Amycolatopsis mediterranei: A Sixty-Year Journey from Strain Isolation to Unlocking Its Potential of Rifamycin Analogue Production by Combinatorial Biosynthesis.

Ever since the isolation of Amycolatopsis mediterranei in 1957, this strain has been the focus of research worldwide. In the last 60 years or more, our understanding of the taxonomy, development of cloning vectors and conjugation system, physiology, genetics, genomics, and biosynthetic pathway of rifamycin B production in A. mediterranei has substantially increased. In particular, the development of cloning vectors, transformation system, characterization of the rifamycin biosynthetic gene cluster, and the regulation of rifamycin B production by the pioneering work of Heinz Floss have made the rifamycin polyketide biosynthetic gene cluster (PKS) an attractive target for extensive genetic manipulations to produce rifamycin B analogues which could be effective against multi-drug-resistant tuberculosis. Additionally, a better understanding of the regulation of rifamycin B production and the application of newer genomics tools, including CRISPR-assisted genome editing systems, might prove useful to overcome the limitations associated with low production of rifamycin analogues.

Beyond rational-biosensor-guided isolation of 100 independently evolved bacterial strain variants and comparative analysis of their genomes.

BACKGROUND: In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum L-histidine producer. Since all known genetic targets contributing to L-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to L-histidine biosynthesis. RESULTS: In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and L-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10-80% more L-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled L-histidine titer (29 mM) and product yield (0.13 C-mol C-mol-1) in comparison to the starting variant. CONCLUSIONS: This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance.

Hot isopropanol quenching procedure for automated microtiter plate scale 13C-labeling experiments.

BACKGROUND: Currently, the generation of genetic diversity for microbial cell factories outpaces the screening of strain variants with omics-based phenotyping methods. Especially isotopic labeling experiments, which constitute techniques aimed at elucidating cellular phenotypes and supporting rational strain design by growing microorganisms on substrates enriched with heavy isotopes, suffer from comparably low throughput and the high cost of labeled substrates. RESULTS: We present a miniaturized, parallelized, and automated approach to 13C-isotopic labeling experiments by establishing and validating a hot isopropanol quenching method on a robotic platform coupled with a microbioreactor cultivation system. This allows for the first time to conduct automated labeling experiments at a microtiter plate scale in up to 48 parallel batches. A further innovation enabled by the automated quenching method is the analysis of free amino acids instead of proteinogenic ones on said microliter scale. Capitalizing on the latter point and as a proof of concept, we present an isotopically instationary labeling experiment in Corynebacterium glutamicum ATCC 13032, generating dynamic labeling data of free amino acids in the process. CONCLUSIONS: Our results show that a robotic liquid handler is sufficiently fast to generate informative isotopically transient labeling data. Furthermore, the amount of biomass obtained from a sub-milliliter cultivation in a microbioreactor is adequate for the detection of labeling patterns of free amino acids. Combining the innovations presented in this study, isotopically stationary and instationary automated labeling experiments can be conducted, thus fulfilling the prerequisites for 13C-metabolic flux analyses in high-throughput.

Chemotactic response of Vibrio coralliilyticus to mucus from various coral species.

Vibrio coralliilyticus, a prominent pathogenic bacteria, is known to cause tissue damage in the coral Pocillopora damicornis and is attracted towards the coral via chemotaxis. However, the potential of V. coralliilyticus to infect most of the other coral hosts via chemotaxis is unknown. In this study, we used capillary assays to quantify the chemotactic response of V. coralliilyticus to the mucus of four tank-cultivated coral species (Cataphyllia jardine, Mussidae sp., Nemenzophyllia turbida, and Euphyllia ancora), and mucus from three wild coral species (Acropora sp., Porites sp., and Montipora sp.). The bacteria showed a positive chemotactic response to each coral mucus tested, with the highest response recorded to the mucus of Acropora sp. and the lowest response to the mucus of Montipora sp. A microfluidic chip was then used to assess the chemotactic preference of V. coralliilyticus to the mucus of the tank cultivated corals. Here too, the bacterium showed positive response, but with a slightly different ranking order. The strong chemotactic response of V. coralliilyticus towards the mucus tested could indicate a broader host range of V. coralliilyticus, and by extension, indicate a threat to weakened coral reefs worldwide.

Interpretation of x-ray absorption spectroscopy in the presence of surface hybridization.

