Characterization of rhizobia for beneficial traits that promote nodulation in legumes under abiotically stressed conditions.

The growing interest in using rhizobia as inoculants in sustainable agricultural systems has prompted the screening of rhizobia species for beneficial traits that enhance nodulation and nitrogen fixation under abiotic stressed conditions. This study reports phenotypic and phylogenetic characterization of rhizobia strains previously isolated from the root nodules of several indigenous and exotic legumes growing in South Africa and other countries. The Rhizobia strains were screened for their ability to tolerate various abiotic stresses (temperature 16, 28, and 36 °C; acidity/alkalinity pH 5, 7, and 9; heavy metals 50, 100, and 150 mM AlCl3.6H2O; and salinity 50, 100, and 150 mM NaCl). Phylogenetic characterization of the isolates was determined using multilocus sequence analysis of the 16S rRNA, recA, acdS, exoR, nodA, and nodC genes. The analysis indicated that the isolates are phylogenetically related to Sinorhizobium, Bradyrhizobium, Rhizobium, Mesorhizobium, and Aminobacter genera and exhibited significant variations in their tolerance to abiotic stresses. Amid the increasing threats of the global stresses, these current results provide baseline information in the selection of rhizobia for use as inoculants under extreme temperatures, acidity/alkalinity, and salinity stress conditions in South Africa.

Biomedicine Innovations and Its Nanohydrogel Classifications.

As one of the most cutting-edge and promising polymer crosslinked network nanoparticle systems. Polymer nano-sized hydrogels (nanogels) have been a hot topic in the biomedical field over the last few decades. Due to their unique characteristics, which include their relatively high drug encapsulation efficiency, ease of preparation, high tunability, low toxicity, high stability in serum and responsive behavior to a range of stimuli to facilitate drug release. Nanogels are thought to be the next generation of drug delivery systems that can completely change the way that drug delivery systems have an impact on patients' lives. Nanogels have demonstrated significant potential in a variety of fields, including chemotherapy, diagnosis, organ targeting, and delivery of bioactive molecules of different dimensions. However, the lack of substantial clinical data from nanogels becomes one of the major barriers to translating the nanogel concept into a practical therapeutic application for many disease conditions. In addition, nanogel safety profiles have been the major concern that hinders it advancement to the clinical trial phase. This review aims to emphasize the unique properties of nanogels as delivery systems for a variety of bioactive molecules over other nano-delivery systems. Also, this review attempts to give insight into the recent progress in nanogels as a carrier in the field of nanomedicine to overcome complex biological barriers. Relevant scientific data and clinical rationale for the development and the potential use of nanogel as a carrier for targeted therapeutic interventions are discussed. Finally, the concluding points of this review highlight the importance of understanding the long-term toxicity profile of nanogel within the biological system to fully understand their biocompatibility.

Characterization and immobilization of Pycnoporus cinnabarinus carboxylic acid reductase, PcCAR2.

Carboxylic acid reductases (CARs) are well-known for their eminent selective one-step synthesis of carboxylic acids to aldehydes. To date, however, few CARs have been identified and characterized, especially from fungal sources. In this study, the CAR from the white rot fungus Pycnoporus cinnabarinus (PcCAR2) was expressed in Escherichia coli. PcCAR2's biochemical properties were explored in vitro after purification, revealing a melting temperature of 53 °C, while the reaction temperature optimum was at 35 °C. In the tested buffers, the enzyme showed a pH optimum of 6.0 and notably, a similar activity up to pH 7.5. PcCAR2 was immobilized to explore its potential as a recyclable biocatalyst. PcCAR2 showed no critical loss of activity after six cycles, with an average conversion to benzaldehyde of more than 85% per cycle. Immobilization yield and efficiency were 82% and 76%, respectively, on Ni-sepharose. Overall, our findings contribute to the characterization of a thermotolerant fungal CAR, and established a more sustainable use of the valuable biocatalyst.

Transcriptome and proteome of the corm, leaf and flower of Hypoxis hemerocallidea (African potato).

