMC3878842  PMID: 24330621
Diterpene synthase; Agrobacterium; Transient protein expression; Nicotiana benthamiana
6.  Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids 
Current Organic Chemistry  2014;18(8):1050-1072.
Isoprenoids constitute the largest class of natural products with greater than 55,000 identified members. They play essential roles in maintaining proper cellular function leading to maintenance of human health, plant defense mechanisms against predators, and are often exploited for their beneficial properties in the pharmaceutical and nutraceutical industries. Most impressively, all known isoprenoids are derived from one of two C5-precursors, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). In order to study the enzyme transformations leading to the extensive structural diversity found within this class of compounds there must be access to the substrates. Sometimes, intermediates within a biological pathway can be isolated and used directly to study enzyme/pathway function. However, the primary route to most of the isoprenoid intermediates is through chemical catalysis. As such, this review provides the first exhaustive examination of synthetic routes to isoprenoid and isoprenoid precursors with particular emphasis on the syntheses of intermediates found as part of the 2C-methylerythritol 4-phosphate (MEP) pathway. In addition, representative syntheses are presented for the monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Finally, in some instances, the synthetic routes to substrate analogs found both within the MEP pathway and downstream isoprenoids are examined.
doi:10.2174/1385272819666140501001101
PMCID: PMC4082188  PMID: 25009443
Enzyme mechanism; isoprenoids; terpenes; MEP pathway.
7.  Different Roles of the Mevalonate and Methylerythritol Phosphate Pathways in Cell Growth and Tanshinone Production of Salvia miltiorrhiza Hairy Roots 
PLoS ONE  2012;7(11):e46797.
Salvia miltiorrhiza has been widely used in the treatment of coronary heart disease. Tanshinones, a group of diterpenoids are the main active ingredients in S. miltiorrhiza. Two biosynthetic pathways were involved in tanshinone biosynthesis in plants: the mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in the plastids. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme of the MVA pathway. The 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) are the key enzymes of the MEP pathway. In this study, to reveal roles of the MVA and the MEP pathways in cell growth and tanshinone production of S. miltiorrhiza hairy roots, specific inhibitors of the two pathways were used to perturb metabolic flux. The results showed that the MVA pathway inhibitor (mevinolin, MEV) was more powerful to inhibit the hairy root growth than the MEP pathway inhibitor (fosmidomycin, FOS). Both MEV and FOS could significantly inhibit tanshinone production, and FOS was more powerful than MEV. An inhibitor (D, L-glyceraldehyde, DLG) of IPP translocation strengthened the inhibitory effects of MEV and FOS on cell growth and tanshinone production. Application of MEV resulted in a significant increase of expression and activity of HMGR at 6 h, and a sharp decrease at 24 h. FOS treatment resulted in a significant increase of DXR and DXS expression and DXS activity at 6 h, and a sharp decrease at 24 h. Our results suggested that the MVA pathway played a major role in cell growth, while the MEP pathway was the main source of tanshinone biosynthesis. Both cell growth and tanshinone production could partially depend on the crosstalk between the two pathways. The inhibitor-mediated changes of tanshinone production were reflected in transcript and protein levels of genes of the MVA and MEP pathways.
doi:10.1371/journal.pone.0046797
PMCID: PMC3510226  PMID: 23209548
8.  2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity 
ACS chemical biology  2012;7(10):1702-1710.
There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites and isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP) and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2Cmethyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme.
doi:10.1021/cb300243w
PMCID: PMC3477264  PMID: 22839733
cyclodiphosphate synthase; IspF; methylerythritol phosphate; MEP pathway regulation
9.  A Whole-Cell Phenotypic Screening Platform for Identifying Methylerythritol Phosphate Pathway-Selective Inhibitors as Novel Antibacterial Agents 
Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition.
doi:10.1128/AAC.00987-12
PMCID: PMC3421842  PMID: 22777049
10.  Inhibition Studies on Enzymes Involved in Isoprenoid Biosynthesis: Focus on Two Potential Drug Targets: DXR and IDI-2 Enzymes 
Current enzyme inhibition  2011;7(2):10.2174/157340811796575317.