X-ray absorption spectroscopy (XAS) yields direct access to the electronic and geometric structure of hybrid inorganic-organic interfaces formed upon adsorption of complex molecules at metal surfaces. The unambiguous interpretation of corresponding spectra is challenged by the intrinsic geometric flexibility of the adsorbates and the chemical interactions with the interface. Density-functional theory (DFT) calculations of the extended adsorbate-substrate system are an established tool to guide peak assignment in X-ray photoelectron spectroscopy of complex interfaces. We extend this to the simulation and interpretation of XAS data in the context of functional organic molecules on metal surfaces using dispersion-corrected DFT calculations within the transition potential approach. For the prototypical case of 2H-porphine adsorbed on Ag(111) and Cu(111) substrates, we follow the two main effects of the molecule/surface interaction onto the X-ray absorption signatures: (1) the substrate-induced chemical shift of the 1s core levels that dominates in physisorbed systems and (2) the hybridization-induced broadening and loss of distinct resonances that dominate in more chemisorbed systems.

Interplay between Steps and Oxygen Vacancies on Curved TiO2(110).

A vicinal rutile TiO2(110) crystal with a smooth variation of atomic steps parallel to the [1-10] direction was analyzed locally with STM and ARPES. The step edge morphology changes across the samples, from [1-11] zigzag faceting to straight [1-10] steps. A step-bunching phase is attributed to an optimal (110) terrace width, where all bridge-bonded O atom vacancies (Obr vacs) vanish. The [1-10] steps terminate with a pair of 2-fold coordinated O atoms, which give rise to bright, triangular protrusions (St) in STM. The intensity of the Ti 3d-derived gap state correlates with the sum of Obr vacs plus St protrusions at steps, suggesting that both Obr vacs and steps contribute a similar effective charge to sample doping. The binding energy of the gap state shifts when going from the flat (110) surface toward densely stepped planes, pointing to differences in the Ti(3+) polaron near steps and at terraces.

Interfacial charge rearrangement and intermolecular interactions: Density-functional theory study of free-base porphine adsorbed on Ag(111) and Cu(111).

We employ dispersion-corrected density-functional theory to study the adsorption of tetrapyrrole 2H-porphine (2H-P) at Cu(111) and Ag(111). Various contributions to adsorbate-substrate and adsorbate-adsorbate interactions are systematically extracted to analyze the self-assembly behavior of this basic building block to porphyrin-based metal-organic nanostructures. This analysis reveals a surprising importance of substrate-mediated van der Waals interactions between 2H-P molecules, in contrast to negligible direct dispersive interactions. The resulting net repulsive interactions rationalize the experimentally observed tendency for single molecule adsorption.

Improving 5-(hydroxymethyl)furfural (HMF) tolerance of Pseudomonas taiwanensis VLB120 by automated adaptive laboratory evolution (ALE).

The aldehyde 5-(hydroxymethyl)furfural (HMF) is of great importance for a circular bioeconomy. It is a renewable platform chemical that can be converted into a range of useful compounds to replace petroleum-based products such as the green plastic monomer 2,5-furandicarboxylic acid (FDCA). However, it also exhibits microbial toxicity for example hindering the efficient biotechnological valorization of lignocellulosic hydrolysates. Thus, there is an urgent need for tolerance-improved organisms applicable to whole-cell biocatalysis. Here, we engineer an oxidation-deficient derivative of the naturally robust and emerging biotechnological workhorse P. taiwanensis VLB120 by robotics-assisted adaptive laboratory evolution (ALE). The deletion of HMF-oxidizing enzymes enabled for the first time evolution under constant selection pressure by the aldehyde, yielding strains with consistently improved growth characteristics in presence of the toxicant. Genome sequencing of evolved clones revealed loss-of function mutations in the LysR-type transcriptional regulator-encoding mexT preventing expression of the associated efflux pump mexEF-oprN. This knowledge allowed reverse engineering of strains with enhanced aldehyde tolerance, even in a background of active or overexpressed HMF oxidation machinery, demonstrating a synergistic effect of two distinct tolerance mechanisms.

Genome reduction in Paenibacillus polymyxa DSM 365 for chassis development.

