Advanced Biotechnological Interventions in Mitigating Drought Stress in Plants.

This comprehensive article critically analyzes the advanced biotechnological strategies to mitigate plant drought stress. It encompasses an in-depth exploration of the latest developments in plant genomics, proteomics, and metabolomics, shedding light on the complex molecular mechanisms that plants employ to combat drought stress. The study also emphasizes the significant advancements in genetic engineering techniques, particularly CRISPR-Cas9 genome editing, which have revolutionized the creation of drought-resistant crop varieties. Furthermore, the article explores microbial biotechnology's pivotal role, such as plant growth-promoting rhizobacteria (PGPR) and mycorrhizae, in enhancing plant resilience against drought conditions. The integration of these cutting-edge biotechnological interventions with traditional breeding methods is presented as a holistic approach for fortifying crops against drought stress. This integration addresses immediate agricultural needs and contributes significantly to sustainable agriculture, ensuring food security in the face of escalating climate change challenges.

Extracellular polymeric substances in electroactive biofilms play a crucial role in improving the efficiency of microbial fuel and electrolysis cells.

In microbial electrochemical cells (MECs), electroactive microbial biofilms can transmit electrons from organic molecules to anodes. To further understand the production of anodic biofilms, it is essential to investigate the composition and distribution of extracellular polymeric substance (EPS) in the MECs. In this study, the structure of EPS was examined in microbial electrolysis cells from mixed cultures forming biofilm using carbon fiber fabric anode. EPS was extracted from the anode biofilm of microbial electrolysis cells inoculated with mixed microbial culture. The anode biofilm yielded 0.4 mg of EPS, of which 51.2% was humic substance, 16.2% was protein, 12.6% was carbohydrates, and 20% consisted of undetermined substances. Using epifluorescence microscopy, the composition of bacterial cells and their location inside EPS were studied, and the distribution of microbial communities was compared based on current density results in the presence of various carbohydrates. On the electrode surface, bacteria and EPS gathered or overlapped in various locations can affect microbial electrochemical performance. Our findings showed that EPS formation in electroactive biofilms would be important for enhanced efficiency of electricity- or hydrogen-producing microbial electrolysis cells.

Metagenomic analysis revealed the potential of lactic acid bacteria in improving natural saline-alkali land / El análisis metagenómico reveló el potencial de las bacterias del ácido láctico para mejorar las tierras salino-álcalis naturales

Management and improving saline-alkali land is necessary for sustainable agricultural development. We conducted a field experiment to investigate the effects of spraying lactic acid bacteria (LAB) on the cucumber and tomato plant soils. Three treatments were designed, including spraying of water, viable or sterilized LAB preparations to the soils of cucumber and tomato plants every 20 days. Spraying sterilized or viable LAB could reduce the soil pH, with a more obvious effect by using viable LAB, particularly after multiple applications. Metagenomic sequencing revealed that the soil microbiota in LAB-treated groups had higher alpha-diversity and more nitrogen-fixing bacteria compared with the water-treated groups. Both viable and sterilized LAB, but not water application, increased the complexity of the soil microbiota interactive network. The LAB-treated subgroups were enriched in some KEGG pathways compared with water or sterilized LAB subgroups, such as environmental information processing–related pathways in cucumber plant; and metabolism-related pathways in tomato plant, respectively. Redundancy analysis revealed association between some soil physico-chemical parameters (namely soil pH and total nitrogen) and bacterial biomarkers (namely Rhodocyclaceae, Pseudomonadaceae, Gemmatimonadaceae, and Nitrosomonadales). Our study demonstrated that LAB is a suitable strategy for decreasing soil pH and improving the microbial communities in saline-alkali land.(AU)

A fast method to distinguish between fermentative and respiratory metabolisms in single yeast cells.

