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Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi- assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression.

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Pyrethrins are natural insecticides biosynthesised by Asteraceae plants, such as Tanacetum cinerariifolium and have a long history, dating back to ancient times. Pyrethrins are often used as low-persistence and safe insecticides to control household, horticultural, and agricultural insect pests. Despite its long history of use, pyrethrin biosynthesis remains a mystery, presenting a significant opportunity to improve yields and meet the growing demand for organic agriculture. To achieve this, both genetic modification and non-genetic methods, such as chemical activation and priming, are indispensable. Plants use pyrethrins as a defence against herbivores, but pyrethrin biosynthesis pathways are shared with plant hormones and signal molecules. Hence, the insight that pyrethrins may play broader roles than those traditionally expected is invaluable to advance the basic and applied sciences of pyrethrins.

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Although protein aggregation can cause cytotoxicity, such aggregates can also form to mitigate cytotoxicity from misfolded proteins, although the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein-linked protein (3×GFP) induces giant aggregates and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control in Saccharomyces cerevisiae. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70 family proteins (Ssa1 and Ssa2 in yeast), causing the heat-shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause a critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat-shock response while preventing an overload to the degradation system.

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Mannan and outer structural yeast cell wall polysaccharides have recently garnered attention for their health defense and cosmetic applications. In addition, many studies have confirmed that yeast cell wall mannans exhibit various biological activities, such as antioxidant, immune regulation, reducing hyperlipidemia, and gut health promotion. This paper elucidates yeast cell wall mannan structural features, biological activities, underlying molecular mechanisms, and biosynthesis. Moreover, mannan-overproducing strategies through yeast strain engineering are emphasized and discussed. This review will provide a scientific basis for yeast cell wall mannan research and industrial applications.

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The overproduction of proteins of the endoplasmic reticulum (ER) of plant cells in prokaryotic heterologous gene expression system remains a technical challenge. Recent advances in genetically modified insect cell technology and virus engineering methods have paved the way to produce recombinant ER plant proteins, including those harboring posttranslational modifications, and therefore, to yield ER plant proteins that are natively folded and fully functional. The present contribution focuses on the baculovirus-expression system flashBAC, which overcomes certain technical hurdles found in other insect cell-based expression systems such as the generation of a bacmid and the negative selection of recombinant clones.

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Parental overproduction is hypothesized to hedge against uncertainty over food availability and stochastic death of offspring and to improve brood fitness. Understanding the evolution of overproduction requires quantifying its benefits to parents across a wide range of ecological conditions, which has rarely been done. Using a multiple hypotheses approach and 30 years of data, we evaluated the benefits of overproduction in the Blue-footed booby, a seabird that lays up to three eggs asynchronously, resulting in an aggressive brood hierarchy that facilitates the death of last-hatched chicks under low food abundance. Results support the resource-tracking hypothesis, as low prey abundance (estimated from sea surface temperature and chlorophyll-a concentration) led to rapid brood reduction. The insurance hypothesis was supported in broods of three, where last-hatched chicks' survival increased after a sibling's death. Conversely, in broods of two, results suggested that parents abandoned last-hatched chicks following first-hatched chicks' deaths. No direct evidence supported the facilitation hypothesis: the presence of a last-hatched chick during development did not enhance its sibling's fitness in the short or long term. The value of last-hatched offspring to parents, as "extra" or "insurance" varied with indices of food abundance, brood size, and parental age. Ninety percent of overproduction benefits came from enabling parents to capitalize on favorable conditions by fledging additional offspring. Our study provides insight into the forces driving overproduction, explaining the adaptiveness of this apparently wasteful behavior and allowing us to better predict how overproduction's benefits might be modified by ocean warming.

