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Betulinic acid (BA) is a lupinane-type pentacyclic triterpenoid natural product derived from lupeol that has favorable anti-inflammatory and anti-tumor activities. Currently, BA is mainly produced via botanical extraction, which significantly limits its widespread use. In this study, we investigated the de novo synthesis of BA in Saccharomyces cerevisiae, and to facilitate the synthesis and storage of hydrophobic BA, we adopted a dual-engineering strategy involving peroxisomes and lipid droplets to construct the BA biosynthetic pathway. By expressing Betula platyphylla-derived lupeol C-28 oxidase (BPLO) and Arabidopsis-derived ATR1, we succeeded in developing a BA-producing strain and following multiple expression optimizations of the linker between BPLO and ATR1, the BA titer reached 77.53 mg/L in shake flasks and subsequently reached 205.74 mg/L via fed-batch fermentation in a 5-L bioreactor. In this study, we developed a feasible approach for the de novo synthesis of BA and its direct precursor lupeol in engineered S. cerevisiae.

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While inhomogeneous diffusivity has been identified as a ubiquitous feature of the cellular interior, its implications for particle mobility and concentration at different length scales remain largely unexplored. In this work, we use agent-based simulations of diffusion to investigate how heterogeneous diffusivity affects the movement and concentration of diffusing particles. We propose that a nonequilibrium mode of membrane-less compartmentalization arising from the convergence of diffusive trajectories into low-diffusive sinks, which we call 'diffusive lensing,' is relevant for living systems. Our work highlights the phenomenon of diffusive lensing as a potentially key driver of mesoscale dynamics in the cytoplasm, with possible far-reaching implications for biochemical processes.

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Subcellular metal distribution assessments are the most adequate biomonitoring approach to evaluate metal toxicity, instead of total metal assessments This study aimed to assess subcellular metal distributions and associations to the main metal exposure biomarker, metallothionein (MT), in two bromeliad species (Tillandsia usneoides and Tillandsia stricta) exposed established in industrial, urban, and port areas in the metropolitan region of Rio de Janeiro, southeastern Brazil, through an active biomonitoring approach conducted one year. Metals and metalloids in three subcellular fractions (insoluble, thermolabile and thermostable) obtained from the MT purification process were determined by inductively coupled plasma mass spectrometry (ICP-MS). Lower MT concentrations were observed both during the dry sampling periods, associated to the crassulacean acid metabolism (CAM) and during the COVID-19 pandemic, due to reduced urban mobility, decreasing pollutant emissions. The percentage of non-bioavailable metals detected in the insoluble fraction increased throughout the sampling period for both species. Several metals (Cr, Co, Cu, Cd, Mn, Ni, Se, and Zn), most associated with vehicle emissions, the main pollutant source in urban centers, were detected in the thermostable fraction and are, thus, associated with MT through the MT-metal detoxification route. Insoluble metal concentrations were higher in T. stricta, indicating that this species seems less susceptible to cellular metal exposure damage. A potential protective effect of Se and Fe was detected against Pb, suggested by a strong negative correlation, which may be attributed to antioxidant roles and similar uptake routes, respectively.

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Subcellular compartmentalization of metabolic pathways plays a crucial role in metabolic engineering. The peroxisome has emerged as a highly valuable and promising compartment for organelle engineering, particularly in the fields of biological manufacturing and agriculture. In this review, we summarize the remarkable achievements in peroxisome engineering in yeast, the industrially popular biomanufacturing chassis host, to produce various biocompounds. We also review progress in plant peroxisome engineering, a field that has already exhibited high potential in both biomanufacturing and agriculture. Moreover, we outline various experimentally validated strategies to improve the efficiency of engineered pathways in peroxisomes, as well as prospects of peroxisome engineering.

