Genome Shuffling in Clostridium diolis DSM 15410 for Improved 1,3-Propanediol Production

Several microorganisms are known for their efficient anaerobic conversion of glycerol to 1,3-propanediol, with Clostridium diolis DSM 15410 as one of the better performers in terms of molar yield and volumetric productivity. However, this performance is still insufficient to compete with established chemical processes. Previous studies have shown that high concentrations of 1,3-propanediol, glycerol, and fermentation side products can limit the productivity of C. diolis DSM 15410. Here, we describe the use of genome shuffling for improved 1,3-propanediol fermentation by the strict anaerobe C. diolis DSM 15410. By using chemical mutagenesis, strains with superior substrate and product tolerance levels were isolated and higher product yields were obtained. These superior strains were then used for genome shuffling and selection for 1,3-propanediol and organic acid tolerance. After four rounds of genome shuffling and selection, significant improvements were observed, with one strain attaining a 1,3-propanediol volumetric yield of 85 g/liter. This result represents an 80% improvement compared to the yield from the parental wild-type strain.The use of biomass instead of petrochemical feedstock could facilitate the sustainable production of many chemicals, but this approach has proven economically feasible in only a few cases (4, 17, 20, 21). The microbial production of 1,3-propanediol (1,3-PD) provides an interesting case study because this monomer is used to produce several plastics, including the relatively new and highly versatile polytrimethylene terephthalate, which has significantly increased demand for 1,3-PD (12, 23, 24). Polytrimethylene terephthalate is currently produced from petrochemical feedstock in a process that involves the conversion of ethylene oxide into 3-hydroxypropionaldehyde by hydroformylation under high pressure and then further into 1,3-PD by hydrogenation using a nickel or rubidium catalyst (21).Several companies have investigated the sustainable production of 1,3-PD from biomass. For example, DuPont and Genencor transferred the relevant 1,3-PD biosynthetic genes from Klebsiella pneumonia into Escherichia coli and further modified carbohydrate metabolism and transport so that 1,3-PD could be synthesized from glucose (10, 11; M. Emptage, S. L. Haynie, L. A. Laffend, J. Pucci, and G. M. Whited, 2001, Patent Cooperation Treaty international application WO 2001/01/12833). Large-scale production of 1,3-PD by this approach is likely to be too expensive due to the high input costs of vitamin B₁₂ and antibiotics, so the use of glycerol as an alternative feedstock has been investigated, although this strategy requires additional enzymes and a shift from aerobic to anaerobic conditions (15, 25, 30).Clostridium diolis DSM 15410 (formerly C. butyricum DSM 5431) can produce 1,3-PD from glycerol under anaerobic conditions and is therefore a desirable alternative to E. coli given the relative costs of industrial aerobic and anaerobic fermentation (6, 8, 9, 22). However, the efficiency of conversion is not yet high enough for an industrial process. The production of 1,3-PD by C. diolis is limited by inhibition from both substrates and products, as well as organic acids produced as fermentation by-products (13).Classical strain improvement has significantly increased 1,3-PD production, but this is a slow process and the mutations are predominantly neutral or detrimental (2). We have therefore approached the problem using genome shuffling, which is more efficient and reliable for engineering complex phenotypes, as demonstrated in several other examples of microbial strain development (16, 26, 31). Genome shuffling offers the advantages of accumulated beneficial mutations and the removal of unnecessary mutations due to simultaneous changes at different positions throughout the genome and, therefore, yields microbes of superior fitness (29). We applied both the classical approach and genome shuffling to C. diolis DSM 15410 to improve the production of 1,3-PD, which is a necessary prerequisite for the fermentation process. To our knowledge, this study is the first example of genome shuffling in a strictly anaerobic microorganism.     

