Research Articles (Biotechnology)

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Identification of superior cellulase secretion phenotypes in haploids derived from natural Saccharomyces cerevisiae isolates
(Oxford University Press, 2019) Davison, Steffi A.; den Haan, Riaan; van Zyl, Willem Heber
The yeast Saccharomyces cerevisiae is considered an important host for consolidated bioprocessing and the production of high titres of recombinant cellulases is required for efficient hydrolysis of lignocellulosic substrates to fermentable sugars. Since recombinant protein secretion profiles vary highly among different strain backgrounds, careful selection of robust strains with optimal secretion profiles is of crucial importance. Here, we construct and screen sets of haploid derivatives, derived from natural strain isolates YI13, FINI and YI59, for improved general cellulase secretion. This report details a novel approach that combines secretion profiles of strains and phenotypic responses to stresses known to influence the secretion pathway for the development of a phenotypic screen to isolate strains with improved secretory capacities. A clear distinction was observed between the YI13 haploid derivatives and industrial and laboratory counterparts, Ethanol Red and S288c, respectively. By using sub-lethal concentrations of the secretion stressor tunicamycin and cell wall stressor Congo Red, YI13 haploid derivative strains demonstrated tolerance profiles related to their heterologous secretion profiles. Our results demonstrated that a new screening technique combined with a targeted mating approach could produce a pool of novel strains capable of high cellulase secretion.
Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)
(Elsevier, 2015) Maropola, Mapula Kgomotso Annah; Ramond, Jean-Baptiste; Trindade, Marla
Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols.
Sustainable agriculture through the enhancement of microbial biocontrol agents: Current challenges and new perspectives
(MDPI, 2023) Gokul, Arun; Mabaso, Jabulani; Klein, Ashwil
The future of pesticide usage in agriculture is uncertain due to its unsustainability, adverse environmental impacts, and its association in enhanced phytopathogen resistance. Hence, this situation urges the development of new sustainable practices in agriculture. A promising approach involves endophytes, which are non-pathogenic microorganisms inhabiting the interior parts of plants. However, due to the vast diversity and complexity of plant microbiomes, a major gap has formed with regards to endophytic research and its application in phytopathogen biocontrol. The gap has mainly been increasing due to the difficulty of isolating underrepresented endophytes and due to limitation of previous genetic tools availability to further research and understand plant-microbe interaction, endophytic biocontrol capabilities and their biocontrol compounds.
Analysis of the differentially expressed proteins and metabolic pathways of honeybush (cyclopia subternata) in response to water deficit stress
(MDPI, 2023) Mahlare, Mary-Jane S; Husselmann, Lizex; Lewu, Muinat N
Honeybush (Cyclopia spp.) is a rich source of antioxidant properties and phenolic compounds. Water availability plays a crucial role in plant metabolic processes, and it contributes to overall quality. Thus, this study aimed to investigate changes in molecular functions, cellular components, and biological processes of Cyclopia subternata exposed to different water stress conditions, which include well-watered (as Control, T1), semi-water stressed (T2), and water-deprived (T3) potted plants. Samples were also collected from a well-watered commercial farm first cultivated in 2013 (T13) and then cultivated in 2017 (T17) and 2019 (T19). Differentially expressed proteins extracted from C. subternata leaves were identified using LC-MS/MS spectrometry. A total of 11 differentially expressed proteins (DEPs) were identified using Fisher’s exact test (p < 0.00100). Only α-glucan phosphorylase was found to be statistically common between T17 and T19 (p < 0.00100). Notably, α-glucan phosphorylase was upregulated in the older vegetation (T17) and downregulated in T19 by 1.41-fold. This result suggests that α-glucan phosphorylase was needed in T17 to support the metabolic pathway. In T19, five DEPs were upregulated, while the other six were downregulated.
