摘要
萜类化合物广泛分布于植物及微生物初级代谢物和次生代谢物中,其种类繁多,不仅在植物的生命活动中扮演重要的作用,而且还被广泛的应用于工业、医药卫生等方面,如倍半萜中的青蒿素、双萜中的紫杉醇可分别作为抗疟、抗癌的有效药物。但是萜类在生物体内的产量很低,而且分离和纯化操作复杂。由于萜类结构复杂,化学合成需要的步骤烦琐,非常困难。由于酵母自身存在甲羟戊酸(Mevalonate,即MVA)途径,可自我合成萜类前体,为利用代谢工程改造细胞代谢途径为提高萜类产量提供了一个很好的操作平台,然而酵母中萜类前体库不足够提供高产,有必要调节其参与萜类前体生物合成的代谢途径以提高产量。
本研究中,我们选择酿酒酵母作为宿主细胞,以酿酒酵母中的萜类生物合成途径为主要研究对象,建立遗传改造的酿酒酵母的分析技术平台,将最先进的分析技术用于测量代谢物及其在一定条件下含量的变化,有助于阐释遗传干预对感兴趣的途径的影响,同时对微生物的应激反应进行整体性无偏的描述,以进一步理解通过这个途径的代谢流的控制分子机制,得到如下结果:
(1)首次将基因敲除的分子生物学方法,引入酿酒酵母的次生代谢途径萜类生物合成途径中,以调节其代谢,增加其萜类前体库供给。酿酒酵母中的萜类生物合成途径主要分成六异戊二烯基焦磷酸合成途径以及麦角甾醇生物合成途径两个部分。为了增强通往萜类前体的代谢流,构建敲除盒,将负责FPP转为角鲨烯,竞争FPP的erg9基因,位于萜类前体下游的coq1基因分别敲除,减少旁路消耗。用
(2) PCR方法构建敲除盒LoxP-kanMX/URA3–loxP,分别敲除erg9基因和coq1基因,剔除筛选标记ura3后,分别得到新菌型BY4743- erg9和BY4743- coq1。在BY4743- erg9基础上,再利用敲除盒LoxP-URA3–loxP,敲除coq1基因,经PCR验证,并剔除筛选标记ura3,得到新菌型BY4743- erg9- coq1。同时考察其基础时间生长曲线的变化,为下一步的基因-生理机能机制深入研究,提供基础。野生菌株生长最快,缺失株生长较慢,双缺失菌生长最慢,形成的菌落也较小。
(3)为了考察上述遗传改造对萜类前体积累的效果,测定酿酒酵母的萜类生物合成途径上重要的指标性成分GGPP (二萜成分的前体),分析它的变化,表征其随erg9, coq1基因缺失的关联性,为阐明酿酒酵母中萜类合成途径的调节分子机制提供现实依据。本研究首次建立LC-MS方法测定遗传改造后酿酒酵母中GGPP含量的变化。应用4.6 mm×25 cm SB-Aq C18 5-μm (Agilent Tech., USA)色谱柱进行液相分离,流动相75%(10mM三丁胺水溶液用15mM醋酸调成pH 4.95):25%甲醇,质谱负离子模式扫描质荷比范围50–600,选择离子检测GGPP([M–H]?),质荷比449.2。标准曲线线性范围0.1–50 ng/ml。在这个范围内的,日内相对标准偏差<10%,日间相对标准偏差<14%,精密度范围96.5-105.4%。结果显示,遗传改造有效,BY4743- erg9- coq1的积累量在72小时后增加非常显著。遗传改造后的菌株与野生型菌株比较,含量有显著差异(P<0.01);随时间的长短,含量显著增高。说明erg9与coq1对GGPP的代谢显著影响。
(4)为了研究遗传改造对萜类生物合成途径代谢流的影响,建立GC–MS方法测定萜类生物合成途径上各代谢物及其在酿酒酵母中的应用。本研究首次建立一个精确灵敏的非放射性的方法,同时定量酿酒酵母中萜类合成途径上的GPP,FPP,GGPP,角鲨烯,麦角甾醇和羊毛甾醇,考察代谢流的变化。这个方法建立在GPP,FPP和GGPP去磷酸化成相应的烯萜醇,用GC-SIM-MS分析。本研究考察了三种转化方法:酸解法、碱解法、酶解法。酸解法和碱解法的转化率都低于15%,不能应用于此,酶解法在不同的缓冲体系中的转化率相差很大,50mM Bis-Tris propane/HCl, 1mM MgCl2 , pH 7.0,转化率<1%; 0.1M 2-amino-2-methyl propanol/NaOH, 1mM MgCl2 , pH 10.35,转化率<1%; 50 mM Tris-HCl, 10 mM MgCl2 , pH9.0,转化率<1%; 0.1 M glycine/1 M NaCl/40% MeOH, pH 10.4,转化率<2%, 1 M diethanolamine, 0.5 mM MgCl2 , pH 9.8,转化率可>88%,符合研究的需要。本研究综合比较全发酵液提取法、细胞液离子交换柱提取法、细胞液超声提取法三种提取方法,正己烷、石油醚、乙酸乙酯、氯仿:乙酸乙酯(1:10)四种提取溶剂,0.5min、1.5min、3min这样3个涡旋时间,比较其效率,确定最终的前处理方法为:细胞液超声提取法,正己烷提取,涡旋3min。应用TRACE TR-5MS柱(30m×0.25mm×0.25μm)进行气相分离,单重四极杆质谱选择离子监测GOH,FOH,GGOH,角鲨烯,麦角甾醇和羊毛甾醇,以保留时间和碎片信息进行定性定量分析。结果显示,方法准确有效,标准曲线线性良好,日内和日间精密度良好,加样回收率准确度高。将此方法应用于酿酒酵母中,发现不同基因对于酿酒酵母中萜类合成途径上的各个代谢物的代谢过程的作用是不一样的,双缺失erg9和coq1,使得GPP、FPP、GGPP大量积累,而角鲨烯、麦角甾醇、羊毛甾醇的含量可维持细胞正常生理功能。。
(5)通过考察不同遗传改造的酿酒酵母基础时间生长曲线的变化,发现其生理活动发生了变化。为了考察遗传改造对细胞生理活动的影响,建立荧光分析和化学发光方法测定分子操作后,与细胞生理特性和基因功能相关代谢物及其含量的变化,有助于阐明萜类代谢途径的基因敲除后,对细胞功能的影响。利用罗丹明6G,测得BY4743- erg9- coq1离子外排作用低于野生型和单缺失菌,并且酿酒酵母的外排作用是能量依赖型的。利用JC-1,测得erg9与coq1对线粒体膜电位的影响巨大,两者的缺失打破线粒体膜电位分布的平衡,但24h后,不同遗传改造的酿酒酵母建立的新的膜电位平衡无明显差别。利用DCFA-DA,测得erg9与coq1的缺失引发大量内源性活性氧的产生。利用荧光素酶的化学发光,测得BY4743- erg9- coq1ATP的含量低于野生型和单敲除菌。
当前的工作力求将代谢工程,化学分析,数据处理等多个领域结合,在细胞水平分析代谢物的变化以详细地认识基因—代谢产物、基因—代谢流之间关联的分子机制,分析生理状态的变化以认识基因功能。本研究涉及多种技术的联用。这些方法包括分子生物学技术,微生物代谢活性瞬时淬灭,化合物的酶法衍生化、相关胞内代谢产物的提取,色谱与质谱的偶联(GC-MS, LC-MS),荧光与化学发光的应用以及统计学上的数据挖掘等等。多种技术的创新和联用必将提供大量的代谢途径信息,有助于反映有机体的生理活动,理解潜在的调节机制,对以酿酒酵母为平台生产多种重要价值的萜类化合物具有重要意义。
Isoprenoids with more than 40,000 described compounds are the largest and most structurally diverse group of plant metabolites. Isoprenoids play various biological roles in plants. Depending on the number of isoprene units, isoprenoids can be classified into several groups, such as monoterpenes, sesquiterpenes and diterpenes (respectively 2, 3 and 4 C5 units).
