Improved Squalene Production via Modulation of the Methylerythritol 4-Phosphate Pathway and Heterologous Expression of Genes from Streptomyces peucetius ATCC 27952 in Escherichia coli

Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.Squalene, an industrially important compound obtained primarily from the liver oil of deep-sea sharks and whales, is an important ingredient in skin cosmetics due to its photoprotective role (2, 7). The decreased cancer risk associated with high olive oil consumption could result from high squalene content (12, 16). Squalene has a chemopreventive effect on colon cancer (14). Moreover, squalene has wide applications in fine chemicals, magnetic tape, and low-temperature lubricants and as an additive in animal feed (1).The use of shark liver oil is limited, due to the presence of environmental pollutants, such as polychlorinated biphenyls, heavy metals, and methylmercury residues, as well as an unpleasant fishy odor and taste (17, 19). Moreover, the presence of similar compounds, such as cholesterol, in the oils from marine animal liver can make squalene purification difficult. In addition, squalene production is limited by uncertain availability because of international concern for the protection of marine animals. Squalene has also been obtained from plant sources (4, 10, 11, 18), but very few methods can produce sufficient quantities at the desired purity level for pharmaceutical and industrial applications (6). The use of engineered microbial cell factories for the biosynthesis of squalene may be a suitable alternative to address these issues.In the genome project for Streptomyces peucetius ATCC 27952, a cluster of genes which comprises five open reading frames, encoding hopanoid biosynthesis, has been detected and annotated. Even though these open reading frames share sequence homology with genes involved in hopanoid biosynthesis, no plausible hopanoid products have been isolated from S. peucetius in all laboratory cultures. Therefore, the hopanoid biosynthetic gene cluster of S. peucetius was considered “cryptic” in the present study. We were interested in activating the so-called “cryptic” hopanoid biosynthetic gene cluster of S. peucetius to produce pharmaceutically important compounds by using genetic engineering tools. Isoprenoid production in Escherichia coli has been extensively studied and reviewed (5, 8, 9, 15, 20, 21), but very few reports detail squalene formation in E. coli by the use of exogenous genes (13). In the present study, we introduced three cryptic genes (hopABD) from the hopanoid biosynthesis gene cluster from S. peucetius that catalyzed squalene production and also modulated the 2-C-methyl-d-erythritol 4-phosphate pathway in E. coli to enhance squalene production (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic representation of the engineered squalene biosynthetic pathway in E. coli BL21(DE3). DXS and IDI were overexpressed for the modulation of the MEP pathway to increase and balance the IPP pool, HopD (farnesyl diphosphate synthase) was overexpressed to increase FPP, and HopA and HopB (HopAB) (squalene synthases) were overexpressed to overproduce squalene. The recombinant enzymes overexpressed in E. coli are boxed.     

Amplification of Plant Beneficial Microbial Communities During Conversion of Coconut Leaf Substrate to Vermicompost by <Emphasis Type="Italic">Eudrilus</Emphasis> sp.

The population densities of 15 microbial communities in the coconut leaves + cow manure mixture (10:1 ratio, w/w) and pure cow manure, gut contents of the earthworm, Eudrilus sp., reared on the above substrates and vermicompost produced by the worm were studied. The enumeration was done by dilution plate and most probable number method using several selective and semi-selective microbial media. In the vermicompost produced from coconut leaves + cow manure (CLV) mixture, 9 out of 15 microbial communities, particularly the plant beneficial ones, were amplified whereas five communities were amplified in case of pure cow manure (CMV). The CLV contained significantly high population of fungi, free-living nitrogen fixers, phosphate solubilizers, fluorescent pseudomonads, and silicate solubilizers. The CMV was preponderant with aerobic heterotrophic bacteria, actinomycetes, and Trichoderma spp. Spore formers were present in similar numbers in both the vermicomposts. Presence of Azotobacter was detected only in CMV. The results obtained in this study suggest coconut leaf litter to be a good alternative for cow manure for the production of vermicompost, especially in the areas where coconut is grown in plenty.     

Association Mapping of Quantitative Disease Resistance in a Natural Population of Loblolly Pine (Pinus taeda L.)