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In nature, the formation of specialized (secondary) metabolites is associated with the late stages of fungal development. Enzymes involved in the biosynthesis of secondary metabolites in fungi are located in distinct subcellular compartments including the cytosol, peroxisomes, endosomes, endoplasmic reticulum, different types of vesicles, the plasma membrane and the cell wall space. The enzymes traffic between these subcellular compartments and the secretion through the plasma membrane are still unclear in the biosynthetic processes of most of these metabolites. Recent reports indicate that some of these enzymes initially located in the cytosol are later modified by posttranslational acylation and these modifications may target them to membrane vesicle systems. Many posttranslational modifications play key roles in the enzymatic function of different proteins in the cell. These modifications are very important in the modulation of regulatory proteins, in targeting of proteins, intracellular traffic and metabolites secretion. Particularly interesting are the protein modifications by palmitoylation, prenylation and miristoylation. Palmitoylation is a thiol group-acylation (S-acylation) of proteins by palmitic acid (C16) that is attached to the SH group of a conserved cysteine in proteins. Palmitoylation serves to target acylated proteins to the cytosolic surface of cell membranes, e.g., to the smooth endoplasmic reticulum, whereas the so-called toxisomes are formed in trichothecene biosynthesis. Palmitoylation of the initial enzymes involved in the biosynthesis of melanin serves to target them to endosomes and later to the conidia, whereas other non-palmitoylated laccases are secreted directly by the conventional secretory pathway to the cell wall space where they perform the last step(s) of melanin biosynthesis. Six other enzymes involved in the biosynthesis of endocrosin, gliotoxin and fumitremorgin believed to be cytosolic are also targeted to vesicles, although it is unclear if they are palmitoylated. Bioinformatic analysis suggests that palmitoylation may be frequent in the modification and targeting of polyketide synthetases and non-ribosomal peptide synthetases. The endosomes may integrate other small vesicles with different cargo proteins, forming multivesicular bodies that finally fuse with the plasma membrane during secretion. Another important effect of palmitoylation is that it regulates calcium metabolism by posttranslational modification of the phosphatase calcineurin. Mutants defective in the Akr1 palmitoyl transferase in several fungi are affected in calcium transport and homeostasis, thus impacting on the biosynthesis of calcium-regulated specialized metabolites. The palmitoylation of secondary metabolites biosynthetic enzymes and their temporal distribution respond to the conidiation signaling mechanism. In summary, this posttranslational modification drives the spatial traffic of the biosynthetic enzymes between the subcellular organelles and the plasma membrane. This article reviews the molecular mechanism of palmitoylation and the known fungal palmitoyl transferases. This novel information opens new ways to improve the biosynthesis of the bioactive metabolites and to increase its secretion in fungi.

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The Integrated Stress Response (ISR) is an essential homeostatic signaling network that controls the cell's biosynthetic capacity. Four ISR sensor kinases detect multiple stressors and relay this information to downstream effectors by phosphorylating a common node: the alpha subunit of the eukaryotic initiation factor eIF2. As a result, general protein synthesis is repressed while select transcripts are preferentially translated, thus remodeling the proteome and transcriptome. Mounting evidence supports a view of the ISR as a dynamic signaling network with multiple modulators and feedback regulatory features that vary across cell and tissue types. Here, we discuss updated views on ISR sensor kinase mechanisms, how the subcellular localization of ISR components impacts signaling, and highlight ISR signaling differences across cells and tissues. Finally, we consider crosstalk between the ISR and other signaling pathways as a determinant of cell health.

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Single-cell C4 (SCC4) plants with bienertioid anatomy carry out photosynthesis in a single cell. Chloroplast movement is the underlying phenomenon, where chloroplast unusual positioning 1 (CHUP1) plays a key role. This study aimed to characterize CHUP1 and CHUP1-like proteins in an SCC4 photosynthetic plant, Bienertia sinuspersici. Also, a comparative analysis of SCC4 CHUP1 was made with C3, C4, and CAM model plants including an extant basal angiosperm, Amborella. The CHUP1 gene exists as a single copy from the basal angiosperms to SCC4 plants. Our analysis identified that Chenopodium quinoa, a recently duplicated allotetraploid, has two copies of CHUP1. In addition, the numbers of CHUP1-like and its associated proteins such as CHUP1-like_a, CHUP1-like_b, HPR, TPR, and ABP varied between the species. Hidden Markov Model analysis showed that the gene size of CHUP1-like_a and CHUP1-like_b of SCC4 species, Bienertia, and Suaeda were enlarged than other plants. Also, we identified that CHUP1-like_a and CHUP1-like_b are absent in Arabidopsis and Amborella, respectively. Motif analysis identified several conserved and variable motifs based on the orders (monocot and dicot) as well as photosynthetic pathways. For instance, CAM plants such as pineapple and cactus shared certain motifs of CHUP1-like_a irrespective of their distant phylogenetic relationship. The free ratio model showed that CHUP1 maintained purifying selection, whereas CHUP1-like_a and CHUP1-like_b have adaptive functions between SCC4 plants and quinoa. Similarly, rice and maize branches displayed functional diversification on CHUP1-like_b. Relative gene expression data showed that during the subcellular compartmentalization process of Bienertia, CHUP1 and actin-binding proteins (ABP) genes showed a similar pattern of expression. Altogether, the results of this study provide insight into the evolutionary and functional details of CHUP1 and its associated proteins in the development of the SCC4 system in comparison with other C3, C4, and CAM model plants.

