WO2013133772A1

WO2013133772A1 - An integrated solid phase extraction coupled ultra-performance liquid chromatography-mass spectrometry (spe-uplc-ms) method for the profiling of dxp metabolites and system diagnostics of dxp pathway

Info

Publication number: WO2013133772A1
Application number: PCT/SG2013/000098
Authority: WO
Inventors: Heng Phon Too; Kang ZHOU; Gregory N STEPHANOPOULOS
Original assignee: National University Of Singapore; Massachusetts Institute Of Technology
Priority date: 2012-03-08
Filing date: 2013-03-08
Publication date: 2013-09-12

Abstract

A method for extracting at least a first 1-deoxy-D-xylulose 5-phosphate (DXP) pathway metabolite and at least a second DXP pathway metabolite from a sample and detecting the at least first and the at least second extracted DXP pathway metabolites in the sample simultaneously is disclosed. Also disclosed is a method for identifying a modulator, such as an inhibitor, of an enzyme in the DXP pathway for the purposes of drug discovery.

Description

AN INTEGRATED SOLID PHASE EXTRACTION COUPLED ULTRA- PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (SPE-UPLC-MS) METHOD FOR THE PROFILING OF DXP METABOLITES

AND SYSTEM DIAGNOSTICS OF DXP PATHWAY

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 61/608,289, filed on March 8, 2012. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Isoprenoids and terpenoids belong to the largest group of natural products found in living organisms. These lipids have highly diverse, complex and multicyclic structures. Some of these natural products have therapeutic value for antibacterial, antineoplastic, and other pharmaceutical uses. An example of one such natural product is taxol, which is currently used clinically for the treatment of many solid tumors.

[0003] The metabolic engineering of Escherichia coli and other

biotechnologically relevant organisms to produce natural products is an emerging solution to the shortage in the supply of isoprenoids. With E. coli, high isoprenoid production is achieved by manipulating the 1-deoxy-D-xylulose 5 -phosphate (DXP) pathway. The DXP pathway is of critical importance because the isoprenoid building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), are synthesized by this pathway. To date, no method has been reported to detect all of the DXP pathway metabolites. The lack of a precise, high-performance and robust approach to profile all the DXP pathway metabolites simultaneously has hampered the development of a systematic approach to tuning the DXP pathway. Thus, there is a need for an efficient method to detect and quantify the metabolites involved in the DXP pathway.

SUMMARY OF THE INVENTION

[0004] One embodiment of the invention is a method for extracting at least a first 1-deoxy-D-xylulose 5 -phosphate (DXP) pathway metabolite and at least a second DXP pathway metabolite from a sample and detecting the at least first and the at least second extracted DXP pathway metabolites in the sample simultaneously, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of DXP, MEP, HMBPP, MEC, CDP-ME and CDP-MEP. The method comprises extracting the at least first and the at least second DXP pathway metabolites from the sample by solid-phase extraction, thereby obtaining an extracted sample; and simultaneously detecting at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in the extracted sample by liquid

chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio.

[0005] Another embodiment of the invention is a method for simultaneously detecting at least a first DXP pathway metabolite and at least a second DXP pathway metabolite in a sample, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of DXP, MEP, HMBPP, MEC, CDP-ME and CDP-MEP. The method comprises simultaneously detecting at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in a sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio.

[0006] Yet another embodiment of the invention is a method for identifying a modulator of an enzyme in the DXP pathway. The method comprises exposing a bacterial cell in a medium to a potential modulator of an enzyme in the DXP pathway, thereby providing a sample. At least a first and at least a second DXP pathway metabolite are extracted from the sample by solid-phase extraction, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of DXP, MEP, HMBPP, MEC, CDP-ME and CDP-MEP, thereby obtaining an extracted sample. The intensity of at least a first signal produced by the at least first DXP pathway metabolite and the intensity of at least a second signal produced by the at least second DXP pathway metabolite in the extracted sample are measured by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid

chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio. The measured intensity of the at least first signal is compared to a signal produced by the at least first DXP pathway metabolite in an extracted sample corresponding to a bacterial cell in a medium that was not treated with the potential modulator, and the at least second signal is compared to a signal produced by the at least second DXP pathway metabolite in the extracted sample corresponding to the bacterial cell in a medium that was not treated with the potential modulator. A change in the measured intensity of the at least first or the at least second signal indicates modulation of an enzyme in the DXP pathway.

[0007] Another embodiment of the invention is a method for determining the rate constant of a flux in the DXP pathway of a bacterium in a medium by simultaneously measuring the amount of at least a first DXP pathway metabolite and at least a second DXP pathway metabolite, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of 1-deoxy-D- xylulose 5-phosphate, 2C-methyl-D-erythritol 4-phosphate, 4-diphosphocytidyl-2C- methyl D-erythritol, 4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, 2C- methyl-D-erythritol 2,4-diphosphate and hydroxylmethylbutenyl diphosphate. The method comprises incubating a first bacterial cell in a medium for a first period of time, thereby providing a first sample; and incubating a second bacterial cell in a medium for a second period of time different than the first period of time, thereby providing a second sample. The intensities of at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in the first sample are simultaneously measured by liquid chromatography, mass spectrometry or liquid chromatography in

conjunction with mass spectrometry, wherein each of the at least first and the at least second signals corresponds to an amount of the at least first and the at least second metabolites, respectively, in the first sample after the first period of time and is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio. The intensities of the at least first signal produced by the at least first DXP pathway metabolite and the at least second signal produced by the at least second DXP pathway metabolite in the second sample are simultaneously measured by liquid chromatography, mass spectrometry or liquid chromatography in

conjunction with mass spectrometry, wherein each of the at least first and the at least second signals corresponds to an amount of the at least first and the at least second metabolites, respectively, in the second sample after at least the second period of time and is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio. A rate constant of a flux is calculated using the measured intensity of the at least first and the at least second signals after the first period of time and after the second period of time, thereby determining the rate constant of a flux in the DXP pathway.

[0008] The method comprises providing a bacterial cell in a medium, thereby providing a sample; simultaneously measuring the intensity of at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in a first sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals corresponds to an amount of the at least first and the at least second metabolites, respectively, in the sample at a first time point and is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio; and simultaneously measuring the intensity of the at least first signal produced by the at least first DXP pathway metabolite and the at least second signal produced by the at least second DXP pathway metabolite in a second sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals corresponds to an amount of the at least first and the at least second metabolites, respectively, in the sample at at least a second time point and is characterized using liquid

chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio. The measured intensity of the at least first and the at least second signals at the first time point and the at least second time point are used to calculate the rate constant of a flux in the DXP pathway.

[0009] Another embodiment of the invention is a method for characterizing the effect of a potential modulator of the DXP pathway, for example, to improve production of an isoprenoid. The method comprises exposing a bacterial cell in a medium to at least one potential modulator of the DXP pathway, or expressing at least one potential modulator of the DXP pathway in a bacterial cell in a medium, thereby providing a sample. At least a first and at least a second DXP pathway metabolite are extracted from the sample by solid-phase extraction, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of 1 -deoxy-D-xylulose 5-phosphate, 2C-methyl-D-erythritol 4-phosphate. 4-diphosphocytidyl-2C-methyl D-erythritol, 4-diphosphocytidyl-2C-methyl D- erythritol 2-phosphate, 2C-methyl-D-erythritol 2,4-diphosphate and

hydroxylmethylbutenyl diphosphate, thereby obtaining an extracted sample. The intensity of at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in the extracted sample are simultaneously measured by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry. Each of the at least first and the at least second signals is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio. The measured intensity of the at least first signal is compared to a signal produced by the at least first DXP pathway metabolite in an extracted sample corresponding to a bacterial cell in a medium not treated with or not expressing the at least one potential modulator, and the at least second signal is compared to a signal produced by the at least second DXP pathway metabolite in the extracted sample corresponding to the bacterial cell in a medium not treated with or not expressing the at least one potential modulator. A change in the measured intensity of the at least first or the at least second signal indicates modulation of the DXP pathway.

[0010] The integrated SPE-UPLC-MS method disclosed herein allows the direct monitoring of DXP metabolites, including isoprenoids, in cells and in vitro, and provides an invaluable tool for the rational engineering of the DXP pathway. With the SPE-UPLC-MS method disclosed herein, the DXP pathway metabolites can be detected with a limit of quantification (LOQ) of 0.02 μΜ, except for CDP-MEP, for which the LOQ is 0.1 μΜ. The linearity (R² >0.99) and repeatability (CV <20%) of the method was confirmed and was satisfactory. The SPE-UPLC-MS method disclosed herein can be used to measure isoprenoid production in E. coli

overexpressing four putative genes of the DXP pathway. Only DXP, MEP, CDP-ME and MEC accumulated intracellularly upon induction of overexpression.

Unexpectedly, DXP and MEC, supposedly unable to cross the cell membrane, were detected in the broth at high concentrations upon the induction of the recombinant DNA. The SPE-UPLC-MS method disclosed herein can also be used to detect DXP pathway metabolites in wild-type bacteria, in which the concentrations of the metabolites are much lower than in, for example, E. coli engineered to over-express the metabolites. In addition, the method was shown to be applicable to the discovery of broad spectrum antibiotics against bacteria in which the DXP pathway is essential. [0011] Methods of characterizing the effect of a potential modulator of the DXP pathway can be used, for example, to improve production of an isoprenoid (e.g., lycopene, amorphadiene), or to directly assess and improve the solubility of enzymes in the DXP pathway. The methods disclosed herein have been used to show that protein solubility can limit metabolic flux through the DXP pathway; use of a conventional lycopene reporter system failed to reveal this information.

BRIEF DESCRIPTION OF THE FIGURES

[0012] The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the

accompanying figures.

[0013] Figure 1 shows enzymes and chemical structures of metabolites involved in the DXP pathway (DXP: 1-deoxy-D-xylulose 5-phosphate, MEP: 2C-methyl-D- erythritol 4-phosphate, CDP-ME: 4-diphosphocytidyl-2C-methyl D-erythritol, CDP- MEP: 4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, MEC: 2C-methyl-D- erythritol 2,4-diphosphate, HMBPP: hydroxylmethylbutenyl diphosphate).

[0014] Figures 2A-2E are chromatograms showing the effects of UPLC gradient on retention time of the metabolites in the DXP pathway (the mass/charge (m/z) ratio for each metabolite was extracted from a total ion chromatogram and the resulting traces were overlaid).

[0015] Figure 3 A is a bar graph of lycopene concentration 24 hours after isopropyl β-D-l -thiogalactopyranoside (IPTG) induction as a function of the concentration of IPTG (the data is an average of triplicates and standard errors are indicated on the graph).

[0016] Figure 3B is a bar graph of the intracellular concentrations of DXP, MEP, CDP-ME and MEC 5 hours after IPTG induction as a function of IPTG concentration (the data is an average of triplicates and standard errors are indicated on the graph).

[0017] Figure 3C is a bar graph of the extracellular concentration of MEC 5 hours after IPTG induction as a function of IPTG concentration (the data is an average of triplicates and standard errors are indicated on the graph). [0018] Figure 4A is a graph of lycopene production 24 hours after IPTG induction as a function of extracellular MEC concentration 5 hours after IPTG induction (the data is an average of triplicates and standard errors are indicated on the graph).

[0019] Figure 4B is a bar graph, and shows lycopene production as a function of time in the presence of SIDF or SIDFG (SIDF: BL21 Gold (DE3) harboring pET- SIDFG and pACLYC; SIDFG: BL21 Gold (DE3) harboring pET-ACLYC; the data is an average of triplicates and standard errors are indicated on the graph).

[0020] Figure 4C is a bar graph, and shows extracellular MEC concentration as a function of time in the presence of SIDF or SIDFG (the data is an average of triplicates and standard errors are indicated on the graph).

[0021] Figure 4D is a bar graph, and shows intracellular MEC concentration as a function of time in the presence of SIDF or SIDFG (the data is an average of triplicates and standard errors are indicated on the graph).

[0022] Figure 4E is a bar graph, and shows intracellular HMBPP concentration as a function of time in the presence of SIDF and SIDFG (no detectable levels of HMBPP were detected in the presence of SIDF; the data is an average of triplicates and standard errors are indicated on the graph).

[0023] Figure 5 is a bar graph of the concentrations of DXP, MEP, CDP-ME and MEC in Chromobacterium violaceum (C. viol.), Pseudomonas aeruginosa (P. aeru.) and Bacillus subtilis (B. subt.) at middle exponential growth phase (the data is an average of triplicates and standard errors are indicated on the graph).

[0024] Figure 6 is a bar graph of the concentrations of DXP and MEP in in vitro reactions as a function of fosmidomycin (fos.) concentration (the data is an average of triplicates and standard errors are indicated on the graph).

[0025] Figure 7 is a bar graph of the intracellular concentrations of DXP, MEP, CDP-ME and MEC as a function of fosmidomycin (fos.) concentration 8 hours after induction with 0.1 mM IPTG (the data is an average of triplicates and standard errors are indicated on the graph).

[0026] Figure 8A is a graph of the concentration of intracellular MEC as a function of time (the data is an average of duplicates). [0027] Figure 8B is a graph of the concentration of extracellular MEC as a function of time (the data is an average of duplicates).

[0028] Figure 9 shows metabolites involved in the DXP pathway (the solid arrows indicate known fluxes and the dotted arrows indicate the fluxes discovered by the SPE-UPLC-MS method disclosed herein; "ki" to "k₉" indicate the kinetic parameters of each step).

[0029] Figure 1 OA is a bar graph, and shows the solubility of DXP enzymes expressed in BL21 strain using a T7 promoter at 20 °C and 37 °C (quantification of the data was based on an image of SDS-PAGE gels; presented data are the average of triplicates and standard errors are indicated on the plot).

