Comparative study of multiple approaches for identifying cultivable microalgae population diversity from freshwater samples.

The vast diversity of microalgae imposes the challenge of identifying them through the most common and economical identification method, morphological identification, or through using the more recent molecular-level identification tools. Here we report an approach combining enrichment and metagenomic molecular techniques to enhance microalgae identification and identify microalgae diversity from environmental water samples. From this perspective, we aimed to identify the most suitable culturing media and molecular approach (using different primer sets and reference databases) for detecting microalgae diversity. Using this approach, we have analyzed three water samples collected from the River Nile on several enrichment media. A total of 37 microalgae were identified morphologically to the genus level. While sequencing the three-primer sets (16S rRNA V1-V3 and V4-V5 and 18S rRNA V4 region) and aligning them to three reference databases (GG, SILVA, and PR2), a total of 87 microalgae were identified to the genus level. The highest eukaryotic microalgae diversity was identified using the 18S rRNA V4 region and alignment to the SILVA database (43 genera). The two 16S rRNA regions sequenced added to the eukaryotic microalgae identification, 26 eukaryotic microalgae. Cyanobacteria were identified through the two sequenced 16S rRNA regions. Alignment to the SILVA database served to identify 14 cyanobacteria to the genera level, followed by Greengenes, 11 cyanobacteria genera. Our multiple-media, primer, and reference database approach revealed a high microalgae diversity that would have been overlooked if a single approach had been used over the other.

Development of an in vitro regeneration system from immature inflorescences and CRISPR/Cas9-mediated gene editing in sudangrass.

BACKGROUND: Sudangrass (Sorghum sudanense) is a major biomass producer for livestock feed and biofuel in many countries. It has a wide range of adaptations for growing on marginal lands under biotic and abiotic stresses. The immature inflorescence is an explant with high embryogenic competence and is frequently used to regenerate different sorghum cultivars. Caffeic acid O-methyl transferase (COMT) is a key enzyme in the lignin biosynthesis pathway, which limits ruminant digestion of forage cell walls and is a crucial barrier in the conversion of plant biomass to bioethanol. Genome editing by CRISPR/Cas9-mediated mutagenesis without a transgenic footprint will accelerate the improvement and facilitate regulatory approval and commercialization of biotech crops. METHODS AND RESULTS: We report the overcome of the recalcitrance in sudangrass transformation and regeneration in order to use genome editing technique. Hence, an efficient regeneration system has been established to induce somatic embryogenesis from the immature inflorescence of two sudangrass cultivars on four MS-based media supplemented with different components. Our results indicate an interaction between genotype and medium composition. The combination of Giza-1 cultivar and M4 medium produces the maximum frequency of embryogenic calli of 80% and subsequent regeneration efficiency of 22.6%. Precise mutagenesis of the COMT gene is executed using the CRISPR/Cas9 system with the potential to reduce lignin content and enhance forage and biomass quality in sudangrass. CONCLUSION: A reliable regeneration and transformation system has been established for sudangrass using immature inflorescence, and the CRISPR/Cas9 system has demonstrated a promising technology for genome editing. The outcomes of this research will pave the road for further improvement of various sorghum genotypes to meet the global demand for food, feed, and biofuels, achieving sustainable development goals (SDGs).

Sustainable production of Origanum syriacum L. using fish effluents improved plant growth, yield, and essential oil composition.

The concept of Integrating Aquaculture with Agriculture (IAA) is considered the right path towards achieving sustainable agriculture in semi and arid areas. With the increase of global water scarcity, the double utilization of water for both fish and crop production is gaining more attention since it ensures maximizing the productivity of every unit of water used. This study investigated the effect of fish effluent irrigation on the herbage growth, essential oil content, and composition of Origanum syriacum L. The experiment followed a randomized complete design of three irrigation treatments with three replicates, i.e., control with 100% chemical fertilizers (CT), full irrigation with fish effluent (FT), and the mixed treatment (MT) with 50% CT and 50% FT. Study findings showed that FT reached 49 branches/plant, gained maximum plant height (58.8cm), and highest fresh and dry herbage yield reaching 17.76 and 6.722 tons ha-1, respectively, in the second cut. Essential oil content reached the maximum in FT at 64.02dm3 ha-1 and 143.5dm3 ha-1, while the lowest in CT at 15.95dm3 ha-1 and 109.33dm3 ha-1 for the first cut and second, respectively. Carvacrol was the main constituent of the excreted essential oil, representing a maximum of 80.87% for FT in the first cut and 74.69% for MT in the second cut. It was closely followed by p-Cymene (10.75% - CT, 6.38% - FT) and γ-Terpinene (5.06% - CT, 8.49% - FT) for the first and second cut respectively. The importance of these major chemical components stems from their use in both the food and pharmaceutical industries.