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Bermudagrass is a perennial herb with the potential to remediate Pb pollution in soils, and it has mechanical resistance to shearing. However, the effects of mowing on Pb absorption and accumulation in bermudagrass are still unclear. In this study, we investigated the effects of different quantities (0, 1, 2, 4 applications) of mowing treatments under 200 mg L-1 Pb application on Pb accumulation and transport in bermudagrass and explored the related mechanisms. Compared to the Pb treatment, all of the mowing treatments greatly decreased root Pb concentration/accumulation, significantly enhanced Pb concentrations/accumulations in stubble stems and stubble leaves, and ultimately promoted Pb enrichment and transport. Of the treatments in this study, two applications of mowing best promoted Pb enrichment, and four applications of mowing best promoted Pb transport efficiency. Furthermore, mowing mediated the microdistribution and physiological patterns of Pb in bermudagrass and affected the Pb transport by changing the subcellar distribution patterns and chemical forms of Pb in various tissues. Additionally, mowing promoted the transport of all mineral elements and showed a synergistic relationship with Pb absorption and transport. The change in mineral element metabolism patterns may be an important reason why mowing promoted Pb accumulation in bermudagrass. Our study provides the first comprehensive evidence regarding mowing facilitating the absorption, accumulation and transport of Pb in bermudagrass. Moderate mowing may be an effective strategy to assist in soil Pb remediation using bermudagrass.

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Fungicides containing manganese (Mn) applied to control plant diseases increase the concentration of Mn in soils, which may potentiate Mn toxicity in acid soils. Some species of wild grasses, such as those from the Pampa biome located in South America, or even those introduced into this biome, may possess different mechanisms of tolerance to excess Mn. The present study aimed to evaluate the subcellular distribution and physiological and biochemical responses of exotic and native grasses from the Pampa biome, cultivated in Mn excess. The experiment was conducted in nutrient solution in a greenhouse, in an entirely randomized design, bifactorial 4 × 4, consisting of four Mn concentrations (2 [control], 300, 600 and 900 µM) and four species (two exotic: Avena strigosa and Lolium multiflorum; and two native: Paspalum notatum and Paspalum plicatulum). At 27 days of exposure to the treatments, biomass and growth rates, leaf gas exchange with the environment, photosynthetic pigment concentrations of malondialdehyde and H2O2, antioxidant enzyme activities (SOD and POD), and subcellular distribution of Mn were evaluated. Most of the grasses showed high concentration of Mn in tissues, mainly, in the shoot. In the presence of 900 µM Mn, more than 80% of the absorbed Mn was compartmentalized in the cell walls and vacuoles of the cells. Compartmentalization of Mn excess into metabolically less active organelles is the main tolerance factor in grasses. Physiological and biochemical responses were stimulated in the presence of 300 µM Mn, while 900 µM Mn negatively affected biochemical-physiological responses of grasses. The species L. multiflorum was most sensitive to excess Mn, while P. notatum was the most tolerant.