[0030] Figure 10B is a bar graph, and shows the solubility of DXP enzymes expressed in Ml 5 strain using a T5 promoter at 20 °C and 37 °C (quantification of the data was based on an image of SDS-PAGE gels; presented data are the average of triplicates and standard errors are indicated on the plot).

[0031] Figure I OC is a bar graph, and shows the solubility of DXP enzymes expressed in DH 10B strain using an araBAD promoter at 20 °C and 37 °C

(quantification of the data was based on an image of SDS-PAGE gels; presented data are the average of triplicates and standard errors are indicated on the plot).

[0032] Figure 10D is an image of an SDS-PAGE gel corresponding to the 20 °C data depicted in Figure I OC, and shows the soluble (S) and insoluble (IS) protein fractions of DXP enzymes expressed in DH 10B strain using an araBAD promoter at 20 °C.

[0033] Figure 1 1 is a bar graph of DXP concentration, and shows that the activity of insoluble DXS was much lower than that of an equal amount of soluble DXS, as measured by quantifying the quantity of DXP produced in vitro (presented data are average of triplicates and standard errors are indicated on the plot).

[0034] Figure 12A is a bar graph, and shows the effect of 100 mM K₂P0₄, 50 mM HEPES, 1 mM betaine and 500 mM sorbitol on the solubility of DXS (presented data are average of triplicates and standard errors are indicated on the plot; Student's t-test was used to calculate the p values in the statistical analysis). [0035] Figure 12B is an image of an SDS-PAGE gel, and shows the effect on protein solubility of 500 mM sorbitol in soluble (S) and insoluble (IS) protein fractions.

[0036] Figure 12C is two bar graphs, and shows the effects of sorbitol addition on production of DXP; addition of sorbitol increased DXP production in the cells expressing functional DXS but not in cells expressing a non-functional mutant of DXS, R398A (presented data are average of triplicates and standard errors are indicated on the plot; Student's t-test was used to calculate the p values in the statistical analysis).

[0037] Figure 13A shows enzymes and metabolites involved in the mevalonate (MVA) pathway (MVAP: mevalonate phosphate; MVAPP: mevalonate diphosphate; IPP: isopentenyl diphosphate; DMAPP: dimethylallyl diphosphate; ERG 12:

mevalonate kinase; ERG8: mevalonate phosphate kinase; ERG 19: mevalonate diphosphate decarboxylase).

[0038] Figure 13B is a bar graph, and shows the effect of 500 mM sorbitol on the solubility of ERG 12 (presented data are average of triplicates and standard errors are indicated on the plot; Student's t-test was used to calculate the p values in the statistical analysis).

[0039] Figure 13C is a bar graph, and shows the effect of 500 mM sorbitol on production of MVAP (presented data are average of triplicates and standard errors are indicated on the plot; Student's t-test was used to calculate the p values in the statistical analysis). Because ERG8 and ERG 19 are not present in E. coli and were not recombinantly expressed, MVAP accumulation in ERG12-expressing E. coli directly indicated activity of ERG 12.

[0040] Figure 14 is a bar graph of transcription fold change of the indicated efflux pump-encoding genes upon ispG overexpression, and shows that efflux pumps were not repressed in the strain overexpressing dxs-idi-ispDF-ispG (presented data are average of triplicates and standard errors are indicated on the plot), bcr, mdtJ, mdtH, mdtA, mdtG, cmr, emrK, emrE, fsr, mdtL and acrA are known genes encoding E. coli efflux pumps. Transcription of the following efflux pump-encoding genes were too low to be accurately quantified: cusC, macA, mdtK, mdtA, mdtM and emrD. [0041] Figure 15 is a bar graph of the concentration of the indicated DXP metabolites before and 2 hours after inhibition with 25 μg/mL fosmidomycin

(presented data are average of triplicates and standard errors are indicated on the plot). Twenty five micrograms per milliliter fosmidomycin was used to inhibit MEC biosynthesis at 4 hours after induction, and the metabolites of BL21 Gold (DE3) harboring pET-SIDF and pAC-LYC were analyzed before and 2 hour after

fosmidomycin inhibition.

[0042] Figure 16A shows enzymes and metabolites involved in the synthesis of amorphadiene.

[0043] Figure 16B shows expression cassettes in plasmid pHT-ads and pWH-DI.

[0044] Figure 16C is a bar graph, and shows the quantity of Pgrac and PxylA transcribed as a function of IPTG and xylose concentrations.

[0045] Figure 17 is a graph, and shows the concentration of amorphadiene produced as a function of IPTG and xylose concentrations.

[0046] Figure 18 A shows that introduction of an N-terminal polyarginine tag increased expression but not transcription of ADS.

[0047] Figure 18B is a line graph, and shows the concentration of amorphadiene produced by cells expressing 1A1 pHT-ads and 1 Al pHT-6xR.ads.

[0048] Figure 19A shows that addition of pyruvate and potassium phosphate increased production of amorphadiene in B. subtilis.

[0049] Figure 1 B is a bar graph, and shows the effect of 0.8% pyruvate and 3.2% K2HPO4 on transcription of dxs-idi and 6xR.ads.

[0050] Figure 19C is a bar graph, and shows the effect of 0.8% pyruvate and 3.2% K2HPO4 on the amount of DXP metabolites.

[0051] Figure 19D is a bar graph, and shows the effect of 0.8% pyruvate and 3.2% K2HPO4 on the amount of DAHP, PEP, CTP and ATP.

DETAILED DESCRIPTION OF THE INVENTION

[0052] A description of example embodiments of the invention follows.

[0053] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. [0054] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a metabolite" can include a plurality of metabolites. Further, the plurality can comprise more than one of the same metabolite or a plurality of different metabolites.

[0055] A solid phase extraction ultra performance liquid chromatography with mass spectrometry (SPE-UPLC-MS) method has been developed for the extraction and quantification of all the DXP pathway metabolites in a biological sample. For example, anion-exchange solid phase extraction (SPE) to extract the DXP pathway metabolites from a biological sample and ultra-performance liquid chromatography- mass spectrometry (UPLC-MS) can be used to reliably and simultaneously quantify the extracted metabolites with good sensitivity.

[0056] Isoprenoids are a large family of compounds (having more than 55,000 members) that can be used as fragrances, insecticides, nutraceuticals and

pharmaceuticals. The supply of these molecules has been limited by the scarce plant resources from which they were originally extracted and by the difficulty in total chemical synthesis due to their structural complexity. Despite the structural diversity of isoprenoids, they are all derived from IPP and DMAPP and the ability of cells to synthesize IPP and DMAPP largely determines the amount of isoprenoids that can be produced. IPP and DMAPP are either synthesized by the mevalonate (MVA) pathway or the DXP pathway. Figure 1 shows enzymes and chemical structures of metabolites involved in the DXP pathway. Figure 1 also shows the mechanism of fosmidomycin inhibition of the DXP pathway. Figure 16A shows enzymes and metabolites of the DXP pathway involved in the synthesis of amorphadiene.

[0057] In the DXP pathway, pyruvate (PYR) and glyceraldehyde 3-phosphate (GAP) are first condensed by dxs with thiamine to produce 1 -deoxy-D-xylulose 5- phosphate (DXP). DXP is then reduced and isomerized by a single enzyme, dxr, with NADPH to form 2C-methyl-D-erythritol 4-phosphate (MEP). MEP reacts with CTP in the presence of ispD to produce 4-diphosphocytidyl-2C-methyl D-erythritol (CDP- ME). CDP-ME is phosphorylated by an ATP dependent kinase, ispE, to form 4- diphosphocytidyl-2C-methyl D-erythritol 2-phosphate (CDP-MEP). CMP is eliminated from CDP-MEP and the molecule is cyclized by ispF to form 2C-methyl- D-erythritol 2,4-diphosphate (MEC). The ring structure of MEC is opened and reduced by an iron-sulfur cluster containing enzyme, ispG, by a yet to be fully characterized mechanism to form hydroxylmefhylbutenyl diphosphate (HMBPP). HMBPP is further reduced by another iron-sulfur cluster containing enzyme, ispH, to produce a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Unlike the mevalonate pathway, all the DXP intermediates are phosphorylated and highly acidic. This unique physiochemical property allowed a single step purification of these metabolites using anionic SPE.

[0058] One embodiment of the invention is a method for extracting at least a first DXP pathway metabolite and at least a second DXP pathway metabolite from a sample and detecting the at least first and the at least second extracted DXP pathway metabolites in the sample simultaneously, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of DXP, MEP, HMBPP, MEC, CDP-ME and CDP-MEP. The method comprises extracting the at least first and the at least second DXP pathway metabolites from the sample by solid-phase extraction, thereby obtaining an extracted sample; and simultaneously detecting at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in the extracted sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio.

(0059] "Metabolite," as used herein, refers to a substrate, intermediate or product of metabolism. Therefore, a metabolite of the DXP pathway includes the substrates, intermediates and products involved in the conversion of pyruvate and GAP to an isoprenoid.

[0060] In some embodiments, the at least first DXP pathway metabolite is DXP, and the at least second DXP pathway metabolite is MEP. In some embodiments, the at least first and the at least second DXP pathway metabolites include DXP, MEP, CDP-ME, CDP-MEP, MEC and HMBPP. In some embodiments, the at least first and the at least second DXP pathway metabolites include DXP, MEP, CDP-ME and MEC. In some embodiments, the at least first and the at least second DXP pathway metabolites include DXP, MEP, CDP-ME, MEC and HMBPP. In some

embodiments, the at least first and the at least second DXP pathway metabolites include DXP, MEP, CDP-ME and MEC.

[0061] Other metabolites that can be extracted and/or detected using the methods described herein include DAHP, PEP, CTP and ATP.

[0062] In some embodiments of the methods described herein, the sample is an extract of a bacterial cell (e.g. , a bacterial cell exposed to a potential modulator of an enzyme of the DXP pathway, a bacterial cell expressing a potential modulator of the DXP pathway), medium in which a bacterial cell (e.g., a bacterial cell exposed to a potential modulator of an enzyme of the DXP pathway, a bacterial cell expressing a potential modulator of the DXP pathway) was incubated, or an extract of a bacterial cell (e.g., a bacterial cell exposed to a potential modulator of an enzyme of the DXP pathway, a bacterial cell expressing a potential modulator of the DXP pathway) and medium in which the bacterial cell was incubated. Thus, when an extracted sample is analyzed, the extracted sample can contain intracellular metabolites, for example, when the sample is an extract of a bacterial cell, extracellular metabolites, for example, when the sample is a sample of medium in which a bacterial cell was incubated, or a combination of intracellular and extracellular metabolites. DXP and MEC, in particular, can be effluxed from cells. Therefore, in some embodiments, the sample is a sample of medium in which a bacterial cell was incubated, the at least first DXP pathway metabolite is DXP, and the at least second DXP pathway metabolite is MEC. Preferably, the sample is an extract of a bacterial cell or medium in which a bacterial cell was incubated.

[0063] In some embodiments of the methods described herein, the sample is an extract of a bacterial cell. When the sample is an extract of a bacterial cell, the methods described herein can further comprise preparing an extract of the bacterial cell prior to extracting the sample by solid-phase extraction. [0064] A bacterial cell can be a wild-type bacterial cell or a genetically engineered bacterial cell, for example, a bacterial cell genetically engineered to over- express a DXP pathway enzyme {e.g., ispG).

[0065] Exemplary bacteria include gram negative bacteria, for example,

Escherichia coli, Chromobacteri m violacewn and Psudomonas aeruginosa, and gram positive bacteria, for example, Staphylococcus aureus and Bacillus subtilis. In some embodiments of the methods disclosed herein, the bacterium or bacterial cell is E. coli, C. violaceum, P. aeruginosa or B. subtilis. In some embodiments of the methods disclosed herein, the bacterium or bacterial cell is E. coli. In other embodiments of the methods disclosed herein, the bacterium or bacterial cell is B. subtilis.

[0066] In some embodiments of a method for extracting at least a first DXP pathway metabolite and at least a second DXP pathway metabolite from a sample and detecting the at least first and the at least second extracted DXP pathway metabolites in the sample simultaneously, detecting the at least first and the at least second DXP pathway metabolites in the sample includes quantifying the amount of each of the at least first and the at least second DXP pathway metabolites in the sample and the method further comprises measuring the intensity of the at least the first signal and the at least second signal of the at least first and the at least second DXP pathway metabolites; and comparing the measured intensity of the at least first signal to a standard curve of the at least first DXP pathway metabolite and comparing the at least second signal to a standard curve of the at least second DXP pathway metabolite, thereby quantifying the amount of the at least first and the at least second DXP pathway metabolites in the sample.

[0067] Liquid chromatography can be used to separate a mixture of compounds based on their chemical and/or physical properties. Compounds can be detected by liquid chromatography using, for example, absorbance spectroscopy, mass spectroscopy, or other methods known to those of skill in the art. Upon identification of a specific set of separation conditions, the separated compounds in the mixture can subsequently be identified based on a unique retention time. [0068] Mass spectrometry can be used to determine the masses of particles and compounds in a mixture. Typically, mass spectrometry works by ionizing a compound to generate a charged compound or a compound fragment, and measuring the mass/charge ratio of each species. Upon identification of a specific set of ionization conditions, each species in the mixture can subsequently be identified based on a unique mass/charge ratio.

[0069] Liquid chromatography in conjunction with mass spectrometry, also referred to as LC-MS, can be used to separate compounds in a mixture based on their chemical and/or physical properties. The separated compounds can then be detected by ionizing each compound to generate a charged compound or a compound fragment, and measuring the mass/charge ratio of each species. Upon identification of a specific set of separation and ionization conditions, the compounds in the mixture can subsequently be identified based on a unique retention time and unique mass/charge ratio.

