High yield 1,3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii

Background: Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii Delta dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply. Results: By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA, and ethanol dehydrogenase coded by adhE) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii Delta dhaD resulted in the strain C. werkmanii Delta dhaD Delta ldhA.adhE::ChlFRT which provided the maximum theoretical yield of 1.00 +/- 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 +/- 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism. Conclusions: In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached

The rational search for selective anticancer derivatives of the peptide Trichogin GA IV: a multi-technique biophysical approach

Peptaibols are peculiar peptides produced by fungi as weapons against other microorganisms. Previous studies showed that peptaibols are promising peptide-based drugs because they act against cell membranes rather than a specific target, thus lowering the possibility of the onset of multi-drug resistance, and they possess non-coded alpha-amino acid residues that confer proteolytic resistance. Trichogin GA IV (TG) is a short peptaibol displaying antimicrobial and cytotoxic activity. In the present work, we studied thirteen TG analogues, adopting a multidisciplinary approach. We showed that the cytotoxicity is tuneable by single amino-acids substitutions. Many analogues maintain the same level of non-selective cytotoxicity of TG and three analogues are completely non-toxic. Two promising lead compounds, characterized by the introduction of a positively charged unnatural amino-acid in the hydrophobic face of the helix, selectively kill T67 cancer cells without affecting healthy cells. To explain the determinants of the cytotoxicity, we investigated the structural parameters of the peptides, their cell-binding properties, cell localization, and dynamics in the membrane, as well as the cell membrane composition. We show that, while cytotoxicity is governed by the fine balance between the amphipathicity and hydrophobicity, the selectivity depends also on the expression of negatively charged phospholipids on the cell surface

On-chip inverted emulsion method for fast giant vesicle production, handling, and analysis

Liposomes and giant unilamellar vesicles (GUVs) in particular are excellent compartments for constructing artificial cells. Traditionally, their use requires bench-top vesicle growth, followed by experimentation under a microscope. Such steps are time-consuming and can lead to loss of vesicles when they are transferred to an observation chamber. To overcome these issues, we present an integrated microfluidic chip which combines GUV formation, trapping, and multiple separate experiments in the same device. First, we optimized the buffer conditions to maximize both the yield and the subsequent trapping of the vesicles in micro-posts. Captured GUVs were monodisperse with specific size of 18 ± 4 µm in diameter. Next, we introduce a two-layer design with integrated valves which allows fast solution exchange in less than 20 s and on separate sub-populations of the trapped vesicles. We demonstrate that multiple experiments can be performed in a single chip with both membrane transport and permeabilization assays. In conclusion, we have developed a versatile all-in-one microfluidic chip with capabilities to produce and perform multiple experiments on a single batch of vesicles using low sample volumes. We expect this device will be highly advantageous for bottom-up synthetic biology where rapid encapsulation and visualization is required for enzymatic reactions

Neuronal SIRT1 Regulates Metabolic and Reproductive Function and the Response to Caloric Restriction.

Sirt1 is an NAD-dependent, class III deacetylase that functions as a cellular energy sensor. In addition to its well-characterized effects in peripheral tissues, emerging evidence suggests that neuronal Sirt1 activity plays a role in the central regulation of energy balance and glucose metabolism. In this study, we generated mice expressing an enzymatically inactive form (N-MUT) or wild-type (WT) SIRT1 (N-OX) in mature neurons. N-OX male and female mice had impaired glucose tolerance, and N-MUT female, but not male, mice had improved glucose tolerance compared with that of WT littermates. Furthermore, glucose tolerance was improved in all mice with caloric restriction (CR) but was greater in the N-OX mice, who had better glucose tolerance than their littermates. At the reproductive level, N-OX females had impaired estrous cycles, with increased cycle length and more time in estrus. LH and progesterone surges were absent on the evening of proestrus in the N-OX mice, suggesting a defect in spontaneous ovulation, which was confirmed by the ovarian histology revealing fewer corpora lutea. Despite this defect, the mice were still fertile when mated to WT mice on the day of proestrus, indicating that the mice could respond to normal pheromonal or environmental cues. When subjected to CR, the N-OX mice went into diestrus arrest earlier than their littermates. Together, these results suggested that the overexpression of SIRT1 rendered the mice more sensitive to the metabolic improvements and suppression of reproductive cycles by CR, which was independent of circadian rhythms

Evaluation of agro-industrial by-products as protein sources for duck production in the Mekong Delta of Vietnam