The demand for highly robust and metabolically versatile microbes is of utmost importance for replacing fossil-based processes with biotechnological ones. Such an example is the implementation of Paenibacillus polymyxa DSM 365 as a novel platform organism for the production of value-added products such as 2,3-butanediol or exopolysaccharides. For this, a complete genome sequence is the first requirement towards further developing this host towards a microbial chassis. A genome sequencing project has just been reported for P. polymyxa DSM 365 showing a size of 5,788,318 bp with a total of 47 contigs. Herein, we report the first complete genome sequence of P. polymyxa DSM 365, which consists of 5,889,536 bp with 45 RNAs, 106 tRNAs, 5,370 coding sequences and an average GC content of 45.6%, resulting in a closed genome of P. polymyxa 365. The additional nucleotide data revealed a novel NRPS synthetase that may contribute to the production of tridecaptin. Building on these findings, we initiated the top-down construction of a chassis variant of P. polymyxa. In the first stage, single knock-out mutants of non-essential genomic regions were created and evaluated for their biological fitness. As a result, two out of 18 variants showed impaired growth. The remaining deletion mutants were combined in two genome-reduced P. polymyxa variants which either lack the production of endogenous biosynthetic gene clusters (GR1) or non-essential genomic regions including the insertion sequence ISPap1 (GR2), with a decrease of the native genome of 3.0% and 0.6%, respectively. Both variants, GR1 and GR2, showed identical growth characteristics to the wild-type. Endpoint titers of 2,3-butanediol and EPS production were also unaffected, validating these genome-reduced strains as suitable for further genetic engineering.

Corrigendum: Genome reduction in Paenibacillus polymyxa DSM 365 for chassis development.

[This corrects the article DOI: 10.3389/fbioe.2024.1378873.].

Metabolic engineering of Corynebacterium glutamicum for the production of anthranilate from glucose and xylose.

Anthranilate and its derivatives are important basic chemicals for the synthesis of polyurethanes as well as various dyes and food additives. Today, anthranilate is mainly chemically produced from petroleum-derived xylene, but this shikimate pathway intermediate could be also obtained biotechnologically. In this study, Corynebacterium glutamicum was engineered for the microbial production of anthranilate from a carbon source mixture of glucose and xylose. First, a feedback-resistant 3-deoxy-arabinoheptulosonate-7-phosphate synthase from Escherichia coli, catalysing the first step of the shikimate pathway, was functionally introduced into C. glutamicum to enable anthranilate production. Modulation of the translation efficiency of the genes for the shikimate kinase (aroK) and the anthranilate phosphoribosyltransferase (trpD) improved product formation. Deletion of two genes, one for a putative phosphatase (nagD) and one for a quinate/shikimate dehydrogenase (qsuD), abolished by-product formation of glycerol and quinate. However, the introduction of an engineered anthranilate synthase (TrpEG) unresponsive to feedback inhibition by tryptophan had the most pronounced effect on anthranilate production. Component I of this enzyme (TrpE) was engineered using a biosensor-based in vivo screening strategy for identifying variants with increased feedback resistance in a semi-rational library of TrpE muteins. The final strain accumulated up to 5.9 g/L (43 mM) anthranilate in a defined CGXII medium from a mixture of glucose and xylose in bioreactor cultivations. We believe that the constructed C. glutamicum variants are not only limited to anthranilate production but could also be suitable for the synthesis of other biotechnologically interesting shikimate pathway intermediates or any other aromatic compound derived thereof.

Sodium-Controlled Interfacial Resistive Switching in Thin Film Niobium Oxide for Neuromorphic Applications.

A double layer 2-terminal device is employed to show Na-controlled interfacial resistive switching and neuromorphic behavior. The bilayer is based on interfacing biocompatible NaNbO3 and Nb2O5, which allows the reversible uptake of Na+ in the Nb2O5 layer. We demonstrate voltage-controlled interfacial barrier tuning via Na+ transfer, enabling conductivity modulation and spike-amplitude- and spike-timing-dependent plasticity. The neuromorphic behavior controlled by Na+ ion dynamics in biocompatible materials shows potential for future low-power sensing electronics and smart wearables with local processing.

Hippocampal subfield plasticity is associated with improved spatial memory.

Physical exercise studies are generally underrepresented in young adulthood. Seventeen subjects were randomized into an intervention group (24.2 ± 3.9 years; 3 trainings/week) and 10 subjects into a passive control group (23.7 ± 4.2 years), over a duration of 6 months. Every two months, performance diagnostics, computerized spatial memory tests, and 3 Tesla magnetic resonance imaging were conducted. Here we find that the intervention group, compared to controls, showed increased cardiorespiratory fitness, spatial memory performance and subregional hippocampal volumes over time. Time-by-condition interactions occurred in right cornu ammonis 4 body and (trend only) dentate gyrus, left hippocampal tail and left subiculum. Increases in spatial memory performance correlated with hippocampal body volume changes and, subregionally, with left subicular volume changes. In conclusion, findings support earlier reports of exercise-induced subregional hippocampal volume changes. Such exercise-related plasticity may not only be of interest for young adults with clinical disorders of hippocampal function, but also for sedentary normal cohorts.

