Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose

Xylose utilization is of commercial interest for efficient conversion of abundant plant material to ethanol. Perhaps the most important ethanol-producing organism, Saccharomyces cerevisiae, however, is incapable of xylose utilization. While S. cerevisiae strains have been metabolically engineered to utilize xylose, none of the recombinant strains or any other naturally occurring yeast has been able to grow anaerobically on xylose. Starting with the recombinant S. cerevisiae strain TMB3001 that overexpresses the xylose utilization pathway from Pichia stipitis, in this study we developed a selection procedure for the evolution of strains that are capable of anaerobic growth on xylose alone. Selection was successful only when organisms were first selected for efficient aerobic growth on xylose alone and then slowly adapted to microaerobic conditions and finally anaerobic conditions, which indicated that multiple mutations were necessary. After a total of 460 generations or 266 days of selection, the culture reproduced stably under anaerobic conditions on xylose and consisted primarily of two subpopulations with distinct phenotypes. Clones in the larger subpopulation grew anaerobically on xylose and utilized both xylose and glucose simultaneously in batch culture, but they exhibited impaired growth on glucose. Surprisingly, clones in the smaller subpopulation were incapable of anaerobic growth on xylose. However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose.     

Validation of the TracmorD Triaxial Accelerometer to Assess Physical Activity in Preschool Children

Objectives: To assess validity evidence of Tracmor_D to determine energy used for physical activity in 3‐4‐year‐old children. Design and Methods: Participants were randomly selected from GECKO Drenthe cohort (n = 30, age 3.4 ± 0.3 years). Total energy expenditure (TEE) was measured using the doubly labeled water method. Sleeping metabolic rate (SMR) was measured by indirect calorimetry (Deltatrac). TEE and SMR were used to calculate physical activity level (PAL) and activity energy expenditure (AEE). Physical activity was monitored using a DirectLife triaxial accelerometer, Tracmor_D with activity counts per minute (ACM) and activity counts per day (ACD) as outcome measures. Results: The best predictor for PAL was ACM with gender and weight, the best predictor for AEE was ACM alone (backward regression, R² = 0.50, P = 0.010 and R² = 0.31, P = 0.011, respectively). With ACD, the prediction model for PAL included ACD, height, gender, and sleep duration (R² = 0.48, P = 0.033), the prediction model for AEE included ACD, gender and sleep duration (R² = 0.39, P = 0.042). The accelerometer was worn for 5 days, but 3 days did not give a different estimated PAL. Conclusion: Tracmor_D provides moderate‐to‐strong validity evidence that supports its use to evaluate energy used for physical activity in 3‐4‐year‐old children.     

Somatic embryogenesis from ovaries,developing ovules and immature zygotic embryos,and improved embryo development of <Emphasis Type="Italic">Castanea sativa</Emphasis>

Somatic embryogenesis of European chestnut (Castanea sativa Mill.) was obtained using juvenile tissue cultured on P24 medium with 5 M 2,4-dichlorophenoxyacetic acid plus 0.5 M 6-benzylaminopurine (BA) for three weeks and then cultured on 0.89 M BA. Induction frequency with ovaries ranged from 2.0 to 19.1 % and was observed in tissue collected 2 to 8 weeks postanthesis, ovules used as a starting tissue gained 0.8 to 7.8 %, 3 to 9 weeks postanthesis. Zygotic embryos collected 5 to 10 weeks postanthesis formed 10.5 to 57.1 % somatic embryos, respectively. The culture lines were maintained via secondary embryogenesis on P24 medium with 0.89 M BA. Development and maturation were stimulated on P24 medium with increased agar concentration (1.1 %). Five plantlets were transferred to substrate and acclimatized successfully in greenhouse.     

Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells

Rat liver cells isolated by the collagenase-hyaluronidase perfusion method were treated with membrane-impermeable protein reagents (7-diazonium, 1–3-naphthalene disulfonate, diazotized sulfanilic acid, 8-anilino-naphthalene disulfonate), trypsin, phospholipase A, phospholipase C, and phospholipase D. The treated cells were incubated with [1-¹⁴C]palmitate and the ¹⁴CO₂ produced was taken as a measure of fatty acid uptake by the cells. ¹⁴CO₂ production by the cells was not inhibited after treatments with the membrane-impermeable protein reagents or phospholipase D. Treatments with small amounts of trypsin or phospholipases A or C caused inhibition of CO₂ production from tracer amounts of palmitate. The inhibition by trypsin was partially, and that by phospholipase A was fully, reversed by increasing the amount of palmitic acid in the incubation medium. The oxidation of shorter-chain fatty acids such as octanoic acid was not decreased but increased after treating the cells with trypsin or phospholipase A. The membrane-impermeable reagents inhibited the oxidation of palmitate to CO₂ by liver cells isolated by mechanical dispersion. These reagents also inhibited the long-chain acyl CoA ligase activity of liver microsomes. From these results it is suggested that the inhibition of CO₂ production by intact liver cells from palmitate after enzyme treatments, is due to partial removal or modification of a normal transport component for long-chain fatty acids on the plasma membrane. The possibility of proteins (or lipoproteins) buried below the surface layer of plasma membrane in fatty acid uptake by liver cells is indicated.     

The human collagen beta(1-O)galactosyltransferase,GLT25D1, is a soluble endoplasmic reticulum localized protein

Background  

Glycosyl transferases transfer glycosyl groups onto their substrate. Localization partially defines their function. Glycosyl transferase 25 domain 1 (GLT25D1) was recently shown to have galactosyltransferase activity towards collagens and another well known substrate, mannose binding lectin (MBL). To gain more insight in the role of galactosylation of lysines in the Gly-X-Lys repeats of collagenous proteins, we investigated the subcellular localization of GLT25D1.     