Whole-genome sequencing of African swine fever virus from wild boars in the Kaliningrad region reveals unique and distinguishing genomic mutations
(Frontiers Media, 2023) Mazloum, Ali; van Schalkwyk, Antoinette; Shotin, Andrey
Since the first report of outbreaks of African swine fever (ASF) in Georgia in 2007, the disease has expanded into Europe, Russia, and Asia, spreading rapidly via contact with infected animals including domestic pigs and wild boars. The vast expansion of this Genotype II African swine fever virus (ASFV) across wide-ranging territories and hosts inevitably led to the acquisition of novel mutations. These mutations could be used to track the molecular epidemiology of ASFV, provided that they are unique to strains restricted within a certain area. Whilst whole-genome sequencing remains the gold standard for examining evolutionary changes, sequencing of a single locus with significant variation and resolution power could be used as a rapid and cost-e􀀀ective alternative to characterize multiple isolates from a single or related outbreak.
Increasing extracellular cellulase activity of the recombinant Saccharomyces cerevisiae by engineering cell wall-related proteins for improved consolidated processing of carbon neutral lignocellulosic biomass
(Elsevier, 2022) Li, Jie; Zeng, Yu; Wang, Wei-Bin
Sustainable bioproduction using carbon neutral feedstocks, especially lignocellulosic biomass, has attracted increasing attention due to concern over climate change and carbon reduction. Consolidated bioprocessing (CBP) of lignocellulosic biomass using recombinant yeast of Saccharomyces cerevisiae is a promising strategy for lignocellulosic biorefinery. However, the economic viability is restricted by low enzyme secretion levels. For more efficient CBP, MIG1 spsc01 isolated from the industrial yeast which encodes the glucose repression regulator derivative was overexpressed. Increased extracellular cellobiohydrolase (CBH) activity was observed with unexpectedly decreased cell wall integrity. Further studies revealed that disruption of CWP2, YGP1, and UTH1, which are functionally related to MIG1 spsc01, also enhanced CBH secretion. Subsequently, improved cellulase production was achieved by simultaneous disruption of YGP1 and overexpression of SED5, which remarkably increased extracellular CBH activity of 2.2-fold over the control strain. These results provide a novel strategy to improve the CBP yeast for bioconversion of carbon neutral biomass.
Microbiome enrichment from contaminated marine sediments unveils novel bacterial strains for petroleum hydrocarbon and heavy metal bioremediation
(Elsevier, 2023) Dell’Anno, Filippo; Joaquim van Zyl, Leonardo; Trindade, Marla
Petroleum hydrocarbons and heavy metals are some of the most widespread contaminants affecting marine ecosystems, urgently needing effective and sustainable remediation solutions. Microbial-based bioremediation is gaining increasing interest as an effective, economically and environmentally sustainable strategy. Here, we hypothesized that the heavily polluted coastal area facing the Sarno River mouth, which discharges >3 tons of polycyclic aromatic hydrocarbons (PAHs) and ~15 tons of heavy metals (HMs) into the sea annually, hosts unique microbiomes including marine bacteria useful for PAHs and HMs bioremediation. We thus enriched the microbiome of marine sediments, contextually selecting for HM-resistant bacteria. The enriched mixed bacterial culture was subjected to whole-DNA sequencing, metagenome-assembled-genomes (MAGs) annotation, and further sub-culturing to obtain the major bacterial species as pure strains. We obtained two novel isolates corresponding to the two most abundant MAGs (Alcanivorax xenomutans strain-SRM1 and Halomonas alkaliantarctica strain-SRM2), and tested their ability to degrade PAHs and remove HMs. Both strains exhibited high PAHs degradation (60–100%) and HMs removal (21–100%) yield, and we described in detail >60 genes in their MAGs to unveil the possible genetic basis for such abilities.
