Biosynthetic Systems Research Articles

Cysteine-Persulfide Sulfane Sulfur-Ligated Zn Complex of Sulfur-Carrying SufU in the SufCDSUB System for Fe-S Cluster Biosynthesis.

SufU, a component of the SufCDSUB Fe-S cluster biosynthetic system, serves as a Zn-dependent sulfur-carrying protein that delivers inorganic sulfur in the form of cysteine persulfide from SufS to SufBCD. To understand this sulfur delivery mechanism, we studied the X-ray crystal structure of SufU and its sulfur-carrying state (persulfurated SufU) and performed functional analysis of the conserved amino acid residues around the Zn sites. Interestingly, sulfur-carrying SufU with Cys41-persulfide (Cys41-Sγ-Sδ-) exhibited a unique Zn coordination structure, in which electrophilic Sγ is ligated to Zn and nucleophilic/anionic Sδ is bound to distally conserved Arg125. This structure is distinct from those of other Cys-persulfide-Sδ-ligated metals of metalloproteins, such as hybrid cluster proteins and SoxAX. Functional analysis of SufU variants with Zn-ligand and Arg125 substitutions revealed that both Zn and Arg125 are critical for the function of SufU with SufS. The Zn-persulfide structure of SufU provides insight into the sulfur-transfer process, suggesting that persulfide-Sδ- is stabilized via bridging by Zn and Arg125 of SufU.

The Effects of Omeprazole on the Neuron-like Spiking of the Electrical Potential of Proteinoid Microspheres.

This study examines a new approach to hybrid neuromorphic devices by studying the impact of omeprazole-proteinoid complexes on Izhikevich neuron models. We investigate the influence of these metabolic structures on five specific patterns of neuronal firing: accommodation, chattering, triggered spiking, phasic spiking, and tonic spiking. By combining omeprazole, a proton pump inhibitor, with proteinoids, we create a unique substrate that interfaces with neuromorphic models. The Izhikevich neuron model is used because it is computationally efficient and can accurately simulate the various behaviours of cortical neurons. The results of our simulations show that omeprazole-proteinoid complexes have the ability to affect neuronal dynamics in different ways. This suggests that they could be used as adjustable components in bio-inspired computer systems. We noticed a notable alteration in the frequency of spikes, patterns of bursts, and rates of adaptation, especially in chattering and triggered spiking behaviours. The findings indicate that omeprazole-proteinoid complexes have the potential to serve as adaptable elements in neuromorphic systems, presenting novel opportunities for information processing and computation that have origins in neurobiological principles. This study makes a valuable contribution to the expanding field of biochemical neuromorphic devices and establishes a basis for the development of hybrid bio-synthetic computational systems.

Differential regulation of heparan sulfate biosynthesis in fibroblasts cocultured with normal vs. cancerous prostate cells.

Heparan sulfate proteoglycans (HSPGs) regulate a wide range of biological activities in both physiological and pathological conditions. Altered expression or deregulated function of HSPGs and their heparan sulfate (HS) chains significantly contribute to carcinogenesis as well and crucially depends on the functioning of the complex system of HS biosynthetic/modifying enzymes termed as "GAGosome". Here, we aimed at investigating the expression profile of the system in a cell culture model of stroma-epithelial crosstalk and searching for transcription factors potentially related to the regulation of expression of the genes involved. Coculture of BjTERT-fibroblasts with normal PNT2 human prostate epithelial cells resulted in significant downregulation (2-4-fold) of transcriptional activity of HS metabolism-involved genes (EXT1/2, NDST1/2, GLCE, HS2ST1, HS3ST1/2, HS6ST1/2, SULF1/2, HPSE) in both cell types, whereas coculture with prostate cancer cells (LNCaP, PC3, DU145) demonstrated no significant interchanges. Human Transcription Factor RT2 Profiler PCR array and manual RT-PCR verification supposed FOS, MYC, E2F, SRF, NR3C1 as potential candidates for regulation and/or coordination of HS biosynthesis. Taken together, transcriptional activity of HS biosynthetic system in normal fibroblasts and prostate epithelial cells during their coculture might be controlled by their intercellular communication, reflecting of adaptation of these cells to each other. The regulation is attenuated or abrogated if normal fibroblasts interact with prostate cancer cells making the cancer cells independent of the limiting effects of fibroblasts, thus contributing to possibility of unlimited growth and progression. Overall, these data demonstrate an ability of cell-cell interactions to affect transcriptional activity of HS biosynthesis-involved genes.