Increase of intracellular Ca2+ by adenine and uracil nucleotides in human midbrain-derived neuronal progenitor cells

Nucleotides play an important role in brain development and may exert their action via ligand-gated cationic channels or G protein-coupled receptors. Patch-clamp measurements indicated that in contrast to AMPA, ATP did not induce membrane currents in human midbrain derived neuronal progenitor cells (hmNPCs). Various nucleotide agonists concentration-dependently increased [Ca²⁺]_i as measured by the Fura-2 method, with the rank order of potency ATP > ADP > UTP > UDP. A Ca²⁺-free external medium moderately decreased, whereas a depletion of the intracellular Ca²⁺ storage sites by cyclopiazonic acid markedly depressed the [Ca²⁺]_i transients induced by either ATP or UTP. Further, the P2Y₁ receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y_1,2,12 antagonistic suramin only slightly blocked the action of ATP, but strongly inhibited that of UTP. In agreement with this finding, UTP evoked the release of ATP from hmNPCs in a suramin-, but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of P2Y_1,2,4-immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y₂ receptor-activation and thereby causes [Ca²⁺]_i transients by stimulating a P2Y₁-like receptor.     

Inactivation of Vibrio parahaemolyticus in pure culture, whole live and half shell oysters (Crassostrea virginica) by X-ray

Aims:  To study the inactivation effect of different doses of X-ray on Vibrio parahaemolyticus in pure culture, inoculated whole live and half shell oysters and to evaluate the efficacy of X-ray doses on reduction of inherent microflora on oysters.
Methods and Results:  X-ray was produced using RS 2400 generator system (Rad Source Technologies Inc.). Pure culture of V. parahaemolyticus , inoculated half and whole shell oysters with V. parahaemolyticus were treated with 0·0, 0·1, 0·5, 0·75, 1·0, 1·5, 2·0, 3·0 and 5·0 kGy X-ray. Surviving bacteria in the pure culture and inoculated oysters, before and after treatment, were enumerated using overlay plating (in TSA then TCBS) and most probable number (MPN) methods. A greater than 6·0 log reduction of V. parahaemolyticus was observed with 0·75, 2·0 and 5·0 kGy X-ray for pure culture, half shell and whole shell oysters, respectively. Treatment with 0·75, 2·0 and 5·0 kGy X-ray reduced the MPN to <3 for pure culture, half and whole shell oysters, respectively. Treatment with 1·0 kGy X-ray significantly ( P <  0·05) reduced the inherent micro-organisms on whole shell oysters from 4·7 ± 0·1 to less than the detectable limit (<1·0 log CFU g⁻¹).
Conclusions:  X-ray (1–5 kGy) significantly ( P <  0·05) reduced V . parahaemolyticus and inherent microflora on oysters to less than detectable limit (<1·0 log CFU g⁻¹).
Significance and Impact of the Study:  Treatment with X-ray could control pathogenic bacteria and extend the shelf life of oysters.     

Development of pentaplex PCR and genetic analysis of X chromosomal STRs in Punjabi population of Pakistan

The X-STRs are important tools in forensic application, particularly in complex cases of kinship testing. In deficiency paternity testing when alleged father cannot be typed, investigation of X-STR markers yields the desired information. Blood samples were collected from unrelated individual (118 females and 94 males) and 84 trios families (father, mother and daughter). DNA extraction from whole blood was performed with Phenol chloroform method. Five X-linked STR markers DXS6800, DXS7133, DXS6797, DXS981 and GATA165B12 were selected. The amplicons were analyzed through ABI 3100 Genetic Analyzer. Pentaplex PCR system was developed for multilocus amplification at the same time. For each locus 4–9 alleles were noted. Altogether, 32 alleles were observed from five markers. Eighty-four trios families were analysed to check the mutation rate and no mutation was observed. Stutter peaks were observed maximum at locus DXS6797 (12.44%) while the minimum at locus DXS7133 (4.5%). For sensitivity study, amplification of X chromosomal short tandem repeats loci was successfully performed using 0.15 ng quantity of DNA as template. In conclusion; this pentaplex represents a convenient method to study X chromosome markers. It works with reasonable amounts of DNA and is suitable for paternity cases.     

Thidiazuron-induced high-frequency direct shoot organogenesis of <Emphasis Type="Italic">Cannabis sativa</Emphasis> L.

Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yr-old mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0 μM thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibberellic acid into a medium containing 0.5 μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l⁻¹ activated charcoal and 2.5 μM indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000 μmol m⁻² s⁻¹). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 μmol m⁻² s⁻¹ and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500 μmol m⁻² s⁻¹. Stomatal conductance and transpiration increased with light intensity up to highest level (2000 μmol m⁻² s⁻¹) tested. Intercellular CO₂ concentration (C _i) and the ratio of intercellular CO₂ concentration to ambient CO₂ (C _i/C _a) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.     