Isoprenoids are functionally important in many different parts of cell metabolism such as photosynthesis (carotenoids, chlorophylls, plastoquinone), respiration (ubiquinone), hormonal regulation of metabolism (sterols), regulation of growth and development (gibberellic acid, abscisic acid, brassinosteroids, cytokinins, prenylated proteins), defense against pathogen attack, intracellular signal transduction (Ras proteins), vesicular transport within the cell (Rab proteins) as well as defining membrane structures(sterols, dolichols, carotenoids). Many isoprenoids also have considerable medical and commercial interest as flavors, fragrances (such as limonene, menthol, camphor), food colorants (carotenoids) or pharmaceuticals (such as bisabolol, artemisinin, lycopene, taxol).
Isoprenoids are widely present in plant tissues, and extraction from plants has been the traditional option for the large-scale production of these compounds. However, in many cases this method is neither feasible nor economical. Among the drawbacks in using plants as a source for isoprenoid production are influence of geographical location and weather on the composition and concentration of isoprenoids in the plant tissues, low concentration and poor yields for the recovery of isoprenoids from plants, and the high costs associated with extraction and purification. Chemical synthesis of isoprenoids has also been reported, and currently most of the industrially interesting carotenoids are produced via chemical synthesis. However, because of the complex structures of isoprenoids, chemical synthesis, involving many steps, is difficult. Side reactions, unwanted side products, and low yield are other disadvantages. In vitro enzymatic production of isoprenoids through the action of plant isoprenoid synthases is also impractical due to the dependency on the expensive precursors, as well as poor in vitro conversion.
There is therefore much interest in using microorganisms as cell factories for the production of isoprenoids. The intracellular pools of isoprenoid precursors in microorganisms appear, to be however, not enough to provide high level production. It may therefore be necessary to deregulate the pathways involved in the biosynthesis of isoprenoid precursors in order to improve production.
Yeast therefore has a high inherent capacity for the biosynthesis of isoprenoid precursors that may be directed to the production of heterologous compounds. Besides, tools from other related areas are being incorporated into the metabolic engineer’s repertoire. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites, quantification of these metabolites using modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (GC-MS, LC-MS), as well as the mathematical analyses.
In this study, we chose S. cerevisiae as a host cell for the accumulation of isoprenoid precursors to extend an understanding of the mechanisms by which the flux through the pathway is controlled. The results are as follows:
(1) In order to enhance the flux to isoprenoid precursors, both the erg9 gene which is responsible for conversion of FPP to squalene and the coq1 gene which lies in downstream pathway of GPP, FPP, GGPP were knockout by two disruption cassettes for gene replacement.