The corm of Hypoxis hemerocallidea, commonly known as the African potato, is used in traditional medicine to treat several medical conditions such as urinary infections, benign prostate hyperplasia, inflammatory conditions and testicular tumours. The metabolites contributing to the medicinal properties of H. hemerocallidea have been identified in several studies and, more recently, the active terpenoids of the plant were profiled. However, the biosynthetic pathways and the enzymes involved in the production of the terpene metabolites in H. hemerocallidea have not been characterised at a transcriptomic or proteomic level. In this study, total RNA extracted from the corm, leaf and flower tissues of H. hemerocallidea was sequenced on the Illumina HiSeq 2500 platform. A total of 143,549 transcripts were assembled de novo using Trinity and 107,131 transcripts were functionally annotated using the nr, GO, COG, KEGG and SWISS-PROT databases. Additionally, the proteome of the three tissues were sequenced using LC-MS/MS, revealing aspects of secondary metabolism and serving as data validation for the transcriptome. Functional annotation led to the identification of numerous terpene synthases such as nerolidol synthase, germacrene D synthase, and cycloartenol synthase amongst others. Annotations also revealed a transcript encoding the terpene synthase phytoalexin momilactone A synthase. Differential expression analysis using edgeR identified 946 transcripts differentially expressed between the three tissues and revealed that the leaf upregulates linalool synthase compared to the corm and the flower tissues. The transcriptome as well as the proteome of Hypoxis hemerocallidea presented here provide a foundation for future research.

Bioethanolic yeasts from dung beetles: tapping the potential of extremophilic yeasts for improvement of lignocellulolytic feedstock fermentation.

Bioethanol from abundant and inexpensive agricultural and industrial wastes possesses the potential to reduce greenhouse gas emissions. Bioethanol as renewable fuel addresses elevated production costs, as well as food security concerns. Although technical advancements in simultaneous saccharification and fermentation have reduced the cost of production, one major drawback of this technology is that the pre-treatment process creates environmental stressors inhibitory to fermentative yeasts subsequently reducing bioethanol productivity. Robust fermentative yeasts with extreme stress tolerance remain limited. This review presents the potential of dung beetles from pristine and unexplored environments as an attractive source of extremophilic bioethanolic yeasts. Dung beetles survive on a recalcitrant lignocellulose-rich diet suggesting the presence of symbiotic yeasts with a cellulolytic potential. Dung beetles inhabiting extreme stress environments have the potential to harbour yeasts with the ability to withstand inhibitory environmental stresses typically associated with bioethanol production. The review further discusses established methods used to isolate bioethanolic yeasts, from dung beetles.

Adaptive Gene Content and Allele Distribution Variations in the Wild and Domesticated Populations of Saccharomyces cerevisiae.

Recent studies on population genomics of Saccharomyces cerevisiae have substantially improved our understanding of the genetic diversity and domestication history of the yeast. However, the origin of the domesticated population of S. cerevisiae and the genomic changes responsible for ecological adaption of different populations and lineages remain to be fully revealed. Here we sequenced 64 African strains from various indigenous fermented foods and forests in different African countries and performed a population genomic analysis on them combined with a set of previously sequenced worldwide S. cerevisiae strains representing the maximum genetic diversity of the species documented so far. The result supports the previous observations that the wild and domesticated populations of S. cerevisiae are clearly separated and that the domesticated population diverges into two distinct groups associated with solid- and liquid-state fermentations from a single ancestor. African strains are mostly located in basal lineages of the two domesticated groups, implying a long domestication history of yeast in Africa. We identified genes that mainly or exclusively occur in specific groups or lineages and genes that exhibit evident group or lineage specific allele distribution patterns. Notably, we show that the homing endonuclease VDE is generally absent in the wild but commonly present in the domesticated lineages of S. cerevisiae. The genes with group specific allele distribution patterns are mostly enriched in functionally similar or related fundamental metabolism processes, including the evolutionary conserved TOR signaling pathway.

Synthesis and application of cationised cellulose for removal of Cr(VI) from acid mine-drainage contaminated water.

Background: Acid mine drainage (AMD) leads to contamination of surface and ground water by high levels of toxic metals including chromium. In many cases, these waters are sources of drinking water for communities, and treatment is therefore required before consumption to prevent negative health effects. Methods: Cationised hemp cellulose was prepared by etherification with two quaternary ammonium salts: 3-chloro-2-hydroxypropyl trimethyl ammonium chloride (CHPTAC) and glycidyltrimethylammonium chloride (GTMAC) and examined for (i) the efficiency of Cr(VI) removal under acid mine-drainage (AMD) conditions, and (ii) antibacterial activity. Adsorbents were characterised by electron microscopy, Fourier transform infrared (FTIR), CP-MAS 13C nuclear magnetic resonance (NMR) spectroscopy, elemental composition and surface charge. Results: FTIR and solid state 13C NMR confirmed the introduction of quaternary ammonium moieties on cellulose. 13C NMR also showed that cationisation decreased the degree of crystallisation and lateral dimensions of cellulose fibrils. Nevertheless, 47 %  - 72 % of Cr(VI) ions were removed from solutions at pH 4, by 0.1 g of CHPTAC and GTMAC-cationised cellulose, respectively. Adsorption kinetics followed the pseudo-second order model and isotherms were best described by the Freundlich and Dubinin-Radushkevich models. When GTMAC-modified cellulose was applied to AMD contaminated water (pH 2.7); however, Cr(VI) removal decreased to 22% likely due to competition from Al and Fe ions. Cationised materials displayed considerable antibacterial effects, reducing the viability of Escherichia coli by up to 45 % after just 3 hours of exposure. Conclusions: Together, these results suggest that cationised cellulose can be applied in the treatment of Cr(VI)-contaminated mine water particularly if pre-treatments to reduce Fe and Al concentrations are applied.