Seed germination and seedling establishment in pistacia atlantica desf. and pistacia lentiscus l. under drought
(Russian Journal of Plant Physiology, 2023) Ludidi, N; Lefi, E; Ben, Hamed S; Saidi, E
The rehabilitation of degraded Mediterranean areas requires reintroduction of key-stone woody species. However, seed germination and seedling establishment for native species are poor. Germination behavior of Pistacia atlantica Desf. and Pistacia lentiscus L. in response to temperature (15, 20, 25 and 30°C), salinity (0, 10, 25 and 50 mM NaCl) and scarification (seeds with/without pulp) were analyzed. The response of seedling establishment to water deficit was related to soil moisture at 100 and 50% of the field capacity. Scarification facilitated germination while salinity should not exceed 50 mM NaCl and temperature must be between 20 and 25°C for P. lentiscus and 25°C for P. atlantica. In P. atlantica, seedling establishment was susceptible to water deficit and it showed high-water requirement.
Enhanced anti-bacterial activity of biogenic silver nanoparticles synthesized from terminalia mantaly extracts
(Dove Press, 2019) Majoumouo, Michele Stella; Sibuyi, Nicole Remaliah Samantha; Tincho, Marius Belmondo
The global increase in outbreaks and mortality rates associated with multidrug- resistant (MDR) bacteria is a major health concern and calls for alternative treatments. Natural-derived products have shown potential in combating the most dreadful diseases, and therefore serve as an effective source of bioactive compounds that can be used as antibacterial agents. These compounds are able to reduce metal ions and cap nanoparticles to form biogenic nanoparticles (NPs) with remarkable anti-bacterial activities. This study explores the use of Terminalia mantaly (TM) extracts for the synthesis of biogenic silver NPs (TM-AgNPs) and the evaluation of their antibacterial activity. TM-AgNPs were synthetized by the reduction of AgNO3 with aqueous andmethanolic TM extracts. UV–visible (UV-vis) spectrophotometry, Dynamic Light Scattering (DLS), Transmission Electron Microscopy, and Fourier Transform Infrared (FTIR) analyses were used to characterise the TM-AgNPs. Anti-bacterial activity of the TM extracts and TM-AgNPs was evaluated against eight bacterial strains using the broth microdilution assay.
Unique aliphatic amidase from a psychrotrophic and haloalkaliphilic nesterenkonia isolate
(American Society for Microbiology, 2011) Nel, A. J. M.; Tuffin, I. M.; Sewell, B. T.
Nesterenkonia strain AN1 was isolated from a screening program for nitrile- and amide-hydrolyzing microorganisms in Antarctic desert soil samples. Strain AN1 showed significant 16S rRNA sequence identity to known members of the genus. Like known Nesterenkonia species, strain AN1 was obligately alkaliphilic (optimum environmental pH, 9 to 10) and halotolerant (optimum environmental Na+ content, 0 to 15% [wt/vol]) but was also shown to be an obligate psychrophile with optimum growth at approximately 21°C.
Identification and characterization of a heat-inducible Hsp70 gene from Sorghum bicolor which confers tolerance to thermal stress
(Springer, 2015) Mulaudzi-Masuku, Takalani; Mutepe, Rendani Daphney; Mukhoro, Ofhani Christopher
This study describes the first detailed molecular characterization of the heat shock protein 70 (Hsp70) gene from Sorghum bicolor, MN1618 designated as SbHsp70-1. The full-length cDNA of SbHsp70-1 consists of 2524 bp with a 1950 bp open reading frame, which encodes a protein of 649 amino acids. SbHsp70-1 is a cytoplasmic protein with high homology to other plant Hsp70s, especially grain crops. Recombinant SbHsp70-1 was able to bind and hydrolyse ATP in a dose-dependent manner, suggesting that SbHsp70-1 functions as an ATPase.