(2) Upon genetic modification, the change in the concentration of GGPP was measured via LC-MS at batch cultivations time. LC separations were performed on a 4.6 mm×25 cm SB-Aq C18 5-μm column (Agilent Tech., USA) with a mobile phase consisted of 75% eluent A (10mM tributylamine aqueous solution adjusted pH to 4.95 with 15mM acetic acid) and 25% eluent B (methanol), and mass spectra were operated in negative ion mode over a range of m/z 50–600, and selective ion monitors (SIM) were at m/z 449.1-449.2 for GGPP ([M–H]?). The calibration curves were linear over the range of 0.1–50 ng/ml. In this range, relative standard deviations (R.S.D.) were <10% for intra-day precision and <14% for interday precision. The accuracy was within the range of 96.5-105.4%. The disruption of both erg9 and coq1 at 72 h of the fermentation period improved the accumulation of GGPP at most
(3) A precise and sensitive nonradioactive method was developed for the simultaneous quantification of the isoprenoid precursors, geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), squalene, ergosterol and lanosterol in recombinant and wild-type S. cerevisiae. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography–selective ion-monitoring mass spectrometry (GC-SIM-MS). The analysis of GOH, FOH, GGOH, squalene, ergosterol and lanosterol illustrates robustness and reliability of this method outlined. Quantification of the analytes was performed by external calibration with reference substances and internal standardization. The recovery of the procedure has been evaluated.
(4) The application of fluorescence and chemiluminescence analysis to the measurement of metabolites and the changes in metabolite concentrations under the molecular manipulation characterized cellular physiology and gene function, which would help us illuminate the effects of perturbation in pathways of interest, as well as unbiased characterizations of microbial stress responses as a whole. Functional analysis revealed greater energy-dependent efflux activity of membrane transporters, lower intracellular ATP level and mitochondrial membrane potential, more endogenous reactive oxygen species generation in response to gene disruption.
The current work is based on the integration of genetic engineering, chemical analysis and data processing, which may provide a convenient format for considerable knowledge of metabolic pathway, as well as a platform for the production of a broad range of high value isoprenoids in yeast. This combination of rigorous analysis and quantitative molecular biology methods has endowed metabolic engineering with an effective synergism that crosses traditional disciplinary bounds. Meantime, the application of metabolic flux analysis contributes to reflect the activities of organisms, understand the underlying regulation mechanisms.
引文
[1] Akashi T, Aoki T, Ayabe S (1999) Cloning and functional expression of a cytochrome P450 cDNA encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice. Plant Physiol 121:821-828
[2] Antoniewicz MR, Kelleher JK, Stephanopoulos G (2007) Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis. Anal Chem 79:7554-7559
[3] Anzellotti D, Ibrahim RK (2004) Molecular characterization and functional expression of flavonol 6-hydroxylase. BMC Plant Biol 4:20
[4] Asadollahi MA, Maury J, Moller K, Nielsen KF, Schalk M, Clark A, Nielsen J (2007) Production of plant sesquiterpenes in Saccharomyces cerevisiae: Effect of ERG9 repression on sesquiterpene biosynthesis. Biotechnol Bioeng 99:666-677
[5] Ashby MN, Edwards PA (1990) Elucidation of the deficiency in two yeast coenzyme Q mutants. Characterization of the structural gene encoding hexaprenyl pyrophosphate synthetase. J Biol Chem 265:13157-13164
[6] Aura AM, Mattila I, Seppanen-Laakso T, Miettinen J, Oksman-Caldentey aMO K-M (2008) Microbial metabolism of catechin stereoisomers by human faecal microbiota: comparison of targeted analysis and a non-targeted metabolomics method. Phytochem Lett 1:18-22.
[7] Baidoo EE, Benke PI, Neususs C et al. (2008) Capillary electrophoresis-fourier transform ion cyclotron resonance mass spectrometry for the identification of cationic metabolites via a pH-mediated stacking-transient isotachophoretic method. Anal Chem 80:3112-3122
[8] Bajad SU, Lu W, Kimball EH, Yuan J, Peterson C, Rabinowitz JD (2006) Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry. J Chromatogr A 1125:76-88
[9] Banhegyi G, Braun L, Csala M, Puskas F, Mandl J (1997) Ascorbate metabolism and its regulation in animals. Free Radic Biol Med 23: 793–803.
[10] Baran R, Kochi H, Saito N et al. (2006) MathDAMP: a package for differential analysis of metabolite profiles. BMC Bioinformatics 7:530
[11] Becker JV, Armstrong GO, van der Merwe MJ, Lambrechts MG, Vivier MA, Pretorius IS (2003) Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol. FEMS Yeast Res 4:79-85
[12] Beckwith-Hall BM, Brindle JT, Barton RH et al. (2002) Application of orthogonal signal correction to minimise the effects of physical and biological variation in high resolution 1H NMR spectra of biofluids. Analyst 127:1283-1288
[13] Beekwilder J, Wolswinkel R, Jonker H, Hall R, de Vos CH, Bovy A (2006) Production of resveratrol in recombinant microorganisms. Appl Environ Microbiol 72:5670-5672
[14] Bollard ME, Holmes E, Lindon JC et al. (2001) Investigations into biochemical changes due to diurnal variation and estrus cycle in female rats using high-resolution (1)H NMR spectroscopy of urine and pattern recognition. Anal Biochem 295:194-202
[15] Branduardi P, Fossati T, Sauer M, Pagani R, Mattanovich D, Porro D (2007) Biosynthesis of vitamin C by yeast leads to increased stress resistance. PLoS ONE 2:e1092
[16] Brauer MJ, Yuan J, Bennett BD et al. (2006) Conservation of the metabolomic response to starvation across two divergent microbes. Proc Natl Acad Sci U S A 103:19302-19307
[17] Brindle JT, Antti H, Holmes E et al. (2002) Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics. Nat Med 8:1439-1444
[18] Bruce SJ, Jonsson P, Antti H et al. (2008) Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis. Anal Biochem 372:237-249
[19] Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45-66
[20] Chemler JA, Yan Y, Koffas MA (2006) Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae. Microb Cell Fact 5:20
[21] Coulier L, Bas R, Jespersen S, Verheij E, van der Werf MJ, Hankemeier T (2006) Simultaneous quantitative analysis of metabolites using ion-pair liquid chromatography–electrospray ionization mass spectrometry. Anal Chem 78:6573-6582.