Saccharomyces cerevisiae adjusts its metabolism based on nutrient availability, typically transitioning from glucose fermentation to ethanol respiration as glucose becomes limiting. However, our understanding of the regulation of metabolism is largely based on population averages, whereas nutrient transitions may cause heterogeneous responses. Here we introduce iCRAFT, a method that couples the ATP Förster resonance energy transfer (FRET)-based biosensor yAT1.03 with Antimycin A to differentiate fermentative and respiratory metabolisms in individual yeast cells. Upon Antimycin A addition, respiratory cells experienced a sharp decrease of the normalized FRET ratio, while respiro-fermentative cells showed no response. Next, we tracked changes in metabolism during the diauxic shift of a glucose pre-grown culture. Following glucose exhaustion, the entire cell population experienced a progressive rise in cytosolic ATP produced via respiration, suggesting a gradual increase in respiratory capacity. Overall, iCRAFT is a robust tool to distinguish fermentation from respiration, offering a new single-cell opportunity to study yeast metabolism.

Metagenomic analysis revealed the potential of lactic acid bacteria in improving natural saline-alkali land.

Management and improving saline-alkali land is necessary for sustainable agricultural development. We conducted a field experiment to investigate the effects of spraying lactic acid bacteria (LAB) on the cucumber and tomato plant soils. Three treatments were designed, including spraying of water, viable or sterilized LAB preparations to the soils of cucumber and tomato plants every 20 days. Spraying sterilized or viable LAB could reduce the soil pH, with a more obvious effect by using viable LAB, particularly after multiple applications. Metagenomic sequencing revealed that the soil microbiota in LAB-treated groups had higher alpha-diversity and more nitrogen-fixing bacteria compared with the water-treated groups. Both viable and sterilized LAB, but not water application, increased the complexity of the soil microbiota interactive network. The LAB-treated subgroups were enriched in some KEGG pathways compared with water or sterilized LAB subgroups, such as environmental information processing-related pathways in cucumber plant; and metabolism-related pathways in tomato plant, respectively. Redundancy analysis revealed association between some soil physico-chemical parameters (namely soil pH and total nitrogen) and bacterial biomarkers (namely Rhodocyclaceae, Pseudomonadaceae, Gemmatimonadaceae, and Nitrosomonadales). Our study demonstrated that LAB is a suitable strategy for decreasing soil pH and improving the microbial communities in saline-alkali land.

An Engineered Plant Metabolic Pathway Results in High Yields of Hydroxytyrosol Due to a Modified Whole-Cell Biocatalysis in Bioreactor.

Hydroxytyrosol (HT) is a phenolic substance primarily present in olive leaves and olive oil. Numerous studies have shown its advantages for human health, making HT a potentially active natural component with significant added value. Determining strategies for its low-cost manufacturing by metabolic engineering in microbial factories is hence still of interest. The objective of our study was to assess and improve HT production in a one-liter bioreactor utilizing genetically modified Escherichia coli strains that had previously undergone fed-batch testing. Firstly, we compared the induction temperatures in small-scale whole-cell biocatalysis studies and then examined the optimal temperature in a large volume bioreactor. By lowering the induction temperature, we were able to double the yield of HT produced thereby, reaching 82% when utilizing tyrosine or L-DOPA as substrates. Hence, without the need to further modify our original strains, we were able to increase the HT yield.

Streamlined assembly of cloning and genome editing vectors for genus Clostridium.

Reported herein is a new set of vectors designed to streamline molecular cloning and genome editing by exploiting modern cloning methods. The new vectors build on the existing pMTL8000 vectors that have been a staple of Clostridium research for more than a decade. The introduction of two pairs of type IIS restriction sites flanking an insulated multiple cloning site in both a cloning vector and a CRISPR-Cas9 gene editing vector enables plasmid construction in a "one-pot" reaction, avoiding the more laborious steps of conventional cloning. A synthetic lacZα expression cassette introduced between the cloning sites enables visual detection of background colonies. In addition, distinct selection markers on each vector permit selection of the desired clones according to antibiotic resistance. An example of strain development using the new vectors is demonstrated.

Functional small peptides for enhanced protein delivery, solubility, and secretion in microbial biotechnology.