Design and synthesis of acyldepsipeptide-1 analogues: antibacterial activity and cytotoxicity screening
(Arabian Journal of Chemistry, 2023) Sibuyi, Nicole R.S; Cobongela, Sinazo Z.Z; Makatini, Maya M
Acyldepsipeptides (ADEPs) are receiving more attention as prospective antimicrobial agents due to their unique mode of action and chemical properties. However, their therapeutic potential is limited by their poor pharmacokinetic properties. Chemical modifications have been successful in improving the biocompatibility and bioavailability of ADEPs. In the current study, ADEP1 was modified by introducing a disulphide linkage, replacement of the octa-2,4,6-trienoic acid (OTEA) with either adamantane (Ada) or palmitic acid (Pal), and lastly, comparing the use of D versus L amino acids. The antibacterial effects of the ADEP1 analogues were investigated in Gram-positive and Gram-negative strains using agar well diffusion and microdilution assays. Cytotoxicity was evaluated in human embryonic kidney (HEK)-293 and colon cancer (Caco-2) cells by the MTS assay. Using solid phase peptide synthesis (SPPS), the percentage yield of the synthetic peptides was increased to > 37% with > 96% purity.
The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is coexpressed with pathogen defense related genes
(Public Library of Science, 2010) Meier, Stuart; Ruzvidzo, Oziniel; Morse, Monique
Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3′,5′-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. We have identified an Arabidopsis receptor type wall associated kinase–like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431–700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently co-expressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors.
A simple, high-throughput modeling approach reveals insights into the mechanism of gametophytic self-incompatibility
(Nature Research, 2016) Ashkani, Jahanshah; Rees, D. J. G.
Specificity in the GSI response results from the S-haplotype-specific molecular interaction of S-locus F-box (SLF/SFB) and SRNase proteins in the self-incompatibility locus (S-locus). The answer to the question of how these two components of the S-locus (SRNase and SLF/SFB) interact has been gathered from several models. Since there is not enough evidence as to which one is the definitive model, none of them can be ruled out. Despite the identification of interacting protein elements, the mechanism by which SLF/SFB and SRNase interact to differently trigger the self-incompatibility among families and subfamilies remain uncertain. The high-throughput modeling approach demonstrates structural visions into the possible existence of a Collaborative Non-Self Recognition model in apple. These findings postulate several prospects for future investigation providing useful information to guide the implementation of breeding strategies.
Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form
(Wiley, 2007) Thuku, Ndoria R.; Weber, Brandon W.; Varsani, Arvind
Nitrilases convert nitriles to the corresponding carboxylic acids and ammonia. The nitrilase from Rhodococcus rhodochrous J1 is known to be inactive as a dimer but to become active on oligomerization. The recombinant enzyme undergoes post-translational cleavage at approximately residue 327, resulting in the formation of active, helical homo-oligomers. Determining the 3D structure of these helices using electron microscopy, followed by fitting the stain envelope with a model based on homology with other members of the nitrilase superfamily, enables the interacting surfaces to be identified. This also suggests that the reason for formation of the helices is related to the removal of steric hindrance arising from the 39 C-terminal amino acids from the wild-type protein. The helical form can be generated by expressing only residues 1–327.
Characterization and expression analysis of heme oxygenase genes from sorghum bicolor
(SAGE Publications, 2019) Mulaudzi-Masuku, Takalani; Ikebudu, Vivian; Muthevhuli, Mpho
Heme oxygenases (HOs) have a major role in phytochrome chromophore biosynthesis, and chromophores in turn have antioxidant properties. Plant heme oxygenases are divided into the HO1 sub-family comprising HO1, HO3, and HO4, and the HO2 sub-family, which consists of 1 member, HO2. This study identified and characterized 4 heme oxygenase members from Sorghum bicolor. Multiple sequence alignments showed that the heme oxygenase signature motif (QAFICHFYNI/V) is conserved across all SbHO proteins and that they share above 90% sequence identity with other cereals. Quantitative real-time polymerase chain reaction revealed that SbHO genes were expressed in leaves, stems, and roots, but most importantly their transcript level was induced by osmotic stress, indicating that they might play a role in stress responses. These findings will strengthen our understanding of the role of heme oxygenases in plant stress responses and may contribute to the development of stress tolerant crops.
