Determination of nicotine and its metabolites by capillary electrophoresis and mass spectrometry.

In England an estimated, 284,000 patients are admitted to NHS hospitals each year due to disease caused by smoking. It is estimated that half of all teenagers who are currently smoking will die from diseases caused by tobacco if they continue to smoke. An estimated one quarter of smokers will die after 70 years of age and one quarter before, with those dying before 70 losing on average 23 years of life. It is the addictive nature of nicotine that exacerbates the toxicities of the other components of cigarette smoke. The Royal College of Physicians has affirmed that the way in which nicotine causes addiction is similar to drugs such as heroin and cocaine. Thus studies of nicotine metabolism are of great importance since they determine the extent of which the addictive nature of nicotine can influence smoking behaviour and hence the onset of smoking related illnesses. In this area of research capillary electrophoresis (CE) is still in its infancy. But with its high resolution and high number of theoretical plates achieved, CE makes for an attractive separating device for coupling with a mass spectrometer (MS). The overall aims of this work were to produce a sensitive, highly selective, and simple CE-sample stacking/MS assay for the measurement of nicotine and its metabolites in urine, to develop a highly sensitive transient isotachophoretic/MS method, that yields detection limits comparable to that observed by HPLC/MS and with a separation efficiency to match that of CE-sample stacking/MS, and finally to characterise the metabolic activity of cytochrome P450 (e.g. CYP2D6) in the placenta, with respect to nicotine, from a representative in-vitro human trophoblast-like cell line, BeWo, via HPLC/MS and CE/UV. Analysis of urine samples was accompanied by sample clean up via SPE to ensure the appropriate removal of inorganic salts. An optimised hydrodynamic and electrokinetic injection method (HE injection) was used for CE-sample stacking/MS. HE-sample stacking/MS brought about lower detection limits (LODs of nicotine and cotinine, by CE-sample stacking/ MS (via HE injection), were found to be 0.11 and 2.25 mug/ mL, respectively) when compared to sample stacking/MS via hydrodynamic or electrokinetic injection alone. The added selectivity that the selected ion monitoring mode of MS provided ensured the clear identification of nicotine and its metabolites in urine. A counterflow transient isotachophoresis (tITP) method was developed, with MS detection, for the analysis of even lower analyte concentrations. Limits of detection for both nicotine (0.03 mug/ mL) and cotinine (0.34 mug/ mL), via HE-tlTP/MS, were considerably lower than those obtained by HE injection-sample stacking/MS, suggesting that HE-tlTP/MS could be used complementary to HE injection-sample stacking/MS. When HPLC/MS and CE/UV were used to analyse cytochrome p450 activity in the human trophoblast-like BeWo cell line, it was clear from our data that nicotine metabolism was observed. Thus it is probable that CYP mediated processes play an important role in nicotine metabolism in the placenta. When compared to HPLC/MS both HE-sample stacking/MS and HE-tlTP/MS exhibited higher resolutions and peak efficiencies; with HE-tITP/MS exhibiting comparable limits of detection and quantitation to those obtained by HPLC/MS with respect to nicotine, but not cotinine. The major improvements to the coaxial interface performance and sensitivity enhancements, has made CE/MS an attractive alternative to HPLC/MS for the determination of nicotine and its' metabolites from biological matrices

Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency.

Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate - an intermediate of the shikimate pathway - into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass

Remodeling the Isoprenoid Pathway in Tobacco by Expressing the Cytoplasmic Mevalonate Pathway in Chloroplasts

Metabolic engineering to enhance production of isoprenoid metabolites for industrial and medical purposes is an important goal. The substrate for isoprenoid synthesis in plants is produced by the mevalonate pathway (MEV) in the cytosol and by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. A multi-gene approach was employed to insert the entire cytosolic MEV pathway into the tobacco chloroplast genome. Molecular analysis confirmed the site-specific insertion of seven transgenes and homoplasmy. Functionality was demonstrated by unimpeded growth on fosmidomycin, which specifically inhibits the MEP pathway. Transplastomic plants containing the MEV pathway genes accumulated higher levels of mevalonate, carotenoids, squalene, sterols, and triacyglycerols than control plants. This is the first time an entire eukaryotic pathway with six enzymes has been transplastomically expressed in plants. Thus, we have developed an important tool to redirect metabolic fluxes in the isoprenoid biosynthesis pathway and a viable multigene strategy for engineering metabolism in plants