[0070] Techniques for detecting a signal produced by a compound in a sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry are known to those of ordinary skill in the art. In some embodiments, the DXP pathway metabolites are detected by liquid chromatography in conjunction with mass spectrometry. Preferably, the liquid chromatography is ultra- performance liquid chromatography (UPLC), and the mass spectrometry is time-of- flight (TOF) mass spectrometry.

[0071] When a mixture of compounds is separated and analyzed using liquid chromatography or liquid chromatography in conjunction with mass spectrometry, the separated compounds (e.g. , the at least first and the at least second metabolites) are not detected by the instrument at precisely the same instant in time. It should be understood that "simultaneously detecting" refers to a scenario in which the separated compounds (e.g. , the at least first and the at least second metabolites) are, for example, simultaneously injected into the instrument as a single sample or aliquot of a sample. "Simultaneously detecting" is merely meant to distinguish from a scenario in which, for example, a separate injection is made for each metabolite to be analyzed or detected. [0072] Solid phase extraction (SPE) is a separation process by which compounds that are dissolved or suspended in a liquid mixture are separated from other compounds in the mixture according to their chemical and/or physical properties. SPE typically involves a liquid mobile phase and a solid stationary phase. If the compounds of interest in the liquid mixture are retained by the stationary phase, the stationary phase can be rinsed with an eluent to elute the compounds of interest. Solid phase extraction techniques are known to those of ordinary skill in the art. For example, Qu, J., Y. Qu, and R.M. Straubinger, Ultra-sensitive quantification of corticosteroids in plasma samples using selective solid-phase extraction and reversed-phase capillary high-performance liquid chromatography/tandem mass spectrometry. Anal Chem, 2007. 79(10): p. 3786-93; Batt, A.L., M.S. Kostich, and J.M. Lazorchak. Analysis of ecologically relevant pharmaceuticals in wastewater and surface water using selective solid-phase extraction and UPLC-MS/MS. Anal Chem, 2008. 80(13): p. 5021 -30; and Chiuminatto, U., et al., Automated online solid phase extraction ultra high performance liquid chromatography method coupled with tandem mass spectrometry for determination of forty-two therapeutic drugs and drugs of abuse in human urine. Anal Chem, 2010. 82(13): p. 5636-45, the entire contents of which are incorporated herein by reference, use solid phase extraction to isolate analytes from biological samples.

[0073] Preferably, SPE in the methods described herein is anion-exchange chromatography. A preferred resin for use in the solid phase extractions described herein is an amino-functionalized resin, such as an aminopropyl resin, LC-NH₂ (available from Sigma).

[0074] Another embodiment of the invention is a method for simultaneously detecting at least a first DXP pathway metabolite and at least a second DXP pathway metabolite in a sample, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of DXP, MEP, HMBPP, MEC, CDP-ME and CDP-MEP. The method comprises simultaneously detecting at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in a sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio.

[0075] In one aspect of a method for simultaneously detecting at least a first DXP pathway metabolite and at least a second DXP pathway metabolite in a sample, simultaneously detecting the at least first signal produced by the at least first DXP pathway metabolite and the at least a second signal produced by the at least second DXP pathway metabolite includes simultaneously measuring the intensity of the at least first signal produced by the at least first DXP pathway metabolite and the at least second signal produced by the at least second DXP pathway metabolite.

[0076] When a mixture of compounds is separated and analyzed using liquid chromatography or liquid chromatography in conjunction with mass spectrometry, the separated compounds (e.g. , the at least first and the at least second metabolites) are not measured by the instrument at precisely the same instant in time. It should be understood that "simultaneously measuring" refers to a scenario in which the separated compounds (e.g. , the at least first and the at least second metabolites) are, for example, simultaneously injected into the instrument as a single sample or aliquot of a sample. "Simultaneously measuring" is merely meant to distinguish from a scenario in which, for example, a separate injection is made for each metabolite to be analyzed or detected.

[0077] Yet another embodiment of the invention is a method for identifying a modulator of an enzyme in the DXP pathway. The method comprises exposing a bacterial cell in a medium to a potential modulator of an enzyme in the DXP pathway, thereby providing a sample. At least a first and at least a second DXP pathway metabolite are extracted from the sample by solid-phase extraction, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of DXP, MEP, HMBPP, MEC, CDP-ME and CDP-MEP, thereby obtaining an extracted sample. The intensity of at least a first signal produced by the at least first DXP pathway metabolite and the intensity of at least a second signal produced by the at least second DXP pathway metabolite in the extracted sample are measured by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid

[0078] In some embodiments of a method for identifying a modulator of an enzyme in the DXP pathway, the modulator is an inhibitor of an enzyme in the DXP pathway; the at least first metabolite is a substrate of the enzyme; the at least second metabolite is a product of the enzyme; and the change is an increase in the measured intensity of the at least first signal and a decrease in the measured intensity of the at least second signal.

[0079] In other embodiments of a method for identifying a modulator of an enzyme in the DXP pathway, the modulator upregulates an enzyme in the DXP pathway; the at least first metabolite is a substrate of the enzyme; the at least second metabolite is a product of the enzyme; and the change is a decrease in the measured intensity of the at least first signal and an increase in the measured intensity of the at least second signal.

[0080J In some embodiments of a method for identifying a modulator of an enzyme in the DXP pathway, the modulator of the enzyme in the DXP pathway is an antibiotic and the potential modulator is a potential antibiotic, for example, fosmidomycin. [0081] Another embodiment of the invention is a method for determining the rate constant of a flux in the DXP pathway of a bacterium in a medium by simultaneously measuring the amount of at least a first DXP pathway metabolite and at least a second DXP pathway metabolite, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of 1 -deoxy-D- xylulose 5-phosphate, 2C-methyl-D-erythritol 4-phosphate, 4-diphosphocytidyl-2C- methyl D-erythritol, 4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, 2C- methyl-D-erythritol 2,4-diphosphate and hydroxylmethylbutenyl diphosphate. The method comprises incubating a first bacterial cell in a medium for a first period of time, thereby providing a first sample; and incubating a second bacterial cell in a medium for a second period of time different than the first period of time, thereby providing a second sample. The intensities of at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in the first sample are simultaneously measured by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals corresponds to an amount of the at least first and the at least second metabolites, respectively, in the first sample after the first period of time and is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio. The intensities of the at least first signal produced by the at least first DXP pathway metabolite and the at least second signal produced by the at least second DXP pathway metabolite in the second sample are simultaneously measured by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals corresponds to an amount of the at least first and the at least second metabolites, respectively, in the second sample after at least the second period of time and is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio. A rate constant of a flux is calculated using the measured intensity of the at least first and the at least second signals after the first period of time and after the second period of time, thereby determining the rate constant of a flux in the DXP pathway.

[0082] The method for determining the rate constant of a flux in the DXP pathway can be used to identify a rate limiting flux in the DXP pathway. Figure 9 shows metabolites and fluxes involved in the DXP pathway. A rate limiting flux can be identified, for example, by comparing the rate constants, obtained according to the method described herein, of two or more fluxes in the DXP pathway. Therefore, in some embodiments of a method for determining the rate constant of a flux in the DXP pathway, the flux is a rate limiting flux in the DXP pathway.

[0083J Another embodiment of the invention is a method for determining the rate constant of a flux in the DXP pathway of a bacterium in a medium by simultaneously measuring the amount of at least a first DXP pathway metabolite and at least a second DXP pathway metabolite, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of 1-deoxy-D- xylulose 5-phosphate, 2C-methyl-D-erythritol 4-phosphate, 4-diphosphocytidyl-2C- methyl D-erythritol, 4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, 2C- methyl-D-erythritol 2,4-diphosphate and hydroxylmethylbutenyl diphosphate. The method comprises simultaneously measuring the intensity of at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in a first sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals corresponds to an amount of the at least first and the at least second metabolites, respectively, in the bacterium or the medium at a first time point and is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio;

simultaneously measuring the intensity of the at least first signal produced by the at least first DXP pathway metabolite and the at least second signal produced by the at least second DXP pathway metabolite in a second sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals corresponds to an amount of the at least first and the at least second metabolites, respectively, in the bacterium or the medium at at least a second time point and is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio; and calculating a rate constant of a flux using the measured intensity of the at least first and the at least second signals at the first time point and the at least second time point, thereby determining the rate constant of a flux in the DXP pathway.

(0084] Another embodiment of the invention is a method for characterizing the effect of a potential modulator of the DXP pathway, for example, to improve production of an isoprenoid. The method comprises exposing a bacterial cell in a medium to at least one potential modulator of the DXP pathway, and/or expressing at least one potential modulator of the DXP pathway, thereby providing a sample;

extracting at least a first and at least a second DXP pathway metabolite from the sample by solid-phase extraction, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of 1 -deoxy- D-xylulose 5-phosphate, 2C-methyl-D-erythritol 4-phosphate, 4-diphosphocytidyl- 2C-methyl D-erythritol, 4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, 2C- methyl-D-erythritol 2,4-diphosphate and hydroxylmethylbutenyl diphosphate, thereby obtaining an extracted sample; simultaneously measuring the intensity of at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in the extracted sample by liquid chromatography, mass spectrometry or liquid

chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio; and comparing the measured intensity of the at least first signal to a signal produced by the at least first DXP pathway metabolite in an extracted sample corresponding to a bacterial cell in a medium not treated with or not expressing the at least one potential modulator, and comparing the at least second signal to a signal produced by the at least second DXP pathway metabolite in the extracted sample corresponding to the bacterial cell in a medium not treated with or not expressing the at least one potential modulator, wherein a change in the measured intensity of the at least first or the at least second signal indicates modulation of the DXP pathway, thereby characterizing a potential modulator of the DXP pathway.

[0085] In some embodiments, the method for characterizing the effect of a potential modulator of the DXP pathway comprises expressing at least one potential modulator of the DXP pathway in a bacterial cell in a medium. In other

embodiments, the method for characterizing the effect of a potential modulator of the DXP pathway comprises exposing a bacterial cell in a medium to at least one potential modulator of the DXP pathway. In yet other embodiments, the method for characterizing the effect of a potential modulator of the DXP pathway comprises expressing at least one potential modulator of the DXP pathway in a bacterial cell in a medium and exposing a bacterial cell in a medium to at least one potential modulator of the DXP pathway.

[0086] In some embodiments, the method for characterizing the effect of a potential modulator of the DXP pathway comprises over-expressing at least one potential modulator of the DXP pathway, and the at least one potential modulator is an enzyme of the DXP pathway. Preferably, the method for characterizing the effect of a potential modulator of the DXP pathway comprises over-expressing at least one enzyme of the DXP pathway selected from the group consisting of dxs, dxr, ispD, ispE, ispF, ispG, ispH, idi and ads. In some embodiments, the at least one enzyme is ispG. In other embodiments, the at least one enzyme includes dxs, idi and ads.

[0087] In some embodiments of the method for characterizing the effect of a potential modulator, the bacterial cell is exposed to one or more of the following potential modulators: an osmolyte, a buffering agent (e.g. , potassium phosphate, dipotassium phosphate, HEPES, etc. ), a potential modulator of an enzyme in the DXP pathway, a co-factor of the DXP pathway, or a metabolite of the DXP pathway. [0088] "Osmolyte," as used herein refers to compounds that affect osmosis.

Typically, osmolytes are soluble in the solution within a cell or in the cellular medium, and protect cells from desiccation. Non-limiting examples of osmolytes include sorbitol, betaine, trimethylamine-iV-oxide, dimethylsulfoniopriopionate, triemethylglycine, sarcosine and taurine. A preferred osmolyte is sorbitol.

[0089] Potential modulators of an enzyme in the DXP pathway include the potential modulators discussed above with respect to a method for identifying a modulator of an enzyme in the DXP pathway.

[0090] "Co-factor," as used herein, refers to a non-protein chemical compound that binds to a protein and is required for the protein's biological activity. Non- limiting examples of co-factors of the DXP pathway include ATP (adenosine triphosphate) and CTP (cytidine triphosphate).

[0091] Metabolites and preferred metabolites of the DXP pathway include those set forth herein.

EXEMPLIFICATION

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

[0092] The engineered DXP pathway has been demonstrated to be a powerful synthetic platform for microbial production of isoprenoids. However, current strategies in manipulation of the DXP pathway are still mostly combinatorial in nature, largely due to the lack, of methods for conveniently monitoring changes of the pathway in metabolite level. Although an LC-MS/MS method was recently reported for measuring some of the DXP metabolites, a more sensitive and convenient method is still desirable because the LC-MS/MS method had only moderate sensitivity (~5 μΜ for most metabolites), and involved liquid-liquid extraction in sample preparation.

[0093] Described herein is the use of UPLC-MS for the simultaneous detection of many of the DXP metabolites. Coupled to an anion-exchange solid phase extraction (SPE) protocol, UPLC-MS was able to simultaneously quantify many of the DXP metabolites in biological samples with good sensitivity (LOQ < 0.1 μΜ) and reliability (intraday variation CV < 12%). Sample preparation was also quick (5 minutes) and easy (drying free), demonstrating the superiority of the SPE UPLC-MS method described herein. In the analyzed biological samples, all the DXP metabolites except CDP-MEP were detected. A strain overexpressing dxs and ispE (the enzyme to produce CDP-MEP) was constructed for the purpose of quantifying the amount of endogenous CDP-MEP. However, the metabolite was still undetectable. Although not wishing to be bound by any particular theory, it is likely that ispE and ispF formed a protein complex in E. coli and consumed most CDP-MEP once it was produced.

[0094] Beyond the method development, this study has also significantly contributed to the body of knowledge of the DXP metabolism in isoprenoid production. In a genetically engineered lycopene-producing E. coli, MEC was found to be actively effluxed by a previously unrecognized flux in the DXP pathway.