Insights into phosphoethanolamine cellulose synthesis and secretion across the Gram-negative cell envelope

Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, a hexameric semicircle of the periplasmic BcsB protein, and the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a periplasmic cellulase of unknown biological function. While cellulose synthesis and translocation by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three BcsG copies near the nascent cellulose polymer. Further, the semicircle’s terminal BcsB subunit tethers the N-terminus of a single BcsC protein in a trans-envelope secretion system. BcsC’s TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of translocation incompetent cellulose. Lastly, protein engineering introduces cellulose pEtN modification in orthogonal cellulose biosynthetic systems. These findings advance our understanding of pEtN cellulose modification and secretion.

Learning metabolic dynamics from irregular observations by Bidirectional Time-Series State Transfer Network.

Modeling microbial metabolic dynamics is important for the rational optimization of both biosynthetic systems and industrial processes to facilitate green and efficient biomanufacturing. Classical approaches utilize explicit equation systems to represent metabolic networks, enabling the quantification of pathway fluxes to identify metabolic bottlenecks. However, these white-box models, despite their diverse applications, have limitations in simulating metabolic dynamics and are intrinsically inaccurate for industrial strains that lack information on network structures and kinetic parameters. On the other hand, black-box models do not rely on prior mechanistic knowledge of strains but are built upon observed time-series trajectories of biosynthetic systems in action. In practice, these observations are typically irregular, with discontinuously observed time points across multiple independent batches, each time point potentially containing missing measurements. Learning from such irregular data remains challenging for existing approaches. To address this issue, we present the Bidirectional Time-Series State Transfer Network (BTSTN) for modeling metabolic dynamics directly from irregular observations. Using evaluation data sets derived from both ideal dynamic systems and a real-world fermentation process, we demonstrate that BTSTN accurately reconstructs dynamic behaviors and predicts future trajectories. This approach exhibits enhanced robustness against missing measurements and noise, as compared to the state-of-the-art methods.IMPORTANCEIndustrial biosynthetic systems often involve strains with unclear genetic backgrounds, posing challenges in modeling their distinct metabolic dynamics. In such scenarios, white-box models, which commonly rely on inferred networks, are thereby of limited applicability and accuracy. In contrast, black-box models, such as statistical models and neural networks, are directly fitted or learned from observed time-series trajectories of biosynthetic systems in action. These methods typically assume regular observations without missing time points or measurements. If the observations are irregular, a pre-processing step becomes necessary to obtain a fully filled data set for subsequent model training, which, at the same time, inevitably introduces errors into the resulting models. BTSTN is a novel approach that natively learns from irregular observations. This distinctive feature makes it a unique addition to the current arsenal of technologies modeling metabolic dynamics.

Characterization of the SUF FeS cluster synthesis machinery in the amitochondriate eukaryote Monocercomonoides exilis

Monocercomonoides exilis is the first known amitochondriate eukaryote. Loss of mitochondria in M.exilis ocurred after the replacement of the essential mitochondrial iron-sulfur cluster (ISC) assembly machinery by a unique, bacteria-derived, cytosolic SUF system. It has been hypothesized that the MeSuf pathway, in cooperation with proteins of the cytosolic iron-sulfur protein assembly (CIA) system, is responsible for the biogenesis of FeS clusters in M.exilis, yet biochemical evidence is pending. Here, we address the M.exilis MeSuf system and show that SUF genes, individually or in tandem, support the loading of iron-sulfur (FeS) clusters into the reporter protein IscR in Escherichia coli. The Suf proteins MeSufB, MeSufC, and MeSufDSU interact invivo with one another and with Suf proteins of E.coli. Invitro, the M.exilis Suf proteins form large complexes of varying composition and hence may function as a dynamic biosynthetic system in the protist. The putative FeS cluster scaffold MeSufB-MeSufC (MeSufBC) forms multiple oligomeric complexes, some of which bind FeS clusters and form selectively only in the presence of adenosine nucleotides. The multi-domain fusion protein MeSufDSU binds a PLP cofactor and can form higher-order complexes with MeSufB and MeSufC. Our work demonstrates the biochemical property of M. exilis Suf proteins to act as a functional FeS cluster assembly system and provides insights into the molecular mechanism of this unique eukaryotic SUF system.