In microbial biotechnology, there is a constant demand for functional peptides to give new functionality to engineered proteins to address problems such as direct delivery of functional proteins into bacterial cells, enhanced protein solubility during the expression of recombinant proteins, and efficient protein secretion from bacteria. To tackle these critical issues, we selected three types of functional small peptides: cell-penetrating peptides (CPPs) enable the delivery of diverse cargoes into bacterial cytoplasm for a variety of purposes, protein-solubilizing peptide tags demonstrate remarkable efficiency in solubilizing recombinant proteins without folding interference, and signal peptides play a key role in enabling the secretion of recombinant proteins from bacterial cells. In this review, we introduced these three functional small peptides that offer effective solutions to address emerging problems in microbial biotechnology. Additionally, we summarized various engineering efforts aimed at enhancing the activity and performance of these peptides, thereby providing valuable insights into their potential for further applications.

Activating a dormant metabolic pathway for high-temperature l-alanine production in Bacillus licheniformis.

l-Alanine is an important amino acid widely used in food, medicine, materials, and other fields. Here, we develop Bacillus licheniformis as an efficient l-alanine microbial cell factory capable of realizing high-temperature fermentation. By enhancing the glycolytic pathway, knocking out the by-product pathways and overexpressing the thermostable alanine dehydrogenase, the engineered B. licheniformis strain BLA3 produced 93.7 g/L optically pure l-alanine at 50°C. Subsequently, d-alanine dependence of an alanine racemase-deficient strain is relieved by adaptive laboratory evolution, implying that a dormant alternative pathway for d-alanine synthesis is activated in the evolved strain. The d-amino acid aminotransferase Dat1 is shown to be a key enzyme in the dormant alternative pathway. Molecular mechanism of the d-alanine dependence is revealed via mutational analysis. This study demonstrates a novel technology for high-temperature l-alanine production and shows that activating dormant metabolic pathway(s) is an effective strategy of metabolic engineering.

What makes Yarrowia lipolytica well suited for industry?

Yarrowia lipolytica possesses natural and engineered traits that make it a good host for the industrial bioproduction of chemicals, fuels, foods, and pharmaceuticals. In recent years, academic and industrial researchers have assessed its potential, developed synthetic biology techniques, improved its features, scaled its processes, and identified its limitations. Both publications and patents related to Y. lipolytica have shown a drastic increase during the past decade. Here, we discuss the characteristics of this yeast that make it suitable for industry and the remaining challenges for its wider use at large scale. We present evidence herein that shows the importance and potential of Y. lipolytica in bioproduction such that it may soon be one of the preferred choices of industry.

Lachnospiraceae are emerging industrial biocatalysts and biotherapeutics.

The Lachnospiraceae is a family of anaerobic bacteria in the class Clostridia with potential to advance the bio-economy and intestinal therapeutics. Some species of Lachnospiraceae metabolize abundant, low-cost feedstocks such as lignocellulose and carbon dioxide into value-added chemicals. Others are among the dominant species of the human colon and animal rumen, where they ferment dietary fiber to promote healthy gut and immune function. Here, we summarize recent studies of the physiology, cultivation, and genetics of Lachnospiraceae, highlighting their wide substrate utilization and metabolic products with industrial applications. We examine studies of these bacteria as Live Biotherapeutic Products (LBPs), focusing on in vivo disease models and clinical studies using them to treat infection, inflammation, metabolic syndrome, and cancer. We discuss key research areas including elucidation of intra-specific diversity and genetic modification of candidate strains that will facilitate the exploitation of Lachnospiraceae in industry and medicine.

Characterization of Sphingobacterium sp. Ab3 Lipase and Its Coexpression with LEA Peptides.

Sphingobacterium sp. is a yellowish Gram-negative bacterium that is usually characterized by high concentrations of sphingophospholipids as lipid components. As microbial enzymes have been in high demand in industrial fields in the past few decades, this study hopes to provide significant information on lipase activities of Sphingobacterium sp., since limited studies have been conducted on the Sphingobacterium sp. lipase. A microbe from one collected Artic soil sample, ARC4, was identified as psychrotolerant Sphingobacterium sp., and it could grow in temperatures ranging from 0°C to 24°C. The expression of Sphingobacterium sp. lipase was successfully performed through an efficient approach of utilizing mutated group 3 late embryogenesis abundant (G3LEA) proteins developed from Polypedilum vanderplanki. Purified enzyme was characterized using a few parameters, such as temperature, pH, metal ion cofactors, organic solvents, and detergents. The expressed enzyme is reported to be cold adapted and has the capability to work efficiently under neutral pH (pH 5.0 to 7.0), cofactors like Na+ ion, and the water-like solvent methanol. Addition of nonionic detergents greatly enhanced the activity of purified enzyme. IMPORTANCE The mechanism of action of LEA proteins has remained unknown to many; in this study we reveal their presence and improved protein expression due to the molecular shielding effect reported by others. This paper should be regarded as a useful example of using such proteins to influence an existing expression system to produce difficult-to-express proteins.

