Construction of new <Emphasis Type="Italic">Pichia pastoris</Emphasis> X-33 strains for production of lycopene and β-carotene

In this study, we used the non-carotenogenic yeast Pichia pastoris X33 as a receptor for β-carotene-encoding genes, in order to obtain new recombinant strains capable of producing different carotenoidic compounds. We designed and constructed two plasmids, pGAPZA-EBI* and pGAPZA-EBI*L*, containing the genes encoding lycopene and β-carotene, respectively. Plasmid pGAPZA-EBI*, expresses three genes, crtE, crtB, and crtI*, that encode three carotenogenic enzymes, geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, respectively. The other plasmid, pGAPZA-EBI*L*, carried not only the three genes above mentioned, but also the crtL* gene, that encodes lycopene β-cyclase. The genes crtE, crtB, and crtI were obtained from Erwinia uredovora, whereas crtL* was cloned from Ficus carica (JF279547). The plasmids were integrated into P. pastoris genomic DNA, and the resulting clones Pp-EBI and Pp-EBIL were selected for either lycopene or β-carotene production and purification, respectively. Cells of these strains were investigated for their carotenoid contents in YPD media. These carotenoids produced by the recombinant P. pastoris clones were qualitatively and quantitatively analyzed by high-resolution liquid chromatography, coupled to photodiode array detector. These analyses confirmed that the recombinant P. pastoris clones indeed produced either lycopene or β-carotene, according to the integrated vector, and productions of 1.141 μg of lycopene and 339 μg of β-carotene per gram of cells (dry weight) were achieved. To the best of our knowledge, this is the first time that P. pastoris has been genetically manipulated to produce β-carotene, thus providing an alternative source for large-scale biosynthesis of carotenoids.     

A comparative study of variation in codon 33 of the<Emphasis Type="Italic"> rpoS</Emphasis> gene in<Emphasis Type="Italic"> Escherichia coli</Emphasis> K12 stocks: implications for the synthesis of σ<Superscript>s</Superscript>

The Escherichia coli rpoS gene encodes an RNA polymerase sigma factor (sigma S or ^S) required for the expression of stationary-phase genes. In the first published rpoS sequence from E. coli K-12 codon 33 is given as CAG. However, several subsequent independent studies found the amber codon TAG at this position ( rpoSAm). Besides this amber codon, other codons such as TAT have also been found at this location in rpoS. Comparative genome analysis now leads us to propose TAG as the parental codon 33 in rpoS in E. coli K-12. Five different stocks of the strain W3110, which differ in the levels of ^S protein they express, were investigated. We sequenced the rpoS gene from these, and found a T at nucleotide position 97 in four out of the five stocks and a G at position 99 in three out of the five. W1485, a parental strain of W3110, and W3350, a derivative of W3110, are also rpoSAm mutants. Such rpoSAm mutants would be expected to show no RpoS activity. The retention of partial or intermediate ^S activity by suppressor-free rpoSAm mutants is therefore puzzling. We propose that a functional, N-terminally truncated, ^S (1–53^S) can be translated from a Secondary Translation Initiation Region (STIR) located downstream of the amber codon 33. It has recently been reported that a fragment of RpoS (1–53^S) that lacks the first 53 amino acids is functional when synthesized in vivo. Taken together, our results support the hypothesis that the original codon 33 of the rpoS gene in E. coli K-12 strains is the amber codon TAG.Communicated by W. Goebel     

High-level expression of biologically active glycoprotein hormones in <Emphasis Type="Italic">Pichia pastoris</Emphasis> strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated <Superscript>15</Superscript>N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active ¹⁵N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH₄Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man_8–15GlcNAc₂). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Man_up-to-30GlcNAc₂). The latter finding made the X-33 strain not very suitable for generating ¹⁵N-labeled material. Therefore, ¹⁵N-phCG was expressed in the GS115 strain using the new optimized protocol. The ¹⁵N-enrichment was evaluated by ¹⁵N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that ¹⁵N-phCG/GS115 had the same folding as urinary hCG. Furthermore, ¹⁵N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays. Véronique Blanchard and Rupali A. Gadkari contributed equally.     