Genetic resistance to disease incited by necrotrophic pathogens is not well understood in plants. Whereas resistance is often quantitative, there is limited information on the genes that underpin quantitative variation in disease resistance. We used a population genomic approach to identify genes in loblolly pine (Pinus taeda) that are associated with resistance to pitch canker, a disease incited by the necrotrophic pathogen Fusarium circinatum. A set of 498 largely unrelated, clonally propagated genotypes were inoculated with F. circinatum microconidia and lesion length, a measure of disease resistance, data were collected 4, 8, and 12 weeks after inoculation. Best linear unbiased prediction was used to adjust for imbalance in number of observations and to identify highly susceptible and highly resistant genotypes (“tails”). The tails were reinoculated to validate the results of the full population screen. Significant associations were detected in 10 single nucleotide polymorphisms (SNPs) (out of 3938 tested). As hypothesized for genes involved in quantitative resistance, the 10 SNPs had small effects and proposed roles in basal resistance, direct defense, and signal transduction. We also discovered associated genes with unknown function, which would have remained undetected in a candidate gene approach constrained by annotation for disease resistance or stress response.GENETIC interactions between host and pathogen populations result in abundant natural variation in the genes involved in host disease resistance. Most of the studies leading to identification and cloning of disease resistance genes are focused on major gene disease resistance (Johal and Briggs 1992; Dangl and Jones 2001; Jones and Dangl 2006). In cases where resistance is associated with single genes, genetic effects are large in magnitude and detection is straightforward. In contrast, quantitative disease resistance is typically conditioned by many genes with relatively small effects. Quantitative resistance is generally considered to be more durable but also more difficult to investigate relative to major gene resistance, since the effects of individual genes are small and phenotyping experiments must be performed with high levels of precision. As a consequence, the genes and mechanisms of quantitative disease resistance are poorly understood, in part due to the smaller effect of individual genes on the resistance phenotype. Interactions between plants and necrotrophic pathogens often exhibit quantitative resistance (Balint-Kurti et al. 2008; Poland et al. 2009).Pitch canker disease of loblolly pine and other pine species is incited by the necrotrophic pathogen Fusarium circinatum and is manifest as resinous lesions in stems and branches (Dwinell et al. 1985; Enebak and Stanosz 2003; Carey et al. 2005; Sakamoto and Gordon 2006). There is evidence for heritable resistance to pitch canker in loblolly pine (Kayihan et al. 2005) as well as other pine species (Hodge and Dvorak 2000, 2007). In this article we report the first population-wide phenotypic screen of a clonally propagated population of loblolly pine for association testing (Eckert et al. 2010). Clonal propagation of this population enabled precise phenotyping, which was required to obtain the resolution needed to identify candidates for quantitative disease resistance loci.Pine species in general exhibit high levels of nucleotide variation and low linkage disequilibrium (LD) (Brown et al. 2004). An association genetic approach relies on the premise that historical, unrecorded recombination events over many generations have reduced LD between markers and quantitative trait loci such that only those marker-trait pairs that are tightly linked remain detectable; this may enable “fine mapping” to identify genes underlying quantitative variation (Flint-Garcia et al. 2003; Neale and Savolainen 2004). Association-based approaches have been used to identify candidate genes underlying traits in plants (Zhao et al. 2007; Stich et al. 2008; Wang et al. 2008; Yahiaoui et al. 2008; Inostroza et al. 2009; Stracke et al. 2009), based in part on applications in humans (D''alfonso et al. 2002; McGuffin et al. 2003; Easton et al. 2007; Lee et al. 2007), livestock (Martinez et al. 2006; Charlier et al. 2008; Goddard and Hayes 2009), and Drosophila (Kennington et al. 2007; Norry et al. 2007; Jiang et al. 2009). Recent association studies in tree species have evaluated single candidate genes or a modest number of candidate genes for association (Thumma et al. 2005; Gonzalez-Martinez et al. 2007, 2008; Ingvarsson et al. 2008; Eckert et al. 2009a). Association mapping has been used to identify disease resistance genes in several crop species including sugarcane, maize, barley, and potato (Flint-Garcia et al. 2005; Wei et al. 2006; Yu and Buckler 2006; Malosetti et al. 2007; Stich et al. 2008; Inostroza et al. 2009; Murray et al. 2009). The population analyzed in this study was genotyped at 3938 SNP loci that were selected without regard to the functional annotation of ESTs from which they were derived. Thus, we reasoned that the status of any particular marker as a candidate disease resistance gene would be determined by association testing, as opposed to previous studies in which markers were typically evaluated on the basis of their presumed roles in disease resistance in other species.Several different, but not mutually exclusive hypotheses have been proposed regarding the genetic origins of quantitative resistance (Poland et al. 2009), providing a useful framework for understanding evolution of resistance to necrotrophic pathogens. These six hypotheses proposed by Poland et al. (2009) predict that quantitative disease resistance is conditioned by: (1) genes regulating morphological and developmental phenotypes; (2) mutations in genes involved in basal defense causing small, incremental levels of resistance; (3) components of chemical warfare, through the action of genes producing antibiotic or antifungal compounds; (4) genes involved in defense signal transduction pathways; (5) weak forms of defeated R genes; and/or (6) genes not yet known to be involved in disease resistance.In this study, our main objective was to evaluate the genetic architecture of pitch canker disease resistance: to quantify the extent to which genes contribute to variation in the disease phenotype, to evaluate the hypothesis that disease resistance was quantitative, and to identify candidate genes for resistance as well as quantify their magnitude of effect. In the process of identifying candidate genes for resistance we were also able to evaluate support for hypotheses recently put forth by Poland et al. (2009) regarding the biological roles and origins of quantitative resistance genes.     