Genome-Scale Metabolic Modeling Combined with Transcriptome Profiling Provides Mechanistic Understanding of Streptococcus thermophilus CH8 Metabolism.

Streptococcus thermophilus is a lactic acid bacterium adapted toward growth in milk and is a vital component of starter cultures for milk fermentation. Here, we combine genome-scale metabolic modeling and transcriptome profiling to obtain novel metabolic insights into this bacterium. Notably, a refined genome-scale metabolic model (GEM) accurately representing S. thermophilus CH8 metabolism was developed. Modeling the utilization of casein as a nitrogen source revealed an imbalance in amino acid supply and demand, resulting in growth limitation due to the scarcity of specific amino acids, in particular sulfur amino acids. Growth experiments in milk corroborated this finding. A subtle interdependency of the redox balance and the secretion levels of the key metabolites lactate, formate, acetoin, and acetaldehyde was furthermore identified with the modeling approach, providing a mechanistic understanding of the factors governing the secretion product profile. As a potential effect of high expression of arginine biosynthesis genes, a moderate secretion of ornithine was observed experimentally, augmenting the proposed hypothesis of ornithine/putrescine exchange as part of the protocooperative interaction between S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in yogurt. This study provides a foundation for future community modeling of food fermentations and rational development of starter strains with improved functionality. IMPORTANCE Streptococcus thermophilus is one the main organisms involved in the fermentation of milk and, increasingly, also in the fermentation of plant-based foods. The construction of a functional high-quality genome-scale metabolic model, in conjunction with in-depth transcriptome profiling with a focus on metabolism, provides a valuable resource for the improved understanding of S. thermophilus physiology. An example is the model-based prediction of the most significant route of synthesis for the characteristic yogurt flavor compound acetaldehyde and identification of metabolic principles governing the synthesis of other flavor compounds. Moreover, the systematic assessment of amino acid supply and demand during growth in milk provides insights into the key challenges related to nitrogen metabolism that is imposed on S. thermophilus and any other organism associated with the milk niche.

A Candidate Gene Cluster for the Bioactive Natural Product Gyrophoric Acid in Lichen-Forming Fungi.

Natural products of lichen-forming fungi are structurally diverse and have a variety of medicinal properties. Despite this, they have limited implementation in industry mostly because the corresponding genes are unknown for most of their natural products. Here, we implement a long-read sequencing and bioinformatic approach to identify the putative biosynthetic gene cluster of the bioactive natural product gyrophoric acid (GA). Using 15 high-quality genomes representing nine GA-producing species of the lichen-forming fungal genus Umbilicaria, we identify the most likely GA cluster and investigate the cluster gene organization and composition across the nine species. Our results show that GA clusters are promiscuous within Umbilicaria, and only three genes are conserved across species, including the polyketide synthase (PKS) gene. In addition, our results suggest that the same cluster codes for different, but structurally similar compounds, namely, GA, umbilicaric-, and hiascic acid, bringing new evidence that lichen metabolite diversity is also generated through regulatory mechanisms at the molecular level. Ours is the first study to identify the most likely GA cluster and, thus, provides essential information to open new avenues for biotechnological approaches to producing and modifying GA and similar lichen-derived compounds. GA PKS is the first tridepside PKS to be identified. IMPORTANCE The implementation of natural products in the pharmaceutical industry relies on the possibility of modifying the natural product (NP) pathway to optimize yields and pharmacological effects. Characterization of genes and pathways underlying natural product biosynthesis is a major bottleneck for exploiting the medicinal properties of the natural products. Genome mining is a promising and relatively cost- and time-effective approach to utilize unexplored NP resources for drug discovery. In this study, we identify the most likely gene cluster for the lichen-forming fungal depside gyrophoric acid in nine Umbilicaria species. This compound shows cytotoxic and antiproliferative properties against several cancer cell lines and is also a broad-spectrum antimicrobial agent. This information paves the way for generating GA analogs with modified properties by selective activation/deactivation of genes.

