A Constitutive Expression System for Cellulase Secretion in Escherichia coli and Its Use in Bioethanol Production

The production of biofuels from lignocellulosic biomass appears to be attractive and viable due to the abundance and availability of this biomass. The hydrolysis of this biomass, however, is challenging because of the complex lignocellulosic structure. The ability to produce hydrolytic cellulase enzymes in a cost-effective manner will certainly accelerate the process of making lignocellulosic ethanol production a commercial reality. These cellulases may need to be produced aerobically to generate large amounts of protein in a short time or anaerobically to produce biofuels from cellulose via consolidated bioprocessing. Therefore, it is important to identify a promoter that can constitutively drive the expression of cellulases under both aerobic and anaerobic conditions without the need for an inducer. Using lacZ as reporter gene, we analyzed the strength of the promoters of four genes, namely lacZ, gapA, ldhA and pflB, and found that the gapA promoter yielded the maximum expression of the β-galactosidase enzyme under both aerobic and anaerobic conditions. We further cloned the genes for two cellulolytic enzymes, β-1,4-endoglucanase and β-1,4-glucosidase, under the control of the gapA promoter, and we expressed these genes in Escherichia coli, which secreted the products into the extracellular medium. An ethanologenic E. colistrain transformed with the secretory β-glucosidase gene construct fermented cellobiose in both defined and complex medium. This recombinant strain also fermented wheat straw hydrolysate containing glucose, xylose and cellobiose into ethanol with an 85% efficiency of biotransformation. An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol.     

Identification of genome-wide single-nucleotide polymorphisms (SNPs) associated with tolerance to chromium toxicity in spring wheat (Triticum aestivum L.)

Plant and Soil - Extreme climate events, including flooding and prolonged drought, may establish long-lasting (legacy) effects on soil abiotic and biotic properties, potentially influencing soil...     

Novel mutation G324C in <Emphasis Type="Italic">WNT1</Emphasis> mapped in a large Pakistani family with severe recessively inherited <Emphasis Type="Italic">Osteogenesis Imperfecta</Emphasis>

Introduction

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease with skeletal fragility and variable extra-skeletal manifestations. To date several point mutations in 18 different genes causing different types of OI have been identified. Mutations in WNT1 compromise activity of the osteoblasts leading to disturbed bone mass accrual, fragility fractures and progressive skeletal abnormalities. The present study was conducted to determine the underlying genetic cause of an autosomal recessive skeletal dysplasia in a large consanguineous family from Chinute, Pakistan.

Materials and methods

Blood was collected from 24 individuals of affected family along with clinical data. Homozygosity mapping was performed to confirm consanguinity. SNPs were identified, followed by whole exome and Sanger sequencing. In silico characterization of WNT1 mutation was performed using multiple platforms.

Results

Nine affected family members exhibited severe bone deformities, recurrent fractures, short stature and low bone mineral density. SNP array data revealed homozygous segments >?1 Mb in length accounting for 2.1–12.7% of the genome in affected individuals and their siblings and a single 6,344,821 bp homozygous region in all affected individuals on chromosome 12q12-q13. This region includes two potential OI candidate genes WNT1 and VDR. We did whole-exome sequencing for both genes in two patients and identified a novel damaging missense mutation in exon 4 of WNT1: c.1168G?>?T (NM_005430) resulting in p.G324C. Sanger sequencing confirmed segregation of mutation with the disease in family.

Conclusion

We report a novel mutation responsible for OI and our investigation expands the spectrum of disease-causing WNT1 mutations and the resulting OI phenotypes.

     

Bioremediation of hexachlorocyclohexane (HCH) in soil using spent mushroom compost of Pleurotus ostreatus

Abstract

Hexachlorocyclohexane (HCH), a highly chlorinated pesticide, was used worldwide in the 1950s and 1960s. HCH toxic residues are still detected in environmental compartments. Thus, effective, viable and eco-friendly strategy is required for its remediation. In this study, degradation of four HCH isomers was evaluated by amending contaminated soil using four treatments of spent mushroom compost (SMC) of Pleurotus ostraetus. The soil was incubated for 5 weeks and was sampled every seven days. Quantitative attenuation in HCH was calculated using gas chromatography–electron capture detector (GC-ECD) and metabolite was identified using gas chromatography–mass selective detector (GC-MSD). Maximum reduction 58%, 26%, 45%, and 64% for α-, β-, γ- and δ-HCH isomers, respectively, using SMC and soil (both unsterilized) showed that this treatment was the best for bioremediation of HCH in soil. However, when one of the factors, either soil or SMC, was sterilized, a significant reduction in HCH degradation was noticed. The second most reduction of isomers was seen during treatment where unsterilized SMC was added in sterilized soil followed by treatment where SMC was sterilized but soil was not. Abiotic control did not remove any significant quantities of HCH. Simple first-order (SFO) kinetic confirmed that SMC reduced the half-live manifolds as compared to biotic control. Only one metabolite δ-PCCH was identified during the course of study.     