Δ1-11-oxa-11-deoxycortisol: A new antiglucocorticoid with activity in vivo

A new steroid-like compound, Δ¹-11-oxa-11-deoxycortisol, was tested in a one-week growth suppression, thymus suppression and adrenal weight suppression bioassay for possible glucocorticoid antagonist activity in $\text{vivo}$. We hypothesized that this compound would have antiglucocorticoid activity based on previous studies of 11-deoxycortisol and Δ^1,9(11)-11-deoxycortisol, which were optimal glucocorticoid antagonists $\text{in vivo}$ in adrenalectomized rats, but which lost antiglucocorticoid activity in intact animals, apparently due to adrenal 11β-hydroxylation. Thus, Δ¹-11-oxa-11-deoxycortisol, a compound which cannot undergo llβ-hydroxylation, was synthesized and tested as an antiglucocorticoid. This analog had an affinity for the rat thymus glucocorticoid receptor similar to that of its parent compounds (Ki 0.9-3.1×10^?7M). A dose of 1 $\text{mg}\text{rat}$ antagonized the effect of 15μg of dexamethasone in the growth suppression assay (p<0.05) and in the thymus suppression assay (p<0.06), but did not antagonize dexamethasone-induced adrenal weight suppression. Δ¹-11-oxa-11-deoxycortisol did not exhibit glucocorticoid activity in any of the three assays. These data suggest that Δ¹-11-oxa-11-deoxycortisol may be a pure competitive antagonist of dexamethasone.     

Prostaglandin E(2)-stimulated secretion of protein in the salivary glands of the lone star tick via a phosphoinositide signaling pathway

Previous studies identified a prostaglandin E(2) (PGE(2)) receptor in the salivary glands of partially fed female lone star ticks, Amblyomma americanum (L.). In the present studies, protein secretion from dispersed salivary gland acini was shown to be specific for PGE(2), as compared with PGF(2alpha) or the thromboxane analog U-46619, in accordance with their respective binding affinities for the PGE(2) receptor. Furthermore, the selective PGE(2) EP1 receptor agonist, 17-phenyl trinor PGE(2), was as effective as PGE(2) in stimulating secretion of anticoagulant protein. Calcium ionophore A-23187 (1 to 100 microM) stimulated secretion of anticoagulant protein in a dose-dependent manner but the voltage-gated Ca(2+)-channel blocker verapamil (1 to 1000 microM) and the receptor-mediated Ca(2+)-entry antagonist, SK&F 96365 (1 and 10 microM), and 5mM ethylene glycol bis(beta-aminoethyl ether)-N,NN', N'-tetraacetic acid (EGTA) had no appreciable effect on inhibiting PGE(2)-stimulated secretion of anticoagulant protein. PGE(2) (0.1 microM) and the non-hydrolyzable analog of guanosine triphosphate (GTP), GTPgammaS (10 microM), directly activated phospholipase C (PLC) in a membrane-enriched fraction of the salivary glands after PLC was first incubated with the PGE(2) EP1 receptor antagonist AH-6809, which presumably antagonized endogenous PGE(2) (0.3 microM) in the broken-cell-membrane-enriched fraction. TMB-8, an antagonist of intracellular inositol trisphosphate (IP(3)) receptors, inhibited PGE(2)-stimulated secretion. The results support the hypothesis that PGE(2) stimulates secretion of tick salivary gland protein via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+).     

Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan

Background

Aluminum (Al) toxicity is an important limitation to food security in tropical and subtropical regions. High Al saturation on acid soils limits root development, reducing water and nutrient uptake. In addition to naturally occurring acid soils, agricultural practices may decrease soil pH, leading to yield losses due to Al toxicity. Elucidating the genetic and molecular mechanisms underlying maize Al tolerance is expected to accelerate the development of Al-tolerant cultivars.

Results

Five genomic regions were significantly associated with Al tolerance, using 54,455 SNP markers in a recombinant inbred line population derived from Cateto Al237. Candidate genes co-localized with Al tolerance QTLs were further investigated. Near-isogenic lines (NILs) developed for ZmMATE2 were as Al-sensitive as the recurrent line, indicating that this candidate gene was not responsible for the Al tolerance QTL on chromosome 5, qALT5. However, ZmNrat1, a maize homolog to OsNrat1, which encodes an Al³⁺ specific transporter previously implicated in rice Al tolerance, was mapped at ~40 Mbp from qALT5. We demonstrate for the first time that ZmNrat1 is preferentially expressed in maize root tips and is up-regulated by Al, similarly to OsNrat1 in rice, suggesting a role of this gene in maize Al tolerance. The strongest-effect QTL was mapped on chromosome 6 (qALT6), within a 0.5 Mbp region where three copies of the Al tolerance gene, ZmMATE1, were found in tandem configuration. qALT6 was shown to increase Al tolerance in maize; the qALT6-NILs carrying three copies of ZmMATE1 exhibited a two-fold increase in Al tolerance, and higher expression of ZmMATE1 compared to the Al sensitive recurrent parent. Interestingly, a new source of Al tolerance via ZmMATE1 was identified in a Brazilian elite line that showed high expression of ZmMATE1 but carries a single copy of ZmMATE1.

Conclusions

High ZmMATE1 expression, controlled either by three copies of the target gene or by an unknown molecular mechanism, is responsible for Al tolerance mediated by qALT6. As Al tolerant alleles at qALT6 are rare in maize, marker-assisted introgression of this QTL is an important strategy to improve maize adaptation to acid soils worldwide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-153) contains supplementary material, which is available to authorized users.