Isoprenoid compounds constitute an immensely diverse group of acyclic, monocyclic and polycyclic compounds that play important roles in all living organisms. Despite the diversity of their structures, this plethora of natural products arises from only two 5-carbon precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This review will discuss the enzymes in the mevalonate (MVA) and methylerythritol phosphate (MEP) biosynthetic pathways leading to IPP and DMAPP with a particular focus on MEP synthase (DXR) and IPP isomerase (IDI), which are potential targets for the development of antibiotic compounds. DXR is the second enzyme in the MEP pathway and the only one for which inhibitors with antimicrobial activity at pharmaceutically relevant concentrations are known. All of the published DXR inhibitors are fosmidomycin analogues, except for a few bisphosphonates with moderate inhibitory activity. These far, there are no other candidates that target DXR. IDI was first identified and characterised over 40 years ago (IDI-1) and a second convergently evolved isoform (IDI-2) was discovered in 2001. IDI-1 is a metalloprotein found in Eukarya and many species of Bacteria. Its mechanism has been extensively studied. In contrast, IDI-2 requires reduced flavin mononucleotide as a cofactor. The mechanism of action for IDI-2 is less well defined. This review will describe how lead inhibitors are being improved by structure-based drug design and enzymatic assays against DXR to lead to new drug families and how mechanistic probes are being used to address questions about the mechanisms of the isomerases.
doi:10.2174/157340811796575317
PMCID: PMC3856697  PMID: 24339799
DXR; IDI; isomerase; isopentenyl; isoprenoid; MEP; mevalonate; MVA; reductoisomerase
11.  Retinoid production using metabolically engineered Escherichia coli with a two-phase culture system 
Background
Retinoids are lipophilic isoprenoids composed of a cyclic group and a linear chain with a hydrophilic end group. These compounds include retinol, retinal, retinoic acid, retinyl esters, and various derivatives of these structures. Retinoids are used as cosmetic agents and effective pharmaceuticals for skin diseases. Retinal, an immediate precursor of retinoids, is derived by β-carotene 15,15'-mono(di)oxygenase (BCM(D)O) from β-carotene, which is synthesized from the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Retinoids are chemically unstable and biologically degraded via retinoic acid. Although extensive studies have been performed on the microbial production of carotenoids, retinoid production using microbial metabolic engineering has not been reported. Here, we report retinoid production using engineered Escherichia coli that express exogenous BCM(D)O and the mevalonate (MVA) pathway for the building blocks synthesis in combination with a two-phase culture system using a dodecane overlay.
Results
Among the BCM(D)O tested in E. coli, the synthetic retinoid synthesis protein (SR), based on bacteriorhodopsin-related protein-like homolog (Blh) of the uncultured marine bacteria 66A03, showed the highest β-carotene cleavage activity with no residual intracellular β-carotene. By introducing the exogenous MVA pathway, 8.7 mg/L of retinal was produced, which is 4-fold higher production than that of augmenting the MEP pathway (dxs overexpression). There was a large gap between retinal production and β-carotene consumption using the exogenous MVA pathway; therefore, the retinal derivatives were analyzed. The derivatives, except for retinoic acid, that formed were identified, and the levels of retinal, retinol, and retinyl acetate were measured. Amounts as high as 95 mg/L retinoids were obtained from engineered E. coli DH5α harboring the synthetic SR gene and the exogenous MVA pathway in addition to dxs overexpression, which were cultured at 29°C for 72 hours with 2YT medium containing 2.0% (w/v) glycerol as the main carbon source. However, a significant level of intracellular degradation of the retinoids was also observed in the culture. To prevent degradation of the intracellular retinoids through in situ extraction from the cells, a two-phase culture system with dodecane was used. The highest level of retinoid production (136 mg/L) was obtained after 72 hours with 5 mL of dodecane overlaid on a 5 mL culture.