Effect of plant growth promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. on compatible solutes,antioxidant status and plant growth of maize under drought stress

Drought is one of the major abiotic stresses affecting yield of dryland crops. Rhizobacterial populations of stressed soils are adapted and tolerant to stress and can be screened for isolation of efficient stress adaptive/tolerant, plant growth promoting rhizobacterial (PGPR) strains that can be used as inoculants for crops grown in stressed ecosystems. The effect of inoculation of five drought tolerant plant growth promoting Pseudomonas spp. strains namely P. entomophila strain BV-P13, P. stutzeri strain GRFHAP-P14, P. putida strain GAP-P45, P. syringae strain GRFHYTP52, and P. monteilli strain WAPP53 on growth, osmoregulation and antioxidant status of maize seedlings under drought stress conditions was investigated. Drought stress induced by withholding irrigation had drastic effects on growth of maize seedlings. However seed bacterization of maize with Pseudomonas spp. strains improved plant biomass, relative water content, leaf water potential, root adhering soil/root tissue ratio, aggregate stability and mean weight diameter and decreased leaf water loss. The inoculated plants showed higher levels of proline, sugars, free amino acids under drought stress. However protein and starch content was reduced under drought stress conditions. Inoculation decreased electrolyte leakage compared to uninoculated seedlings under drought stress. As compared to uninoculated seedlings, inoculated seedlings showed significantly lower activities of antioxidant enzymes, ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX) under drought stress, indicating that inoculated seedlings felt less stress as compared to uninoculated seedlings. The strain GAP-P45 was found to be the best in terms of influencing growth and biochemical and physiological status of the seedlings under drought stress. The study reports the potential of rhizobacteria in alleviating drought stress effects in maize.     

Is gender difference in postnatal thyroid growth associated with specific expression patterns of androgen and estrogen receptors?

Variations in sex steroids bioavailability were linked to the gender difference in the growth of thyroid glands of neonatal rats. In the present study we tested androgen receptor (AR) and estrogen receptor (ER) concentrations by ligand binding assay, and expression of their genes by RT-PCR and Western blot in the thyroid glands of neonatal rats. AR concentration remained elevated from postnatal day (PND) 10 onwards in males, whereas it decreased by PND 20 in females. AR mRNA and protein expressions were higher in males than females, which increased by PND 10, decreased after PND 15 and reached the nadir by PND 20. ER concentration increased by PND 10 and decreased thereafter in both sex. ERα mRNA expression diminished by PND 15 in both sex; while ERβ mRNA decreased by PND 15 to reach the nadir by PND 20 in males, it was augmented by PND 10 in females to reach the peak by PND 15 and diminished by PND 20. ERα protein expression increased by PND 10 and remained elevated till PND 20 in both sex. ERβ protein expression in males increased by PND 10 and decreased by PND 20, while it remained static up to PND 15 and decreased in females. Testosterone stimulated [³H]-thymidine uptake and the expression of IGF-1 and NIS genes in thyrocytes of both sex in vitro, while estradiol stimulated them in females but not in males. We conclude that androgens influence the growth and differentiation of thyrocytes through augmented expression of AR, IGF-1 and NIS in either sex, whereas estrogen imparts the gender difference, which may be at a level beyond the expression of ERs.     