[22] Daum G, Lees ND, Bard M et al (1998) Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 14(16):1471–1510
[23] Dauner M, Sauer U (2000) GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing. Biotechnol Prog 16:642-649
[24] De Vos RC, Moco S, Lommen A, Keurentjes JJ, Bino RJ, Hall RD (2007) Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry. Nat Protoc 2:778-791
[25] Dejong JM, Liu Y, Bollon AP, Long RM, Jennewein S, Williams D, Croteau RB (2006) Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae. Biotechnol Bioeng 93:212-224
[26] Dettmer K, Aronov PA, Hammock BD (2007) Mass spectrometry-based metabolomics. Mass Spectrom Rev 26:51-78
[27] Ding J, Sorensen CM, Zhang Q et al. (2007) Capillary LC coupled with high-mass measurement accuracy mass spectrometry for metabolic profiling. Anal Chem 79:6081-6093
[28] Ding J, Sorensen CM, Zhang QB, Jiang HL, Jaitly N, Livesay EA, Shen YF, Smith RD, Metz TO (2007) Capillary LC coupled with highmass measurement accuracy mass spectrometry for metabolic profiling. Anal Chem 79:6081-6093.
[29] Dixon RA (2004) Phytoestrogens. Annu Rev Plant Physiol Plant Mol Biol 55: 225-261
[30] Dominguez A, Ferminan E, Sanchez M, Gonzalez FJ, Perez-Campo FM, Garcia S, Herrero AB, San Vicente A, Cabello J, Prado M, Iglesias FJ, Choupina A, Burguillo FJ, Fernandez-Lago L, Lopez MC (1998) Non-conventional yeasts as hosts for heterologous protein production. Int Microbiol 1:131-142
[31] Edwards JL, Chisolm CN, Shackman JG, Kennedy RT (2006) Negative mode sheathless capillary electrophoresis electrospray ionization-mass spectrometry for metabolite analysis of prokaryotes. J Chromatogr A 1106:80-88
[32] Edwards JL, Edwards RL, Reid KR, Kennedy RT (2007) Effect of decreasing column inner diameter and use of off-line two-dimensional chromatography on metabolite detection in complex mixtures. J Chromatogr A 1172:127-134
[33] Fiehn O (2002) Metabolomics--the link between genotypes and phenotypes. Plant Mol Biol 48:155-171
[34] Fiehn O (2008) Extending the breadth of metabolite profiling by gas chromatography coupled to mass spectrometry. Trends Analyt Chem 27:261-269
[35] Fisher AJ, Rosenstiel TN, Shirk MC, Fall R (2001) Nonradioactive assay for cellular dimethylallyl diphosphate. Anal Biochem 292:272-279
[36] Forster J, Famili I, Fu P, Palsson BO, Nielsen J (2003) Genome-scale reconstruction of the Saccharomyces cerevisiae metabolic network. Genome Res 13:244-253
[37] Fujii H, Koyama T, Ogura K (1982) Efficient enzymatic hydrolysis of polyprenyl pyrophosphates. Biochim Biophys Acta 712:716-718
[38] Gao P, Shi C, Tian J et al. (2007) Investigation on response of the metabolites in tricarboxylic acid cycle of Escherichi coli and Pseudomonas aeruginosa to antibiotic perturbation by capillary electrophoresis. J Pharm Biomed Anal 44:180-187
[39] Gardner RG, Hampton RY (1999) A highly conserved signal controls degradation of 3-hydroxy-3-methylglutaryl-coenzyme a (HMG-CoA) reductase in eukaryotes. J Biol Chem 274:31671–31678.
[40] Gasser B, Maurer M, Gach J, Kunert R, Mattanovich D (2006) Engineering of Pichia pastoris forimproved production of antibody fragments. Biotechnol Bioeng 94:353-361
[41] Gavaghan CL, Wilson ID, Nicholson JK (2002) Physiological variation in metabolic phenotyping and functional genomic studies: use of orthogonal signal correction and PLS-DA. FEBS Lett 530:191-196
[42] Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741-750
[43] Gellissen G, Hollenberg CP (1997) Application of yeasts in gene expression studies: a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis -- a review. Gene 190:87-97
[44] Giga-Hama Y, Kumagai H (1999) Expression system for foreign genes using the fission yeast Schizosaccharomyces pombe. Biotechnol Appl Biochem 30 (Pt 3):235-244
[45] Gin P, Clarke CF (2005) Genetic evidence for a multi-subunit complex in coenzyme Q biosynthesis in yeast and the role of the Coq1 hexaprenyl diphosphate synthase. J Biol Chem 280:2676-2681
[46] Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG (1996) Life with 6000 genes. Science 274:546, 563-547
[47] Griffin JL, Williams HJ, Sang E, Clarke K, Rae C, Nicholson JK (2001) Metabolic profiling of genetic disorders: a multitissue (1)H nuclear magnetic resonance spectroscopic and pattern recognition study into dystrophic tissue. Anal Biochem 293:16-21
[48] Gullberg J, Jonsson P, Nordstrom A, Sjostrom M, Moritz T (2004) Design of experiments: an efficient strategy to identify factors influencing extraction and derivatization of Arabidopsis thaliana samples in metabolomic studies with gas chromatography/mass spectrometry. Anal Biochem 331:283-295
[49] Guo X, Lidstrom ME (2008) Metabolite profiling analysis of Methylobacterium extorquens AM1 by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry. Biotechnol Bioeng 99:929-940
[50] Guo XF, Lidstrom ME (2008) Metabolite profiling analysis of Methylobacterium extorquens AM1 by comprehensive two dimensional gas chromatography coupled with time-of-flight mass spectrometry. Biotechnol Bioeng 99:929-940.