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Overproduction of desired native or nonnative biochemical(s) in (micro)organisms can be achieved through metabolic engineering. Appropriate rewiring of cell metabolism is performed by making rational changes such as insertion, up-/down-regulation and knockout of genes and consequently metabolic reactions. Finding appropriate targets (including proper sets of reactions to be knocked out) for metabolic engineering to design optimal production strains has been the goal of a number of computational algorithms. We developed FastKnock, an efficient next-generation algorithm for identifying all possible knockout strategies (with a predefined maximum number of reaction deletions) for the growth-coupled overproduction of biochemical(s) of interest. We achieve this by developing a special depth-first traversal algorithm that allows us to prune the search space significantly. This leads to a drastic reduction in execution time. We evaluate the performance of the FastKnock algorithm using various Escherichia coli genome-scale metabolic models in different conditions (minimal and rich mediums) for the overproduction of a number of desired metabolites. FastKnock efficiently prunes the search space to less than 0.2% for quadruple- and 0.02% for quintuple-reaction knockouts. Compared to the classic approaches such as OptKnock and the state-of-the-art techniques such as MCSEnumerator methods, FastKnock found many more beneficial and important practical solutions. The availability of all the solutions provides the opportunity to further characterize, rank and select the most appropriate intervention strategy based on any desired evaluation index. Our implementation of the FastKnock method in Python is publicly available at https://github.com/leilahsn/FastKnock .

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The main objective of this study is to investigate the effect of mixtures of seven widely used human antibiotics (ciprofloxacin, clarithromycin, erythromycin, metronidazole, ofloxacin, sulfamethoxazole, and trimethoprim) on the growth, pH, pigment production, and antibiotics removal of three microalgal species (Auxenochlorella protothecoides, Tetradesmus obliquus, and Chlamydomonas acidophila). Batch assays were conducted with media with antibiotic mixtures at 10, 50, and 100 µg L-1 for each antibiotic. The three microalgae species effectively removed the antibiotics without any growth inhibition, even when exposed to the highest antibiotic concentrations. Biosorption was reported as the primary mechanism for ciprofloxacin, clarithromycin, metronidazole, and ofloxacin, with up to 70% removal, especially in A. protothecoides and C. acidophila. A. protothecoides, a species never investigated for antibiotic removal, was the only microalgae exhibiting bioaccumulation and biodegradation of specific antibiotics, including sulfamethoxazole. Furthermore, in media with the highest antibiotic concentration, all three species exhibited increased chlorophyll (up to 37%) and carotenoid (up to 32%) production, accompanied by a pH decrease of 3 units. Generally, in the present study, it has been observed that physiological responses and the removal of antibiotics by microalgae are interlinked and contingent on the antibiotic levels and types.

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Bacterial cytochrome P450 enzymes catalyze various and often intriguing tailoring reactions during the biosynthesis of natural products. In contrast to the majority of membrane-bound P450 enzymes from eukaryotes, bacterial P450 enzymes are soluble proteins and therefore represent excellent candidates for in vitro biochemical investigations. In particular, cyclodipeptide synthase-associated cytochrome P450 enzymes have recently gained attention due to the broad spectrum of reactions they catalyze, i.e. hydroxylation, aromatization, intramolecular C-C bond formation, dimerization, and nucleobase addition. The latter reaction has been described during the biosynthesis of guanitrypmycins, guatrypmethines and guatyromycines in various Streptomyces strains, where the nucleobases guanine and hypoxanthine are coupled to cyclodipeptides via C-C, C-N, and C-O bonds. In this chapter, we provide an overview of cytochrome P450 enzymes involved in the C-C coupling of cyclodipeptides with nucleobases and describe the protocols used for the successful characterization of these enzymes in our laboratory. The procedure includes cloning of the respective genes into expression vectors and subsequent overproduction of the corresponding proteins in E. coli as well as heterologous expression in Streptomyces. We describe the purification and in vitro biochemical characterization of the enzymes and protocols to isolate the produced compounds for structure elucidation.

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Synaptic overproduction and elimination is a regular developmental event in the mammalian brain. In the cerebral cortex, synaptic overproduction is almost exclusively correlated with glutamatergic synapses located on dendritic spines. Therefore, analysis of changes in spine density on different parts of the dendritic tree in identified classes of principal neurons could provide insight into developmental reorganization of specific microcircuits.The activity-dependent stabilization and selective elimination of the initially overproduced synapses is a major mechanism for generating diversity of neural connections beyond their genetic determination. The largest number of overproduced synapses was found in the monkey and human cerebral cortex. The highest (exceeding adult values by two- to threefold) and most protracted overproduction (up to third decade of life) was described for associative layer IIIC pyramidal neurons in the human dorsolateral prefrontal cortex.Therefore, the highest proportion and extraordinarily extended phase of synaptic spine overproduction is a hallmark of neural circuitry in human higher-order associative areas. This indicates that microcircuits processing the most complex human cognitive functions have the highest level of developmental plasticity. This finding is the backbone for understanding the effect of environmental impact on the development of the most complex, human-specific cognitive and emotional capacities, and on the late onset of human-specific neuropsychiatric disorders, such as autism and schizophrenia.

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Systems biology tools offer new prospects for industrial strain selection. For bacteria that are significant for industrial applications, whole-genome sequencing coupled to flux balance analysis (FBA) can help unpack the complex relationships between genome mutations and carbon trafficking. This work investigates the l-tyrosine (l-Tyr) overproducing model system Corynebacterium glutamicum ATCC 21573 with an eye to more rational and precision strain development. Using genome-wide mutational analysis of C. glutamicum, we identified 27,611 single nucleotide polymorphisms and 479 insertion/deletion mutations. Mutations in the carbon uptake machinery have led to phosphotransferase system-independent routes as corroborated with FBA. Mutations within the central carbon metabolism of C. glutamicum impaired the carbon flux, as evidenced by the lower growth rate. The entry to and flow through the tricarboxylic acid cycle was affected by mutations in pyruvate and α-ketoglutarate dehydrogenase complexes, citrate synthase, and isocitrate dehydrogenase. FBA indicated that the estimated flux through the shikimate pathway became larger as the l-Tyr production rate increased. In addition, protocatechuate export was probabilistically impossible, which could have contributed to the l-Tyr accumulation. Interestingly, aroG and cg0975, which have received previous attention for aromatic amino acid overproduction, were not mutated. From the branch point molecule, prephenate, the change in the promoter region of pheA could be an influential contributor. In summary, we suggest that genome sequencing coupled with FBA is well poised to offer rational guidance for industrial strain development, as evidenced by these findings on carbon trafficking in C. glutamicum ATCC 21573.

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Airborne micro- and nanoplastics are widely spread and pose a risk to human health. The third polymer plastic most commonly produced and present in atmospheric fallout is polystyrene (PS). For these reasons and for a more realistic assessment of biological effects, we examined in-home oxidised (ox-, simulating photoaging) nPS/mPS (0.1 and 1 µm), comparing the effects with virgin ones (v-). On human alveolar cells (A549), we quantified the cellular uptake, using FITC-functionalised nPS/mPS, while cytotoxicity, changes in the acidic compartment, ROS production, mitochondrial function, and DNA damage were assessed to study the effects of internalised v- and ox-nPS/mPS. The results showed that the uptake was dose-dependent and very fast (1 h), since, at the lowest dose (1.25 µg/well), it was 20.8% and 21.8% of nPS and mPS, respectively. Compared to v-, significant ROS increases, DNA damage, and mitochondrial impairment were observed after exposure to ox-nPS/mPS. The enhancement of effects due to environmental aging processes highlighted the true potential impact on human health of these airborne pollutants.

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Overproduction of desired native or nonnative biochemical(s) in (micro)organisms can be achieved through metabolic engineering. Appropriate rewiring of cell metabolism is performed making rational changes such as insertion, up-/down-regulation and knockout of genes and consequently metabolic reactions. Finding appropriate targets (including proper sets of reactions to be knocked out) for metabolic engineering to design optimal production strains has been the goal of a number of computational algorithms. We developed FastKnock, an efficient next-generation algorithm for identifying all possible knockout strategies for the growth-coupled overproduction of biochemical(s) of interest. We achieve this by developing a special depth-first traversal algorithm that allows us to prune the search space significantly. This leads to a drastic reduction in execution time. We evaluate the performance of the FastKnock algorithm using three Escherichia coli genome-scale metabolic models in different conditions (minimal and rich mediums) for the overproduction of a number of desired metabolites. FastKnock efficiently prunes the search space to less than 0.2% for quadruple and 0.02% for quintuple-reaction knockouts. Compared to the classic approaches such as OptKnock and the state-of-the-art techniques such as MCSEnumerator methods, FastKnock found many more useful and important practical solutions. The availability of all the solutions provides the opportunity to further characterize and select the most appropriate intervention strategy based on any desired evaluation index. Our implementation of the FastKnock method in Python is publicly available at https://github.com/leilahsn/FastKnock.