Kinetics of this competing pathway were estimated by inhibiting MEC synthesis with fosmidomycin, and measuring the amounts of accumulated intracellular MEC. Figure 15 shows that a portion of the accumulated MEC was actually effluxed within 2 hours of fosmidomycin inhibition instead of being utilized by the cells, indicating that MEC efflux served as a significant competing pathway, diverting carbon source away from isoprenoid biosynthesis (consistent with the inverse correlation between Figure 3A and C). From a theoretical perspective, the amounts of MEC accumulated in the media at 24 hours after induction was approximately 220 μηιοΙ/L (Figure 4C), which was calculated to be equivalent to 4500 ppm lycopene, and significantly greater than the amount of lycopene produced (1000 ppm, Figure 4B). As a rational strategy, ispG was then overexpressed and the efflux of MEC was successfully reduced, resulting in an increase of the downstream DXP pathway intermediates (HMBPP, etc.) and isoprenoid product (lycopene). Diversion of effluxed MEC into isoprenoid biosynthesis will invariably be valuable to the engineering of microbes for the overproduction of isoprenoids.

Chemicals/materials

[0095] The following intermediates were purchased from Echelon: DXP, MEP, MEC, CDP-ME and HMBPP. Unless otherwise indicated, the remaining reagents were purchased from Sigma. Bacterial strains and plasmids

[0096] E. coli BL21 -Gold (DE3) (Stratagen) [E. coli B F^" ompT hsdS (r_B ^~ m_B ^_) dcm⁺ Tet^r gal λ(ϋΕ3) endA Hte] was used for lycopene production with pACLYC [31 ]. pET-SIDF (also referred to as p20T7MEP) was transformed into BL21 -Gold (DE3) harboring pACLYC. IspG was amplified by polymerase chain reaction with the extracted E. coli genomic DNA. Primers XhoI-RBS-ispG F and ispG-BamHI R were used (primers used in this study are summarized in File S6). The purified PCR product was ligated into pET-SIDF which contained Xhol and BamHI sites. The resulting plasmid was termed as pET-SIDFG, which was subsequently transformed into BL21 -Gold (DE3) with pAC-LYC.

[0097] E. coli DH5ot (Invitrogen) [F- (p801acZAM 15 A(lacZ YA-argF)U 169 recA 1 endAl hsdR17(rk^", mk⁺) phoA supE44 thi-1 gyrA96 relAl λ^'] was used for genomic DNA extraction. E. coli XLIO-Gold (Stratagene) [Tet^r A(mcrA)183 A(mcrCB- hsdSMR-mrr)173 endAl supE44 thi-1 recAl gyrA96 relAl lac Hte [F^' proAB lacI^qZAM 15 Tnl O (Tet^r) Tn5 (Kan^r) Amy]] with pAC-LYC was used for production of recombinant ispE enzymes. IspE was amplified by polymerase chain reaction with the extracted E. coli genomic DNA. Primers SacI-ispE F and ispE-XhoI R were used (File S6). The purified PCR product was ligated into a modified pBAD-B (Invitrogen) which contained a six histidine tag and Sacl, Xhol sites. The resulting plasmids were termed as pBAD-ispE, which was subsequently transformed into E. coli XLIO-Gold with pAC-LYC.

[0098] Wild type Bacillus subtilis (BGSC 1 A 1 ) was requested from BGSC. Wild type Chromobacterium violaceum, Pseadomonas aeruginosa and Staphylococcus aureus were gifts from Prof. Yew Wen Shan (Department of Biochemistry, NUS).

[0099] E. coli BL21 -Gold (DE3) (Stratagen) [E. coli B F^" ompT hsdS (r_B ^" m_B ^~ ) dcm⁺ Tet^r gal λ(ϋΕ3) endA Hte] was used for lycopene production with pACLYC [4]. p20T7MEP was transformed into BL21-Gold (DE3) harboring pACLYC.

[00100] E. coli DH5a (Invitrogen) [F- (p80lacZAM15 A(lacZYA-argF)U169 recAl endAl hsdR17(rk\ mk⁺) phoA supE44 thi-1 gyrA96 relAl λ^"] was used for genomic DNA extraction. E. coli XLIO-Gold (Stratagene) [Tet^r A(mcrA)183 A(mcrCB- hsdSMR-mrr)173 endAl supE44 thi- 1 recAl gyrA96 relAl lac Hte [F^' proAB lacI^qZAM 15 Tnl O (Tet^r) Tn5 ( an^r) Amy]] with pAC-LYC was used for production of recombinant enzymes. Dxs/dxr/ispE were amplified by polymerase chain reaction with the extracted genomic DNA. Primers used were dxs_F:

GCTTAGAGCTCAGTTTTGATATTGCCAAATA (SEQ ID NO: l ), dxs_R: GTAAC CTCGAGTTATGCCAGCCAGGC (SEQ ID NO:2), dxr_F:

GCTTAGAGCTCAAGCAACTCACCATTCTGGG (SEQ ID NO:3), dxr_R:

GTAACCTCGAGTCAGCTTGCGAGACGC (SEQ ID NO:4), ispE F:

GCTTAGAGCTCCGGACACAGTGGCCCTC (SEQ ID NO: 5) and ispE_R:

GTAACCTCGAGTTAAAGCATGGCTCTGTGC (SEQ ID NO:6). The purified PCR product was ligated into a modified pBAD-B (Invitrogen) which contained a six histidine tag and Sacl, Xhol sites. The resulting plasmids were termed as pBAD- dxs/dxr/ispE, which were subsequently transformed into E. coli XL I O-Gold with pAC-LYC.

Purification of recombinant proteins

[00101] Recombinant dxs/dxr/ispE was purified from E. coli XL I O-Gold containing pBAD-dxs/dxr/ispE and pAC-LYC. A colony was picked from an agar plate, inoculated into 2xPY medium (20 g/L Peptone, 10 g/L Yeast extract, and 10 g/L NaCl, pH=7) containing 34 μg/mL chloramphenicol and 100 μg/mL ampicillin, and incubated overnight. Five hundred microliter aliquots of cell culture grown overnight were inoculated into 25 mL 2xPY medium in a 250-mL shake flask. Cells were grown at 37 °C with shaking (300 rpm) until OD595 reached the range of 0.5- 1.0. The cells were then induced with 10 mM L-arabinose and grown at 20 °C for 24 hours. The cell pellets were collected and resuspended in 1 mL B-PER II reagent (Pierce) and homogenized on Mini-Beadbeater-16 (Biospec) with 200 μί glass beads for 1 minute. The cell lysates were centrifuged at 16,000 g for 10 minutes at 4 °C and the supernatant was diluted with 20 mL NPI- 10 (50 mM Na₂H₂P0₄, 300 mM NaCl, 10 mM imidazole, pH 8) and incubated with 200 mg Ni-NTA micro beads (USB) for 1 hour at room temperature. The beads were washed with 40 mL NPI- 10 and eluted with 300 μΐ NPI-250 (50 mM Na₂H₂P0₄, 300 mM NaCl, 250 mM imidazole, pH 8). Synthesis ofCDP-MEP

100102] In a 50-μί reaction, 1.25 mM CDP-ME was converted into CDP-MEP with 10.5 μg recombinant ispE in 40 mM Tris (pH 6), 2.5 mM MgCl₂, 50 mM β- mercaptoethanol and 10 mM ATP. The reaction was incubated at 37 °C for 15 hours and terminated with 450 μΙ acidic extraction solution (acetonitrile/methanol/water 40:40:20 + 0.1 M formic acid). CDP-MEP was confirmed by mass spectrometry and the concentration was estimated by measuring consumed CDP-ME in the reaction. Bacteria growth for lycopene production and IPTG induction

[00103] A colony was picked from an agar plate, inoculated into 2xPY medium (20 g/L Peptone, 10 g/L Yeast extract, and 10 g/L NaCl, pH 7) containing 34 μg/mL chloramphenicol and 100 μg mL ampicillin, and incubated overnight. Fifty-microliter aliquots of cell culture grown overnight were inoculated into 5 mL 2xPY medium in a 50-mL Falcon tube. Cells were grown at 37 °C with shaking (300 rpm) until OD595 reached the range of 0.5-1.0. The cells were then induced with the indicated concentration of IPTG and grown at 28 °C for the indicated time before being collected for metabolite extraction or lycopene assay. 25 μg/mL fosmidomycin was added 4 hours after IPTG induction, if required.

Extraction and quantification of lycopene

[00104] Fifty microliters of cell suspension were sampled 8 hours after IPTG induction, and OD595 was recorded. The cells were centrifuged at 3,000 g for 2 minutes and resuspended in 200 μΐ, acetone. Resuspended cells were vortexed for 10 minutes and then centrifuged at 3,000 g for 2 minutes. One hundred microliters of supernatant was mixed with an equal volume of ethanol and transferred to 96-well optical bottom plates (NUNC). Lycopene content was determined by interpolating from a standard dilution of lycopene (Sigma) based on absorbance at 472 nm (Spectra Max 190, Molecular Devices).

Solid phase extraction of the DXP pathway metabolites from biological samples

[00105] Anion-exchange SPE was used to extract the DXP pathway metabolites from biological samples. The SPE recoveries of all of the metabolites were greater than 40% of the initial concentration of standards used. The LC-NH₂ resin (available from Sigma) used in this study was superior in performance when compared to other similar supports.

[00106] Cell suspension equivalent to 6 mL OD595 = 1.0 cells were withdrawn 4 hours and 8 hours after IPTG induction. The cells were centrifuged at 3,000 g for 5 minutes. The cell pellets were resuspended in 10 mL acidic extraction solution (acetonitrile/methanol/water 40:40:20 + 0.1 M formic acid) for intracellular metabolite extraction. One hundred microliters supernatant were diluted with 900 μί modified acidic extraction solution (acetonitrile: methanol 50:50 + 0.125M formic acid) for extracellular metabolite extraction. The resuspended cell pellets in acidic extraction solution were incubated at -20 °C for 60 minutes with periodic shaking. After centrifugation, the supernatant was purified by loading on a cartridge holding 50 mg Sigma LC-NH₂ resin. Diluted broth supernatant was also purified by LC-NH₂ resin in the same way. The cartridge was eluted by gravity with 400 μί, 1% NH₄OH solution after centrifugation at 3,000 g for 2 minutes and the pH of the eluate was subsequently adjusted to pH 5 with 3 μΐ. acetic acid.

UPLC - MS quantification of the DXP pathway metabolites

[00107] Attempts were initially made to quantify the DXP pathway intermediates by gas chromatography mass spectrometry (GC-MS) after derivatization with trimethylsilyl (TMS). DXP and MEP were readily detected (limit of detection at least 9 pmol per injection). However, all other DXP intermediates were not detectable, likely due to the low volatility of the TMS derivatives). Therefore, ultraperformance liquid chromatography mass spectrometry (UPLC-MS) was explored for the quantification of the DXP pathway intermediates.

[00108] Time-of-flight mass spectrometry was selected because it has high scan speed and excellent mass accuracy. A range of mass/charge ratios (m/z) was scanned in the negative mode and synthetic standards were characterized. All DXP pathway intermediates can be detected and their m/z unambigously determined (see Table 2). Separation of the DXP pathway intermediates was then optimized on a UPLC CI 8 column (1.7 μπι particle size). Ion pairing with tributylamine was found to be an ideal ion-pair reagent, allowing excellent resolution of the various intermediates rapidly (10 minutes per sample; see Table 1 and Figures 2A-2E). [00109] The DXP pathway metabolites were detected by UPLC (Waters

ACQUITY UPLC) - (TOF)MS (Bruker micrOTOF II). In brief, an aqueous solution containing 15 mM acetic acid and 10 mM tributylamine in methanol were used as a mobile phase with a UPLC C 18 column (Waters CSH C 18 1.7μιη 2.1x 50 mm). The elution was done at 0.15 mL/min according to the program described in Table 1.

Electrospray ionization was used and (TOF)MS was operated to scan 50-800 m/z in negative mode with -500V end plate voltage and 4500 V capillary voltage. Nebulizer gas was provided in 1 bar, drying gas temperature was 9 mL/min, and dry gas temperature was 200 °C. Sample injection volume was 5 μί.

Table 1. Mobile phase gradient used for the separation of DXP pathway metabolites in the UPLC method.

Step Time (minutes) Aqueous solution* Methanol

1 0 100% 0

2 1.8 100% 0

3 3.1 60% 40%

4 4.9 60% 40%

5 5.4 10% 90%

6 9.5 10% 90%

7 10 100% 0

* Aqueous solution: 15 mM acetic acid and 10 mM tributylamine

[00110] A range of m/z typically with 0.06 m/z width (the average m/z distribution width with the MS instrument used) was extracted from the acquired data (50-800 m/z) for each intermediate. The range of m/z was determined with 50 μΜ standards of each intermediate prepared individually in water except CDP-MEP which was in elution solution of SPE. Under the assay conditions, all the intermediates were detected in the form [M-H]^'. Retention time was determined for each intermediate with the same 50 μΜ standards and the set m/z extraction range. The integrated area of signal peak at its retention time then was calculated for each intermediate with the software provided by the manufacturer. Based on the integrated area of signal, the concentrations of the DXP pathway metabolites were determined by interpolating from a standard dilution of the intermediates prepared in a biological matrix (cell extract or broth supernatant).