Media optimization of antimicrobial activity production and beta-glucan content of endophytic fungi Xylaria sp. BCC 1067.

Fungi is a notable asset for drug discovery and production of pharmaceuticals; however, slow growth and poor product yields have hindered industrial utilization. Here, the mycelial biomass of Xylaria sp. BCC 1067 was examined in parallel with the assessment of antimicrobial properties by using media-type selection. To enhance both mycelial content and antifungal activity, the media replacement approach was successfully applied to stimulate fungal growth and successively switched to poorer malt-peptone extract media for metabolite production. This simple optimization reduced fungal cultivation time by 7 days and yielded 4-fold increased mycelial mass (32.59 g/L), with approximately 3-fold increased antifungal activity against the model yeast Saccharomyces cerevisiae strain. A high level of ß-glucan (115.84 mg/g of cell dry weight) and additive antibacterial effect against Propionibacterium acnes were also reported. This simple strategy of culture media optimization allows for investigation of novel and rich source of health-promoting substances for effective microbial utilization.

Feeding probiotics and organic acids to honeybees enhances acinal surface area of their hypopharyngeal glands.

Modern bee keeping demands more scientific and environment compatible methodologies to improve honeybee's health and efficiency. The current study was designed to explore the hidden potential of probiotics (Lactobacillus rhamnosus) and organic acids (lactic acid and acetic acid) on bee's growth especially hypopharyngeal gland (HPG). Large sized HPG has the ability to produce more royal jelly than smaller ones. For the purpose, the experimentation was carried out in 7 different treatment groups in which probiotics and organic acids were provided in different proportions. Significant increase in acinal surface area of bees in all the experimental groups was observed. Control bees which were fed with pollens and sugar syrup only for two weeks depicted the mean ± SE value of 0.011 ± 0.001 for acinal surface area. Similarly, worker bees of the experimental group 3 [pollens +50% (w/v) sucrose in 1.96% acetic acid], group 4 [pollens + L. rhamnosus in 50% (w/v) sucrose in distilled water], group 5 [pollens + L. rhamnosus in 50% (w/v) sucrose in 2.99% lactic acid], group 6 [pollens + L. rhamnosus in 50% (w/v) sucrose in 2.91% acetic acid] and group 7 [pollens + L. rhamnosus in 50% (w/v) sucrose in 1.96% acetic acid] showed mean ± SE values of 0.019 ± 0.001, 0.017 ± 0.001, 0.013 ± 0.001, 0.016 ± 0.001 and 0.014 ± 0.001 mm2, respectively. The use of acidifying agents and probiotics resulted in enhanced growth of HPG of Apis mellifera workers. Our findings of the present study will be helpful to obtain higher royal jelly yields.

Coupling wastewater valorization with sustainable biofuel production: Comparison of lab- and pilot-scale biomass yields of Chlorella sorokiniana grown in wastewater under photoautotrophic and mixotrophic conditions.

Microalgae are the important biofuel precursors and their economic cultivation can be boosted under mixotrophic (MT) conditions while employing different industrial wastewaters containing organic carbon. In the current research, the quantitative analysis of microalgal biomass production under MT and photoautotrophic (PT) cultivation conditions both at lab and pilot scales was studied. For the purpose, a pre-identified microalgal species Chlorella sorokiniana was cultivated mixotrophically and photoautotrophically at lab and pilot scales. Artificially prepared wastewater containing 2% (w/v) sugarcane molasses was used for MT cultivation. However, for PT cultivation, atmospheric CO2 was the only carbon source. After 15 days of aerobic incubation, microalgal biomass was harvested and analyzed for biomass productivity. Cultivation conditions and cultivation scale posed significant and non-significant impact, respectively on biomass productivities. However, biomass productivity was comparatively higher for the biomass raised under MT conditions at lab scale. The recorded values of biomass productivity were 88.75 ± 9.51 and 127.68 ± 7.91 mg L-1 d-1 for the biomass raised at lab scale under PT and MT conditions, respectively. Pilot-scale cultivation depicted biomass productivities as 83.49 ± 7.87 and 124.88 ± 3.76 mg L-1 d-1 under PT and MT conditions, respectively. High biomass production under MT conditions may suggest the elevated production of biofuels from microalgae. Future studies on biomass production while utilizing different industrial wastewaters at pilot scale and in open raceway ponds are needed for viable production of microalgae-based fuels.