Creation of an international laboratory network towards global microplastics monitoring harmonisation.

Infrastructure is often a limiting factor in microplastics research impacting the production of scientific outputs and monitoring data. International projects are therefore required to promote collaboration and development of national and regional scientific hubs. The Commonwealth Litter Programme and the Ocean Country Partnership Programme were developed to support Global South countries to take actions on plastics entering the oceans. An international laboratory network was developed to provide the infrastructure and in country capacity to conduct the collection and processing of microplastics in environmental samples. The laboratory network was also extended to include a network developed by the University of East Anglia, UK. All the laboratories were provided with similar equipment for the collection, processing and analysis of microplastics in environmental samples. Harmonised protocols and training were also provided in country during laboratory setup to ensure comparability of quality-controlled outputs between laboratories. Such large networks are needed to produce comparable baseline and monitoring assessments.

Investigating quorum-quenching marine bacilli as potential biocontrol agents for protection of shrimps against Early Mortality Syndrome (EMS).

Early Mortality Syndrome (EMS) has been a major problem for shrimp aquaculture in Southeast Asia due to its epizootic prevalence within the region since the first reported case in 2009. This study explores the application of halophilic marine bacilli isolated from coral mucus and their quorum-quenching abilities as potential biocontrol agents in aquaculture systems to combat the causative agent of EMS, Vibrio parahaemolyticus. N-acylhomoserine lactone (AHL)-degrading (AiiA) activity was first screened by PCR then confirmed by bio-reporter assay, and a combination of 16S rDNA sequence analysis and quantitative phenotype assays including biofilm-formation and temperature-growth responses were used to demonstrate diversity amongst these quorum-quenching isolates. Three phenotypically distinct strains showing notable potential were chosen to undergo co-cultivation as a method for strain improvement via long term exposure to the pathogenic V. parahaemolyticus. The novel approach taken led to significant improvements in antagonism and quorum quenching activities as compared to the ancestral wild-type strains and offers a potential solution as well as pathway to improve existing beneficial microbes for one of the most pressing issues in shrimp aquacultures worldwide.

Acupuncture in management of acute dental pain - A systematic review and meta-analysis.

Acute dental pain is a common issue leading to dental consultation. Besides causal therapy, patients are treated with acupuncture, but efficacy in acute dental pain is still not clarified. We aimed to evaluate results of recent research to estimate the efficacy of acupuncture compared to pain-relieving approaches in treatment of acute dental pain. A systematic review of controlled trials being published between database inception and 2020 were conducted to evaluate the efficacy of acupuncture (alone or as complementary therapy) compared to local anesthesia or conventional analgesic medications in acute dental pain (intraoperatively and postoperatively) and to clarify whether acupuncture reduces the use of postoperative analgesic medications. Of 1672 publications, 23 publications met the inclusion criteria. From these, 11 randomized controlled trials (n = 668) reported on the efficacy of acupuncture on postoperative acute dental pain. Patients, who received acupuncture, showed lower pain scores postoperatively compared to sham acupuncture (Relative Risk -0.77, 95% Confidence interval -1.52 to -0.03). Overall, the results suggest a potential role of acupuncture in improving acute dental pain intraoperatively and postoperatively as well as improving the efficacy of local anesthesia, but the results are limited due to methodological shortcomings emphasizing the necessity for future high-quality research.

Comparison of learning models to predict LDPE, PET, and ABS concentrations in beach sediment based on spectral reflectance.

Microplastic (MP) contamination on land has been estimated to be 32 times higher than in the oceans, and yet there is a distinct lack of research on soil MPs compared to marine MPs. Beaches are bridges between land and ocean and present equally understudied sites of microplastic pollution. Visible-near-infrared (vis-NIR) has been applied successfully for the measurement of reflectance and prediction of low-density polyethylene (LDPE), polyethylene terephthalate (PET), and polyvinyl chloride (PVC) concentrations in soil. The rapidity and precision associated with this method make vis-NIR promising. The present study explores PCA regression and machine learning approaches for developing learning models. First, using a spectroradiometer, the spectral reflectance data was measured from treated beach sediment spiked with virgin microplastic pellets [LDPE, PET, and acrylonitrile butadiene styrene (ABS)]. Using the recorded spectral data, predictive models were developed for each microplastic using both the approaches. Both approaches generated models of good accuracy with R2 values greater than 0.7, root mean squared error (RMSE) values less than 3 and mean absolute error (MAE) < 2.2. Therefore, using this study's method, it is possible to rapidly develop accurate predictive models without the need of comprehensive sample preparation, using the low-cost option ASD HandHeld 2 VNIR Spectroradiometer.