Rational construction of a high-quality and high-efficiency biosynthetic system and fermentation optimization for A82846B based on combinatorial strategies in Amycolatopsis orientalis

BackgroundOritavancin is a new generation of semi-synthetic glycopeptide antibiotics against Gram-positive bacteria, which served as the first and only antibiotic with a single-dose therapeutic regimen to treat ABSSSI. A naturally occurring glycopeptide A82846B is the direct precursor of oritavancin. However, its application has been hampered by low yields and homologous impurities. This study established a multi-step combinatorial strategy to rationally construct a high-quality and high-efficiency biosynthesis system for A82846B and systematically optimize its fermentation process to break through the bottleneck of microbial fermentation production.ResultsFirstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities.ConclusionUnder approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products.

Escherichia coli monothiol glutaredoxin GrxD replenishes Fe-S clusters to the essential ErpA A-type carrier under low iron stress

Iron-sulfur (Fe-S) clusters are required for essential biological pathways, including respiration and isoprenoid biosynthesis. Complex Fe-S cluster biogenesis systems have evolved to maintain an adequate supply of this critical protein cofactor. In Escherichia coli, two Fe-S biosynthetic systems, the “housekeeping” Isc and “stress responsive” Suf pathways, interface with a network of cluster trafficking proteins, such as ErpA, IscA, SufA, and NfuA. GrxD, a Fe-S cluster-binding monothiol glutaredoxin, also participates in Fe-S protein biogenesis in both prokaryotes and eukaryotes. Previous studies in E. coli showed that the ΔgrxD mutation causes sensitivity to iron depletion, spotlighting a critical role for GrxD under conditions that disrupt Fe-S homeostasis. Here, we utilized a global chemoproteomic mass spectrometry (MS) approach to analyse the contribution of GrxD to the Fe-S proteome. Our results demonstrate that 1) GrxD is required for biogenesis of a specific subset of Fe-S proteins under iron-depleted conditions, 2) GrxD is required for cluster delivery to ErpA under iron limitation, 3) GrxD is functionally distinct from other Fe-S trafficking proteins and, 4) GrxD Fe-S cluster binding is responsive to iron limitation. All these results lead to the proposal that GrxD is required to maintain Fe-S cluster delivery to the essential trafficking protein ErpA during iron limitation conditions.

The Progress of the Biotechnological Production of Class IIa Bacteriocins in Various Cell Factories and Its Future Challenges.

Class IIa bacteriocins produced in lactic acid bacteria are short cationic peptides with antimicrobial activity. In the search for new biopreservation agents, class IIa bacteriocins are considered to be the best potential candidates, not only due to their large abundance but also because of their high biological activity and excellent thermal stability. However, regulated by the biosynthetic regulatory system, the natural class IIa bacteriocin yield is low, and the extraction process is complicated. The biotechnological production of class IIa bacteriocins in various cell factories has been attempted to improve this situation. In this review, we focus on the application of biotechnological routes for class IIa bacteriocin production. The drawbacks and improvements in the production of class IIa bacteriocins in various cell factories are discussed. Furthermore, we present the main challenge of class IIa bacteriocins, focusing on increasing their production by constructing suitable cell factories. Recombinant bacteriocins have made considerable progress from inclusion body formation, dissolved form and low antibacterial activity to yield recovery. The development of prospective cell factories for the biotechnological production of bacteriocins is still required, which may facilitate the application of bacteriocins in the food industry.

Haematococcus lacustris Carotenogensis: A Historical Event of Primary to Secondary Adaptations to Earth's Oxygenation.