[00111] To examine the linearity and limit of quantification (LOQ) of the SPE UPLC-MS analysis for the DXP pathway intermediates in cell extracts, a mixture of DXP, MEP, CDP-ME, CDP-MEP, MEC, and HMBPP were spiked into 10 mL of cell extracts (containing no more than 0.01 μΜ DXP intermediates) to final concentrations ranging from 2 μΜ to 0.01 μΜ. The extracts were then loaded onto an SPE cartridge and eluted in ammonium hydroxide. The results, which are shown in Table 2, showed that the UPLC-MS signals of all metabolites were linearly correlated with their initial concentrations in cell extracts (R >0.99). In addition, Table 2 shows that an LOQ of at least 0.02 μΜ was achieved for each metabolite, except for CDP-MEP, for which the LOQ was 0.1 μΜ. Intraday and interday variations of C V less than 20% and, in some cases, less than 12%, allowed precise quantification of the DXP pathway intermediates in biological samples.

Table 2. Retention time, quantification ions, linearity, limit of quantification (LOQ) and repeatability of SPE-UPLC-MS analysis of the DXP pathway metabolites in cell extracts.

Retention

Quantification LOQ

Compound time Linearity Repeatability (0.1 μΜ)*

ions (μΜ)

(mins)

Intraday Interday Interday

Range variation variation variation

R²

(μΜ) (n=5, (n=3, (n=4,

CV%) CV%) CV%)

0.01 - 7.71

DXP 5.6 213.0170±0.03 0.9934 0.01 6.85 9.94

2

215.0330+0.01 - 0.01 - 7.83

MEP 5.2 0.9956 0.01 4.69 7.75

0.03 2

0.01 - 7.62

CDP-ME 6.2 520.0730±0.03 0.9941 0.01 3.90 5.73

0.5

CDP-MEP 7.3 600.0390±0.03 0. 1 - 1 0.9902 0.1 10.91 19.10 1 1.63

0.02 - 5.63

MEC 6.6 276.9884±0.03 0.99 1 0.02 6.20 6.96

2

0.01 - 9.97

HMBPP 7.0 260.9920±0.03 0.9959 0.01 6.54 14.96

2

^♦Concentration of CDP-MEP for intra-day/inter-day variation was 0.25 μΜ.

[00112] Though LOQ of CDP-MEP (0.1 μΜ) was much higher than that of the other metabolites examined, the method described herein was still considerably more sensitive than reported for the quantification of mevalonate metabolites (a similar class of compounds to DXP metabolites) at LOQ of 4.17 μΜ. CDP-MEP was found to be co-eluted with many major cellular phospho-compounds, which is likely to result in lower signal due to the phenomenon of ion suppression. Nonetheless, most of these intermediates were readily detected in the genetically modified strains relevant to metabolic engineering of the DXP pathway.

[00113] The effect of other UPLC-MS conditions on the separation and detection of the DXP pathway metabolites was also investigated. Figures 2A-2E are spectra showing the effects of UPLC gradient on the retention time of each metabolite.

Specifically, 0.8, 0.4, 2.6, 2.6, 0.5 and 2 μΜ solutions of DXP, MEP, CDP-ME, CDP- MEP, MEC and HMBPP, respectively, were prepared in 1 mL acidic extraction solution. The UPLC gradient used to produce the spectrum of Figure 2A mirrors the program described in Table 1 , except 40% of the aqueous solution was used in steps 3 and 4. The spectrum of Figure 2B was obtained using the program described in Table 1 , except 50% of the aqueous solution was used in steps 3 and 4. The spectrum of Figure 2C was obtained using the program described in Table 1 , without modification. The spectrum of Figure 2D was obtained using the program described in Table 1 , except 70% of the aqueous solution was used in steps 3 and 4. The spectrum of Figure 2E was obtained using the program described in Table 1 , except 80% of the aqueous solution was used in steps 3 and 4.

Growth of bacteria other than E. coli

[00114] Glycerol stocks of Bacillus subtilis, Chromobacterium violaceum, Pseudomonas aeruginosa and Staphylococcus aureus were directly inoculated into 2xPY and incubated at 37 °C overnight. Cell pellets of the cells grown overnight were resuspended in 10 mL 2xPY to an OD595 of about 0.2, except Bacillus subtilis was resuspended to an OD595 of about 0.4, in a 50-mL Falcon tube. Cells were grown at 37 °C with shaking (300 rpm) until OD595 readings of 2-3.

Synthesis of MEP in vitro and fosmidomycin inhibition

[00115] In a 20 μΐ reaction, MEP was synthesized from 20 mM DL-GAP and 10 mM pyruvate with 3 μg recombinant dxs and dxr in 40 mM Tris (pH 6), 12.5 mM MgCl₂, 50 mM β-mercaptoethanol, 10 mM ThDP and 10 mM NADPH.

Fosmidomycin was added to a final concentration of 25 μg/mL for dxr inhibition studies. The reaction was incubated at 37 °C for 3 hours and \ 0-μΙ. aliquots of the reaction were sampled and terminated with an acidic extraction solution

(acetonitrile/methanol/water 40:40:20 + 0.1 M formic acid). The samples were purified by SPE and quantified as described below.

Analysis of a lycopene-producing E. coli

[00116] To demonstrate the utility of the SPE-UPLC-MS method, DXP pathway metabolites were measured in a lycopene-producing E. coli. Lycopene is an antioxidant isoprenoid and its production in E. coli is known to be enhanced by overexpression of four enzymes in the DXP pathway (dxs, idi, ispD and ispF) under non-induced conditions. The enzymes ispD and ispF are jointly referred to herein as ispDF. Consistent with other studies, overexpression of the four enzymes under induced conditions (0.01 mM or more IPTG) inhibited lycopene production (see Figure 3A). Next, intracellular concentrations of the DXP pathway metabolites were quantified in cells harvested in the exponential growth phase (5 hours after IPTG induction). Four metabolites, DXP, MEP, CDP-ME and MEC, were detected and the concentrations generally increased with increased concentrations of the inducer, IPTG (Figure 3B). Among the detected metabolites, the most abundant was MEC.

[00117] Because an abrupt decrease of intracellular MEC concentration upon induction with 0.1 mM IPTG was observed, the broth supernatant was also subjected to SPE-UPLC-MS to investigate whether MEC was released from the cells under the conditions examined. A large quantity of MEC, more than the total amount found in the cellular fraction, was detected in the medium (Figure 3C). This was an unexpected observation as highly charged, phosphorylated compounds do not generally diffuse across intact cell membranes. Growth curves of E. coli strains expressing BL21-Gold (DE3) harboring pET-SIDF without and with IPTG induction (0.1 mM) were very similar, suggesting that the viabilities of the cells were not observably different. Preliminary studies showed that the fsr efflux pump may be involved in effluxing MEC from the cells. Although not wishing to be bound by any particular theory, it is possible that MEC reached an intracellular threshold concentration (e.g., approximately 30 μι-nol/L), and a cellular stress response was triggered. The stress response may then activate export pumps to prevent further intracellular accumulation of MEC and may coincidently shut down isoprenoid synthesis by an unknown mechanism. Although the exogenous addition of MEC to levels similar to the amounts effluxed did not affect cell growth and lycopene production, efflux of MEC itself may have diverted carbon source away from biosynthesis of isoprenoid products, resulting in the decrease in isoprenoid biosynthesis. The inverse correlation between lycopene production and the concentration of extracellular MEC at various IPTG inductions was consistent with this hypothesis (Figure 4A).

Rational engineering of the DXP pathway in E. coli for lycopene production

[00118) To test whether lycopene production inhibition was related to MEC efflux, the strain was engineered to co-overexpress ispG (with dxs-idi-ispDF), the enzyme utilizing MEC. Under strong induction conditions (0.1 mM IPTG) in which lycopene production was severely inhibited and MEC efflux was high, co-overexpression of ispG concurrently increased lycopene production (Figure 4B) and reduced efflux of MEC (Figure 4C). The concentration of intracellular MEC was found to be increased with ispG overexpression, a result supporting the proposal of an intimate relationship between ispG and intracellular MEC. HMBPP (a product of ispG) was also readily detected inside the cells overexpressing ispG (HMBPP was not detectable in the parental strain expressing dxs-idi-ispDF, Figure 4E), consistent with the proposal that with higher ispG more MEC was consumed for isoprenoid biosynthesis. In order to gain an insight into the profiles of efflux pumps in these strains, all 20 known efflux pump operons in E. coli were analyzed at the transcription level. The expression levels of these pumps were not regulated by ispG overexpression (Figure 14). Taken together, overexpression of the metabolic enzyme ispG reduced the amount of MEC efflux, possibly by shuttling it directly to downstream products, e.g. , lycopene.

Analysis of bacteria other than E. coli

[00119] Besides E. coli, other bacteria have also been used for production of isoprenoids. The SPE-UPLC-MS method disclosed herein was used to detect DXP pathway metabolites in two gram negative strains (Chromobacterium violaceum and Pseudomonas aeruginosa) and two gram positive strains {Bacillus subtilis and Staphylococcus aureus). Staphylococcus aureus utlizes the MVA pathway but not the DXP pathway, and was included as a negative control. Analysis of the cells harvested in the middle exponential growth phase (OD595 = 2-3) showed that intracellular DXP pathway intermediates can be detected in all strains except S. aureus (Figure 5). The four bacteria used in this study were all wild type, meaning none of the DXP pathway genes were overexpressed. The concentration of the DXP pathway metabolites in these strains was lower than those in engineered E. coli, yet the developed method was sensitive enough to measure these metabolites. None of the DXP pathway metabolites were detected in the broth supernatant of these strains, suggesting that no DXP pathway intermediates were effluxed from the cells under these conditions. Drug discovery targeting the DXP enzymes

[00120] The DXP pathway is absent in humans, but is essential in bacteria.

Therefore, enzymes in the DXP pathway are ideal targets for development of broad spectrum antibiotics. The SPE-UPLC-MS method disclosed herein can be used to identify inhibitors of enzymes of the DXP pathway because activity of each enzyme can be directly monitored.

[00121] To prove this concept, fosmidomycin, a known inhibitor of dxr, was spiked into in vitro reconstituted enzymatic reactions containing dxs and dxr. The decrease of dxr enzymatic activity by fosmidomycin inhibition was readily recognized based on the sharp decrease in MEP formation and DXP accumulation (Figure 6). Figure 7 is a bar graph of the intracellular concentrations of DXP, MEP, CDP-ME and MEC as a function of fosmidomycin concentration 8 hours after induction with 0.1 mM IPTG and shows that inhibition of dxr enzymatic activity by fosmidomycin can also be monitored in growing E. coli using the methods described herein. With a large chemical library, the method described herein could be immensely useful for the discovery of novel inhibitors targeting the DXP pathway enzymes in vivo and in vitro. The ability to monitor the DXP pathway in vivo will invariably provide convenient and productive assays directly related to the physiology of the bacterial growth, accounting for pharmacokinetic and pharmacodynamic limitations of drug diffusion into cells.

Computational modeling of the DXP pathway

[00122] Quantitative mathematical modeling can be used to identify unknown fluxes and regulatory controls of previously unknown metabolic pathways and can provide a rapid and rational approach to engineer the pathway to improve the productivity of the pathway. For example, a feedback inhibition control can be discovered, and the enzymes involved in the feedback inhibition can then be engineered to be insensitive to the regulation, maintaining constant high activity during cell growth. [00123] The SPE-UPLC-MS method disclosed herein enabled mathematical modeling of the DXP pathway for isoprenoid production. The ability to collect reliable, high dimensional time series metabolite data, such as that shown in Figures 8A and 8B, is essential in model construction. Figure 9 shows the metabolites and fluxes involved in the DXP pathway. The unexpected observation of DXP and MEC in the broth supernatant led to the discovery of a novel flux originating from the DXP pathway. The discovery of the efflux of DXP and MEC, in turn, enabled the assignment of a previously unknown kinetic parameter, labeled ki in Figure 9.

Enhancing Solubility of DXP Pathway Enzymes for Isoprenoid Production

[00124] Isoprenoids, a large family of natural compounds including many plant based pharmaceuticals such as artemisinin and paclitaxel, are produced by the deoxyxylulose phosphate (DXP) pathway and/or the mevalonate (MVA) pathway in nature. The current industrial isoprenoid production methods include direct extraction from plants and semi-synthesis using plant metabolites. These processes are all restricted by the supply of specific plant materials, which are often affected by unpredictable factors including variations in weather and market fluctuations. To improve sustainability and production capacity of isoprenoids, heterologous biosynthesis from economical carbon sources in microbes has been intensively studied in the past decade. To date, there have been many successful reports of using the DXP pathway to produce isoprenoid nutraceuticals {e.g., lycopene and other carotenoids) and pharmaceuticals {e.g., artemisinin precursors and paclitaxel precursors).

[00125] This study examined the solubility status of all the DXP enzymes when overexpressed and attempted to demonstrate the importance of protein solubility in the production of secondary metabolites. Computational prediction was initially explored to evaluate the solubility status and empirical verifications were carried out in E. coli. An unanticipated and critical observation is that many DXP enzymes (DXS, ISPA, ISPG and ISPH) were found to be highly insoluble. Interestingly, the enzymes IDI, ISPD and ISPF, thought to be rate-limiting and hence useful for the enhancement of isoprenoids production, were found to be highly soluble. From these observations, it is now necessary to reevaluate the use of the other highly insoluble DXP enzymes for enhancing isoprenoid production. Attempts were also made to optimize the solubility of the insoluble enzymes and to examine the enhancements in isoprenoid production.

Solubility of over-expressed recombinant DXP pathway enzymes

[00126] DXP pathway has so far been characterized to be a linear pathway, producing IPP and DMAPP from pyruvate and GAP, two important metabolites in central metabolism (see Figure 1 ). IPP and DMAPP (C5) are further assembled into geranyl diphosphate (GPP, C IO) and farnesyl diphosphate (FPP, C I 5), precursors for all C I O and C 15 isoprenoids. To date, little is known of the solubility of the enzymes involved in this pathway when overexpressed for the production of isoprenoids.