Reports of Plant-Derived Nanoparticles for Prostate Cancer Therapy
(Plants, 2023) Elbagory, Abdulrahman M.; Meyer, Mervin; Hull, Rodney; Dlamini, Zodwa
Background: Plants have demonstrated potential in providing various types of phytomedicines with chemopreventive properties that can combat prostate cancer. However, despite their promising in vitro activity, the incorporation of these phytochemicals into the market as anticancer agents has been hindered by their poor bioavailability, mainly due to their inadequate aqueous solubility, chemical instability, and unsatisfactory circulation time. To overcome these drawbacks, it has been suggested that the incorporation of phytochemicals as nanoparticles can offer a solution. The use of plant-based chemicals can also improve the biocompatibility of the formulated nanoparticles by avoiding the use of certain hazardous chemicals in the synthesis, leading to decreased toxicity in vivo. Moreover, in some cases, phytochemicals can act as targeting agents to tumour sites.
Ozone and nitric oxide induce cGMP-dependent and independent transcription of defence genes in tobacco
(Wiley, 2009) Pasqualini, Stefania; Meier, Stuart; Gehring, Chris
Here, we analyse the temporal signatures of ozone (O3)-induced hydrogen peroxide (H2O2) and nitric oxide (NO) and the role of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) in transcriptional changes of genes diagnostic for biotic and abiotic stress responses. Within 90 min O3 induced H2O2 and NO peaks and we demonstrate that NO donors cause rapid H2O2 accumulation in tobacco (Nicotiana tabacum) leaf. Ozone also causes highly significant, late (> 2 h) and sustained cGMP increases, suggesting that the second messenger may not be required in all early (< 2 h) responses to O3, but is essential and sufficient for the induction of some O3-dependent pathways.
Identification of a novel alkaliphilic esterase active at low temperatures by screening a metagenomic library from Antarctic desert soil
(American Society for Microbiology, 2009) Heath, Caroline; Hu, Xiao Ping; Cary, Craig
The cold deserts of the McMurdo Dry Valleys, South Victoria Land, Eastern Antarctica, are widely acknowledged as having the harshest soil environments on Earth (6, 8, 26). Despite the apparent hostility of the environment, we and others have reported both unexpectedly high biomass (9) and phylogenetic diversity (1, 19, 24, 29) in Antarctic soils. The presence of numerous novel taxa suggests that these soils might prove to be valuable sources of genetic material for mining novel industrial enzymes active at low temperatures (9, 23). Esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) catalyze the hydrolysis and synthesis of ester compounds. Their applications in industry cover a broad spectrum, including as detergent additives, in food processing, in environmental bioremediation, and in biomass and plant waste degradation for the production of useful organocompounds (3, 16).
Reports of plant-derived nanoparticles for prostate cancer therapy
(MDPI, 2023) Elbagory, Abdulrahman M; Hull, Rodney; Meyer, Mervin; Dlamini, Zodwa
Plants have demonstrated potential in providing various types of phytomedicines with chemopreventive properties that can combat prostate cancer. However, despite their promising in vitro activity, the incorporation of these phytochemicals into the market as anticancer agents has been hindered by their poor bioavailability, mainly due to their inadequate aqueous solubility, chemical instability, and unsatisfactory circulation time. To overcome these drawbacks, it has been suggested that the incorporation of phytochemicals as nanoparticles can offer a solution. The use of plant-based chemicals can also improve the biocompatibility of the formulated nanoparticles by avoiding the use of certain hazardous chemicals in the synthesis, leading to decreased toxicity in vivo. Moreover, in some cases, phytochemicals can act as targeting agents to tumour sites. This review will focus on and summarize the following points: the different types of nanoparticles that contain individual phytochemicals or plant extracts in their design with the aim of improving the bioavailability of the phytochemicals; the therapeutic evaluation of these nanoparticles against prostate cancer both in vitro and in vivo and the reported mode of action and the different types of anticancer experiments used; how the phytochemicals can also improve the targeting effects of these nanoparticles in some instances; and the potential toxicity of these nanoparticles.

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