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The search for novel bioactive compounds to overcome resistance to current therapeutics has become of utmost importance. Streptomyces spp. are one of the main sources of bioactive compounds currently used in medicine. In this work, five different global transcriptional regulators and five housekeeping genes, known to induce the activation or overproduction of secondary metabolites in Streptomyces coelicolor, were cloned in two separated constructs and expressed in 12 different strains of Streptomyces spp. from the in-house CS collection. These recombinant plasmids were also inserted into streptomycin and rifampicin resistant Streptomyces strains (mutations known to enhance secondary metabolism in Streptomyces). Different media with diverse carbon and nitrogen sources were selected to assess the strains' metabolite production. Cultures were then extracted with different organic solvents and analysed to search for changes in their production profiles. An overproduction of metabolites already known to be produced by the biosynthesis wild-type strains was observed such as germicidin by CS113, collismycins by CS149 and CS014, or colibrimycins by CS147. Additionally, the activation of some compounds such as alteramides in CS090a pSETxkBMRRH and CS065a pSETxkDCABA or inhibition of the biosynthesis of chromomycins in CS065a in pSETxkDCABA when grown in SM10 was demonstrated. Therefore, these genetic constructs are a relatively simple tool to manipulate Streptomyces metabolism and explore their wide secondary metabolites production potential.

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Background: Hyperactivated airway mucosa cells overproduce mucin and cause severe breathing complications. Here, we aimed to identify the effects of saponins derived from Panax ginseng on inflammation and mucin overproduction. Methods: NCI-H292 cells were pre-incubated with 16 saponins derived from P. ginseng, and mucin overproduction was induced by treatment with phorbol 12-myristate 13-acetate (PMA). Mucin protein MUC5AC was quantified by enzyme-linked immunosorbent assay, and mRNA levels were analyzed using quantitative polymerase chain reaction (qPCR). Moreover, we performed a transcriptome analysis of PMA-treated NCI-H292 cells in the absence or presence of Rg5, and differential gene expression was confirmed using qPCR. Phosphorylation levels of signaling molecules, and the abundance of lipid droplets, were measured by western blotting, flow cytometry, and confocal microscopy. Results: Ginsenoside Rg5 effectively reduced MUC5AC secretion and decreased MUC5AC mRNA levels. A systematic functional network analysis revealed that Rg5 upregulated cholesterol and glycerolipid metabolism, resulting in the production of lipid droplets to clear reactive oxygen species (ROS), and modulated the mitogen-activated protein kinase and nuclear factor (NF)-κB signaling pathways to regulate inflammatory responses. Rg5 induced the accumulation of lipid droplets and decreased cellular ROS levels, and N-acetyl-l-cysteine, a ROS inhibitor, reduced MUC5AC secretion via Rg5. Furthermore, Rg5 hampered the phosphorylation of extracellular signal-regulated kinase and p38 proteins, affecting the NF-κB signaling pathway and pro-inflammatory responses. Conclusion: Rg5 alleviated inflammatory responses by reducing mucin secretion and promoting lipid droplet-mediated ROS clearance. Therefore, Rg5 may have potential as a therapeutic agent to alleviate respiratory disorders caused by hyperactivation of mucosa cells.

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Streptomyces lividans is a potent host for the extracellular overproduction of heterologous proteins. To further improve the usability and productivity of S. lividans, a dual gene expression vector of "pTSKr duet" containing two strong constitutive promoters, scmpPc and kasOp*, was constructed. The success in the overproduction of two secretory enzymes simultaneously without interference with each other indicated that the "pTSKr duet" vector can realize the coexpression of two genes simultaneously and independently. Further, using the two-gene coexpression vector, we screened the effects of the overexpression of five factors that possibly promote secretion on the extracellular overproduction of heterologous secretory proteins. Interestingly, the coexpression of a quality control regulator (CssR) promoted the overproduction level to 1.3-fold for a stable heterologous protein of SMTG (transglutaminase from S. mobaraensis), while other four factors limited the overproduction of SMTG at different degrees.