[51] Hagan SO, Dunn WB, Knowles JD, Broadhurst D, Williams R, Ashworth JJ, Cameron M, Kell DB(2007) Closed-loop, multiobjective optimization of two-dimensional gas chromatography/mass spectrometry for serum metabolomics. Anal Chem 79:464-476.
[52] Halket JM, Waterman D, Przyborowska AM, Patel RK, Fraser PD, Bramley PM (2005) Chemicalderivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS. J Exp Bot 56:219-243
[53] Harada K, Fukusaki E, Kobayashi A (2006) Pressure-assisted capillary electrophoresis mass spectrometry using combination of polarity reversion and electroosmotic flow for metabolomics anion analysis. J Biosci Bioeng 101:403-409
[54] Hegeman AD, Schulte CF, Cui Q et al. (2007) Stable isotope assisted assignment of elemental compositions for metabolomics. Anal Chem 79:6912-6921
[55] Hezari M, Lewis NG, Croteau R (1995) Purification and characterization of taxa-4(5),11(12)-diene synthase from Pacific yew (Taxus brevifolia) that catalyzes the first committed step of taxol biosynthesis. Arch Biochem Biophys 322:437-444
[56] Holmes E, Antti H (2002) Chemometric contributions to the evolution of metabonomics: mathematical solutions to characterising and interpreting complex biological NMR spectra. Analyst 127:1549-1557
[57] Horswill AR, Escalante-Semerena JC (1999) The prpE gene of Salmonella typhimurium LT2 encodes propionyl-CoA synthetase. Microbiology 145 (Pt 6):1381-1388
[58] Huh WK, Kim ST, Kim H, Jeong G, Kang SO (2001) Deficiency of D-erythroascorbic acid attenuates hyphal growth and virulence of Candida albicans. Infect Immun 69: 3939–3946
[59] Huh WK, Lee BH, Kim ST, Kim YR, Rhie GE, Baek YW, Hwang CS, Lee JS, Kang SO (1998) D-Erythroascorbic acid is an important antioxidant molecule in Saccharomyces cerevisiae. Mol Microbiol 30:895-903
[60] Hui JP, Yang J, Thorson JS, Soo EC (2007) Selective detection of sugar phosphates by capillary electrophoresis/mass spectrometry and its application to an engineered E. coli host. Chembiochem 8:1180-1188
[61] Iwatani S, Van Dien S, Shimbo K et al. (2007) Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS. J Biotechnol 128:93-111
[62] Jennewein S, Park H, DeJong JM, Long RM, Bollon AP, Croteau RB (2005) Coexpression in yeast of Taxus cytochrome P450 reductase with cytochrome P450 oxygenases involved in Taxol biosynthesis. Biotechnol Bioeng 89:588-598
[63] Jiang H, Wood KV, Morgan JA (2005) Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae. Appl Environ Microbiol 71:2962-2969
[64] Jonsson P, Gullberg J, Nordstrom A et al. (2004) A strategy for identifying differences in large series of metabolomic samples analyzed by GC/MS. Anal Chem 76:1738-1745
[65] Kanani HH, Klapa MI (2007) Data correction strategy for metabolomics analysis using gas chromatography–mass spectrometry. Metabol Eng 9:39-51.
[66] Kell DB, King RD (2000) On the optimization of classes for the assignment of unidentified reading frames in functional genomics programmes: the need for machine learning. Trends Biotechnol 18:93-98
[67] Khosla C, Keasling JD (2003) Metabolic engineering for drug discovery and development. Nat Rev Drug Discov 2:1019-1025
[68] Kim DH, Kim BG, Lee HJ, Lim Y, Hur HG, Ahn JH (2005) Enhancement of isoflavone synthase activity by co-expression of P450 reductase from rice. Biotechnol Lett 27:1291-1294
[69] Kimball E, Rabinowitz JD (2006) Identifying decomposition products in extracts of cellular metabolites. Anal Biochem 358:273-280
[70] Kind T, Fiehn O (2007) Seven Golden Rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry. BMC Bioinformatics 8:105
[71] Kind T, Tolstikov V, Fiehn O, Weiss RH (2007) A comprehensive urinary metabolomic approach for identifying kidney cancerr. Anal Biochem 363:185-195
[72] Knekt P, Jarvinen R, Seppanen R, Rissanen A, Aromaa A, Heinonen OP, Albanes D, Heinonen M, Pukkala E, Teppo L (1991) Dietary antioxidants and the risk of lung cancer. Am J Epidemiol 134:471-479
[73] Koek MM, Muilwijk B, van Stee LL, Hankemeier T (2008) Higher mass loadability in comprehensive two-dimensional gas chromatography-mass spectrometry for improved analytical performance in metabolomics analysis. J Chromatogr A 1186:420-429
[74] Kopec S, Holzgrabe U (2007) Amino acids: aspects of impurity profiling by means of CE. Electrophoresis 28:2153-2167
[75] Lee R, Ptolemy AS, Niewczas L, Britz-McKibbin P (2007) Integrative metabolomics for characterizing unknown low-abundance metabolites by capillary electrophoresis-mass spectrometry with computer simulations. Anal Chem 79:403-415
[76] Levine M (1986) New concepts in the biology and biochemistry of ascorbic acid. N Engl J Med 314:892-902
[77] Little JL (1999) Artifacts in trimethylsilyl derivatization reactions and ways to avoid them. J Chromatogr A 844:1-22.