Application of solid-state fermentation by microbial biotechnology for bioprocessing of agro-industrial wastes from 1970 to 2020: A review and bibliometric analysis.

This paper reviews the pertinent literature from 1970 to 2020 and presents a bibliometric analysis of research trends in the application of solid-state fermentation in the bioprocessing of agro-industrial wastes. A total 5630 publications of studies on solid-state fermentation that comprised of 5208 articles (92.50%), 340 book chapters (6.04%), 39 preprints (0.69%), 32 proceedings (0.56%), 8 edited books (0.14%) and 3 monographs (0.05%) were retrieved from Dimensions database. A review of the literature indicated that (i) fermentation of solid substrates is variously defined in the literature over the past 50 years, where "solid-state fermentation" is the most dominant research term used, and (ii) key products derived from the valorization of agro-industrial wastes through solid-state fermentation include, among others, enzymes, antioxidants, animal feed, biofuel, organic acids, biosurfactants, etc. Bibliometric analyses with VOSviewer revealed an astronomic increase in publications between 2000 and 2020, and further elucidated the most frequently explored core research topics, the most highly cited publications and authors, and countries/regions with the highest number of citations. The most cited publication between 2010 and 2020 had 382 citations compared to 725 citations for the most cited publication from 1970 to 2020. Ashok Pandey from India was the most published and cited author with 123 publications and 8,613 citations respectively; whereas Bioresource Technology was the most published and cited journal with 233 publications and 12,394 citations. Countries with the most publications and citations are Brazil, France, India, and Mexico. These findings suggest that research in the application of solid-state fermentation for bioprocessing of agro-industrial wastes has gained prominence over the past 50 years. Future perspectives and implications are discussed.

Bacteria isolated from Triticum monococcum ssp. monococcum roots can improve wheat hologenome in agriculture.

BACKGROUND: Triticum monococcum ssp. monococcum is an ancestral wheat species originated from Karacadag Mountain of Turkey more than ten thousand years ago. Because of environmental and anthropogenic effects, food supply and demand are not balanced. Agricultural activities such as breeding, and fertilization are important to sustain the balance. Conventional breeding and fertilization applications usually neglect contribution of plant related hologenomes in agricultural yield. The disruption of plant growth promoting microorganisms results in intensive usage of chemical fertilizers. The harmony between plant and plant-associated microorganisms is important for sustainability. In this study, isolation, biochemical characterization, and impact on plant growth parameters of natural bacteria associated with Triticum monococcum ssp. monococcum hologenome were aimed. METHODS AND RESULTS: The collection of root samples and isolations of the root-associated bacterial species were carried out from local wheat lands. According to interpretation of three identification methods (MALDI-TOF, 16S rDNA, 16S-23S rDNA) eight isolates are Arthrobacter spp. ESU164, Arthrobacter spp. ESU193, Pseudomonas spp. ESU131, Pseudomonas spp. ESU141, Pseudomonas poae strain ESU182, Pseudomonas thivervalensis strain ESU192, Pseudomonas spp. ESU1531, Bacillus subtilis strain ESU181. For each isolate we investigated biochemical properties especially nitrogen fixation, phosphate solubilization, and indole-3-acetic acid production abilities. The results show that all isolates are nitrogen fixers and the best phosphate solubilizer have been reported as Pseudomonas spp. ESU131 with 2.805 ± 0.439. CONCLUSIONS: All isolates are indole-3-acetic acid productors. 2 isolates affected the coleoptile lengths, 7 bacterial isolates showed statistically positive effect on root number, and 5 isolates promote the root lengths and the root fresh weights.