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Microbial production of aromatic compounds is an attractive and sustainable biotechnological approach. With this motivation, here metabolic engineering of Corynebacterium glutamicum for l-tyrosine (l-Tyr) overproduction was attempted by pushing the carbon flux more towards l-Tyr. Translational start codon exchanges of prephenate dehydratase (pheA), anthranilate synthase (trpE), and phenylalanine aminotransferase (pat) genes revealed that reduced expression of pheA was the major contributor to increased l-Tyr titer while codon exchange in trpE was effective to a lower extent. Overexpression of aroE and qsuC, encoding shikimate dehydrogenase and 3-dehydroquinate dehydratase, respectively, and of dapC (cg1253), which is predicted to encode prephenate aminotransferase, were futile to increase l-Tyr titer. Similarly, deletion of the qsuABD gene cluster had also not enhanced titer. As for increasing precursor supply, deletion of ptsG of glucose uptake and overexpression of inositol permease (iolT2) and glucokinase (glcK) were not effective, but with utilization of xylose, enabled by overexpression of xylose isomerase (xylA) and xylulokinase (xylB), titer improved. Highest l-Tyr titer using the construct was 3.1 g/L on glucose and 3.6 g/L on a 1:3 (w/v) mixture of glucose and xylose. This result displays the potential of the constructed strain to produce l-Tyr from lignocellulosic renewable carbon sources.

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Iron-sulfur clusters are ubiquitous cofactors required for fundamental biological processes. Structural and spectroscopic analysis of Fe-S proteins is often limited by low cluster occupancy in recombinantly produced proteins. In this work, we report a systematic comparison of different maturation strategies for three well-established [4Fe-4S] proteins. Aconitase B, HMBPP reductase (IspH), and quinolinate synthase (NadA) were used as model proteins as they have previously been characterized. The protein production strategies include expression of the gene of interest in BL21(DE3) cells, maturation of the apo protein using chemical or semi-enzymatic reconstitution, co-expression with two different plasmids containing the iron-sulfur cluster (isc) or sulfur formation (suf) operon, a cell strain lacking IscR, the transcriptional regulator of the ISC machinery, and an engineered "SufFeScient" derivative of BL21(DE3). Our results show that co-expression of a Fe-S biogenesis pathway influences the protein yield and the cluster content of the proteins. The presence of the Fe-S cluster is contributing to correct folding and structural stability of the proteins. In vivo maturation reduces the formation of Fe-S aggregates, which occur frequently when performing chemical reconstitution. Furthermore, we show that the in vivo strategies can be extended to the radical SAM protein ThnB, which was previously only maturated by chemical reconstitution. Our results shed light on the differences of in vitro and in vivo Fe-S cluster maturation and points out the pitfalls of chemical reconstitution.

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Microbial cell factories (MCFs) and cell-free systems (CFSs) are generally considered as two unrelated approaches for the biosynthesis of biomolecules. In the current study, two systems were combined together for the overproduction of agroclavine (AC), a structurally complex ergot alkaloid. The whole biosynthetic pathway for AC was split into the early pathway and the late pathway at the point of the FAD-linked oxidoreductase EasE, which was reconstituted in an MCF (Aspergillus nidulans) and a four-enzyme CFS, respectively. The final titer of AC of this combined system is 1209 mg/L, which is the highest one that has been reported so far, to the best of our knowledge. The development of such a combined route could potentially avoid the limitations of both MCF and CFS systems, and boost the production of complex ergot alkaloids with polycyclic ring systems.

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Polysaccharides are important natural biomacromolecules. In particular, microbial exopolysaccharides have received much attention. They are produced by a variety of microorganisms, and they are widely used in the food, pharmaceutical, and chemical industries. The Candida glabrata mutant 4-C10, which has the capacity to produce exopolysaccharide, was previously obtained by random mutagenesis. In this study we aimed to further enhance exopolysaccharide production by systemic fermentation optimization. By single factor optimization and orthogonal design optimization in shaking flasks, an optimal fermentation medium composition was obtained. By optimizing agitation speed, aeration rate, and fed-batch fermentation mode, 118.6 g L-1 of exopolysaccharide was obtained by a constant rate feeding fermentation mode, with a glucose yield of 0.62 g g-1 and a productivity of 1.24 g L-1 h-1. Scaling up the established fermentation mode to a 15-L fermenter led to an exopolysaccharide yield of 113.8 g L-1, with a glucose yield of 0.60 g g-1 and a productivity of 1.29 g L-1 h-1.