Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae
Jongedijk, E.J. ; Cankar, K. ; Ranzijn, J. ; Krol, A.R. van der; Bouwmeester, H.J. ; Beekwilder, M.J. - \ 2015
Yeast 32 (2015)1. - ISSN 0749-503X - p. 159 - 171.
monoterpene biosynthesis - escherichia-coli - synthase - precursor
Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028¿mg/l (+)-limonene and 0.060¿mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12¿mg/l (+)-limonene and 0.49¿mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems. Gene Bank Nos were: KM015220 (Perilla limonene synthase; this study); AF317695 (Perilla limonene synthase; Yuba et al., 1996); AF514287.1 (Citrus limonene synthase; Lucker et al., 2002).
N-glycan occupancy of Arabidopsis N-glycoproteins
Song, W. ; Mentink, R. ; Henquet, M.G.L. ; Cordewener, J.H.G. ; Dijk, A.D.J. van; Bosch, H.J. ; America, A.H.P. ; Krol, A.R. van der - \ 2013
Journal of Proteomics 93 (2013). - ISSN 1874-3919 - p. 343 - 355.
lectin affinity-chromatography - mass-spectrometry - glycosylated proteins - linked glycoproteins - identification - plants - glycopeptides - deglycosylation - glycomics - precursor
Most secreted proteins in eukaryotes are modified on the amino acid consensus sequence NxS/T by an N-glycan through the process of N-glycosylation. The N-glycans on glycoproteins are processed in the endoplasmic reticulum (ER) to different mannose-type N-glycans or, when the protein passes through the Golgi apparatus, to different complex glycan forms. Here we describe the capturing of N-glycopeptides from a trypsin digest of total protein extracts of Arabidopsis plants and release of these captured peptides following Peptide N-glycosidase (PNGase) treatment for analysis of N-glycan site-occupancy. The mixture of peptides released as a consequence of the PNGase treatment was analyzed by two dimensional nano-LC–MS. As the PNGase treatment of glycopeptides results in the deamidation of the asparagine (N) in the NxS/T site of the released peptide, this asparagine (N) to aspartic acid (D) conversion is used as a glycosylation ‘signature’. The efficiency of PNGase F and PNGase A in peptide release is discussed. The identification of proteins with a single glycopeptide was limited by the used search algorithm but could be improved using a reference database including deamidated peptide sequences. Additional stringency settings were used for filtering results to minimize false discovery. This resulted in identification of 330 glycopeptides on 173 glycoproteins from Arabidopsis, of which 28 putative glycoproteins, that were previously not annotated as secreted protein in The Arabidopsis Information Resource database (TAIR). Furthermore, the identified glycosylation site occupancy helped to determine the correct topology for membrane proteins. A quantitative comparison of peptide signal was made between wild type and complex-glycan-less (cgl) mutant Arabidopsis from three replicate leaf samples using a label-free MS peak comparison. As an example, the identified membrane protein SKU5 (AT4G12420) showed differential glycopeptide intensity ratios between WT and cgl indicating heterogeneous glycan modification on single protein.
Urinary felinine excretion in intact male cats is increased by dietary cystine
Hendriks, W.H. ; Rutherfurd-Markwick, K.J. ; Weidgraaf, K. ; Morton, R.H. ; Rogers, Q.R. - \ 2008
The British journal of nutrition 100 (2008)4. - ISSN 0007-1145 - p. 801 - 809.
glutathione s-transferases - felis-catus - cysteine dioxygenase - rats - inactivation - deficiency - methionine - precursor - glutamine - arginine
Felinine is a branched-chain sulfur amino acid present in the urine of certain Felidae, including domestic cats. The objective of the present study was to determine if additional cystine and/or dietary N would increase felinine and N-acetylfelinine excretion by intact male cats fed a low-protein (LP) diet. Feeding five adult intact male cats an LP diet (18·8% of metabolisable energy (ME) as protein) v. a high-protein diet (38·6% of ME as protein) resulted in a trend (P¼0·08) for decreased urinary felinine and no change in N-acetylfelinine excretion. In a 23 d study, when the LP diet was supplemented with L-cystine at 9·3 g/kg DM, urinary felinine:creatinine ratio showed a linear two-fold (121 %) increase (P,0·01) from 0·24 (SEM 0·05) to 0·53 (SEM 0·13) after 10 d. Subsequent feeding of the LP diet resulted in a decrease in felinine excretion to base levels. Plasma gglutamylfelinylglycine concentrations were consistent with the excretion of felinine. Supplementation of the LP diet with L-cystine (9·3 g/kg DM), dispensable amino acids and arginine to a second group (n 5) also resulted in a significant (P,0·01) but smaller (þ72 %) increase in the daily felinine:creatinine ratio (0·25 (SEM 0·04) to 0·43 (SEM 0·05)). The degree of felinine N-acetylation within groups was unaffected by dietary addition and withdrawal of amino acids. The results indicate that felinine synthesis is regulated by cystine availability, and that arginine may be physiologically important in decreasing felinine biosynthesis in intact male cats.
Food intake, growth, and reproduction as affected by day length and food availability in the pond snail Lymnaea stagnalis
Maat, A. ter; Zonneveld, C. ; Visser, J.A.G.M. de; Jansen, R.F. ; Montagne-Wajer, K. ; Koene, J.M. - \ 2007
American Malacological Bulletin 23 (2007)1-2. - ISSN 0740-2783 - p. 113 - 120.
caudo-dorsal cells - egg-laying behavior - freshwater snail - ovulation hormone - energy budgets - trade-off - neurohormone - photoperiod - precursor - peptides
With the aim of integrating the physiology and evolutionary ecology of Lymnaea stagnalis (Linnaeus, 1758), we studied the effects of day length and food availability on the energy budget. Snails were assigned to two different photoperiods and three levels of food availability. The snails were kept individually, and food consumption, growth, and egg production were measured for about 2 months. Snails could nearly compensate for a one-day starvation period by increasing the rate of food-intake. However, food-intake rates did not increase further after a starvation period of 2 days. Growth was well described by the Von Bertalanffy growth equation. The ultimate size of snails kept under medium-day conditions (MD; light:dark = 12:12 h) was not affected by food availability. By contrast, the ultimate size of snails kept under long-day conditions (LD; light:dark = 16:8 h) depended on food availability; those fed the lowest quantities grow the least. Dry-weight densities (dry weight/wet weight) of MD snails were considerably above those of LD snails. In MD snails, food availability did not appreciably affect dry-weight density. By contrast, in LD snails, dry-weight density decreased with decreasing food availability. The reproductive output of LD snails declined with declining food availability, but was 2 to 4 times that of MD snails. The difference in reproductive output was largely accounted for by the difference in stored energy, i.e. dry-weight density. To gauge the extent to which the conclusions from our laboratory work applied to free-living snails, a field study was conducted. The wild-caught snails' dry-weight density was also lowest in long-day conditions when most eggs were laid. However, the dry-weight densities during medium and short days were lower than the dry-weight densities of laboratory animals under LD conditions. Thus, in the field, snails stored less energy than in the laboratory.