Application of an Acyl-CoA Ligase from Streptomyces aizunensis for Lactam Biosynthesis

ε-Caprolactam and δ-valerolactam are important commodity chemicals used in the manufacture of nylons, with millions of tons produced annually. Biological production of these highly valued chemicals has been limited due to a lack of enzymes that cyclize ω-amino fatty acid precursors to corresponding lactams under ambient conditions. In this study, we demonstrated production of these chemicals using ORF26, an acyl-CoA ligase involved in the biosynthesis of ECO-02301 in Streptomyces aizunensis. This enzyme has a broad substrate spectrum and can cyclize 4-aminobutyric acid into γ-butyrolactam, 5-aminovaleric acid into δ-valerolactam and 6-aminocaproic acid into ε-caprolactam. Recombinant E. coli expressing ORF26 produced valerolactam and caprolactam when 5-aminovaleric acid and 6-aminocaproic acid were added to the culture medium. Upon coexpressing ORF26 with a metabolic pathway that produced 5-aminovaleric acid from lysine, we were able to demonstrate production of δ-valerolactam from lysine

Flux-Enabled Exploration of the Role of Sip1 in galactose yeast metabolism

13C metabolic flux analysis (13C MFA) is an important systems biology technique that has been used to investigate microbial metabolism for decades. The heterotrimer Snf1 kinase complex plays a key role in the preference Saccharomyces cerevisiae exhibits for glucose over galactose, a phenomenon known as glucose repression or carbon catabolite repression. The SIP1 gene, encoding a part of this complex, has received little attention, presumably, because its knockout lacks a growth phenotype. We present a fluxomic investigation of the relative effects of the presence of galactose in classically glucose-repressing media and/or knockout of SIP1 using a multi-scale variant of 13C MFA known as 2-Scale 13C metabolic flux analysis (2S-13C MFA). In this study, all strains have the galactose metabolism deactivated (gal1Δ background) so as to be able to separate the metabolic effects purely related to glucose repression from those arising from galactose metabolism. The resulting flux profiles reveal that the presence of galactose in classically glucose-repressing conditions, for a CEN.PK113-7D gal1Δ background, results in a substantial decrease in pentose phosphate pathway (PPP) flux and increased flow from cytosolic pyruvate and malate through the mitochondria toward cytosolic branched-chain amino acid biosynthesis. These fluxomic redistributions are accompanied by a higher maximum specific growth rate, both seemingly in violation of glucose repression. Deletion of SIP1 in the CEN.PK113-7D gal1Δ cells grown in mixed glucose/galactose medium results in a further increase. Knockout of this gene in cells grown in glucose-only medium results in no change in growth rate and a corresponding decrease in glucose and ethanol exchange fluxes and flux through pathways involved in aspartate/threonine biosynthesis. Glucose repression appears to be violated at a 1/10 ratio of galactose-to-glucose. Based on the scientific literature, we may have conducted our experiments near a critical sugar ratio that is known to allow galactose to enter the cell. Additionally, we report a number of fluxomic changes associated with these growth rate increases and unexpected flux profile redistributions resulting from deletion of SIP1 in glucose-only medium

Functional responses of methanogenic archaea to syntrophic growth.

Methanococcus maripaludis grown syntrophically with Desulfovibrio vulgaris was compared with M. maripaludis monocultures grown under hydrogen limitation using transcriptional, proteomic and metabolite analyses. These measurements indicate a decrease in transcript abundance for energy-consuming biosynthetic functions in syntrophically grown M. maripaludis, with an increase in transcript abundance for genes involved in the energy-generating central pathway for methanogenesis. Compared with growth in monoculture under hydrogen limitation, the response of paralogous genes, such as those coding for hydrogenases, often diverged, with transcripts of one variant increasing in relative abundance, whereas the other was little changed or significantly decreased in abundance. A common theme was an apparent increase in transcripts for functions using H(2) directly as reductant, versus those using the reduced deazaflavin (coenzyme F(420)). The greater importance of direct reduction by H(2) was supported by improved syntrophic growth of a deletion mutant in an F(420)-dependent dehydrogenase of M. maripaludis. These data suggest that paralogous genes enable the methanogen to adapt to changing substrate availability, sustaining it under environmental conditions that are often near the thermodynamic threshold for growth. Additionally, the discovery of interspecies alanine transfer adds another metabolic dimension to this environmentally relevant mutualism