[00127] As a first attempt, the solubility of the enzymes in the DXP pathway was evaluated by in silico modeling. Revised WH method (Davis, G.D., et al. : "New fusion protein designed to give soluble expression in Escherichi coli" Biotechnol. Bioeng. 1999, 65(4):382-388), one of the most commonly used and accurate methods, was used to predict solubility of the DXP pathway enzymes. Some of the enzymes (DXS, ISPE and ISPG) were predicted by these methods to be insoluble when overexpressed in E. coli (Table 3). Similarly, in vitro expression study showed that a subgroup of the DXP pathway proteins (DXS and isopentenyl diphosphate isomerase (IDI)) were insoluble (Table 3). As in silico prediction did not completely agree with the published in vitro expression data, it was essential to determine the solubility of the enzymes when overexpressed in vivo.

Table 3. Solubility of the enzymes in the DXP pathway predicted in silico and determined in vitro.

In silico predicted In vitro determined

Protein

solubility solubility

DXS insoluble 22%

DXR soluble N.D.*

ISPD soluble 67%

ISPE insoluble 100%

ISPF soluble 84%

ISPG insoluble N.D.*

ISPH soluble 75%

IDI soluble 32% ISPA soluble 85%

*N.D.: not detected.

[00128] To verify that some of the DXP enzymes, when overexpressed, were differentially soluble, each of the enzymes was expressed individually in three distinct expression systems in different strains of E. coli (BL21 strain - T7 promoter, Ml 5 strain - T5 promoter and DH10B strain - araBAD promoter) at two temperatures (37 °C and 20 °C). The standard dosage of inducers were used to trigger expression of the proteins (lOmM L-arabinose or ImM IPTG). Figures 10A-10D show that, in general, solubility of the proteins varied significantly (5% to 90%). The large variances in solubility across proteins suggested that the method for identifying and quantifying protein solubility is unbiased. This protein solubility analysis method was further validated by filtration studies (data not provided). A group of the DXP enzymes (DXS, ISPG, ISPH and ISPA) was found to be highly insoluble (solubility less than 30% in all conditions examined). DXS, the committed enzyme of DXP pathway, previously identified to be rate-limiting for isoprenoid production, was found to be highly insoluble in the present study. Using dxs as a prototype of highly insoluble enzymes, the impact of inclusion body formation on metabolic engineering of E. coli for isoprenoid production was examined.

Enzymatic activity of insoluble recombinant DXS

[00129] Although some inclusion bodies formed with certain enzymes have been reported to be active, it is generally accepted that inclusion bodies contain primarily incorrectly folded proteins and are functionally inactive. To test whether insoluble DXS is catalytically functional, lysates containing recombinant insoluble DXS were characterized by an in vitro assay, in which DXS activity was determined by measuring the formation of DXP. It was found that DXP was produced at low levels (less than 1 μΜ) with insoluble DXS containing lysates. As a comparison, when the same amount of purified soluble DXS was spiked into the lysates, a high level of DXP (~700 μΜ) was produced (Figure 1 1 ), confirming that specific activity of insoluble DXS was significantly lower than that of soluble DXS. This observation suggested that strategies to increase the solubility of DXS may confer higher activity and metabolic flux for isoprenoid production in vivo. Improving solubility of DXS enhanced the production of DXP

[00130] Improvement of recombinant protein solubility has been intensively studied for the purpose of overproducing soluble proteins, and various effective strategies have been reported, such as lowering incubation temperature, use of fusion partner, overexpression of chaperone proteins and protein mutagenesis. Recently, Prasad et al. reported a simple yet effective approach to increase the solubility of recombinant proteins, where sorbitol at high concentration reduced protein aggregation in E. coli (Prasad, S. et al. : "Effect of chemical chaperones in improving the solubility of recombinant proteins" Appl. Environ. Microbiol. 201 1 , 77(13):4603- 4609). To test if this approach could increase the solubility of DXS, high

concentration of sorbitol was added directly to the cells in culture. Figures 12A and 12B show that the solubility of DXS significantly increased upon addition of sorbitol (500 mM) to the culture solution. Figure 12A also shows that other chemicals, including osmolytes (betaine) and buffering agents (HEPES, phosphate) did not improve the solubility of dxs significantly. Sorbitol similarly improved the solubility of some, but not all other DXP enzymes.

[00131 J To demonstrate that improved solubility of DXS results in enhanced production of DXP, cells grown in sorbitol were lysed and the metabolites in the extracts quantified by LC-MS. Figure 12C shows that the concentration of DXP was significantly higher in sorbitol-treated cells as compared to control cells. Addition of sorbitol did not alter the production of DXP with cells overexpressing a nonfunctional DXS, indicating that the effect of sorbitol on cells overexpressing functional enzyme is likely due to the increase in the concentration of soluble DXS. A parallel increase in the concentrations of MEP and MEC were also observed, suggesting that sorbitol increased the flux through the entire DXP pathway in cells overexpressing DXS. Improvement of ERG 12 solubility and overproduction of mevalonate phosphate

[00132] To extend the observation of the effect of sorbitol, a critical enzyme (ERG 12) in the mevalonate pathway (the other isoprenoid precursor producing pathway, see Figure 13 A) was investigated. More than half of overexpressed ERG 12 was insoluble, and Figure 13B shows that sorbitol enhanced the solubility of ERG 12. In line with the hypothesis that increased solubility confers higher enzymatic activity and better productivity of the respective metabolite, the production of mevalonate phosphate (MV AP) was doubled in the presence of high concentrations of sorbitol (Figure 13C). Since the MVA pathway is not endogenous to E. coli, the production and accumulation of MVAP was attributed to the enzymatic activity of ERG 12.

Discussion

[00133] This study addressed an important and often overlooked issue of the solubility of over-expressed recombinant homologous or heterologous enzymes in metabolic engineering. Specifically, the solubility status of overexpressed DXP enzymes and a heterologous enzyme of the MVA pathway was studied, and its impact on the production of critical precursor metabolites (DXP or MVAP), which are building blocks of all isoprenoids. It was unexpected that four out of nine enzymes in DXP pathway (DXS, ISPA, ISPG and ISPH) were highly insoluble, despite being endogenous enzymes. Overexpression of DXS resulted in the accumulation of highly insoluble and non-functional (<1% activity of the equivalent soluble form) enzyme. This observation cautions against the assumption that overexpression of an enzyme necessarily confers higher enzymatic activity. Interestingly, the combinatorial screening study based on this contentious assumption identified three rate-limiting DXP enzymes (IDI, ISPD and ISPF), which incidentally were found to be highly soluble (Figures 10A-10D). It is thus not unreasonable to speculate that the previously thought to be 'non rate-limiting' enzymes found to be insoluble in this study, may serve to enhance the productions of isoprenoids when expressed in soluble forms.

[00134] Using DXS as a model enzyme, four commonly used fusion partners, trxA, nusA, slyD and malE were fused at the N-terminus of DXS in the attempt to increase solubility. The use of these fusion partners did not significantly increase the solubility of DXS. The effectiveness of the fusion partners in enhancing protein solubility is largely protein-dependent and unpredictable. Cysteine residues on THE surface of DXS (C32, C330 and C457), may form non-specific disulfide bonds and result in protein aggregation. Site-directed mutagenesis of these residues also did not improve solubility, suggesting that the aggregation of dxs protein was not due to disulfide bond mediated interactions. [00135] Osmolytes have been shown to improve the solubility of overexpressed proteins in E. coli. Sorbitol at high concentrations significantly improved DXS solubility and the production of the metabolic product (DXP) in E. coli, indicating that solubility of recombinant enzymes is an important factor in the production of secondary metabolites. Consistent with this suggestion was that ERG 12, another model enzyme, also showed improved solubility and secondary metabolite production in the presence of sorbitol. The metabolic intermediates (DXP, MEP and MEC, etc.) instead of final product (lycopene, etc.) were used as read-outs for characterization of DXS because the rate limiting step (ISPG) existed between the intermediates and the isoprenoid products. An alternative to the use of sorbitol is to modify the host microbes (such as manipulation of cellular protein folding system) to render these proteins more soluble.

[00136] In this study, about half of the nine DXP proteins (DXS, ISPG, ISPH and ISPA) were found to be highly insoluble when overexpressed in E. coli. Insoluble DXS, the committed enzyme of the DXP pathway, showed significantly less enzymatic activity when compared to the equivalent amount of soluble enzyme in vitro. High concentration of sorbitol successfully increased the solubility of DXS and resulted in a parallel increase in the metabolic product (DXP). The strategy also improved both solubility and secondary metabolite production of ERG12, a critical enzyme in the mevalonate pathway. This study highlighted the importance of protein solubility in metabolic engineering of microbes for the overproduction of isoprenoids.

[00137] All the DXP genes were amplified from E. coli genomic DNA and cloned into the modified pBAD-B (Invitrogen), pET-1 la (Stratagene) and pQE30 (Qiagen) plasmids with 6xhis tag, Sad, Xhol restriction enzyme sites. Fusion partners (trxA, nusA, malE and slyD) were amplified from E. coli genomic DNA and cloned into pBAD-dxs with Ncol and Sacl sites. Erg 12 was amplified from S. cerevisiae genomic DNA and cloned into the modified pBAD-B plasmid with 6xhis tag, Sacl and Xhol restriction enzyme sites. Dxs mutants R398A, C32A, C330A, C457A and C32A- C330A-C457A were generated according to the 'megaprimer protocol (Sarkar, G. and Sommer S.S.: "The 'megaprimer' method of site-directed mutagenesis"

Biotechniques 1990, 8(4):404-407). All the pET- 1 la, pBAD-B and pQE30 based plasmids were transformed into E. coli BL21-Gold (DE3), E. coli DH10B and E. coli Ml 5, respectively. pAC-LYC was co-transformed with all the plasmids except pBAD-ergl2.

E. coli growth and induction of protein expression

[00138] A colony was picked from agar plate, inoculated into 2xPY medium (20g/L Peptone, l Og/L Yeast extract, and l Og/L NaCl, pH=7) containing proper antibiotics, and incubated overnight. Ten microliter aliquots of overnight grown cell culture were inoculated into lmL 2xPY medium in 14mL Falcon tube. Cells were grown at 37°C/300rpm till OD595 reached the range of 0.5-1.0. The cells were then induced with ImM IPTG (E. coli BL21-Gold (DE3) and E. coli Ml 5) or lOmM L- arabinose (E. coli DH10B) and grown at 37°C or 20°C for indicated time before collected for protein solubility assay or metabolite assay. Additives (sorbitol, betaine, phosphate, HEPES, mevalonate etc.) were also fed to cell culture upon induction if necessary.

Prediction and quantification of protein solubility

[00139J The revised WH algorithm (Davis, G.D., et al. : "New fusion protein designed to give soluble expression in Escherichi coli" Biotechnol. Bioeng. 1999, 65(4):382-388) was used for prediction of protein solubility. Protein solubility was experimentally quantified by centrifugation [21 ] as described below. 24 hours after induction, cell suspension equivalent to l mL OD595=1.0 cells, was centrifuged, and the pellet was resuspended in lOOuL B-PERII reagent (Pierce). The mixtures were vortexed at room temperature for 10 minutes, and centrifuged at 16,000g for 10 minutes. The supernatant containing soluble cell lysates, and the pellets (resuspended in l OOuL 2% w/v SDS) containing insoluble cell lysates were analyzed by SDS- PAGE. The SDS-PAGE gel was visualized by staining with instant blue (Gentaur), and image of the gel was processed and quantified by the software Quantity One (Bio- Rad). Protein solubility was defined as the quantity of the target protein in soluble cell lysates over that in total cell lysates (soluble cell lysates + insoluble cell lysates). Because ERG 12 protein cannot be separated from an abundant endogenous protein on SDS-PAGE, it was detected by western blot analysis with anti-6xhis tag antibody (Penta-his Ab, Qiagen). In vitro quantification of dxs activity

[00140] The DH10B strain - araBAD promoter system was used to produce DXS at 20 °C, whose catalytic activity was characterized in vitro. 24 hours after induction, insoluble cell lysates were prepared as described above except that they were resuspended in lOOuL NPI-10 (50mM NaH₂P0₄, 300mM NaCl, lOmM imidazole. pH=8) instead of 2% w/v SDS. 1 ί of the mixture was then incubated in 20 μΙ_, in vitro reaction solution containing 40mM Tris (pH = 6), lOmM pyruvate, 20mM DL- glyceraldehyde 3-phosphate, ImM thiamine diphosphate, 12.5 mM MgCl₂ and 5 mM β-mercaptoethanol. The reaction was terminated by lmL acidic extraction solution (acetonitrile/methanol/water 40:40:20, lOOmM formic acid) after 2 hours incubation at 37°C, and formation of DXP was quantified by SPE UPLC-MS.

SPE UPLC-MS quantification of DXP and MVAP

[00141 J Concentration of DXP and MVAP in cell culture was quantified by SPE UPLC-MS. 5 hours after induction, 50 μί cell suspension was sampled and diluted in lmL acidic extraction solution (acetonitrile/methanol/water 40:40:20, lOOmM formic acid) and centrifuged at 16,000g for 1 minute. Supernatant was loaded to a cartridge holding 1 1 mg LC-NH2 resin (Sigma) that was activated by 200uL acidic extraction solution. The cartridge was centrifuged at 2,800g for lmin, and eluted with Ι ΟΟμί 1 % w/v NH4OH that was subsequently neutralized by 0.75μί acetic acid. The eluate was analyzed by UPLC (Waters ACQUITY UPLC) - MS (Bruker micrOTOF II) as described below. Aqueous solution (A) containing 15 mM acetic acid and 10 mM tributylamine and methanol (B) were used as mobile phase with a UPLC C I 8 column (Waters CSH C 18 1.7μη 2.1 x 50mm). The elution was done at 0.15 mL/min with gradient (start: 100% A, 1.8min: 100% A, 3. l min: 60% A, 4.9min: 60% A, 5.4min: 10% A, 9.5min: 10% A, lOmin: 100% A). Electrospray ionization was used and (TOF)MS was operated to scan 50-800 m/z in negative mode with -500V end plate voltage and 4500V capillary voltage. Nebulizer gas was provided in l bar, drying gas temperature was 9mL/min, and dry gas temperature was 200°C. Sample injection volume was 5μΙ,. A range of m/z was extracted from the acquired data for DXP (213.0170±0.03, eluted at 5.6min) or MVAP (227.0315±0.03, eluted at 6.7min). The integrated area of signal peak at its retention time then was calculated for the metabolites with the software provided by the manufacturer. Based on the integrated area of signal, concentration of DXP and MVAP were determined by interpolating from a standard dilution of the intermediates prepared in biological matrix.