Functional characterisation of the transcriptome from leaf tissue of the fluoroacetate-producing plant, Dichapetalum cymosum, in response to mechanical wounding.

Dichapetalum cymosum produces the toxic fluorinated metabolite, fluoroacetate, presumably as a defence mechanism. Given the rarity of fluorinated metabolites in nature, the biosynthetic origin and function of fluoroacetate have been of particular interest. However, the mechanism for fluorination in D. cymosum was never elucidated. More importantly, there is a severe lack in knowledge on a genetic level for fluorometabolite-producing plants, impeding research on the subject. Here, we report on the first transcriptome for D. cymosum and investigate the wound response for insights into fluorometabolite production. Mechanical wounding studies were performed and libraries of the unwounded (control) and wounded (30 and 60 min post wounding) plant were sequenced using the Illumina HiSeq platform. A combined reference assembly generated 77,845 transcripts. Using the SwissProt, TrEMBL, GO, eggNOG, KEGG, Pfam, EC and PlantTFDB databases, a 69% annotation rate was achieved. Differential expression analysis revealed the regulation of 364 genes in response to wounding. The wound responses in D. cymosum included key mechanisms relating to signalling cascades, phytohormone regulation, transcription factors and defence-related secondary metabolites. However, the role of fluoroacetate in inducible wound responses remains unclear. Bacterial fluorinases were searched against the D. cymosum transcriptome but transcripts with homology were not detected suggesting the presence of a potentially different fluorinating enzyme in plants. Nevertheless, the transcriptome produced in this study significantly increases genetic resources available for D. cymosum and will assist with future research into fluorometabolite-producing plants.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): South African resilience and survival strategies.

Containment of the COVID-19 pandemic relies on accurate data regarding symptoms, transmission, prevention, nature of the virus, strains, immunological factors, relevant demographic and behavioural factors, and control strategies. In South Africa, epidemiological infection data revealed 622,551 cases and 231 deaths per million population as of 29 August 2020. This study describes the strategies South Africa is applying in containing the COVID-19 outbreak that could be used to inform appropriate monitoring and surveillance in other settings, and to improve global health preparedness.

Identification and characterisation of a fluorinase from Actinopolyspora mzabensis.

The incorporation of fluorine has been shown to improve the biophysical and bioactive properties of several organic compounds. However, sustainable strategies of fluorination are needed. Fluorinases have the unique ability to catalyse a C-F bond, hence, have vast potential to be applied as biocatalysts in the preparation of fine chemicals. But fluorinases are extremely rare in nature with only five representatives isolated thus far. Moreover, the heterologous expression of fluorinases is challenged by low yields of soluble protein. This study describes the identification of a fluorinase from Actinopolyspora mzabensis. Overexpression of the Am-fluorinase in E. coli BL21 (DE3) resulted in the formation of inclusion bodies (IBs). The enzyme was recovered from IBs, solubilised in 8 M urea, and successfully refolded into a biologically active form. Following hydrophobic interaction chromatography, >80 mg of the active fluorinase was obtained at a purity suitable for biocatalytic applications. An additional gel filtration step gave ≥95% pure Am-fluorinase. Using LC-MS/MS, the optimal pH for activity was found at 7.2 while the optimal temperature was 65 °C. At these conditions, the enzyme exhibited an activity of 0.44 ±â€¯0.03 µM min-1 mg-1. Furthermore, the Am-fluorinase showed exceptional stability at 25 °C. Preliminary results suggest that the newly identified Am-fluorinase is relatively thermostable.

Biodiesel's trash is a biorefineries' treasure: the use of "dirty" glycerol as an industrial fermentation substrate.