Structure of the δ-Chain of Chimpanzee Haemoglobin A<Subscript>2</Subscript>

STUDIES of adult¹ and foetal² haemoglobin from the chimpanzee (Pan troglodytes) have shown that the amino-acid compositions of tryptic and chymotryptic peptides of the α, β and γ-chains are indistinguishable from those of man. The primary structures of chimpanzee α, β and γ-chains are therefore almost certainly identical to the homologous human chains. The two types of γ-chains found in man³, ^Gγ and ^Aγ, with glycine and alanine in position γ136, respectively, are likewise present in the chimpanzee².     

A selected probiotic strain of <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> CM33 isolated from breast-fed infants as a potential source of β-galactosidase for prebiotic oligosaccharide synthesis

Lactic acid bacteria from healthy breast-fed infants were isolated and screened for β-galactosidase production in MRS broth. Among 49 isolates that exhibited the yellow clear zone on MRS agar supplemented with bromocresol blue, the isolate CM33 was selected as being the highest β-galactosidase producer and was identified as Lactobacillus fermentum based on its morphological characteristics and 16S rDNA nucleotide sequence. L. fermentum CM33 exhibited a good survival rate under the simulated stomach passage model, comparable to known probiotic strains L. gallinarum JCM2011 and L. agilis JCM1187. L. fermentum CM33 was antagonistic to pathogenic bacteria Listeria monocytogenes, Escherichia coli 0157:H7, Salmonella typhi, and Salmonella enteriditis, using the well diffusion method. In addition, the selected lactobacilli exhibited a high growth rate when cultivated in modified MRS containing commercial galactooligosaccharide (GOS) as a sole carbon source, as well as in glucose. A preliminary study on the enzymatic synthesis of oligosaccharide using crude β-galactosidase revealed the capability for oligosaccharide synthesis by the transgalactosylation activity.     

Effects of antimicrobial activity of silver nanoparticles on in vitro establishment of G × N15 (hybrid of almond × peach) rootstock

In the present investigation were evaluated the antifungal and antibacterial activities of Nano-silver (NS). Two separate experiments were done to evaluate the potential of silver nanoparticles in controlling the contamination of G × N15 micro-propagation. In the first experiment, a factorial experiment based on a completely randomized design with 15 treatments including five different NS concentrations (0, 50, 100, 150 and 200 ppm) and three soaking time of explants (3, 5 and 7 min) with five replications was conducted. In the other experiment, NS was added to Murashige and Skoog (MS) medium with concentrations of 0, 50, 100, 150 and 200 ppm in a completely randomized design. Results showed that the application of 100 and 150 ppm NS both as an immersion and as added directly to the culture medium significantly reduces internal and external contaminations compared with the control group. Using NS in culture medium was more efficient to reduce fungal and bacterial contamination than immersion. High concentrations of NS had an adverse effect on the viability of G × N15 single nodes and this effect was more serious in immersed explants in NS than directly added NS ones regarding the viability of buds and plantlet regeneration. In conclusion, due to high contamination of woody plants especially fruit trees and also adverse environmental effects of mercury chloride, the NS solution can be used as a low risk bactericide in micro-propagation of G × N15 and can be an appropriate alternative to mercury chloride in the future.     

Can the combination of electrochemical regeneration of NAD+, selectivity of L‐α‐amino‐acid dehydrogenase,and reductive amination of α‐keto‐acid be applied to the inversion of configuration of a L‐α‐amino‐acid?

The inversion of configuration of L‐alanine can be carried out by combining its selective oxidation in the presence of NAD⁺ and L‐alanine dehydrogenase, electrochemical regeneration of the NAD⁺ at a carbon felt anode, and reductive amination of pyruvate, i.e., reduction of its imino derivative at a mercury cathode, the reaction mixture being buffered with concentrated ammonium/ammonia (1.28M / 1.28M). The dehydrogenase exhibits astonishing activity and stability under such extreme conditions of pH and ionic strength. The main drawback of the process is its slowness. At best, the complete inversion of a 10 mM solution of L‐alanine requires 140 h. A careful and detailed quantitative analysis of each of the key steps involved shows that the enzyme catalyzed oxidation is so thermodynamically uphill that it can be driven efficiently to completion only when both the coenzyme regeneration and the pyruvate reduction are very effective. The first condition is easily fulfilled. Under the best conditions, it is the rate of the chemical reaction producing the imine which controls the whole process kinetically. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 101–107, 1999.     