Conclusions
In this study, we successfully produced 136 mg/L retinoids, which were composed of 67 mg/L retinal, 54 mg/L retinol, and 15 mg/L retinyl acetate, using a two-phase culture system with dodecane, which produced 68-fold more retinoids than the initial level of production (2.2 mg/L). Our results demonstrate the potential use of E. coli as a promising microbial cell factory for retinoid production.
doi:10.1186/1475-2859-10-59
PMCID: PMC3160355  PMID: 21801353
12.  Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coli 
Science (New York, N.Y.)  2010;330(6000):70-74.
Taxol (paclitaxel) is a potent anticancer drug first isolated from the Taxus brevifolia Pacific yew tree. Currently, cost-efficient production of Taxol and its analogs remains limited. Here, we report a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene—the first committed Taxol intermediate—approximately 1 gram per liter (~15,000-fold) in an engineered Escherichia coli strain. Our approach partitioned the taxadiene metabolic pathway into two modules: a native upstream methylerythritol-phosphate (MEP) pathway forming isopentenyl pyrophosphate and a heterologous downstream terpenoid–forming pathway. Systematic multivariate search identified conditions that optimally balance the two pathway modules so as to maximize the taxadiene production with minimal accumulation of indole, which is an inhibitory compound found here. We also engineered the next step in Taxol biosynthesis, a P450-mediated 5α-oxidation of taxadiene to taxadien-5α-ol. More broadly, the modular pathway engineering approach helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products.
doi:10.1126/science.1191652
PMCID: PMC3034138  PMID: 20929806
13.  Probing phosphorylation by non-mammalian isoprenoid biosynthetic enzymes using 1H–31P–31P correlation NMR spectroscopy†‡ 
Molecular bioSystems  2009;5(9):935-944.
The biogenesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) is accomplished by the methylerythritol phosphate (MEP) pathway in plants, bacteria and parasites, making it a potential target for the development of anti-infective agents and herbicides. The biosynthetic enzymes comprising this pathway catalyze intriguing chemical transformations on diphosphate scaffolds, offering an opportunity to generate novel analogs in this synthetically challenging compound class. Such a biosynthetic approach to generating new diphosphate analogs may involve transformation through discrete diphosphate species, presenting unique challenges in structure determination and characterization of unnatural enzyme-generated diphosphate products produced in tandem. We have developed 1H–31P–31P correlation NMR spectroscopy techniques for the direct characterization of crude MEP pathway enzyme products at low concentrations (200 μM to 5 mM) on a room temperature (non-cryogenic) NMR probe. Coupling the 100% natural abundance of the 31P nucleus with the high intrinsic sensitivity of proton NMR, 1H–31P–31P correlation spectroscopy is particularly useful for characterization of unnatural diphosphate enzyme products in the MEP pathway. As proof of principle, we demonstrate the rapid characterization of natural enzyme products of the enzymes IspD, E and F in tandem enzyme incubations. In addition, we have characterized several unnatural enzyme products using this technique, including new products of cytidyltransferase IspD bearing erythritol, glycerol and ribose components. The results of this study indicate that IspD may be a useful biocatalyst and highlight 1H–31P–31P correlation spectroscopy as a valuable tool for the characterization of other unnatural products in non-mammalian isoprenoid biosynthesis.
doi:10.1039/b903513c
PMCID: PMC3161243  PMID: 19668858
14.  Combining Genotype Improvement and Statistical Media Optimization for Isoprenoid Production in E. coli 
PLoS ONE  2013;8(10):e75164.