"Dirty" glycerol from biodiesel production is having a considerable environmental impact since its disposal is expensive and difficult. The increased biodiesel production in the last two decades has forced glycerol prices down, thereby making it now unprofitable for chemical companies to produce. The problem lies with the impurities of the biodiesel conversion process usually ending up within the crude glycerol fraction. These impurities are often too costly to purify with current processes, particularly for small scale producers. A wide variety of industries, including the paint, tobacco, food and pharmaceutical industries, utilize glycerol as part of their technology or products. However, the crude glycerol from biodiesel production is not of a high enough grade to be used in these industries. Biodiesel-produced crude glycerol is therefore cheap, readily available and presents itself as an attractive carbon source for industrial microbial production systems synthesizing value-added products. This mini-review will look at (a) microbial production processes which use crude glycerol to produce high-value products (product-driven research) and (b) genetic engineering of microbes which is aimed at improving microbial "dirty" glycerol utilization (substrate driven research).

Streptomyces albulus yields ε-poly-L-lysine and other products from salt-contaminated glycerol waste.

Actinomycetes are the most important microorganisms for the industrial production of secondary metabolites with antimicrobial and anticancer properties. However, they have not been implicated in biorefineries. Here, we study the ability of the ε-poly-L-lysine producing Streptomyces albulus BCRC 11814 to utilize biodiesel-derived crude glycerol. S. albulus was cultured in a mineral medium supplemented with up to 10% w/v sodium chloride or potassium chloride, and with crude glycerol as the sole carbohydrate source. Under these conditions, the strain produced 0.1 g ε-poly-L-lysine per 1 g of biomass. RNA sequencing revealed upregulation of the ectoine biosynthetic pathway of S. albulus, which provides proof of halotolerance. S. albulus has several silent secondary metabolite biosynthetic clusters predicted within the genome. Based on the results, we conclude that S. albulus BCRC 11814 is a halotolerant microorganism capable of utilizing biodiesel-derived crude glycerol better than other actinomycetes included in the present study. S. albulus has the potential to be established as microbial platform production host for a range of high-value biological products.

The availability of second generation feedstocks for the treatment of acid mine drainage and to improve South Africa's bio-based economy.

South Africa has a wide range of mining activities making mineral resources important economic commodities. However, the industry is responsible for several environmental impacts; one of which is acid mine drainage (AMD). Despite several years of research, attempts to prevent AMD generation have proven to be difficult. Therefore, treatment of the resulting drainage has been common practice over the years. One of the recommended treatment methods is the use of second generation feedstocks (lignocellulosic biomass). This biomass is also acknowledged to be an important feedstock for bio-refineries as it is abundant, has a high carbon content and is available at minimal cost. It can also potentially be converted to fermentable sugars (e.g. glucose) through different treatment steps, which could further yield other valuable commodities (cellulase, poly-ß-hydroxybutyric acid (PHB) and penicillin V). It is estimated by a generic flowsheet model that 7 tons of grass biomass can produce 1400 kg of glucose which can subsequently produce 205 kg, 438 kg and 270 kg of cellulase, PHB and Penicillin V, respectively. In this paper we investigate the feasibility of grass as feedstock for AMD treatment and the subsequent conversion of this acid pre-treated grass into valuable bio-products.

Enrichment of maize and triticale bran with recombinant Aspergillus tubingensis ferulic acid esterase.

Ferulic acid is a natural antioxidant found in various plants and serves as a precursor for various fine chemicals, including the flavouring agent vanillin. However, expensive extraction methods have limited the commercial application of ferulic acid, in particular for the enrichment of food substrates. A recombinant Aspergillus tubingensis ferulic acid esterase Type A (FAEA) was expressed in Aspergillus niger D15#26 and purified with anion-exchange chromatography (3487 U/mg, Km  = 0.43 mM, Kcat = 0.48/min on methyl ferulate). The 36-kDa AtFAEA protein showed maximum ferulic acid esterase activity at 50 °C and pH 6, suggesting potential application in industrial processes. A crude AtFAEA preparation extracted 26.56 and 8.86 mg/g ferulic acid from maize bran and triticale bran, respectively, and also significantly increased the levels of p-coumaric and caffeic acid from triticale bran. The cost-effective production of AtFAEA could therefore allow for the enrichment of brans generally used as food and fodder, or for the production of fine chemicals (such as ferulic and p-coumaric acid) from plant substrates. The potential for larger-scale production of AtFAEA was demonstrated with the A. niger D15[AtfaeA] strain yielding a higher enzyme activity (185.14 vs. 83.48 U/ml) and volumetric productivity (3.86 vs. 1.74 U/ml/h) in fed-batch than batch fermentation.

Evaluation of Physicochemical Properties of South African Cashew Apple Juice as a Biofuel Feedstock.