(1) Background: Oxygen has exerted a great effect in shaping the environment and driving biological diversity in Earth's history. Green lineage has evolved primary and secondary carotenoid biosynthetic systems to adapt to Earth's oxygenation, e.g., Haematococcus lacustris, which accumulates the highest amount of secondary astaxanthin under stresses. The two systems are controlled by lycopene ε-cyclase (LCYE) and β-cyclase (LCYB), which leave an important trace in Earth's oxygenation. (2) Objectives: This work intends to disclose the underlying molecular evolutionary mechanism of Earth's oxygenation in shaping green algal carotenogensis with a special focus on lycopene cyclases. (3) Methods: The two kinds of cyclases were analyzed by site-directed mutagenesis, phylogeny, divergence time and functional divergence. (4) Results: Green lineage LCYEs appeared at ~1.5 Ga after the first significant appearance and accumulation of atmospheric oxygen, the so-called Great Oxygenation Event (GOE), from which LCYBs diverged by gene duplication. Bacterial β-bicyclases evolved from β-monocyclase. Enhanced catalytic activity accompanied evolutionary transformation from ε-/β-monocyclase to β-bicyclase. Strong positive selection occurred in green lineage LCYEs after the GOE and in algal LCYBs during the second oxidation, the Neoproterozoic Oxygenation Event (NOE). Positively selected sites in the catalytic cavities of the enzymes controlled the mono-/bicyclase activity, respectively. Carotenoid profiling revealed that oxidative adaptation has been wildly preserved in evolution. (5) Conclusions: the functionalization of the two enzymes is a result of primary to secondary adaptations to Earth's oxygenation.

Multifaceted regulation of siderophore synthesis by multiple regulatory systems in Shewanella oneidensis

Siderophore-dependent iron uptake is a mechanism by which microorganisms scavenge and utilize iron for their survival, growth, and many specialized activities, such as pathogenicity. The siderophore biosynthetic system PubABC in Shewanella can synthesize a series of distinct siderophores, yet how it is regulated in response to iron availability remains largely unexplored. Here, by whole genome screening we identify TCS components histidine kinase (HK) BarA and response regulator (RR) SsoR as positive regulators of siderophore biosynthesis. While BarA partners with UvrY to mediate expression of pubABC post-transcriptionally via the Csr regulatory cascade, SsoR is an atypical orphan RR of the OmpR/PhoB subfamily that activates transcription in a phosphorylation-independent manner. By combining structural analysis and molecular dynamics simulations, we observe conformational changes in OmpR/PhoB-like RRs that illustrate the impact of phosphorylation on dynamic properties, and that SsoR is locked in the ‘phosphorylated’ state found in phosphorylation-dependent counterparts of the same subfamily. Furthermore, we show that iron homeostasis global regulator Fur, in addition to mediating transcription of its own regulon, acts as the sensor of iron starvation to increase SsoR production when needed. Overall, this study delineates an intricate, multi-tiered transcriptional and post-transcriptional regulatory network that governs siderophore biosynthesis.

Multi-modular metabolic engineering and efflux engineering for enhanced lycopene production in recombinant Saccharomyces cerevisiae.

In this research, lycopene production in yeast was markedly enhanced by integrating a multi-modular approach, acetate signaling-based down-regulation of competitive pathways, and an efflux optimization strategy.

Antibacterial activity of Nocardia spp. and Streptomyces sp. on multidrug-resistant pathogens causing neonatal sepsis.

Neonatal sepsis leads to severe morbidity and occasionally death among neonates within the first week following birth, particularly in low- and middle-income countries. Empirical therapy includes antibiotics recommended by WHO. However, these have been ineffective against antimicrobial multidrug-resistant bacterial strains such as Klebsiella spp, Escherichia coli, and Staphylococcus aureus species. To counter this problem, new molecules and alternative sources of compounds with antibacterial activity are sought as options. Actinobacteria, particularly pathogenic strains, have revealed a biotechnological potential still underexplored. This study aimed to determine the presence of biosynthetic gene clusters and the antimicrobial activity of actinobacterial strains isolated from clinical cases against multidrug-resistant bacteria implicated in neonatal sepsis. In total, 15 strains isolated from clinical cases of actinomycetoma were used. PCR screening for the PKS-I, PKS-II, NRPS-I, and NRPS-II biosynthetic systems determined their secondary metabolite-producing potential. The strains were subsequently assayed for antimicrobial activity by the perpendicular cross streak method against Escherichia fergusonii Sec 23, Klebsiella pneumoniae subsp. pneumoniae H1064, Klebsiella variicola H776, Klebsiella oxytoca H793, and Klebsiella pneumoniae subsp. ozaenae H7595, previously classified as multidrug-resistant. Finally, the strains were identified by 16S rRNA gene sequence analysis. It was found that 100% of the actinobacteria had biosynthetic systems. The most frequent biosynthetic system was NRPS-I (100%), and the most frequent combination was NRPS-I and PKS-II (27%). All 15 strains showed antimicrobial activity. The strain with the highest antimicrobial activity was Streptomyces albus 94.1572, as it inhibited the growth of the five multidrug-resistant bacteria evaluated.