Probabilistic Risk Assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in Produced Water

The produced water extracted during oil and gas production includes formation water, injected water, small volumes of condensed water, and any chemical added during the oil/water separation process. Produced water contains both organic and inorganic constituents, and several studies have been conducted in the past to assess their risk. The toxicity and persistence of polycyclic aromatic hydrocarbons (PAHs) in produced water is of particular environmental concern, but there are very few studies on human health risk assessment from PAHs of produced water. This article summarizes the results of a conservative human health risk assessment approach for PAHs in produced water discharges to the marine environment. Due to the absence of available toxicity data for PAHs, the cancer slope factors were determined by using the relative potency factors (RPF) and Toxicity Equivalency Factors (TEF). Using the concentration distribution factors, the maximum cancer risks to humans were predicted in the range of 4.07 × 10^?7 to 2.95 × 10^?6. The 95th-percentile values show that the risks are well within the acceptable limits.     

NMR structure of duplex DNA containing the α‐OH‐PdG·dA base pair: A mutagenic intermediate of acrolein

Acrolein, a cell metabolic product and main component of cigarette smoke, reacts with DNA generating α‐OH‐PdG lesions, which have the ability to pair with dATP during replication thereby causing G to T transversions. We describe the solution structure of an 11‐mer DNA duplex containing the mutagenic α‐OH‐PdG·dA base pair intermediate, as determined by solution nuclear magnetic resonance (NMR) spectroscopy and retrained molecular dynamics (MD) simulations. The NMR data support a mostly regular right‐handed helix that is only perturbed at its center by the presence of the lesion. Undamaged residues of the duplex are in anti orientation, forming standard Watson‐Crick base pairs alignments. Duplication of proton signals at and near the damaged base pair reveals the presence of two enantiomeric duplexes, thus establishing the exocyclic nature of the lesion. The α‐OH‐PdG adduct assumes a syn conformation pairing to its partner dA base that is protonated at pH 6.6. The three‐dimensional structure obtained by restrained molecular dynamics simulations show hydrogen bond interactions that stabilize α‐OH‐PdG in a syn conformation and across the lesion containing base pair. We discuss the implications of the structures for the mutagenic bypass of acrolein lesions. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 391–401, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Investigation of electrorheological properties of biodegradable modified cellulose/corn oil suspensions

Considerable scientific and industrial interest is currently being focused on a class of materials known as electrorheological (ER) fluids, which display remarkable rheological behaviour, being able to convert rapidly and repeatedly from a liquid to solid when an electric field (E) is applied or removed. In this study, biodegradable cellulose was modified and converted to their carboxyl salts. Modified cellulose is characterised by Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, energy dispersive spectroscopy (EDS), thermogravimetric analysis (TGA) and conductivity measurements. Suspensions of cellulose (C) and modified cellulose (MC) were prepared in insulated corn oil (CO). The effects of electric field strength, shear rate, shear stress, temperature, etc. of these suspensions onto ER activity were determined. Rheological measurements were carried out via a rotational rheometer with a high-voltage generator to investigate the effects of electric field strength and particle concentration on ER performance.The results show that the ER properties are enhanced by increasing the particle concentration and electric field strength. Also the cellulose-based ER fluids exhibit viscoelastic behaviour under an applied electric field due to the chain formation induced by electric polarization between particles.     

Succinate cytochrome <Emphasis Type="Italic">c</Emphasis> reductase in schistosomiasis: in vitro inhibition by some schistosomicidal drugs

Enzymes in mitochondria play an important role in biological oxidation and energy production. To understand the effect of schistosomiasis on these important processes, succinate cytochrome c reductase (SCR) from control and Schistosoma-infected mice was subjected for investigation. In this article, we report that SCR from Schistosoma-infected mouse showed a significant decrease in its V _max and K _m compared to control using both cytochrome c and 2,6-dichlorophenolindophenol as substrates. Furthermore, the kinetic studies of the purified SCR in the absence and presence of the schistosomicidal drugs praziquantel and Commiphora extract reveal that both drugs have an inhibitory action on the enzyme from the control and Schistosoma-infected mice and praziquantel changes the type of inhibition of SCR towards cytochrome c from mixed type in control to a competitive one in the case of the infection.