Fungal isolates from natural pectic substrates for polygalacturonase and multienzyme production

Pectin rich wastes and waste dump yard soils were screened and eighty pectinolytic fungal isolates were obtained by enrichment culturing and ruthenium red plate assay. Eight isolates with higher zones of pectin hydrolysis were selected and tested for polygalacturonase production. One isolate identified as Aspergillus awamori MTCC 9166 with highest polygalacturonase activity was tested for utilization of raw pectins for enzyme production. Polygalacturonase production was high in raw pectin sources like Orange peel (16.8 U/ml) Jack fruit rind (38 U/ml) Carrot peel (36U/ml) and Beet root peel (24U/ml). Selected Aspergillus awamori MTCC 9166 was found to be having good polygalacturonase, xylanase, cellulase and weak amylase and protease activities. This isolate with multi-enzyme production could have application for enzymes production and degradation of fruit and vegetable waste in the process of urban waste disposal.     

Immobilization of Bacillus amyloliquefaciens MBL27 cells for enhanced antimicrobial protein production using calcium alginate beads

Cell immobilization is one of the common techniques for increasing the overall cell concentration and productivity. Bacillus amyloliquefaciens MBL27 cells were immobilized in calcium alginate beads and it is a promising method for repeated AMP (antimicrobial protein) production. The present study aimed at determining the optimal conditions for immobilization of B. amyloliquefaciens MBL27 cells in calcium alginate beads and the operational stability for enhanced production of the AMP. AMP production with free and immobilized cells was also done. In batch fermentation, maximum AMP production (7300 AU (arbitrary units)/ml against Staphylococcus aureus) was obtained with immobilized cells in shake flasks under optimized parameters such as 3% (w/v) sodium alginate, 136?mM CaCl2 with 350 alginate beads/flask of 2.7-3.0?mm diameter. In repeated cultivation, the highest activity was obtained after the second cycle of use and approx. 94% production was noted up to the fifth cycle. The immobilized cells of B. amyloliquefaciens MBL27 in alginate beads are more efficient for the production of AMP and had good stability. The potential application of AMP as a wound healant and the need for development of economical methods for improved production make whole cell immobilization an excellent alternative method for enhanced AMP production.     

Syndecans as receptors and organizers of the extracellular matrix

Syndecans are type I transmembrane proteins having a core protein modified with glycosaminoglycan chains, most commonly heparan sulphate. They are an ancient group of molecules, present in invertebrates and vertebrates. Among the plethora of molecules that can interact with heparan sulphate, the collagens and glycoproteins of the extracellular matrix are prominent. Frequently, they do so in conjunction with other receptors, most notably the integrins. For this reason, they are often referred to as “co-receptors”. However, just as with integrins, syndecans can interact with actin-associated proteins and signalling molecules, such as protein kinases. Some aspects of syndecan signalling are understood but much remains to be learned. The functions of syndecans in regulating cell adhesion and extracellular matrix assembly are described here. Evidence from null mice suggests that syndecans have roles in postnatal tissue repair, inflammation and tumour progression. Developmental deficits in lower vertebrates in which syndecans are eliminated are also informative and suggest that, in mammals, redundancy is a key issue.     

An alternative high-throughput staining method for detection of neutral lipids in green microalgae for biodiesel applications

A simple and high-throughput method for determining in situ intracellular neutral lipid accumulation in Chlorella ellipsoidea and Chlorococcum infusionum with flow cytometry and confocal microscopy was established by employing different solvents and a lipophilic dye, Nile red. Seven different organic solvents, acetic acid, dimethyl sulfoxide (DMSO), acetone, methanol, ethanol, n-hexane, and chloroform at different concentrations ranging from 0 to 80% (v/v) were tested. The fluorescence signal for neutral lipids was collected with a 586/42 emission filter (PE-A) and the maximum fluorescence intensity (% grandparent) was measured as 74.01 ± 4.82% for Chlorella and 70.1 ± 5.52% for Chlorococcum at 30% acetic acid (v/v). The statistical analysis of Nile red-stained cells showed a high coefficient of variation (CV), standard deviation (SD), mean, and median values in the acetic acid-based staining method, followed by DMSO, n-hexane and chloroform. Confocal microscopy revealed a high rate of accumulation of cytosolic neutral lipids when stained with Nile red and other organic solvents. Higher lipid accumulation in Fesupplemented conditions was also detected and a maximum lipid content of 57.36 ± 0.41% (4-fold) in Chlorella and 48.20 ± 0.43% (4-fold) in Chlorococcum were measured at 0.001 g/L of ferrous sulfate (FeSO₄). High fluorescence intensity (75.16 ± 0.24% in Chlorella and 72.24 ± 1.07% in Chlorococcum) in Fe-treated cells confirmed the efficiency of the staining procedure.