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.
doi:10.1371/journal.pone.0075164
PMCID: PMC3790805  PMID: 24124471
15.  Current Development in Isoprenoid Precursor Biosynthesis and Regulation 
Isoprenoids are one of the largest classes of natural products and all of them are constructed from two precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). For decades, the mevalonic acid (MVA) pathway was proposed to be the only IPP and DMAPP biosynthetic pathway. This review summarizes the newly discovered IPP and DMAPP production pathways since late 1990s, their distribution among different kingdoms, and their roles in secondary metabolite production. These new IPP and DMAPP production pathways include the methylerythritol phosphate (MEP) pathway, a modified MVA pathway, and the 5-Methylthioadenosine shunt pathway. Relative to the studies on the MVA pathway, information on the MEP pathway regulation is limited and the mechanistic details of several of its novel transformations remain to be addressed. Current status on both MEP pathway regulation and mechanistic issues are also presented.
doi:10.1016/j.cbpa.2013.06.020
PMCID: PMC4068245  PMID: 23891475
isoprenoids; MVA; MEP; methylthioadenosine; regulation; biosynthesis
16.  Optimization of the IPP Precursor Supply for the Production of Lycopene, Decaprenoxanthin and Astaxanthin by Corynebacterium glutamicum 
The biotechnologically relevant bacterium Corynebacterium glutamicum, currently used for the million ton-scale production of amino acids for the food and feed industries, is pigmented due to synthesis of the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides. The precursors of carotenoid biosynthesis, isopenthenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate, are synthesized in this organism via the methylerythritol phosphate (MEP) or non-mevalonate pathway. Terminal pathway engineering in recombinant C. glutamicum permitted the production of various non-native C50 and C40 carotenoids. Here, the role of engineering isoprenoid precursor supply for lycopene production by C. glutamicum was characterized. Overexpression of dxs encoding the enzyme that catalyzes the first committed step of the MEP-pathway by chromosomal promoter exchange in a prophage-cured, genome-reduced C. glutamicum strain improved lycopene formation. Similarly, an increased IPP supply was achieved by chromosomal integration of two artificial operons comprising MEP pathway genes under the control of a constitutive promoter. Combined overexpression of dxs and the other six MEP pathways genes in C. glutamicum strain LYC3-MEP was not synergistic with respect to improving lycopene accumulation. Based on C. glutamicum strain LYC3-MEP, astaxanthin could be produced in the milligrams per gram cell dry weight range when the endogenous genes crtE, crtB, and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were coexpressed with the genes for lycopene cyclase and β-carotene hydroxylase from Pantoea ananatis and carotene C(4) oxygenase from Brevundimonas aurantiaca.
doi:10.3389/fbioe.2014.00028
PMCID: PMC4138558  PMID: 25191655
carotenoid production; genome-reduced Corynebacterium glutamicum; MEP pathway; synthetic operons; astaxanthin
17.  Characterization of the Mycobacterium tuberculosis 4-Diphosphocytidyl-2-C-Methyl-d-Erythritol Synthase: Potential for Drug Development▿  
Journal of Bacteriology  2007;189(24):8922-8927.
Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-d-erythritol is formed from 2-C-methyl-d-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-d-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5′-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 μM for MEP and 53.2 μM for CTP. Calculated kcat and kcat/Km values were 0.72 min−1 and 12.3 mM−1 min−1 for MEP and 1.0 min−1 and 18.8 mM−1 min−1 for CTP, respectively.
doi:10.1128/JB.00925-07
PMCID: PMC2168624  PMID: 17921290
18.  Evolutionary diversification and characterization of the eubacterial gene family encoding DXR type II, an alternative isoprenoid biosynthetic enzyme 
Background
Isoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II).
Results
Searches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria.
Conclusions
Our analyses support a single origin of the DXR-II family through functional divergence, in which constitutes an exceptional model of acquisition and maintenance of redundant gene functions between non-homologous genes as a result of convergent evolution. Subsequently, although old episodic events of HGT could not be excluded, the results supported a prevalent role of gene loss in explaining the distribution of DXR-II in specific pathogenic eubacteria. Our results highlight the importance of the functional characterization of evolutionary shortcuts in isoprenoid biosynthesis for screening specific antibacterial drugs and for regulating the production of isoprenoids of human interest.
doi:10.1186/1471-2148-13-180
PMCID: PMC3847144  PMID: 24004839
DXR-II; Isoprenoid metabolism; Horizontal gene transfer; Gene loss; Functional divergence
19.  In silico profiling of Escherichia coli and Saccharomyces cerevisiae as terpenoid factories 
Background
Heterologous 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.
Results
Starting 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.
Conclusions
This 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.
doi:10.1186/1475-2859-12-84
PMCID: PMC3852115  PMID: 24059635
Terpenoids; Isoprenoids; In silico; Elementary mode analysis; Constrained minimal cut sets; Metabolic engineering; Escherichia coli; Saccharomyces cerevisiae
20.  Bioassay-Guided Evolution of Glycosylated Macrolide Antibiotics in Escherichia coli 
PLoS Biology  2007;5(2):e45.
Macrolide antibiotics such as erythromycin are clinically important polyketide natural products. We have engineered a recombinant strain of Escherichia coli that produces small but measurable quantities of the bioactive macrolide 6-deoxyerythromycin D. Bioassay-guided evolution of this strain led to the identification of an antibiotic-overproducing mutation in the mycarose biosynthesis and transfer pathway that was detectable via a colony-based screening assay. This high-throughput assay was then used to evolve second-generation mutants capable of enhanced precursor-directed biosynthesis of macrolide antibiotics. The availability of a screen for macrolide biosynthesis in E. coli offers a fundamentally new approach in dissecting modular megasynthase mechanisms as well as engineering antibiotics with novel pharmacological properties.
An erythromycin-producingE. coli recombinant strain with an antibiotic-overproducing mutation in the mycarose biosynthesis pathway produced second-generation mutants capable of directed biosynthesis of enhanced precursor macrolide antibiotics.
Author Summary
The antibacterial activity of erythromycin, an important polyketide antibiotic precursor, requires the transfer of two unusual sugars called mycarose and desosamine (both glycosyl groups), onto the nonsugar part of the glycoside molecule (macrocyclic aglycone). We reconstituted the biosynthetic pathways of both sugars in Escherichia coli to yield the 6-deoxyerythromycin D antibiotic. By engineering a recombinant strain of E. coli that produces the bioactive macrolide 6-deoxyerythromycin D from propionate, we have developed a fundamentally new tool for enhancing the efficiency of biosynthetic engineering of this class of antibiotics. Initially, this recombinant strain produced barely enough antibiotic activity to establish an activity-based screening assay. We therefore used the assay to screen for antibiotic overproducers. After three rounds of screening, we identified E. coli cells that overproduced the 6-deoxyerythromycin D antibiotic with significant modifications in the mycarose biosynthetic pathway. We used the same activity-based screening system to evolve E. coli mutants capable of more efficient precursor-directed biosynthesis. As the first example of bioassay-guided evolution of an antibiotic pathway in E. coli, these results open the door for harnessing the power of genetics for mechanistic investigations into polyketide synthases and also for biosynthetic engineering.
doi:10.1371/journal.pbio.0050045
PMCID: PMC1790958  PMID: 17298179
21.  Conformational Dynamics of the Flexible Catalytic Loop in Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase 
Chemical biology & drug design  2009;73(1):26-38.
In mycobacteria, the biosynthesis of the precursors to the essential isoprenoids, isopentenyl diphosphate and dimethylallyl pyrophosphate is carried out by the methylerythritol phosphate (MEP) pathway. This route of synthesis is absent in humans, who utilize the alternative mevalonate acid (MVA) route, thus making the enzymes of the MEP pathway of chemotherapeutic interest. One such identified target is the second enzyme of the pathway, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). Only limited information is currently available concerning the catalytic mechanism and structural dynamics of this enzyme, and only recently has a crystal structure of Mycobacterium tuberculosis species of this enzyme been resolved including all factors required for binding. Here, the dynamics of the enzyme is studied in complex with NADPH, Mn2+, in the presence and absence of the fosmidomycin inhibitor using conventional molecular dynamics and an enhanced sampling technique, Reversible Digitally Filtered Molecular Dynamics. The simulations reveal significant differences in the conformational dynamics of the vital catalytic loop between the inhibitor-free and inhibitor-bound enzyme complexes and highlight the contributions of conserved residues in this region. The substantial fluctuations observed suggest that DXR may be a promising target for computer-aided drug discovery through the relaxed complex method.