Optimization of Amorphadiene Synthesis in Bacillus Subtilis via Transcriptional, Translational and Media Modulation

[00142] Microbial production of isoprenoids has been extensively studied in the past decade. Escherichia coli is one of the most commonly used hosts due to its fast growth rate on a variety of carbon sources and the availability of abundant genetic tools. Research with this organism has yielded gram per liter production levels for certain isoprenoid precursors. Besides E. coli, Saccharomyces cerevisiae has been used for microbial production of pharmaceuticals due to its GRAS (generally regarded as safe) status and availability of a very advanced set of tools for genetic transformation. However, S. cerevisiae grows at half the rate as E. coli, thus limiting its isoprenoid productivity. Considering also the non-GRAS status of E. coli, it is of great interest to engineer a fast growing GRAS bacterium for high level production of pharmaceutical isoprenoids. Bacillus subtilis is a good candidate because it is a GRAS bacterium with rapid growth rate and has yielded satisfactory performance in the production of proteins and valuable metabolites from economical carbon feed stocks. To date, there is only one report on producing heterologous isoprenoids in B. subtilis, which demonstrated the synthesis of a carotenoid in B. subtilis with no quantification data on the product yield. The challenge for producing isoprenoid pharmaceuticals in B. subtilis is the lack of well characterized genetic tools for fine- tuning multiple gene expression that is essential for high level heterologous production of isoprenoids.

[00143] Recently, a promoter of Bacillus megaterium gene xylA (PxylA) was demonstrated to be a strong and inducible (by xylose) promoter for protein expression in B. subtilis. If another controllable promoter that is compatible with PxylA in B. subtilis could be identified, then it could be combined with PxylA to independently control expression of two gene cassettes. Pgrac, an IPTG inducible hybrid promoter containing an E. coli lac operator, was selected on the hypothesis that promoters from different species were unlikely to interact. To test the two promoter system, PxylA and Pgrac were employed to express genes for the synthesis of amorphadiene, a critical precursor to the antimalarial drug artemisinin. The genes overexpressed were ads and dxs-idi; ads encodes the synthase to cyclize farnesyl diphosphate into amorphadiene; dxs and idi were two genes in the deoxyxylulose phosphate (DXP) pathway and were found to control availability of farnesyl diphosphate in E. coli (see Figure 16A). Ads was inserted after Pgrac on plasmid pHTOl (termed as pHT-ads, see Figure 16B), and dxs-idi were cloned under the control of PxylA on plasmid pWH1520 (termed as pWH-DI, see Figure 16B). Figure 16C is a graph quantifying transcription of pxy 1 A and pgrae, and shows that PxylA and Pgrac were inducible by xylose and IPTG, respectively, when used together in B. subtilis. Transcriptions of dxs-idi and ads were well controlled over a wide dynamic range (10⁴ to 10⁶ a.u.), and maximal induction of PxylA and Pgrac was attained with 0.1% xylose and 0.1 mM IPTG, respectively.

[00144] With the validated promoter system, amorphadiene synthesis was systematically optimized by fine tuning the expression of the DXP genes (dxs and idi) and ads. This system was able to elicit a wide range of expression of the two gene cassettes by applying different concentration of inducers xylose and IPTG. Figure 17 shows that under the optimal expression condition identified, approximately 2mg/L amorphadiene was produced. Amorphadiene production increased gradually with both IPTG and xylose induction until the above inducers reached concentrations of 0.1 mM and 0.1%, respectively. As maximal transcription levels were achieved at similar inducer concentrations, it seems plausible that amorphadiene synthesis is controlled by the availability of ADS, DXS and IDI enzymes. Furthermore, considering the strength of Pgrac is generally weaker than that of PxylA (Figure 16C), it is plausible that production of amorphadiene was limited by ads even when ads transcription (under control of Pgrac) was maximally induced.

[00145] Once a limiting step has been identified in a pathway (such as the step catalyzed by ads), it is typical to overcome such limitations by overexpressing the corresponding gene through, for example, the use of a stronger promoter. However, replacement of Pgrac with a stronger promoter would require re-characterization of the new promoter in the presence of PxylA to identify conditions of optimal functioning of the combined system. Instead, ads expression was optimized by employing N terminal fusion tags to increase translation efficiency, another critical step in protein production. Two highly charged tags, six-arginine tag (6xR, positive charge) and six-aspartic acid tag (6xD, negative charge), and one protein stabilizing tag, trxA, were examined. Figure 18A shows that the abundance of ADS protein but not ads mRNA was significantly increased by use of the 6xR tag, indicating that translation efficiency of ADS was indeed enhanced by fusing the 6xR tag to N terminus of ads. Figure 18B shows that amorphadiene production, amorphadiene production by cells expressing the 6xR tagged ads increased by 2.5 fold over the untagged ads transcription.

[00146] The growth medium of B. subtilis was also systematically optimized to further increase amorphadiene production. Pyruvate and dipotassium phosphate were chosen as supplements. Both chemicals were confirmed to increase amorphadiene production when added to growth medium individually. Simultaneous addition of both compounds to growth medium was subsequently optimized by examining combination of the compounds over wide range of concentrations, which experiments increased the yield of amorphadiene to approximately 20mg/L (Figure 19A). To gain insight on the molecular mechanism underlying this semi-empirical optimization, transcription and intermediate metabolite concentrations in cells harvested at three representative conditions (no chemical addition, addition of 8g/L pyruvate and addition of 8g/L pyruvate as well as 32g/L dipotassium phosphate were measured). Figure 19B shows that transcription of dxs-idi and ads were not altered by the conditions, suggesting that factors other than genetic expression improved the amorphadiene production. Consistent with the observed increase in amorphadiene production, metabolic intermediates in the DXP pathway were generally increased upon the chemical addition (Figure 19C), indicative of the enhanced DXP pathway flux. A further non-targeted metabolite analysis revealed that co-factors ATP and CTP, and precursor PEP of the DXP pathway were significantly up-regulated upon chemical addition (Figure 19D). Although not wishing to be bound by any particular theory, the data suggests that peripheral metabolism to the DXP pathway is enhanced by adding pyruvate and dipotassium phosphate, which kinetically drove the reactions in the DXP pathway towards isoprenoid synthesis.

[00147] Although the yield achieved in this study (~20mg/L) is considerably lower than that achieved in E. coli (~700mg/L amorphadiene in shake flask scale (Ma et al. "Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases" Met b. Eng. 201 1 , 13(5):588-597)), it has significantly superseded the highest yield reported for isoprenoid production in B. subtilis (40 fold increase as compared to ~0.5mg/L by (Xue and Ahring: "Enhancing isoprene production by genetic modification of the l -deoxy-d-xylulose-5-phosphate pathway in Bacillus subtilis" Appl. Envrion. Microbiol. 201 1 , 77(7):2399-2405). To further increase amorphadiene productivity, the use of computationally guided competing pathway knockout and global transcription machinery engineering can be explored. Further improvements can be expected using higher biomass concentrations and controlled bioreactor conditions. Considering its advantages in simplifying downstream processing, utilizing broad range of substrates (cellulose) and resisting harsh growth conditions (low pH and high salt concentration), the GRAS B. subtilis strain demonstrated in this study is indeed a promising microbial host for

overproduction of isoprenoid pharmaceuticals.

[00148] This study also emphasized the importance of rational design (guided by transcription and metabolite analysis) in microbial isoprenoid production.

Transcription analysis by quantitative PCR enabled the thorough characterization of the constructed promoter system and guided protein translation engineering. The mass spectrometry-based metabolite analysis provided useful information on metabolic changes relating to the effects of media and suggested possible targets for future rational engineering and optimization (focusing on co-factors and precursors of the DXP pathway). The method employed in this study should be applicable to general metabolic engineering of microbes for metabolite overproduction.

Molecular cloning

[00149] Ads was synthesized with optimized codon (Genescript) based on amino acid sequence (AAF98444, NCBI) and subcloned into pHTOl (MoBiTec). Dxs and idi were PCR amplified from B. subtilis genomic DNA and subcloned into pWH1520 (MoBiTec). The 6xR and 6xD tag was added to N terminus of ads by PCR; the tag trxA was PCR amplified from E. coli genomic DNA and subcloned to N terminus of ads. The constructed plasmids were transformed into B. subtilis 1A1 (BGSC).

Cell growth and induction

[00150] Single colony was picked up from agar plate, inoculated into lmL 2xPY medium (20 g/L Peptone, 10 g/L Yeast extract, and 10 g/L NaCl, pH = 7) with proper antibiotics and incubated at 37°C overnight. Pellets of overnight grown cells were resuspended in lmL fresh medium to cell density 0.1 (OD595). The cells were incubated at 37°C/300rpm until cell density reached 0.7 (OD595), and indicated concentration of IPTG and xylose were added to induce protein expression. 200μΙ_. dodecane was added to cell culture for in situ extraction of amorphadiene, and the cells were incubated at 28°C/300rpm. The cells were cultured in 14mL falcon polypropylene tube (BD). Sodium pyruvate and dipotassium phosphate were used in medium optimization, and initial pH of media was adjusted to 7 (the same to 2xPY medium).

Protein analysis

[00151] Cell suspension equivalent to l mL OD595=T .O cells were sampled at 5h after induction, and cell pellets were lysed in 100μΙ_. 2% SDS. The cell lysates were separated by SDS-PAGE and ads protein was detected by western blot with Penta-His antibody (Qiagen).

Transcription/Metabolic intermediate analysis

[00152] The cells were sampled at 5h after induction; mRNA was quantified by reverse transcription quantitative PCR as described in (Zhou et al. : "Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR" BMC Mol. Biol. 201 1 , 12(1 ): 18), and metabolites were quantified by UPLC-MS as described herein. The transcription results were normalized to cell number; the metabolite results were normalized to sample volume. A control experiment was conducted to rule out that the unchanged transcription levels were due to high level genomic DNA contamination. Product analysis

[00153] Five micro-liter dodecane phase was sampled at indicated time and diluted with 45 μί ethyl acetate, which was analyzed by GC-MS as described in Tsuruta et al: "High-level production of amopha-4, 1 1 -diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli" PLoS One 2009, 4(2):e4489. In vitro synthetic amorphadiene standard was used to construct calibration curve for quantification. References:

1. Ajikumar, P. ., et al., Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms. Mol Pharm, 2008. 5(2): p. 167-90.

2. Keasling, J.D., Manufacturing molecules through metabolic engineering.

Science, 2010. 330(6009): p. 1355-8.

3. Ajikumar, P. ., et al., Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science, 2010. 330(6000): p. 70-4.

4. Hunter, W.N., The non-mevalonate pathway of isoprenoid precursor

biosynthesis. J Biol Chem, 2007. 282(30): p. 21573-7.

5. Jin, Y.S. and G. Stephanopoulos, Multi-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli. Metab Eng, 2007. 9(4): p. 337-47.

6. Yuan, L.Z., et al., Chromosomal promoter replacement of the isoprenoid

pathway for enhancing carotenoid production in E. coli. Metab Eng, 2006. 8(1 ): p. 79-90.

7. Martin, V.J., et al., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol, 2003. 21(7): p. 796-802.

8. Kim, S.W. and J.D. Keasling, Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. Biotechnol Bioeng, 2001. 72(4): p. 408-15.

9. Yoon, S.H., et al.. Enhanced lycopene production in Escherichia coli

engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate. Biotechnol Bioeng, 2006. 94(6): p. 1025-32. Rodriguez-Villalon, A., J. Perez-Gil, and M. Rodriguez-Concepcion,

Carotenoid accumulation in bacteria with enhanced supply of isoprenoid precursors by upregidation of exogenous or endogenous pathways. J

Biotechnol, 2008. 135( 1 ): p. 78-84.

Zhou, K., et al., Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. BMC Mol Biol, 201 1. 12(1 ): p. 18.

Pitera, D.J., et al., Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab Eng, 2007. 9(2): p. 193-207.

Cunningham, F.X., Jr., et al., Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942. Plant Cell, 1994. 6(8): p. 1 107-21 .

Rabinowitz, J.D. and E. Kimball, Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Anal Chem, 2007. 79( 16): p. 6167-73.

Luo, B., et al., Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. J Chromatogr A, 2007. 1147(2): p. 153-64.

Xiao, Y., et al., Methylerythritol cyclodiphosphate (MEcPP) in deoxyxylulose phosphate pathway : synthesis from an epoxide and mechanisms. Chem

Commun (Camb), 2010. 46(38): p. 7220-2.

Grawert, T., et al., Structure of active IspH enzyme from Escherichia coli provides mechanistic insights into substrate reduction. Angew Chem Int Ed Engl, 2009. 48(31 ): p. 5756-9.

Ostrovsky, D., A. Shashkov, and A. Sviridov, Bacterial oxidative-stress substance is 2-C-methyl-D-erythritol 2, 4-cyclopyrophosphate. Biochem J, 1993. 295 ( Pt 3): p. 901 -2.