Rising dissolved organic carbon concentrations in coastal waters of northwestern Borneo related to tropical peatland conversion.

Southeast Asia's peatlands are considered a globally important source of terrigenous dissolved organic carbon (DOC) to the ocean. Human disturbance has probably increased peatland DOC fluxes, but the lack of monitoring has precluded a robust demonstration of such a regional-scale impact. Here, we use a time series of satellite ocean color data from northwestern Borneo to show that DOC concentrations in coastal waters have increased between 2002 and 2021 by 0.31 µmol liter-1 year-1 (95% confidence interval, 0.18 to 0.44 µmol liter-1 year-1). We show that this was caused by a ≥30% increase in the concentration of terrigenous DOC and coincided with the conversion of 69% of regional peatland area to nonforest land cover, suggesting that peatland conversion has substantially increased DOC fluxes to the sea. This rise in DOC concentration has also increased the underwater light absorption by dissolved organic matter, which may affect marine productivity by altering underwater light availability.

Increased transfer of trace metals and Vibrio sp. from biodegradable microplastics to catfish Clarias gariepinus.

Microplastic ingestion has been documented in various aquatic species. This causes physical damage, and additionally contaminated microplastics transfer attached pollutants and microbial pathogens to ingesting organisms. Continued metal accumulation can lead to toxicity and adverse health effects; attached microbial pathogens can cause dysbiosis - which lowers host immunity and promotes infections. Catfish, Clarias gariepinus, are a major food source in Southeast Asia, a hotspot of plastic pollution. This study aimed to quantify the transfer of the trace metals copper (Cu) and lead (Pb) -at environmentally relevant concentrations-from microplastics (polyamide 12, PA12, and polylactic acid, PLA) to catfish. Fish were reared for three months and exposed to seven different combinations of feed, supplemented with plastics and metals. At monthly intervals, fish gills, intestines, liver, and edible muscles were analysed for Cu and Pb concentrations using ICP-OES, and the intestines content assessed for Vibrio sp.. Our results showed that biodegradable PLA transferred higher amounts of metals to catfish than expected and also led to increased Vibrio counts in the intestines compared to PA12. Trace metal accumulation was significantly different in varying tissues, with highest concentrations observed in the gills, followed by liver, intestines, and lastly edible muscles. The results of this study further support the existing evidence that microplastics act as efficient shuttles to concentrate and transfer metals. They also indicate that their uptake can cause dysbiosis (increased numbers of Vibrio sp.). Most importantly, however, our study highlights that biodegradable polymers, such as PLA, could actually pose a greater environmental threat when ingested compared to the more common polymers such as PA12.

Priorities to inform research on marine plastic pollution in Southeast Asia.

Southeast Asia is considered to have some of the highest levels of marine plastic pollution in the world. It is therefore vitally important to increase our understanding of the impacts and risks of plastic pollution to marine ecosystems and the essential services they provide to support the development of mitigation measures in the region. An interdisciplinary, international network of experts (Australia, Indonesia, Ireland, Malaysia, the Philippines, Singapore, Thailand, the United Kingdom, and Vietnam) set a research agenda for marine plastic pollution in the region, synthesizing current knowledge and highlighting areas for further research in Southeast Asia. Using an inductive method, 21 research questions emerged under five non-predefined key themes, grouping them according to which: (1) characterise marine plastic pollution in Southeast Asia; (2) explore its movement and fate across the region; (3) describe the biological and chemical modifications marine plastic pollution undergoes; (4) detail its environmental, social, and economic impacts; and, finally, (5) target regional policies and possible solutions. Questions relating to these research priority areas highlight the importance of better understanding the fate of marine plastic pollution, its degradation, and the impacts and risks it can generate across communities and different ecosystem services. Knowledge of these aspects will help support actions which currently suffer from transboundary problems, lack of responsibility, and inaction to tackle the issue from its point source in the region. Being profoundly affected by marine plastic pollution, Southeast Asian countries provide an opportunity to test the effectiveness of innovative and socially inclusive changes in marine plastic governance, as well as both high and low-tech solutions, which can offer insights and actionable models to the rest of the world.