[78] Lommen A, van der Weg G, van Engelen MC, Bor G, Hoogenboom LA, Nielen MW (2007) An untargeted metabolomics approach to contaminant analysis: pinpointing potential unknown compounds. Anal Chim Acta 584:43-49
[79] Martins LF, Yegles M, Wennig R (2008) Simultaneous enantioselective quantification of methadone and of 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine in oral fluid using capillary electrophoresis. J Chromatogr B Analyt Technol Biomed Life Sci 862:79-85
[80] Mas S, Villas-Boas SG, Hansen ME, Akesson M, Nielsen J (2007) A comparison of direct infusion MS and GC-MS for metabolic footprinting of yeast mutants. Biotechnol Bioeng 96:1014-1022
[81] Mashego MR, Rumbold K, De Mey M, Vandamme E, Soetaert W, Heijnen JJ (2007) Microbial metabolomics: past, present and future methodologies. Biotechnol Lett 29:1-16
[82] Mathur A, Hong Y, Kemp BK, Barrientos AA, Erusalimsky JD (2000) Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes. Cardiovasc Res 46:126-138
[83] Maury J, Asadollahi MA, Moller K, Clark A, Nielsen J (2005) Microbial isoprenoid production: an example of green chemistry through metabolic engineering. Adv Biochem Eng Biotechnol 100:19-51
[84] McCaskill D, Croteau R (1993) Procedures for the isolation and quantification of the intermediates of the mevalonic acid pathway. Anal Biochem 215:142-149
[85] Mercke P, Bengtsson M, Bouwmeester HJ, Posthumus MA, Brodelius PE (2000) Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L. Arch Biochem Biophys 381:173-180
[86] Mercke P, Crock J, Croteau R, Brodelius PE (1999) Cloning, expression, and characterization of epi-cedrol synthase, a sesquiterpene cyclase from Artemisia annua L. Arch Biochem Biophys 369:213-222
[87] Metcalf RL (1987) Plant volatiles as insect attractants. CRC Crit Rev Plant Sci 5: 251–301.
[88] Mol R, de Jong GJ, Somsen GW (2005) On-line capillary electrophoresis-mass spectrometry using dopant-assisted atmospheric pressure photoionization: setup and system performance. Electrophoresis 26:146-154.
[89] Mondello L, Tranchida PQ, Dugo P, Dugo G (2008) Comprehensive two-dimensional gas chromatography–mass spectrometry: a review. Mass Spectrom Rev 27:101-124.
[90] Mukhopadhyay A, He Z, Alm EJ et al. (2006) Salt stress in Desulfovibrio vulgaris Hildenborough: an integrated genomics approach. J Bacteriol 188:4068-4078
[91] Murray RDH (1991) Progress in the Chemistry of Organic Natural Products. Springer Wien, New York
[92] Mutka SC, Bondi SM, Carney JR, Da Silva NA, Kealey JT (2006) Metabolic pathway engineering for complex polyketide biosynthesis in Saccharomyces cerevisiae. FEMS Yeast Res 6:40-47
[93] Naidu KA (2003) Vitamin C in human health and disease is still a mystery? An overview. Nutr J 2: 7
[94] Newman DJ, Cragg GM, Snader KM (2000) The influence of natural products upon drug discovery (Antiquity to late 1999). Nat Prod Rep 17:215-234
[95] Nielsen J (1998) Metabolic engineering: techniques for analysis of targets for genetic manipulations. Biotechnol Bioeng 58:125-132
[96] Nielsen J (2001) Metabolic engineering. Appl Microbiol Biotechnol 55:263-283
[97] O’Brien TJ, Ceryak S, Patierno SR (2003) Complexities of chromium carcinogenesis: role of cellular response, repair and recovery mechanisms. Mutat Res 533: 3–36
[98] O'Hagan S, Dunn WB, Knowles JD et al. (2007) Closed-loop, multiobjective optimization of two-dimensional gas chromatography/mass spectrometry for serum metabolomics. Anal Chem 79:464-476
[99] Ohashi Y, Hirayama A, Ishikawa T et al. (2008) Depiction of metabolome changes in histidine-starved Escherichia coli by CE-TOFMS. Mol Biosyst 4:135-147 This research showcases the potential of CE–MS to be used for the comprehensive analysis of charged intracellular metabolites, while also providing a utility for monitoring metabolic perturbations in a microorganism, upon the application of external stimuli.