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The innate immune response constitutes the cell's first line of defense against viruses and culminates in the expression of type I interferon (IFN) and IFN-stimulated genes, inducing an antiviral state in infected and neighboring cells. Efficient signal transduction is a key factor for strong but controlled type I IFN expression and depends on the compartmentalization of different steps of the signaling cascade and dynamic events between the involved compartments or organelles. This compartmentalization of the innate immune players not only relies on their association with membranous organelles but also includes the formation of supramolecular organizing centers (SMOCs) and effector concentration by liquid-liquid phase separation. For their successful replication, viruses need to evade innate defenses and evolve a multitude of strategies to impair type I IFN induction, one of which is the disruption of spatial immune signaling dynamics. This review focuses on the role of compartmentalization in ensuring an adequate innate immune response to viral pathogens, drawing attention to crucial translocation events occurring downstream of pattern recognition and leading to the expression of type I IFN. Furthermore, it intends to highlight concise examples of viral countermeasures interfering with this spatial organization to alleviate the innate immune response.

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The intracellular calcium content in fungal cells is influenced by a large number of environmental and nutritional factors. Sharp changes in the cytosolic calcium level act as signals that are decoded by the cell gene expression machinery, resulting in several physiological responses, including differentiation and secondary metabolites biosynthesis. Expression of the three penicillin biosynthetic genes is regulated by calcium ions, but there is still little information on the role of this ion in the translocation of penicillin intermediates between different subcellular compartments. Using advanced information on the transport of calcium in organelles in yeast as a model, this article reviews the recent progress on the transport of calcium in vacuoles and peroxisomes and its relation to the translocation of biosynthetic intermediates in filamentous fungi. The Penicillium chrysogenum PenV vacuole transporter and the Acremonium chrysogenum CefP peroxisomal transporter belong to the transient receptor potential (TRP) class CSC of calcium ion channels. The PenV transporter plays an important role in providing precursors for the biosynthesis of the tripeptide δ-(-α-aminoadipyl-L-cysteinyl-D-valine), the first intermediate of penicillin biosynthesis in P. chrysogenum. Similarly, CefP exerts a key function in the conversion of isopenicillin N to penicillin N in peroxisomes of A. chrysogenum. These TRP transporters are different from other TRP ion channels of Giberella zeae that belong to the Yvc1 class of yeast TRPs. Recent advances in filamentous fungi indicate that the cytosolic calcium concentration signal is connected to the calcitonin/calcineurin signal transduction cascade that controls the expression of genes involved in the subcellular translocation of intermediates during fungal metabolite biosynthesis. These advances open new possibilities to enhance the expression of important biosynthetic genes in fungi.

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Lead (Pb) is one of the most harmful, toxic pollutants to the ecological environment and humans. Centipedegrass, a fast-growing warm-season turfgrass, is excellent for Pb pollution remediation. Exogenous low-molecular-weight organic acid (LMWOA) treatment is a promising approach for assisted phytoremediation. However, the effects of this treatment on the tolerance and Pb accumulation of centipedegrass are unclear. This study investigated these effects on the physiological growth response and Pb accumulation distribution characteristics of centipedegrass. Applications of 400 µM citric acid (CA), malic acid (MA) and tartaric acid (TA) significantly reduced membrane lipid peroxidation levels of leaves and improved biomass production of Pb-stressed plants. These treatments mainly increased peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) activities and enhanced free protein (Pro), ascorbic acid (AsA) and phytochelatins (PCs) contents, ultimately improving the Pb tolerance of centipedegrass. Their promoting effects decreased as follows: TA>CA>MA. All the treatments decreased root Pb concentrations and increased stem and leaf Pb concentrations, thus increasing total Pb accumulation and TF values. MA had the best and worst effects on Pb accumulation and Pb transportation, respectively. CA had the best and worst effects on Pb transportation and Pb accumulation, respectively. TA exhibited strong effects on both Pb accumulation and transport. Furthermore, all treatments changed the subcellular Pb distribution patterns and distribution models of the chemical forms of Pb in each tissue. The root Pb concentration was more highly correlated with the Pb subcellular fraction distribution pattern, while the stem and leaf Pb concentrations were more highly correlated with the distribution models of the chemical forms of Pb. Overall, TA improved plant Pb tolerance best and promoted both Pb absorption and transportation well and is considered the best candidate for Pb-contaminated soil remediation with centipedegrass. This study provides a new idea for Pb-contaminated soil remediation with centipedegrass combined with LMWOAs.