doi:10.1111/j.1747-0285.2008.00749.x
PMCID: PMC2982673  PMID: 19152632
22.  Isoprenoid Biosynthesis in Synechocystis sp. Strain PCC6803 Is Stimulated by Compounds of the Pentose Phosphate Cycle but Not by Pyruvate or Deoxyxylulose-5-Phosphate 
Journal of Bacteriology  2002;184(18):5045-5051.
The photosynthetic cyanobacterium Synechocystis sp. strain PCC6803 possesses homologs of known genes of the non-mevalonate 2-C-methyl-d-erythritol 2-phosphate (MEP) pathway for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Isoprenoid biosynthesis in extracts of this cyanobacterium, measured by incorporation of radiolabeled IPP, was not stimulated by pyruvate, an initial substrate of the MEP pathway in Escherichia coli, or by deoxyxylulose-5-phosphate, the first pathway intermediate in E. coli. However, high rates of IPP incorporation were obtained with addition of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P), as well as a variety of pentose phosphate cycle compounds. Fosmidomycin (at 1 μM and 1 mM), an inhibitor of deoxyxylulose-5-phosphate reductoisomerase, did not significantly inhibit phototrophic growth of the cyanobacterium, nor did it affect [14C]IPP incorporation stimulated by DHAP plus GA3P. To date, it has not been possible to unequivocally demonstrate IPP isomerase activity in this cyanobacterium. The combined results suggest that the MEP pathway, as described for E. coli, is not the primary path by which isoprenoids are synthesized under photosynthetic conditions in Synechocystis sp. strain PCC6803. Our data support alternative routes of entry of pentose phosphate cycle substrates derived from photosynthesis.
doi:10.1128/JB.184.18.5045-5051.2002
PMCID: PMC135332  PMID: 12193620
23.  Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production 
PLoS ONE  2012;7(11):e47513.
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.
doi:10.1371/journal.pone.0047513
PMCID: PMC3487848  PMID: 23133596
24.  Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered Escherichia coli 
PLoS ONE  2013;8(12):e83290.
Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways.
doi:10.1371/journal.pone.0083290
PMCID: PMC3869766  PMID: 24376679
25.  Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis 
In comparison to other bacteria Bacillus subtilis emits the volatile compound isoprene in high concentrations. Isoprene is the smallest representative of the natural product group of terpenoids. A search in the genome of B. subtilis resulted in a set of genes with yet unknown function, but putatively involved in the methylerythritol phosphate (MEP) pathway to isoprene. Further identification of these genes would give the possibility to engineer B. subtilis as a host cell for the production of terpenoids like the valuable plant-produced drugs artemisinin and paclitaxel. Conditional knock-out strains of putative genes were analyzed for the amount of isoprene emitted. Differences in isoprene emission were used to identify the function of the enzymes and of the corresponding selected genes in the MEP pathway. We give proof on a biochemical level that several of these selected genes from this species are involved in isoprene biosynthesis. This opens the possibilities to investigate the physiological function of isoprene emission and to increase the endogenous flux to the terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate, for the heterologous production of more complex terpenoids in B. subtilis.
doi:10.1007/s00253-007-0953-5
PMCID: PMC1914294  PMID: 17458547
Bacillus subtilis; Isoprene; Methylerythritol phosphate pathway; MEP pathway; Terpenoid; Gas chromatography

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