A novel thermostable and halophilic thioredoxin reductase from the Red Sea Atlantis II hot brine pool.

The highly extreme conditions of the lower convective layer in the Atlantis II (ATII) Deep brine pool of the Red Sea make it an ideal environment for the search for novel enzymes that can function under extreme conditions. In the current study, we isolated a novel sequence of a thioredoxin reductase (TrxR) enzyme from the metagenomic dataset established from the microbial community that resides in the lower convective layer of Atlantis II. The gene was cloned, expressed and characterized for redox activity, halophilicity, and thermal stability. The isolated thioredoxin reductase (ATII-TrxR) was found to belong to the high-molecular-weight class of thioredoxin reductases. A search for conserved domains revealed the presence of an extra domain (Crp) in the enzyme sequence. Characterization studies of ATII-TrxR revealed that the enzyme was halophilic (maintained activity at 4 M NaCl), thermophilic (optimum temperature was 65°C) and thermostable (60% of its activity was retained at 70°C). Additionally, the enzyme utilized NADH in addition to NADPH as an electron donor. In conclusion, a novel thermostable and halophilic thioredoxin reductase has been isolated with a unique sequence that adapts to the harsh conditions of the brine pools making this protein a good candidate for biological research and industrial applications.

Generation of Transgenic Energy Cane Plants with Integration of Minimal Transgene Expression Cassette.

Lignocellulosic biomass has the potential to serve as feedstock and direct replacement for petrochemicals in the fuel, chemical, pharmaceutical and material industries. Energy cane has been identified by the U.S. Department of Energy (DOE) as prime lignocellulosic feedstock as it produces record biomass yields and is able to grow on low-value land with reduced inputs. Molecular improvement of energy cane is an essential step toward the development of a high-value crop and may contribute to improved biomass conversion to value added products. Such improvements require a development of an efficient regeneration and transformation system for the vegetatively propagated energy cane varieties. In this report, an efficient biolistic gene delivery protocol for energy canes (genotype L 79-1002 and Ho 00-961) has been established with immature leaf rolls as explants. Embryonic calli, developed approximately 6 weeks after culture initiation and was used as target for biolistic transfer of a minimum expression cassette of P-ubi::nptII::35S polyA derived from plasmid pJFNPTII. Putative transgenic clones of callus were obtained after selection on callus induction medium supplemented with 30 mg l-1 geneticin. Regeneration was carried out on NB medium, which is modified from MS supplemented with 1.86 mg l-1 naphthaleneacetic acid (NAA) and 0.1mg l-1, 6-benzylaminopurine (BAP) and 20mg l-1 paromomycin. Shoots growing on selection media were transferred to hormone free medium with 20 mg l-1 paromomycin. Putative transgenic lines were first analyzed by PCR. Transgene integration was confirmed by Southern blot analysis. ELISA (Enzyme-Linked Immunosorbent Assay) and Immunochromathography assays confirmed transgene expression.

Generation of transgenic energy cane plants with integration of minimal transgene expression cassette.

Lignocellulosic biomass has the potential to serve as feedstock and direct replacement for petrochemicals in the fuel, chemical, pharmaceutical and material industries. Energy cane has been identified by the U.S. Department of Energy (DOE) as prime lignocellulosic feedstock as it produces record biomass yields and is able to grow on low-value land with reduced inputs. Molecular improvement of energy cane is an essential step toward the development of a high-value crop and may contribute to improved biomass conversion to value added products. Such improvements require a development of an efficient regeneration and transformation system for the vegetatively propagated energy cane varieties. In this report, an efficient biolistic gene delivery protocol for energy canes (genotype L 79-1002 and Ho 00-961) has been established with immature leaf rolls as explants. Embryonic calli, developed approximately 6 weeks after culture initiation and was used as target for biolistic transfer of a minimum expression cassette of P-ubi::nptII::35S polyA derived from plasmid pJFNPTII. Putative transgenic clones of callus were obtained after selection on callus induction medium supplemented with 30 mg l(-1) geneticin. Regeneration was carried out on NB medium, which is modified from MS supplemented with 1.86 mg l(-1) naphthaleneacetic acid (NAA) and 0.1mg l(-1), 6- benzylaminopurine (BAP) and 20mg l(-1) paromomycin. Shoots growing on selection media were transferred to hormone free medium with 20 mg l(-1) paromomycin. Putative transgenic lines were first analyzed by PCR. Transgene integration was confirmed by Southern blot analysis. ELISA (Enzyme-Linked Immunosorbent Assay) and Immunochromathography assays confirmed transgene expression.