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Fungal specialized metabolites play an important role in the environment and have impacted human health and survival significantly. These specialized metabolites are often the end product of a series of sequential and collaborating biosynthetic enzymes that reside within different subcellular compartments. A wide variety of methods have been developed to understand fungal specialized metabolite biosynthesis in terms of the chemical conversions and the biosynthetic enzymes required, however there are far fewer studies elucidating the compartmentalization of the same enzymes. This review illustrates the biosynthesis of specialized metabolites where the localization of all, or some, of the biosynthetic enzymes have been determined and describes the methods used to identify the sub-cellular localization.

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Phosphodiesterase 11A (PDE11A), an enzyme that degrades cyclic nucleotides (cAMP and cGMP), is the only PDE whose mRNA expression in brain is restricted to the hippocampal formation. Previously, we showed that chronic social isolation changes subsequent social behaviors in adult mice by reducing expression of PDE11A4 in the membrane fraction of the ventral hippocampus (VHIPP). Here we seek extend these findings by determining 1) if isolation-induced decreases in PDE11A4 require chronic social isolation or if they occur acutely and are sustained long-term, 2) if isolation-induced decreases occur uniquely in adults (i.e., not adolescents), and 3) how the loss of PDE11 signaling may increase neuroinflammation. Both acute and chronic social isolation decrease PDE11A4 expression in adult but not adolescent mice. This decrease in PDE11A4 is specific to the membrane compartment of the VHIPP, as it occurs neither in the soluble nor nuclear fractions of the VHIPP nor in any compartment of the dorsal HIPP. The effect of social isolation on membrane PDE11A4 is also selective in that PDE2A and PDE10A expression remain unchanged. Isolation-induced decreases in PDE11A4 expression appear to be functional as social isolation elicited changes in PDE11A-relevant signal transduction cascades (i.e., decreased pCamKIIα and pS6-235/236) and behavior (i.e., increased remote long-term memory for social odor recognition). Interestingly, we found that isolation-induced decreases in membrane PDE11A4 correlated with increased expression of interleukin-6 (IL-6) in the soluble fraction, suggesting pro-inflammatory signaling for this cytokine. This effect on IL-6 is consistent with the fact that PDE11A deletion increased microglia activation, although it left astrocytes unchanged. Together, these data suggest that isolation-induced decreases in PDE11A4 may alter subsequent social behavior via increased neuroinflammatory processes in adult mice.

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The presence of specialized cellular compartments in higher plants express an extraordinary degree of intracellular organization, which provides efficient mechanisms to avoid misbalancing of the metabolism. This offers the flexibility by which plants can quickly acclimate to fluctuating environmental conditions. For that, a fine temporal and spatial regulation of metabolic pathways is required and involves several players e.g. organic acids. In this review we discuss different facets of the organic acid metabolism within plant cells with special focus to those related to the interactions between organic acids compartmentalization and the partitioning of carbon and nitrogen. The connections between organic acids and CO2 assimilation, tricarboxylic acid (TCA) cycle, amino acids metabolism, and redox status are highlighted. Moreover, the key enzymes and transporters as well as their function on the coordination of interorganellar metabolic exchanges are discussed.

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Cells and tissues are continuously exposed to both chemical and physical stimuli and dynamically adapt and respond to this variety of external cues to ensure cellular homeostasis, regulated development and tissue-specific differentiation. Alterations of these pathways promote disease progression-a prominent example being cancer. Rho GTPases are key regulators of the remodeling of cytoskeleton and cell membranes and their coordination and integration with different biological processes, including cell polarization and motility, as well as other signaling networks such as growth signaling and proliferation. Apart from the control of GTP-GDP cycling, Rho GTPase activity is spatially and temporally regulated by post-translation modifications (PTMs) and their assembly onto specific protein complexes, which determine their controlled activity at distinct cellular compartments. Although Rho GTPases were traditionally conceived as targeted from the cytosol to the plasma membrane to exert their activity, recent research demonstrates that active pools of different Rho GTPases also localize to endomembranes and the nucleus. In this review, we discuss how PTM-driven modulation of Rho GTPases provides a versatile mechanism for their compartmentalization and functional regulation. Understanding how the subcellular sorting of active small GTPase pools occurs and what its functional significance is could reveal novel therapeutic opportunities.