[100] Oswald M, Fischer M, Dirninger N, Karst F (2007) Monoterpenoid biosynthesis in Saccharomyces cerevisiae. FEMS Yeast Res 7:413-421
[101] Padh H (1990) Cellular functions of ascorbic acid. Biochem Cell Biol 68:1166-1173
[102] Padh H (1991) Vitamin C: newer insights into its biochemical functions. Nutr Rev 49:65-70
[103] Patil KR, Akesson M, Nielsen J (2004) Use of genome-scale microbial models for metabolic engineering. Curr Opin Biotechnol 15:64-69
[104] Pingitore F, Tang Y, Kruppa GH, Keasling JD (2007) Analysis of amino acid isotopomers using FT-ICR MS. Anal Chem 79:2483-2490
[105] Porro D, Sauer M, Branduardi P, Mattanovich D (2005) Recombinant protein production in yeasts. Mol Biotechnol 31:245-259
[106] Pretorius IS, Bauer FF (2002) Meeting the consumer challenge through genetically customized wine-yeast strains. Trends Biotechnol 20:426-432
[107] Rabinowitz JD, Kimball E (2007) Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Anal Chem 79:6167-6173
[108] Ralston L, Subramanian S, Matsuno M, Yu O (2005) Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases. PlantPhysiol 137:1375-1388
[109] Richards JB, Hemming FW (1972) Dolichols, ubiquinones, geranylgeraniol and farnesol as the major metabolites of mevalonate in Phytophthora cactorum. Biochem J 128:1345-1352
[110] Ro DK, Douglas CJ (2004) Reconstitution of the entry point of plant phenylpropanoid metabolism in yeast (Saccharomyces cerevisiae): implications for control of metabolic flux into the phenylpropanoid pathway. J Biol Chem 279:2600-2607
[111] Ro DK, Paradise EM, Ouellet M et al. (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-943
[112] Robin J, Jakobsen M, Beyer M, Noorman H, Nielsen J (2001) Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7-aminodeacetoxycephalosporanic acid production. Appl Microbiol Biotechnol 57:357-362
[113] Rodriguez AP, Leiro RF, Trillo MC, Cerdan ME, Siso MI, Becerra M (2006) Secretion and properties of a hybrid Kluyveromyces lactis-Aspergillus niger beta-galactosidase. Microb Cell Fact 5:41
[114] Rodriguez E, Gramajo H (1999) Genetic and biochemical characterization of the alpha and beta components of a propionyl-CoA carboxylase complex of Streptomyces coelicolor A3(2). Microbiology 145 (Pt 11):3109-3119
[115] Saisho Y, Morimoto A, Umeda T (1997) Determination of farnesyl pyrophosphate in dog and human plasma by high-performance liquid chromatography with fluorescence detection. Anal Biochem 252:89-95
[116] Saito N, Robert M, Kitamura S et al. (2006) Metabolomics approach for enzyme discovery. J Proteome Res 5:1979-1987
[117] Sangster TP, Wingate JE, Burton L, Teichert F, Wilson ID (2007) Investigation of analytical variation in metabonomic analysis using liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom 21:2965-2970
[118] Sauer U (2004) High-throughput phenomics: experimental methods for mapping fluxomes. Curr Opin Biotechnol 15:58-63
[119] Sauer U (2006) Metabolic networks in motion: 13C-based flux analysis. Mol Syst Biol 2:62
[120] Sauer U, Hatzimanikatis V, Bailey JE, Hochuli M, Szyperski T, Wuthrich K (1997) Metabolic fluxes in riboflavin-producing Bacillus subtilis. Nat Biotechnol 15:448-452
[121] Schoenbohm C, Martens S, Eder C, Forkmann G, Weisshaar B (2000) Identification of the Arabidopsis thaliana flavonoid 3'-hydroxylase gene and functional expression of the encoded P450enzyme. Biol Chem 381:749-753
[122] Shellie RA, Welthagen W, Zrostlikova J et al. (2005) Statistical methods for comparing comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry results: metabolomic analysis of mouse tissue extracts. J Chromatogr A 1086:83-90
[123] Shellie RA, Welthagen W, Zrostlikova J, Spranger J, Ristow M, Fiehn O, Zimmermann R (2005) Statistical methods for comparing comprehensive two-dimensional gas chromatography-timeof-flight mass spectrometry results: metabolomic analysis of mouse tissue extracts. J Chromatogr A 1086:83-90.
[124] Shiba Y, Paradise EM, Kirby J, Ro DK, Keasling JD (2007) Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids. Metab Eng 9:160-168
[125] Shintani T, Torimura M, Sato H, Tao H, Manabe T (2005) Rapid separation of microorganisms by quartz microchip capillary electrophoresis. Anal Sci 21:57-60
[126] Smilde AK, van der Werf MJ, Bijlsma S, van der Werff-van der Vat BJ, Jellema RH (2005) Fusion of mass spectrometry-based metabolomics data. Anal Chem 77:6729-6736
[127] Song L (2003) Detection of farnesyl diphosphate accumulation in yeast ERG9 mutants. Anal Biochem 317:180-185
[128] Spickett CM, Smirnoff N, Pitt AR (2000) The biosynthesis of erythroascorbate in Saccharomyces cerevisiae and its role as an antioxidant. Free Radic Biol Med 28: 183–192
[129] Sudbery PE (1996) The expression of recombinant proteins in yeasts. Curr Opin Biotechnol 7: 517–524
[130] Szyperski T (1995) Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism. Eur J Biochem 232:433-448
[131] Szyperski T (1998) 13C-NMR, MS and metabolic flux balancing in biotechnology research. Q Rev Biophys 31:41-106
[132] Takahashi S, Yeo Y, Greenhagen BT et al. (2007) Metabolic engineering of sesquiterpene metabolism in yeast. Biotechnol Bioeng 97:170-181
[133] Takahashi S, Zhao Y, O'Maille PE et al. (2005) Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme catalyzing successive hydroxylations of sesquiterpenes. J Biol Chem 280:3686-3696
[134] Tanaka Y, Higashi T, Rakwal R, Wakida S, Iwahashi H (2007) Quantitative analysis of sulfur-related metabolites during cadmium stress response in yeast by capillaryelectrophoresis-mass spectrometry. J Pharm Biomed Anal 44:608-613
[135] Tanaka Y, Otsuka K, Terabe S (2003) Evaluation of an atmospheric pressure chemical ionization interface for capillary electrophoresis-mass spectrometry. J Pharm Biomed Anal 30:1889-1895.