RNAi suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass.

Sugarcane is a prime bioethanol feedstock. Currently, sugarcane ethanol is produced through fermentation of the sucrose, which can easily be extracted from stem internodes. Processes for production of biofuels from the abundant lignocellulosic sugarcane residues will boost the ethanol output from sugarcane per land area. However, unlocking the vast amount of chemical energy stored in plant cell walls remains expensive primarily because of the intrinsic recalcitrance of lignocellulosic biomass. We report here the successful reduction in lignification in sugarcane by RNA interference, despite the complex and highly polyploid genome of this interspecific hybrid. Down-regulation of the sugarcane caffeic acid O-methyltransferase (COMT) gene by 67% to 97% reduced the lignin content by 3.9% to 13.7%, respectively. The syringyl/guaiacyl ratio in the lignin was reduced from 1.47 in the wild type to values ranging between 1.27 and 0.79. The yields of directly fermentable glucose from lignocellulosic biomass increased up to 29% without pretreatment. After dilute acid pretreatment, the fermentable glucose yield increased up to 34%. These observations demonstrate that a moderate reduction in lignin (3.9% to 8.4%) can reduce the recalcitrance of sugarcane biomass without compromising plant performance under controlled environmental conditions.

Production of hyperthermostable GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants enables complete hydrolysis of methylglucuronoxylan to fermentable sugars for biofuel production.

Overcoming the recalcitrance in lignocellulosic biomass for efficient hydrolysis of the polysaccharides cellulose and hemicellulose to fermentable sugars is a research priority for the transition from a fossilfuel-based economy to a renewable carbohydrate economy. Methylglucuronoxylans (MeGXn) are the major components of hemicellulose in woody biofuel crops. Here, we describe efficient production of the GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants and demonstrate exceptional stability and catalytic activities of the in planta produced enzyme. Fully expanded leaves from homotransplastomic plants contained enzymatically active Xyl10B at a level of 11-15% of their total soluble protein. Transplastomic plants and their seed progeny were morphologically indistinguishable from non-transgenic plants. Catalytic activity of in planta produced Xyl10B was detected with poplar, sweetgum and birchwood xylan substrates following incubation between 40 and 90 °C and was also stable in dry and stored leaves. Optimal yields of Xyl10B were obtained from dry leaves if crude protein extraction was performed at 85 °C. The transplastomic plant derived Xyl10B showed exceptional catalytic activity and enabled the complete hydrolysis of MeGXn to fermentable sugars with the help of a single accessory enzyme (α-glucuronidase) as revealed by the sugar release assay. Even without this accessory enzyme, the majority of MeGXn was hydrolyzed by the transplastomic plant-derived Xyl10B to fermentable xylose and xylobiose.

Expression of bacterial L-aspartate-alpha-decarboxylase in tobacco increases beta-alanine and pantothenate levels and improves thermotolerance.

L- Aspartate-alpha-decarboxylase catalyzes the decarboxylation of L -aspartate to generate Beta-alanine and carbon dioxide. This is an unusual pyruvoyl-dependent enzyme unique to prokaryotes that undergoes limited self-processing. The Escherichia coli pan D gene encoding L- aspartate-alpha-decarboxylase was expressed under a constitutive promoter in transgenic tobacco. Transgene expression was verified by assays based on RNA blots, immunoblots and enzyme activity in vitro. The pan D lines had increased levels of leaf Beta-alanine (1.2- to 4-fold), pantothenate (3.2- to 4.1-fold) and total free amino acids (up to 3.7-fold) compared to wild-type and vector controls. Growth of homozygous lines expressing E. coli L- aspartate-alpha-decarboxylase was less affected than that of the control lines when the plants were stressed for 1 week at 35 degrees C. When transferred from 35 to 30 degrees C for 3 weeks, the Pan D transgenic lines recovered significantly (P