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Regulatory enzymes often have different roles in distinct subcellular compartments. Yet, most drugs indiscriminately saturate the cell. Thus, subcellular drug-delivery holds promise as a means to reduce off-target pharmacological effects. A-kinase anchoring proteins (AKAPs) sequester combinations of signaling enzymes within subcellular microdomains. Targeting drugs to these 'signaling islands' offers an opportunity for more precise delivery of therapeutics. Here, we review mechanisms that bestow protein kinase A (PKA) versatility inside the cell, appraise recent advances in exploiting AKAPs as platforms for precision pharmacology, and explore the impact of methodological innovations on AKAP research.

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The production of crocin, an uncommon and valuable apocarotenoid with strong biological activity, was obtained in a cell suspension culture of saffron (Crocus sativus L.) established from style-derived calli to obtain an in-vitro system for metabolite production. Salycilic acid (SA) was used at different concentrations to elicit metabolite production, and its effect was analyzed after a 4 days of treatment. HPLC-DAD analysis was used for total crocin quantification while the Folin-Ciocâlteu method was applied for phenolic compounds (PC) content. Interestingly, despite cell growth inhibition, a considerable exudation was observed when the highest SA concentration was applied, leading to a 7-fold enhanced production of crocin and a 4-fold increase of phenolics compared to mock cells. The maximum antioxidant activity of cell extracts was evidenced after SA 0.1 mM elicitation. Water-soluble extracts of saffron cells at concentrations of 1, 0.5, and 0.1 µg mL-1 showed significant inhibitory effects on MDA-MB-231 cancer cell viability. The heterologous vacuolar markers RFP-SYP51, GFPgl133Chi, and AleuRFP, were transiently expressed in protoplasts derived from the saffron cell suspensions, revealing that SA application caused a rapid stress effect, leading to cell death. Cell suspension elicitation with SA on the 7th day of the cell growth cycle and 24 h harvest time was optimized to exploit these cells for the highest increase of metabolite production in saffron cells.

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Fungal secondary metabolites are synthesized by complex biosynthetic pathways catalized by enzymes located in different subcellular compartments, thus requiring traffic of precursors and intermediates between them. The ß-lactam antibiotics penicillin and cephalosporin C serve as an excellent model to understand the molecular mechanisms that control the subcellular localization of secondary metabolites biosynthetic enzymes. Optimal functioning of the ß-lactam biosynthetic enzymes relies on a sophisticated temporal and spatial organization of the enzymes, the intermediates and the final products. The first and second enzymes of the penicillin pathway, ACV synthetase and IPN synthase, in Penicillium chrysogenum and Aspergillus nidulans are cytosolic. In contrast, the last two enzymes of the penicillin pathway, phenylacetyl-CoA ligase and isopenicillin N acyltransferase, are located in peroxisomes working as a tandem at their optimal pH that coincides with the peroxisomes pH. Two MFS transporters, PenM and PaaT have been found to be involved in the import of the intermediates isopenicillin N and phenylacetic acid, respectively, into peroxisomes. Similar compartmentalization of intermediates occurs in Acremonium chrysogenum; two enzymes isopenicillin N-CoA ligase and isopenicillin N-CoA epimerase, that catalyse the conversion of isopenicillin N in penicillin N, are located in peroxisomes. Two genes encoding MFS transporters, cefP and cefM, are located in the early cephalosporin gene cluster. These transporters have been localized in peroxisomes by confocal fluorescence microscopy. A third gene of A. chrysogenum, cefT, encodes an MFS protein, located in the cell membrane involved in the secretion of cephalosporin C, although cefT-disrupted mutants are still able to export cephalosporin by redundant transporters. The secretion of penicillin from peroxisomes to the extracellular medium is still unclear. Attempts have been made to identify a gene encoding the penicillin secretion protein among the 48 ABC-transporters of P. chrysogenum. The highly efficient secretion system that exports penicillin against a concentration gradient may involve active penicillin extrusion systems mediated by vesicles that fuse to the cell membrane. However, there is no correlation of pexophagy with penicillin or cephalosporin formation since inactivation of pexophagy leads to increased penicillin or cephalosporin biosynthesis due to preservation of peroxisomes. The penicillin biosynthesis finding shows that in order to increase biosynthesis of novel secondary metabolites it is essential to adequately target enzymes to organelles.