Organizational change of synthetic biology research: Emerging initiatives advancing a bottom-up approach

Bottom-up Synthetic Biology (buSynBio) is an approach focused on the artificial making of minimal functional biosynthetic systems by recombining existent biochemical modules or manufacturing them from scratch. Over the last decade, this emerging orientation has gained new momentum with the development of new bioengineering tools, theories, and technologies. Despite the growing acceptance of buSynBio, few studies have dedicated attention to the analysis of its organizational aspects. This article offers the first systematic investigation of emerging research initiatives in buSynBio and their meaning to bioengineering research. Our analysis is based on a multi-method qualitative study, including expert literature review, bibliometric research and a documentary analysis of online materials such as reports and project descriptions available in official grant data repositories. Our study found that publications of specialized articles on “bottom-up synthetic biology” have increased, both in absolute numbers and normalized to total number of publications. We show how that might be enabled by novel mechanisms of organization that reposition material, intellectual and political resources in synthetic biology. Drawing on theoretical analyses within Science and Technology Studies (STS), we examine 14 research initiatives in 5 selected countries (Germany, United Kingdom, United States, Netherlands, and Switzerland). The bottom-up approach is supported by a variety of “tentative regimes” of scientific governance in different stages of consolidation, but holding in common the establishment of novel basic research in Chemistry, Biology, Engineering and Physics. The study aims to contribute to social science research in synthetic biology by shedding light on the implications of buSynBio as trend driving the current organizational change of bioengineering research.

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system.

Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.

Giant DNA Viruses Infecting Unicellular Protists

Giant DNA Viruses Infecting Unicellular Protists

Dexamethasone Inhibits Heparan Sulfate Biosynthetic System and Decreases Heparan Sulfate Content in Orthotopic Glioblastoma Tumors in Mice.

Glioblastoma (GB) is an aggressive cancer with a high probability of recurrence, despite active chemoradiotherapy with temozolomide (TMZ) and dexamethasone (DXM). These systemic drugs affect the glycosylated components of brain tissue involved in GB development; however, their effects on heparan sulfate (HS) remain unknown. Here, we used an animal model of GB relapse in which SCID mice first received TMZ and/or DXM (simulating postoperative treatment) with a subsequent inoculation of U87 human GB cells. Control, peritumor and U87 xenograft tissues were investigated for HS content, HS biosynthetic system and glucocorticoid receptor (GR, Nr3c1). In normal and peritumor brain tissues, TMZ/DXM administration decreased HS content (5-6-fold) but did not affect HS biosynthetic system or GR expression. However, the xenograft GB tumors grown in the pre-treated animals demonstrated a number of molecular changes, despite the fact that they were not directly exposed to TMZ/DXM. The tumors from DXM pre-treated animals possessed decreased HS content (1.5-2-fold), the inhibition of HS biosynthetic system mainly due to the -3-3.5-fold down-regulation of N-deacetylase/N-sulfotransferases (Ndst1 and Ndst2) and sulfatase 2 (Sulf2) expression and a tendency toward a decreased expression of the GRalpha but not the GRbeta isoform. The GRalpha expression levels in tumors from DXM or TMZ pre-treated mice were positively correlated with the expression of a number of HS biosynthesis-involved genes (Ext1/2, Ndst1/2, Glce, Hs2st1, Hs6st1/2), unlike tumors that have grown in intact SCID mice. The obtained data show that DXM affects HS content in mouse brain tissues, and GB xenografts grown in DXM pre-treated animals demonstrate attenuated HS biosynthesis and decreased HS content.