Cashew apple juice (CAJ) is one of the feedstocks used for biofuel production and ethanol yield depends on the physical and chemical properties of the extracted juice. As far as can be ascertained, information on physical and chemical properties of South African cashew apple juice is limited in open literature. Therefore, this study provides information on the physical and chemical properties of the South African cashew apple juice. Physicochemical characteristics of the juice, such as specific gravity, pH, sugars, condensed tannins, Vitamin C, minerals, and total protein, were measured from a mixed variety of cashew apples. Analytical results showed the CAJ possesses specific gravity and pH of 1.050 and 4.52, respectively. The highest sugars were glucose (40.56 gL(-1)) and fructose (57.06 gL(-1)). Other chemical compositions of the juice were condensed tannin (55.34 mgL(-1)), Vitamin C (112 mg/100 mL), and total protein (1.78 gL(-1)). The minerals content was as follows: zinc (1.39 ppm), copper (2.18 ppm), magnesium (4.32 ppm), iron (1.32 ppm), sodium (5.44 ppm), and manganese (1.24 ppm). With these findings, South African CAJ is a suitable biomass feedstock for ethanol production.

Tools for metabolic engineering in Streptomyces.

During the last few decades, Streptomycetes have shown to be a very important and adaptable group of bacteria for the production of various beneficial secondary metabolites. These secondary metabolites have been of great interest in academia and the pharmaceutical industries. To date, a vast variety of techniques and tools for metabolic engineering of relevant structural biosynthetic gene clusters have been developed. The main aim of this review is to summarize and discuss the published literature on tools for metabolic engineering of Streptomyces over the last decade. These strategies involve precursor engineering, structural and regulatory gene engineering, and the up or downregulation of genes, as well as genome shuffling and the use of genome scale metabolic models, which can reconstruct bacterial metabolic pathways to predict phenotypic changes and hence rationalize engineering strategies. These tools are continuously being developed to simplify the engineering strategies for this vital group of bacteria.

Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material.

The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.

Bioflocculant production by a consortium of Streptomyces and Cellulomonas species and media optimization via surface response model.

Species of actinobacteria previously isolated from Tyume River in the Eastern Cape Province of South Africa and identified by 16S rDNA sequence as Cellulomonas and Streptomyces species were evaluated as a consortium for the production of bioflocculant. Sucrose, peptone and magnesium chloride were the nutritional sources which supported optimal production of bioflocculant resulting in flocculation activities of 91%, 82% and 78% respectively. Response surface design revealed sucrose, peptone and magnesium chloride as critical media components following Plackett-Burman design, while the central composite design showed optimum concentration of the critical nutritional source as 16.0 g/L (sucrose), 1.5 g/L (peptone) and 1.6g/L (magnesium chloride) yielding optimal flocculation activity of 98.9% and bioflocculant yield of 4.45 g/L. FTIR spectrometry of the bioflocculant indicated the presence of carboxyl, hydroxyl and amino groups, typical for heteropolysaccharide, while SEM imaging revealed an interwoven clump-like structure. The molecular weight distribution of the constituents of the bioflocculants ranged 494.81-18,300.26 Da thus, an indication of heterogeneity in composition. Additionally, the chemical analyses of the purified bioflocculant revealed the presence of polysaccharides and proteins with neutral sugar, amino sugar and uronic acids in the following concentration: 5.7 mg, 9.3mg and 17.8 mg per 100mg. The high flocculation activity of the bioflocculant suggests commercial potential.

HCN production and hydroxynitrile lyase: a natural activity in plants and a renewed biotechnological interest.

Over 3,000 plant species are cyanogenic. Cyanogenesis is defined as the hydroxynitrile lyase catalysed release of a cyanide group in the form of HCN and the corresponding aldehyde or ketone. When a plant is attacked, HCN released is a self defence mechanism. A special characteristic of enzymatic reactions is that all enzymatic reactions are reversible-hydroxynitrile lyases can also be used for the synthesis of enantiomerically pure cyanohydrins which are of great importance in industry. This article presents a comprehensive review of the role of hydroxynitrile lyases, both in nature and industry, and an insightful. Areas covered include: history, discovery and natural sources of the hydroxynitrile lyase. Molecular cloning for mass production of this enzyme, including detailed information about several successful recombinant hydroxynitrile lyases is also included.

Draft Genome Sequence of Streptomyces albulus Strain CCRC 11814, an {varepsilon}-Poly-L-Lysine-Producing Actinomycete.

Here, we report the draft genome sequence of Streptomyces albulus strain CCRC 11814, a soil-dwelling, Gram-positive bacterium. S. albulus produces ε-poly-l-lysine, which has diverse antimicrobial activity. The genome is 9.43 Mb in size, with a G+C content of 72.2%, and contains 9,177 protein-coding sequences.