Predicting the cumulative effect of multiple disturbances on seagrass connectivity

The rate of exchange, or connectivity, among populations effects their ability to recover after disturbance events. However, there is limited information on the extent to which populations are connected or how multiple disturbances affect connectivity, especially in coastal and marine ecosystems. We used network analysis and the outputs of a biophysical model to measure potential functional connectivity and predict the impact of multiple disturbances on seagrasses in the central Great Barrier Reef World Heritage Area (GBRWHA), Australia. The seagrass networks were densely connected, indicating that seagrasses are resilient to the random loss of meadows. Our analysis identified discrete meadows that are important sources of seagrass propagules and that serve as stepping stones connecting various different parts of the network. Several of these meadows were close to urban areas or ports and likely to be at risk from coastal development. Deep water meadows were highly connected to coastal meadows and may function as a refuge, but only for non‐foundation species. We evaluated changes to the structure and functioning of the seagrass networks when one or more discrete meadows were removed due to multiple disturbance events. The scale of disturbance required to disconnect the seagrass networks into two or more components was on average >245 km, about half the length of the metapopulation. The densely connected seagrass meadows of the central GBRWHA are not limited by the supply of propagules; therefore, management should focus on improving environmental conditions that support natural seagrass recruitment and recovery processes. Our study provides a new framework for assessing the impact of global change on the connectivity and persistence of coastal and marine ecosystems. Without this knowledge, management actions, including coastal restoration, may prove unnecessary and be unsuccessful.     

Improvement of both lipid and biomass productivities of Qatar Chlorocystis isolate for biodiesel production and food security

Microalgae are considered a very promising alternative for biofuel production. Several strategies were developed to modulate and improve algae metabolites production to meet the requirements for biodiesel production. Most previous research evidenced that the increase of the lipid content is accompanied by a decrease of the biomass production, which increases the cost of the downstream processing. Hence, the challenge is to find special culture conditions that increase the lipid and the biomass productivities simultaneously. In the present work, we developed a strategy for the improvement of biomass and lipid productivities in a novel local microalga isolate, Chlorocystis sp. QUCCCM14, which was not previously known as a promising strain. Indeed, culturing QUCCCM14 using f/2 medium with 10× NaH₂PO₄ (0.15 g L^?1 NaNO₃ and 5.6 mg L^?1 NaH₂PO₄) resulted in an improvement of 3.178 folds the lipid productivity reaching 56.121 mg L^?1 day^?1 and enhanced the biomass productivity reaching 141.363 mg L^?1 day^?1, simultaneously. Comparative analyses of the FAME profiles demonstrated that fed‐batch culture with phosphate or nitrate separately leads to a high production of the omega 3 fatty acids (Linolenic acid), whereas fed‐batch culture with phosphate and nitrate simultaneously increased the production of fatty acids suitable for biodiesel production.     

Herbivore and phytohormone induced defensive response in kale against cabbage butterfly, Pieris brassicae Linn

The constitutive and induced resistance were studied in two varieties (Khanyari and Kawdari) of kale, Brassica oleracea var. acephala in response to cabbage butterfly, Pieris brassicae infestation and exogenous application of jasmonic acid (JA) and salicylic acid (SA). Phenols, condensed tannins, flavonoids and proteins were measured at six days after JA (1?mM) and SA (1?mM) application and/or insect infestation. Plant damage and larval weights were also recorded. Khanyari variety showed highest amounts of phenols (208.23?μg/g FW), condensed tannin (347.76?μg/g FW), flavonoids (175.61?μg/g FW) and proteins (0.71?mg/g FW) in plants pre-treated with JA and infested with insects. The PAL activity was high in response to JA application followed by insect infestation. Insects reared on Khanyari and Kawdari plants pre-treated with JA prior showed significantly reduced larval weights (97.88 and 102.46?mg, respectively). Damage was low in plants pre-treated with JA in Khanyari at 3, 6 and 9?days after treatment (10.52%, 8.52%, and 5.30%, respectively). Thus, JA can play an important role in plant defense in kale against P. brassicae.     

Partial reactions of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase

After removal of tightly bound NAD(+) by using charcoal, a preparation of d-glucose 6-phosphate-1 l-myoinositol 1-phosphate cyclase catalysed the reduction of 5-keto-d-glucitol 6-phosphate and 5-keto-d-glucose 6-phosphate by [4-(3)H]NADH to give [5-(3)H]-glucitol 6-phosphate and [5-(3)H]glucose 6-phosphate respectively. The position of the tritium atom in the latter was shown by degradation. Both enzyme-catalysed reductions were strongly inhibited by 2-deoxy-d-glucose 6-phosphate, a powerful competitive inhibitor of inositol cyclase. The charcoal-treated enzyme preparation also converted 5-keto-d-glucose 6-phosphate into [(3)H]myoinositol 1-phosphate in the presence of [4-(3)H]NADH, but less effectively. These partial reactions of inositol cyclase are interpreted as providing strong evidence for the formation of 5-keto-d-glucose 6-phosphate as an enzyme-bound intermediate in the conversion of d-glucose 6-phosphate into 1 l-myoinositol 1-phosphate. The enzyme was partially inactivated by NaBH(4) in the presence of NAD(+). Glucose 6-phosphate did not increase the inactivation, and there was no inactivation in the absence of NAD(+). There was no evidence for Schiff base formation during the cyclization. d-Glucitol 6-phosphate (l-sorbitol 1-phosphate) was a good inhibitor of the overall reaction. It did not inactivate the enzyme. The apparent molecular weight of inositol cyclase as determined by Sephadex chromatography was 2.15x10(5).