[136] Tang Y, Pingitore F, Mukhopadhyay A, Phan R, Hazen TC, Keasling JD (2007) Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris hildenborough using gas chromatography-mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry. J Bacteriol 189:940-949
[137] Tang YJ, Ashcroft JM, Chen D et al. (2007) Charge-associated effects of fullerene derivatives on microbial structural integrity and central metabolism. Nano Lett 7:754-760
[138] Tang YJ, Meadows AL, Kirby J, Keasling JD (2007) Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling. J Bacteriol 189:894-901
[139] Taylor J, King RD, Altmann T, Fiehn O (2002) Application of metabolomics to plant genotype discrimination using statistics and machine learning. Bioinformatics 18 Suppl 2:S241-248
[140] The authors have utilized two analytical techniques, GC–MS and DI–MS, to define discriminating metabolites in yeast mutants. They evaluated the two techniques, which are complementary, and are used to show differences in metabolic footprints for yeast mutants.
[141] The authors present an improved GC–GC setup for metabolomic profiling, which provides a more straightforward data analysis platform than conventional setups. Their method can be very useful for complex microbial, plant, and mammalian samples, which contain extremely large concentration differences among the metabolite composition.
[142] Theobald U, Mailinger W, Baltes M, Rizzi M, Reuss M (1997) In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae. I. Experimental observations. Biotechnol Bioeng 55:305–16
[143] Theobald U, Mailinger W, Reuss M, Rizzi M (1993) In vivo analysis of glucose-induced fast changes in yeast adenine nucleotide pool applying a rapid sampling technique. Anal Biochem 214:31-37
[144] Tian L, Dixon RA (2006) Engineering isoflavone metabolism with an artificial bifunctional enzyme. Planta 224:496-507
[145] Tong H, Holstein SA, Hohl RJ (2005) Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells. Anal Biochem 336:51-59
[146] Toya Y, Ishii N, Hirasawa T et al. (2007) Direct measurement of isotopomer of intracellularmetabolites using capillary electrophoresis time-of-flight mass spectrometry for efficient metabolic flux analysis. J Chromatogr A 1159:134-141
[147] Trommer H, Bottcher R, Poppl A, Hoentsch J, Wartewig S, Neubert RH (2002) Role of ascorbic acid in stratum corneum lipid models exposed to UV irradiation. Pharm Res 19:982-990
[148] Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M (2006) Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact 160:1-40
[149] van der Werf MJ, Jellema RH, Hankemeier T (2005) Microbial metabolomics: replacing trial-and-error by the unbiased selection and ranking of targets. J Ind Microbiol Biotechnol 32:234-252
[150] van der Werf MJ, Overkamp KM, Muilwijk B, Coulier L, Hankemeier T (2007) Microbial metabolomics: toward a platform with full metabolome coverage. Anal Biochem 370:17-25
[151] This paper shows how satisfactory coverage of metabolites can be obtained by combining various separation techniques with mass spectrometry. The technical description of separation and detection is extremely useful.
[152] Vanhulle S, Enaud E, Trovaslet M et al. (2008) Coupling occurs before breakdown during biotransformation of Acid Blue 62 by white rot fungi. Chemosphere 70:1097-1107
[153] Vannelli T, Wei Qi W, Sweigard J, Gatenby AA, Sariaslani FS (2007) Production of p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli by expression of heterologous genes from plants and fungi. Metab Eng 9:142-151
[154] Vaseghi S, Baumeister A, Rizzi M, Reuss M (1999) In vivo dynamics of the pentose phosphate pathway in Saccharomyces cerevisiae. Metab Eng 1:128-140
[155] Veen M, Lang C (2004) Production of lipid compounds in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 63(6):635–646
[156] Verhoeckx KC, Bijlsma S, Jespersen S et al. (2004) Characterization of anti-inflammatory compounds using transcriptomics, proteomics, and metabolomics in combination with multivariate data analysis. Int Immunopharmacol 4:1499-1514
[157] Vivier MA, Pretorius IS (2002) Genetically tailored grapevines for the wine industry. Trends Biotechnol 20:472-478
[158] Wahl SA, Dauner M, Wiechert W (2004) New tools for mass isotopomer data evaluation in (13)C flux analysis: mass isotope correction, data consistency checking, and precursor relationships. Biotechnol Bioeng 85:259-268
[159] Wheeler GL, Jones MA, Smirnoff N (1998) The biosynthetic pathway of vitamin C in higher plants. Nature 393: 365–369.
[160] Wiechert W (2001) 13C metabolic flux analysis. Metab Eng 3:195-206
[161] Withers ST, Keasling JD (2007) Biosynthesis and engineering of isoprenoid small molecules. Appl Microbiol Biotechnol 73:980-990
[162] Wittmann C (2007) Fluxome analysis using GC-MS. Microb Cell Fact 6:6
[163] Wu L, Mashego MR, van Dam JC et al. (2005) Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13C-labeled cell extracts as internal standards. Anal Biochem 336:164-171
[164] Yan Y, Huang L, Koffas MA (2007) Biosynthesis of 5-deoxyflavanones in microorganisms. Biotechnol J 2:1250-1262
[165] Yan Y, Kohli A, Koffas MA (2005) Biosynthesis of natural flavanones in Saccharomyces cerevisiae. Appl Environ Microbiol 71:5610-5613
[166] Zhang Y, Li SZ, Li J, Pan X, Cahoon RE, Jaworski JG, Wang X, Jez JM, Chen F, Yu O (2006) Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and Mammalian cells. J Am Chem Soc 128:13030-13031