Reconstruction of the Native Biosynthetic System of Carotenoids in E. coli─Biosynthesis of a Series of Carotenoids Specific to Paprika Fruit

Capsanthin, capsorubin, cucurbitaxanthin A, and capsanthin 3,6-epoxide, a series of carotenoids specific to the red fruit of paprika (Capsicum annuum), were produced in pathway-engineered Escherichia coli cells. These cells functionally expressed multiple genes for eight carotenogenic enzymes, two of which, paprika capsanthin/capsorubin synthase (CaCCS) and zeaxanthin epoxidase (CaZEP), were designed to be located adjacently. The biosynthesis of these carotenoids, except for capsanthin, was the first successful attempt in E. coli. In a previous study, the levels of capsanthin synthesized were low despite the high expression of the CaCCS gene, which may have been due to the dual activity of CaCCS as a lycopene β-cyclase and CCS. An enhanced interaction between CaCCS and CaZEP that supplies antheraxanthin and violaxanthin, substrates for CaCCS, was considered to be crucial for an efficient reaction. To achieve this, we adapted S·tag and S-protein binding. The S·tag Thrombin Purification Kit (Novagen) is merchandized for in vitro affinity purification, and S·tag-fused proteins in the E. coli lysate are specifically trapped by S-proteins fixed on the agarose carrier. Furthermore, S-proteins have been reported to oligomerize via C-terminal swapping. In the present study, CaCCS and CaZEP were individually fused to the S·tag and designed to interact on oligomerized S-protein scaffolds in E. coli, which led to the biosynthesis of not only capsanthin and capsorubin but also cucurbitaxanthin A and capsanthin 3,6-epoxide. The latter reaction by CaCCS was assigned for the first time. This approach reinforces the scaffold's importance for multienzyme pathways when native biosynthetic systems are reconstructed in microorganisms.

Glycoside hydrolase processing of the Pel polysaccharide alters biofilm biomechanics and Pseudomonas aeruginosa virulence

Pel exopolysaccharide biosynthetic loci are phylogenetically widespread biofilm matrix determinants in bacteria. In Pseudomonas aeruginosa, Pel is crucial for cell-to-cell interactions and reducing susceptibility to antibiotic and mucolytic treatments. While genes encoding glycoside hydrolases have long been linked to biofilm exopolysaccharide biosynthesis, their physiological role in biofilm development is unclear. Here we demonstrate that the glycoside hydrolase activity of P. aeruginosa PelA decreases adherent biofilm biomass and is responsible for generating the low molecular weight secreted form of the Pel exopolysaccharide. We show that the generation of secreted Pel contributes to the biomechanical properties of the biofilm and decreases the virulence of P. aeruginosa in Caenorhabditis elegans and Drosophila melanogaster. Our results reveal that glycoside hydrolases found in exopolysaccharide biosynthetic systems can help shape the soft matter attributes of a biofilm and propose that secreted matrix components be referred to as matrix associated to better reflect their influence.

Exposure to Heated Tobacco Products Aerosol Causes Acute Stress Responses in the Lung of Mouse.

In the present study, we evaluated the acute response of mice exposed to IQOS aerosol, a brand-name heated tobacco product (HTP), in the lung tissue. First, the thiobarbituric acid-reactive substances (TBA-RS) value was measured as an index to assess oxidative stress, and a significant increase was observed after exposure, followed by a significant increase in the total lung GSH concentration. The stress responses induced by IQOS aerosols was then analyzed by focusing on the changes in Nrf2 and ATF4, which are transcription factors that induce the expression of genes involved in GSH biosynthesis or metabolism. Although Nrf2 activation was not observed, significant accumulation of ATF4 in the nuclear fraction was noted three hours after exposure to IQOS aerosols. Upon an examination of changes in factors in the GSH biosynthetic system, a significant increase in cystine concentration in the lung tissue was measured, and an increase in xCT expression level was observed in the cell membrane fraction three-six hours after IQOS exposure. Furthermore, characteristic changes in HO-1, a stress-response protein regulated by ATF4, was discovered six hours after IQOS exposure. Moreover, analysis of the upstream ATF4 regulatory system revealed that phosphorylation of eIF2α was enhanced in the lung cytoplasmic fraction three hours after exposure to IQOS aerosols. These findings suggest that ER stress might be induced as an early response to IQOS aerosol exposure, accompanied by the activation of the eIF2α-ATF4 axis. These intracellular changes have also been reported after exposure to combustible cigarette smoke. Thus, the acute response found in the lungs of mice in the present study demonstrate that the inhalation of aerosols from IQOS elicits a biological response similar to that of combustible cigarette smoke. In conclusion, our results provide evidence that the biological effects of HTPs, such as IQOS, cannot be ignored in the lungs.