This thesis is based on four studies to determine the nutritive value and ileal and total tract amino acid digestibility of agro-industrial by-products, and the effects of inclusion of these by-products in diets on the biological performance of growing crossbred Muscovy and common ducks in the Mekong Delta, Vietnam. The results show that brewery waste (BW) was characterised by high protein content,good balance of essential amino acids, but high crude fibre content. The best growth performance was observed when 50% of the concentrate was replaced by BW for both Muscovy and common ducks. The crossbred Muscovies had better performance and gave higher economic benefit than the local Muscovies, and common ducks gave the lowest profit. BW could be offered either in mixed or separate feeding systems without any effects on performance and carcass yield (Paper I and II). Soya waste (SW) and ensiled shrimp waste (ESW) are good protein sources, but low in dry matter (DM) and methionine content (SW) and high in chitin content (ESW). The best performance in terms of growth rate and carcass quality was found with replacement of up to 60% of soybean meal by SW, and only 20% of fish meal by ESW, which gave the lowest feed costs (Paper III). The inclusion of ESW in diets for adult crossbred common ducks resulted in a linear decrease of the apparent total tract digestibility of dry matter, organic matter, ether extract, N free extract and N retention with increasing levels of ESW. The digestibility of individual amino acids followed a similar pattern to the nutrient digestibility. The apparent total tract digestibility of most individual amino acids was higher than the apparent ileal value (Paper IV). It can be concluded that from nutritive and economic apects the three agro-industrial byproducts evaluated have proven to be acceptable feed ingredients in duck diets. Replacement up to levels of 20% (ESW), 50% (BW) and 60% (SW) of fish meal, concentrate and soybean meal, respectively, in diets for growing crossbred ducks can be recommended under the conditions of the Mekong Delta

The effects of graded levels of calorie restriction : II. Impact of short term calorie and protein restriction on circulating hormone levels, glucose homeostasis and oxidative stress in male C57BL/6 mice

This work was supported by BBSRC BB009953/1 awarded to JRS and SEM. PK and CD were funded by the Erasmus exchange programme. JRS, SEM, DD, CG, LC, JJDH, YW, DELP, DL and AD are members of the BBSRC China Partnership Award, BB/J020028/1.Peer reviewedPublisher PD

Tug-of-war in motility assay experiments

The dynamics of two groups of molecular motors pulling in opposite directions on a rigid filament is studied theoretically. To this end we first consider the behavior of one set of motors pulling in a single direction against an external force using a new mean-field approach. Based on these results we analyze a similar setup with two sets of motors pulling in opposite directions in a tug-of-war in the presence of an external force. In both cases we find that the interplay of fluid friction and protein friction leads to a complex phase diagram where the force-velocity relations can exhibit regions of bistability and spontaneous symmetry breaking. Finally, motivated by recent work, we turn to the case of motility assay experiments where motors bound to a surface push on a bundle of filaments. We find that, depending on the absence or the presence of a bistability in the force-velocity curve at zero force, the bundle exhibits anomalous or biased diffusion on long-time and large-length scales

Recombinant subunit vaccines against Neospora caninum

The intracellular protozoan parasite Neospora caninum is an important cause of bovine abortion and congenital infection in many countries. Currently there is no effective control method available, and development of a vaccine has been suggested as a possible strategy to prevent the disease neosporosis. In this thesis an experimental recombinant subunit vaccine, consisting of the N. caninum antigen NcSRS2 and immunostimulating complexes (iscoms), was evaluated. The iscom is an adjuvant formulation into which antigens can be incorporated by hydrophobic interactions. Many recombinant proteins are hydrophilic, and special methods are needed for binding them to the iscoms and to achieve optimal adjuvant effects. A novel method to combine recombinant proteins with iscoms utilizing the strong interaction between biotin and streptavidin was evaluated. Recombinant NcSRS2 was expressed in Escherichia coli as two different fusion proteins. One of them was biotinylated in E. coli and was bound to streptavidin-coated iscom matrix (iscom particles without any antigen). The other fusion protein contained recombinant streptavidin and was bound to biotinylated iscom matrix. These two strategies both resulted in efficient binding to the iscom matrix. Thus, this method might offer a convenient and effective alternative for association of other hydrophilic recombinant antigens with iscoms for evaluation in immunisation experiments. Both the immunogenicity and the protective effect of the NcSRS2-iscoms were investigated by immunisation and challenge infection of mice. A real-time PCR assay was developed, and used for quantification of parasite DNA in the brain and blood of infected mice. The NcSRS2-iscoms induced production of antibodies that recognised NcSRS2 of parasite origin, suggesting that the conformation of the recombinant protein was similar to that of the native antigen. Antigen-specific cellular responses were also induced as demonstrated by in vitro proliferation and cytokine production. After challenge infection, the immunised mice had significantly lower levels of N. caninum DNA in their brains and blood than did non-immunised control mice. These mice were also less affected by the disease, as judged by clinical symptoms and changes in body weight. These results, together with results from other studies, indicate that recombinant NcSRS2 might be considered as a potential candidate antigen for a future subunit vaccine against N. caninum infection in cattle