Gika, H.G., et al., Does the mass spectrometer define the marker? A

comparison of global metabolite profiling data generated simultaneously via UPLC-MS on two different mass spectrometers. Anal Chem, 2010. 82(19): p. 8226-34.

Qu, J., Y. Qu, and R.M. Straubinger, Ultra-sensitive quantification of corticosteroids in plasma samples using selective solid-phase extraction and reversed-phase capillary high-performance liquid chromatography/tandem mass spectrometry. Anal Chem, 2007. 79(10): p. 3786-93.

Batt, A.L., M.S. Kostich, and J.M. Lazorchak, Analysis of ecologically relevant pharmaceuticals in wastewater and surface water using selective solid-phase extraction and UPLC-MS/MS. Anal Chem, 2008. 80(13): p. 5021- 30.

Chiuminatto, U., et al., Automated online solid phase extraction ultra high performance liquid chromatography method coupled with tandem mass spectrometry for determination of forty-two therapeutic drugs and drugs of abuse in human urine. Anal Chem, 2010. 82(13): p. 5636-45.

Henneman, L., et al., Detection of nonsterol isoprenoids by HPLC-MS/MS. Anal Biochem, 2008. 383(1 ): p. 18-24.

Zhang, ., From purification of large amounts of phospho-compounds (nucleotides) to enrichment of phospho-peptides using anion-exchanging resin. Anal Biochem, 2006. 357(2): p. 225-31.

Xue, J. and B.K. Ahring, Enhancing Isoprene Production by Genetic

Modification of the l-Deoxy-D-Xylulose-5 -Phosphate Pathway in Bacillus subtilis. Appl Environ Microbiol, 201 1. 77(7): p. 2399-405.

Cheng, Y.Q., M. Yang, and A.M. Matter, Characterization of a gene cluster responsible for the biosynthesis of anticancer agent FK228 in

Chromobacterium violaceum No. 968. Appl Environ Microbiol, 2007. 73(1 1 ): p. 3460-9.

Freedman, D.L., et al., Biodegradation of chloromethane by Pseudomonas aeruginosa strain NB1 under nitrate-reducing and aerobic conditions. Appl Environ Microbiol, 2004. 70(8): p. 4629-34.

Ding, D., et al., Application of constraint-based methods in Staphylococcus aureus metabolic network. Riv Biol, 2009. 102(3): p. 385-97. 29. Koppisch, A.T., et al., E. coli MEP synthase: steady-state kinetic analysis and substrate binding. Biochemistry, 2002. 41(1): p. 236-43.

30. Qu, J., Y. Qu, and R.M. Straubinger, Ultra-sensitive quantification of

corticosteroids in plasma samples using selective solid-phase extraction and reversed-phase capillary high-performance liquid chromatography/ tandem mass spectrometry. Anal Chem, 2007. 79(10): p. 3786-93.

31. Batt, A.L., M.S. ostich, and J.M. Lazorchak, Analysis of ecologically

relevant pharmaceuticals in wastewater and surface water using selective solid-phase extraction and UPLC-MS/MS. Anal Chem, 2008. 80(13): p. 5021- 30.

32. Chiuminatto, U., et al., Automated online solid phase extraction ultra high performance liquid chromatography method coupled with tandem mass spectrometry for determination of forty-two therapeutic drugs and drugs of abuse in human urine. Anal Chem, 2010. 82(13): p. 5636-45

[00154] The relevant teachings of all references identified herein are incorporated by reference in their entirety.

[00155] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

claimed is:

A method for extracting at least a first 1 -deoxy-D-xylulose 5-phosphate (DXP) pathway metabolite and at least a second DXP pathway metabolite from a sample and detecting the at least first and the at least second extracted DXP pathway metabolites in the sample simultaneously, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of 1 -deoxy-D-xylulose 5-phosphate, 2C-methyl-D-erythritol 4-phosphate, 4-diphosphocytidyl-2C-methyl D-erythritol, 4-diphosphocytidyl- 2C-methyl D-erythritol 2-phosphate, 2C-methyl-D-erythritol 2,4-diphosphate and hydroxylmethylbutenyl diphosphate, the method comprising:

(a) extracting the at least first and the at least second DXP pathway

metabolites from the sample by solid-phase extraction, thereby obtaining an extracted sample; and

(b) simultaneously detecting at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in the extracted sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals is characterized using liquid

chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio, thereby extracting at least a first and at least a second DXP pathway metabolite from a sample and detecting the at least first and the at least second extracted DXP pathway metabolites in the sample simultaneously.

The method of Claim 1 , wherein detecting the at least first and the at least second DXP pathway metabolites in the sample includes quantifying the amount of each of the at least first and the at least second DXP pathway metabolites in the sample, the method further comprising:

(a) measuring the intensity of the at least the first signal and the at least second signal of the at least first and the at least second DXP pathway metabolites; and

(b) comparing the measured intensity of the at least first signal to a

standard curve of the at least first DXP pathway metabolite and comparing the at least second signal to a standard curve of the at least second DXP pathway metabolite, thereby quantifying the amount of the at least first and the at least second DXP pathway metabolites in the sample.

3. The method of Claim 1 , wherein the sample is an extract of a bacterial cell or medium in which a bacterial cell was incubated.

4. The method of Claim 3, wherein the sample is medium in which a bacterial cell was incubated, the at least first DXP pathway metabolite is 1-deoxy-D- xylulose 5-phosphate, and the at least second DXP pathway metabolite is 2C- methyl-D-erythritol 4-phosphate.

5. The method of Claim 1 , wherein the solid phase extraction is anion-exchange chromatography.

6. The method of Claim 1 , wherein the DXP pathway metabolites are detected by liquid chromatography in conjunction with mass spectrometry.

7. The method of Claim 6, wherein the liquid chromatography is ultra- performance liquid chromatography and the mass spectrometry is time-of- flight mass spectrometry.

8. The method of Claim 1 , wherein the at least first and the at least second DXP pathway metabolites include 1-deoxy-D-xylulose 5-phosphate, 2C-methyl-D- erythritol 4-phosphate, 4-diphosphocytidyl-2C-methyl D-erythritol, 4- diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, 2C-methyl-D-erythritol 2,4-diphosphate and hydroxylmethylbutenyl diphosphate.

9. The method of Claim 1 , wherein the at least first and the at least second DXP pathway metabolites include 1 -deoxy-D-xylulose 5-phosphate, 2C-methyl-D- erythritol 4-phosphate, 4-diphosphocytidyl-2C-methyl D-erythritol and 2C- methyl-D-erythritol 2,4-diphosphate.

10. A method for simultaneously detecting at least a first 1 -deoxy-D-xylulose 5- phosphate (DXP) pathway metabolite and at least a second DXP pathway metabolite in a sample, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of 1 -deoxy- D-xylulose 5-phosphate, 2C-methyl-D-erythritol 4-phosphate, 4- diphosphocytidyl-2C-methyl D-erythritol, 4-diphosphocytidyl-2C-methyl D- erythritol 2-phosphate, 2C-methyl-D-erythritol 2,4-diphosphate and

hydroxylmethylbutenyl diphosphate, the method comprising:

simultaneously detecting at least a first signal produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in a sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio, thereby simultaneously detecting the at least first and the at least second DXP pathway metabolites in the sample.

1 1. The method of Claim 10, wherein simultaneously detecting the at least first signal produced by the at least first DXP pathway metabolite and the at least a second signal produced by the at least second DXP pathway metabolite includes simultaneously measuring the intensity of the at least first signal produced by the at least first DXP pathway metabolite and the at least second signal produced by the at least second DXP pathway metabolite.

12. A method for identifying a modulator of an enzyme in the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway, the method comprising:

(a) exposing a bacterial cell in a medium to a potential modulator of an enzyme in the DXP pathway, thereby providing a sample;

(b) extracting at least a first and at least a second DXP pathway metabolite from the sample by solid-phase extraction, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of 1 -deoxy-D-xylulose 5-phosphate, 2C- methyl-D-erythritol 4-phosphate, 4-diphosphocytidyl-2C-methyl D- erythritol, 4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, 2C-methyl-D-erythritol 2,4-diphosphate and hydroxylmethylbutenyl diphosphate, thereby obtaining an extracted sample;

(c) simultaneously measuring the intensity of at least a first signal

produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in the extracted sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio; and

(d) comparing the measured intensity of the at least first signal to a signal produced by the at least first DXP pathway metabolite in an extracted sample corresponding to a bacterial cell in a medium that was not treated with the potential modulator and comparing the at least second signal to a signal produced by the at least second DXP pathway metabolite in the extracted sample corresponding to the bacterial cell in a medium that was not treated with the potential modulator, wherein a change in the measured intensity of the at least first or the at least second signal indicates modulation of an enzyme in the DXP pathway, thereby identifying a modulator of an enzyme in the DXP pathway.

13. The method of Claim 12, wherein the modulator is an inhibitor of the enzyme in the DXP pathway; the at least first metabolite is a substrate of the enzyme; the at least second metabolite is a product of the enzyme; and the change is an increase in the measured intensity of the at least first signal and a decrease in the measured intensity of the at least second signal.

14. The method of Claim 12, wherein the bacterial cell is a wild-type bacterial cell.

15. The method of Claim 14, wherein the wild-type bacterial cell is an

Escherichia coli cell, a Chromobacterium violaceum cell, a Pseudomonas aeruginosa cell or a Bacillus subtilis cell.

16. The method of Claim 12, wherein the modulator of the enzyme in the DXP pathway is an antibiotic and the potential modulator is a potential antibiotic.

17. A method for determining the rate constant of a flux in the 1-deoxy-D- xylulose 5-phosphate (DXP) pathway of a bacterium in a medium by simultaneously measuring the amount of at least a first DXP pathway metabolite and at least a second DXP pathway metabolite, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of 1 -deoxy-D-xylulose 5-phosphate, 2C-methyl-D- erythritol 4-phosphate, 4-diphosphocytidyl-2C-methyl D-erythritol, 4- diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, 2C-methyl-D-erythritol 2,4-diphosphate and hydroxylmethylbutenyl diphosphate, the method comprising:

(a) incubating a first bacterial cell in a medium for a first period of time, thereby providing a first sample;

(b) incubating a second bacterial cell in a medium for a second period of time different than the first period of time, thereby providing a second sample;

(c) simultaneously measuring the intensities of at least a first signal

produced by the at least first DXP pathway metabolite and at least a second signal produced by the at least second DXP pathway metabolite in the first sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals corresponds to an amount of the at least first and the at least second metabolites, respectively, in the first sample after the first period of time and is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio;

(b) simultaneously measuring the intensities of the at least first signal

produced by the at least first DXP pathway metabolite and the at least second signal produced by the at least second DXP pathway metabolite in the second sample by liquid chromatography, mass spectrometry or liquid chromatography in conjunction with mass spectrometry, wherein each of the at least first and the at least second signals corresponds to an amount of the at least first and the at least second metabolites, respectively, in the second sample after at least the second period of time and is characterized using liquid chromatography by a unique retention time, using mass spectrometry by a unique mass/charge ratio or by liquid chromatography in conjunction with mass spectrometry by a unique retention time and a unique mass/charge ratio; and

(c) calculating a rate constant of a flux using the measured intensity of the at least first and the at least second signals after the first period of time and the second period of time, thereby determining the rate constant of a flux in the DXP pathway.

18. The method of Claim 17, wherein the flux is a rate limiting flux in the DXP pathway.

19. A method for characterizing the effect of a potential modulator of the DXP pathway, the method comprising:

(a) exposing a bacterial cell in a medium to at least one potential

modulator of the DXP pathway, and/or expressing at least one potential modulator of the DXP pathway in a bacterial cell in a medium, thereby providing a sample;

(c) extracting at least a first and at least a second DXP pathway metabolite from the sample, the medium, or the sample and the medium by solid- phase extraction, wherein at least the at least first and the at least second DXP pathway metabolites are selected from the group consisting of 1 -deoxy-D-xylulose 5-phosphate, 2C-methyl-D-erythritol 4-phosphate, 4-diphosphocytidyl-2C-methyl D-erythritol, 4- diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, 2C-methyl-D- erythritol 2,4-diphosphate and hydroxylmethylbutenyl diphosphate, thereby obtaining an extracted sample;

(d) simultaneously measuring the intensity of at least a first signal

(e) comparing the measured intensity of the at least first signal to a signal produced by the at least first DXP pathway metabolite in an extracted sample corresponding to a bacterial cell in a medium not treated with or not expressing the at least one potential modulator, and comparing the at least second signal to a signal produced by the at least second DXP pathway metabolite in the extracted sample corresponding to the bacterial cell in a medium not treated with or not expressing the at least one potential modulator, wherein a change in the measured intensity of the at least first or the at least second signal indicates modulation of the DXP pathway, thereby characterizing a potential modulator of the DXP pathway.

20. The method of Claim 19, wherein the method comprises expressing at least one potential modulator of the DXP pathway in a bacterial cell in a medium.

21. The method of Claim 20, wherein the method comprises over-expressing at least one potential modulator of the DXP pathway in a bacterial cell in a medium, and the at least one potential modulator is an enzyme of the DXP pathway.

22. The method of Claim 21 , wherein the enzyme of the DXP pathway is selected from the group consisting of dxs, dxr, ispD, ispE, ispF, ispG, ispH, idi and ads.

23. The method of Claim 19, wherein the method comprises exposing a bacterial cell in a medium to at least one potential modulator of the DXP pathway. The method of Claim 23, wherein the at least one potential modulator is an osmolyte, a buffering agent, a potential modulator of an enzyme in the DXP pathway, a co-factor of the DXP pathway, or a metabolite of the DXP pathway.