Study of the Mechanism of Interaction of Oligonucleotides with the 3′-Terminal Region of tRNA<Superscript>Phe</Superscript> by Computer Modeling

Three-dimensional atomic models of complexes between yeast tRNA^Phe and 10- or 15-mer oligonucleotides complementary to the 3′-terminal tRNA sequence have been constructed using computer modeling. It has been found that rapidly formed primary complexes appear when an oligonucleotide binds to the coaxial acceptor and T stems of the tRNA^Phe along the major groove, which results in the formation of a triplex. Long stems allow the formation of a sufficiently strong complex with the oligonucleotide, which delivers its 3′-terminal nucleotides to the vicinity of the T loop adjoining the stem. These nucleotides destabilize the loop structure and initiate conformational rearrangements involving local tRNA^Phe destruction and formation of the final tRNA^Phe-oligonucleotide complementary complex. The primary complex formation and the following tRNA^Phe destruction constitute the “molecular wedge” mechanism. An effective antisence oligonucleotide should consist of three segments—(1) complex initiator, (2) primary complex stabilizer, and (3) loop destructor—and be complementary to the (free end)/loop-stem-loop tRNA structural element.     

The fate of fertiliser P in soil under pasture and uptake by subterraneum clover – a field study using <Superscript>33</Superscript>P-labelled single superphosphate

Background and aims

Single superphosphate (SSP) is a major source of phosphorus (P) used in grazing systems to improve pasture production. The aim of this experiment was to determine the fate of fertiliser P in clover pastures under field conditions.

Methods

A procedure was developed to radiolabel SSP granules with a ³³P radiotracer, which was then applied to the soil surface (equivalent to ~12 kg P ha^?1) of a clover pasture. Recovery of fertiliser P was determined in clover shoots, fertiliser granules and soil fractions (surface layer: 0–4 cm and sub-surface layer: 4–8 cm).

Results

The P diffusion patterns of the ³³P-labelled SSP granules were not significantly different to those of commercial SSP granules (P?>?0.05). Recovery of fertiliser P in clover shoots was 30–35 %. A considerable proportion of the fertiliser P (~28 %) was recovered in the surface soil layer and was largely inorganic P.

Conclusions

Recovery of fertiliser P by clover plants was up to 35 % in the year of application. Much of the fertiliser P in soil fractions was inorganic P, which highlights the importance of inorganic P forms and dynamics in soils under clover pasture on a single season timeframe at these sites.

     

Computational studies of the binding site of α<Subscript>1A</Subscript>-adrenoceptor antagonists

Aimed at achieving a good understanding of the 3-dimensional structures of human α_1A-adrenoceptor (α_1A-AR), we have successfully developed its homology model based on the crystal structure of β₂-AR. Subsequent structural refinements were performed to mimic the receptor’s natural membrane environment by using molecular mechanics (MM) and molecular dynamics (MD) simulations in the GBSW implicit membrane model. Through molecular docking and further simulations, possible binding modes of subtype-selective α_1A-AR antagonists, Silodosin, RWJ-69736 and (+)SNAP-7915, were examined. Results of the modeling and docking studies are qualitatively consistent with available experimental data from mutagenesis studies. The homology model built should be very useful for designing more potent subtype-selective α_1A-AR antagonists and for guiding further mutagenesis studies. Figure The superposition of β₂-AR crystal structure (gold ribbons) and α_1A-AR homology model (blue ribbons)     

Determination of the lifetime of D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack> and HD<Superscript>−</Superscript> ions

A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D^? ₂ and HD^? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively.