The GE296_RS03820 and GE296_RS03830 genes are involved in capsular polysaccharide biosynthesis in Riemerella anatipestifer.

Riemerella anatipestifer is a pathogenic bacterium that causes duck serositis and meningitis, leading to significant harm to the duck industry. To escape from the host immune system, the meningitis-causing bacteria must survive and multiply in the bloodstream, relying on specific virulence factors such as capsules. Therefore, it is essential to study the genes involved in capsule biosynthesis in R. anatipestifer. In this study, we successfully constructed gene deletion mutants Δ3820 and Δ3830, targeting the GE296_RS03820 and GE296_RS03830 genes, respectively, using the RA-LZ01 strain as the parental strain. The growth kinetics analysis revealed that these two genes contribute to bacterial growth. Transmission and scanning electron microscopy (TEM and SEM) and silver staining showed that Δ3820 and Δ3830 produced the altered capsules and compounds of capsular polysaccharides (CPSs). Serum resistance test showed the mutants also exhibited reduced C3b deposition and decreased resistance serum killing. In vivo, Δ3820 and Δ3830 exhibited markedly declining capacity to cross the blood-brain barrier, compared to RA-LZ01. These findings indicate that the GE296_RS03820 and GE296_RS03830 genes are involved in CPSs biosynthesis and play a key role in the pathogenicity of R. anatipestifer. Furthermore, Δ3820 and Δ3830 mutants presented a tendency toward higher survival rates from RA-LZ01 challenge in vivo. Additionally, sera from ducklings immunized with the mutants showed cross-immunoreactivity with different serotypes of R. anatipestifer, including 1, 2, 7 and 10. Western blot and SDS-PAGE assays revealed that the altered CPSs of Δ3820 and Δ3830 resulted in the exposure of some conserved proteins playing the key role in the cross-immunoreactivity. Our study clearly demonstrated that the GE296_RS03820 and GE296_RS03830 genes are involved in CPS biosynthesis in R. anatipestifer and the capsule is a target for attenuation in vaccine development.

Oleaginous fungi: a promising source of biofuels and nutraceuticals with enhanced lipid production strategies.

Oleaginous fungi have attracted a great deal of interest for their potency to accumulate high amounts of lipids (more than 20% of biomass dry weight) and polyunsaturated fatty acids (PUFAs), which have a variety of industrial and biological applications. Lipids of plant and animal origin are related to some restrictions and thus lead to attention towards oleaginous microorganisms as reliable substitute resources. Lipids are traditionally biosynthesized intra-cellularly and involved in the building structure of a variety of cellular compartments. In oleaginous fungi, under certain conditions of elevated carbon ratio and decreased nitrogen in the growth medium, a change in metabolic pathway occurred by switching the whole central carbon metabolism to fatty acid anabolism, which subsequently resulted in high lipid accumulation. The present review illustrates the bio-lipid structure, fatty acid classes and biosynthesis within oleaginous fungi with certain key enzymes, and the advantages of oleaginous fungi over other lipid bio-sources. Qualitative and quantitative techniques for detecting the lipid accumulation capability of oleaginous microbes including visual, and analytical (convenient and non-convenient) were debated. Factors affecting lipid production, and different approaches followed to enhance the lipid content in oleaginous yeasts and fungi, including optimization, utilization of cost-effective wastes, co-culturing, as well as metabolic and genetic engineering, were discussed. A better understanding of the oleaginous fungi regarding screening, detection, and maximization of lipid content using different strategies could help to discover new potent oleaginous isolates, exploit and recycle low-cost wastes, and improve the efficiency of bio-lipids cumulation with biotechnological significance.

Production of Secreted Antibody in Baculovirus Expression Vector System.

Monoclonal antibodies have widespread applications in disease treatment and antigen detection. They are traditionally produced using mammalian cell expression system, which is not able to satisfy the increasing demand of these proteins at large scale. Baculovirus expression vector system (BEVS) is an attractive alternative platform for the production of biologically active monoclonal antibodies. In this chapter, we demonstrate the production of an HIV-1 broadly neutralizing antibody b12 in BEVS. The processes including transfer vector construction, recombinant baculovirus generation, and antibody production and detection are described.

Production of Recombinant Viral Antigens Using the Baculovirus-Insect Cell Expression System.

Insect cell expression has been successfully used for the production of viral antigens as part of commercial vaccine development. As expression host, insect cells offer advantage over bacterial system by presenting the ability of performing post-translational modifications (PTMs) such as glycosylation and phosphorylation thus preserving the native functionality of the proteins especially for viral antigens. Insect cells have limitation in exactly mimicking some proteins which require complex glycosylation pattern. The recent advancement in insect cell engineering strategies could overcome this limitation to some extent. Moreover, cost efficiency, timelines, safety, and process adoptability make insect cells a preferred platform for production of subunit antigens for human and animal vaccines. In this chapter, we describe the method for producing the SARS-CoV2 spike ectodomain subunit antigen for human vaccine development and the virus like particle (VLP), based on capsid protein of porcine circovirus virus 2 (PCV2d) antigen for animal vaccine development using two different insect cell lines, SF9 & Hi5, respectively. This methodology demonstrates the flexibility and broad applicability of insect cell as expression host.

Recombinant AAV Production.

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.

Protein Production at the 100L Scale.

The Baculovirus Expression Vector System (BEVS) has revolutionized the field of recombinant protein expression by enabling efficient and high yield production. The platform offers many advantages including manufacturing speed, flexible design, and scalability. In this chapter, we describe the methods including strategies and considerations to successfully optimize and scale-up using BEVS as a tool for production (Fig. 1). As an illustrative case study, we present an example focused on the production of a viral glycoprotein.

Recombinant Protein Production in Suspension Mammalian Cells Using the BacMam Baculovirus Expression System.

Mammalian cell lines are one of the best options when it comes to the production of complex proteins requiring specific glycosylation patterns. Plasmid DNA transfection and stable cell lines are frequently used for recombinant protein production, but they are expensive at large scale or can become time-consuming, respectively. The BacMam baculovirus (BV) is a safe and cost-effective platform to produce recombinant proteins in mammalian cells. The process of generating BacMam BVs is straightforward and similar to the generation of "insect" BVs, with different commercially available platforms. Although there are several protocols that describe recombinant protein expression with the BacMam BV in adherent cell lines, limited information is available on suspension cells. Therefore, it is of relevance to define the conditions to produce recombinant proteins in suspension cell cultures with BacMam BVs that facilitate bioprocess transfer to larger volumes. Here, we describe a method to generate a high titer BacMam BV stock and produce recombinant proteins in suspension HEK293 cells.

Sources and control of impurity during one-pot enzymatic production of dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) has a promising market due to its capacity to regulate human hormone levels as well as preventing and treating various diseases. We have established a chemical esterification coupled biocatalytic-based scheme by lipase-catalyzed 4-androstene-3,17-dione (4-AD) hydrolysis to obtain the intermediate product 5-androstene-3,17-dione (5-AD), which was then asymmetrically reduced by a ketoreductase from Sphingomonas wittichii (SwiKR). Co-enzyme required for KR is regenerated by a glucose dehydrogenase (GDH) from Bacillus subtilis. This scheme is more environmentally friendly and more efficient than the current DHEA synthesis pathway. However, a significant amount of 4-AD as by-product was detected during the catalytic process. Focused on the control of by-products, we investigated the source of 4-AD and identified that it is mainly derived from the isomerization activity of SwiKR and GDH. Increasing the proportion of glucose in the catalytic system as well as optimizing the catalytic conditions drastically reduced 4-AD from 24.7 to 6.5% of total substrate amount, and the final yield of DHEA achieved 40.1 g/L. Furthermore, this is the first time that both SwiKR and GDH have been proved to be promiscuous enzymes with dehydrogenase and ketosteroid isomerase (KSI) activities, expanding knowledge of the substrate diversity of the short-chain dehydrogenase family enzymes. KEY POINTS: • A strategy of coupling lipase, ketoreductase, and glucose dehydrogenase in producing DHEA from 4-AD • Both SwiKR and GDH are identified with ketosteroid isomerase activity. • Development of catalytic strategy to control by-product and achieve highly selective DHEA production.

A review on microbes mediated resource recovery and bioplastic (polyhydroxyalkanoates) production from wastewater.

BACKGROUND: Plastic is widely utilized in packaging, frameworks, and as coverings material. Its overconsumption and slow degradation, pose threats to ecosystems due to its toxic effects. While polyhydroxyalkanoates (PHA) offer a sustainable alternative to petroleum-based plastics, their production costs present significant obstacles to global adoption. On the other side, a multitude of household and industrial activities generate substantial volumes of wastewater containing both organic and inorganic contaminants. This not only poses a threat to ecosystems but also presents opportunities to get benefits from the circular economy. Production of bioplastics may be improved by using the nutrients and minerals in wastewater as a feedstock for microbial fermentation. Strategies like feast-famine culture, mixed-consortia culture, and integrated processes have been developed for PHA production from highly polluted wastewater with high organic loads. Various process parameters like organic loading rate, organic content (volatile fatty acids), dissolved oxygen, operating pH, and temperature also have critical roles in PHA accumulation in microbial biomass. Research advances are also going on in downstream and recovery of PHA utilizing a combination of physical and chemical (halogenated solvents, surfactants, green solvents) methods. This review highlights recent developments in upcycling wastewater resources into PHA, encompassing various production strategies, downstream processing methodologies, and techno-economic analyses. SHORT CONCLUSION: Organic carbon and nitrogen present in wastewater offer a promising, cost-effective source for producing bioplastic. Previous attempts have focused on enhancing productivity through optimizing culture systems and growth conditions. However, despite technological progress, significant challenges persist, such as low productivity, intricate downstream processing, scalability issues, and the properties of resulting PHA.

Efficient and markerless gene integration with SlugCas9-HF in Kluyveromyces marxianus.

The nonconventional yeast Kluyveromyces marxianus has potential for industrial production, but the lack of advanced synthetic biology tools for precise engineering hinders its rapid development. Here, we introduce a CRISPR-Cas9-mediated multilocus integration method for assembling multiple exogenous genes. Using SlugCas9-HF, a high-fidelity Cas9 nuclease, we enhance gene editing precision. Specific genomic loci predisposed to efficient integration and expression of heterologous genes are identified and combined with a set of paired CRISPR-Cas9 expression plasmids and donor plasmids to establish a CRISPR-based biosynthesis toolkit. This toolkit enables genome integration of large gene modules over 12 kb and achieves simultaneous quadruple-locus integration in a single step with 20% efficiency. As a proof-of-concept, we apply the toolkit to screen for gene combinations that promote heme production, revealing the importance of HEM4Km and HEM12Sc. This CRISPR-based toolkit simplifies the reconstruction of complex pathways in K. marxianus, broadening its application in synthetic biology.

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches.

BACKGROUND: Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. RESULTS: In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. CONCLUSIONS: Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

Natural dyes developed by microbial-nanosilver to produce antimicrobial and anticancer textiles.

Developing special textiles (for patients in hospitals for example) properties, special antimicrobial and anticancer, was the main objective of the current work. The developed textiles were produced after dyeing by the novel formula of natural (non-environmental toxic) pigments (melanin amended by microbial-AgNPs). Streptomyces torulosus isolate OSh10 with accession number KX753680.1 was selected as a superior producer for brown natural pigment. By optimization processes, some different pigment colors were observed after growing the tested strain on the 3 media. Dextrose and malt extract enhanced the bacteria to produce a reddish-black color. However, glycerol as the main carbon source and NaNO3 and asparagine as a nitrogen source were noted as the best for the production of brown pigment. In another case, starch as a polysaccharide was the best carbon for the production of deep green pigment. Peptone and NaNO3 are the best nitrogen sources for the production of deep green pigment. Microbial-AgNPs were produced by Fusarium oxysporum with a size of 7-21 nm, and the shape was spherical. These nanoparticles were used to produce pigments-nanocomposite to improve their promising properties. The antimicrobial of nanoparticles and textiles dyeing by nanocomposites was recorded against multidrug-resistant pathogens. The new nanocomposite improved pigments' dyeing action and textile properties. The produced textiles had anticancer activity against skin cancer cells with non-cytotoxicity detectable action against normal skin cells. The obtained results indicate to application of these textiles in hospital patients' clothes.

Optimization, gene cloning, expression, and molecular docking insights for enhanced cellulase enzyme production by Bacillus amyloliquefaciens strain elh1.

BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71. CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.

Disruptive effects of plasticizers bisphenol A, F, and S on steroidogenesis of adrenocortical cells.

Introduction: Endocrine disrupting chemicals (EDCs) are known to interfere with endocrine homeostasis. Their impact on the adrenal cortex and steroidogenesis has not yet been sufficiently elucidated. This applies in particular to the ubiquitously available bisphenols A (BPA), F (BPF), and S (BPS). Methods: NCI-H295R adrenocortical cells were exposed to different concentrations (1nM-1mM) of BPA, BPF, BPS, and an equimolar mixture of them (BPmix). After 72 hours, 15 endogenous steroids were measured using LC-MS/MS. Ratios of substrate and product of CYP-regulated steps were calculated to identify most influenced steps of steroidogenesis. mRNA expression of steroidogenic enzymes was determined by real-time PCR. Results: Cell viability remained unaffected at bisphenol concentrations lower than 250 µM. All tested bisphenols and their combination led to extensive alterations in the quantified steroid levels. The most profound fold changes (FC) in steroid concentrations after exposure to BPA (>10µM) were seen for androstenedione, e.g. a 0.37±0.11-fold decrease at 25µM (p≤0.0001) compared to vehicle-treated controls. For BPF, levels of 17-hydroxyprogesterone were significantly increased by 25µM (FC 2.57±0.49, p≤0.001) and 50µM (FC 2.65±0.61, p≤0.0001). BPS treatment led to a dose-dependent decrease of 11-deoxycorticosterone at >1µM (e.g. FC 0.24±0.14, p≤0.0001 at 10µM). However, when combining all three bisphenols, additive effects were detected: e.g. 11-deoxycortisosterone was decreased at doses >10µM (FC 0.27±0.04, p≤0.0001, at 25µM), whereas 21-deoxycortisol was increased by 2.92±0.20 (p≤0.01) at 10µM, and by 3.21±0.45 (p≤0.001) at 50µM. While every measured androgen (DHEA, DHEAS, androstenedione, testosterone, DHT) was lowered in all experiments, estradiol levels were significantly increased by BPA, BPF, BPS, and BPmix (e.g. FC 3.60±0.54, p≤0.0001 at 100µM BPF). Calculated substrate-product ratios indicated an inhibition of CYP17A1-, and CYP21A2 mediated conversions, whereas CYP11B1 and CYP19A1 showed higher activity in the presence of bisphenols. Based on these findings, most relevant mRNA expression of CYP genes were analysed. mRNA levels of StAR, CYP11B1, and CYP17A1 were significantly increased by BPF, BPS, and BPmix. Discussion: In cell culture, bisphenols interfere with steroidogenesis at non-cytotoxic levels, leading to compound-specific patterns of significantly altered hormone levels. These results justify and call for additional in-vivo studies to evaluate effects of EDCs on adrenal gland functionality.

Human cytomegalovirus infection impairs neural differentiation via repressing sterol regulatory element binding protein 2-mediated cholesterol biosynthesis.

Congenital human cytomegalovirus (HCMV) infection is a major cause of abnormalities and disorders in the central nervous system (CNS) and/or the peripheral nervous system (PNS). However, the complete pathogenesis of neural differentiation disorders caused by HCMV infection remains to be fully elucidated. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells (MSCs) with a high proliferation and neurogenic differentiation capacity. Since SHEDs originate from the neural crest of the early embryonic ectoderm, SHEDs were hypothesized to serve as a promising cell line for investigating the pathogenesis of neural differentiation disorders in the PNS caused by congenital HCMV infection. In this work, SHEDs were demonstrated to be fully permissive to HCMV infection and the virus was able to complete its life cycle in SHEDs. Under neurogenic inductive conditions, HCMV infection of SHEDs caused an abnormal neural morphology. The expression of stem/neural cell markers was also disturbed by HCMV infection. The impairment of neural differentiation was mainly due to a reduction of intracellular cholesterol levels caused by HCMV infection. Sterol regulatory element binding protein-2 (SREBP2) is a critical transcription regulator that guides cholesterol synthesis. HCMV infection was shown to hinder the migration of SREBP2 into nucleus and resulted in perinuclear aggregations of SREBP2 during neural differentiation. Our findings provide new insights into the prevention and treatment of nervous system diseases caused by congenital HCMV infection.

Endophytic Aspergillus fumigatiaffinis: Novel paclitaxel production and optimization insights.

This study investigated the potential of endophytic fungi to produce paclitaxel (Taxol®), a potent anticancer compound widely employed in chemotherapy. This research aimed to identify, confirm, and characterize endophytic fungi capable of paclitaxel (PTX) production and assess their paclitaxel yield. Additionally, it aimed to investigate factors influencing paclitaxel production. A total of 100 endophytic fungal isolates were collected and identified from the roots of Artemisia judaica. Aspergillus fumigatiaffinis exhibited the highest PTX production (26.373 µg L-1) among the isolated endophytic fungi. The strain was identified as A. fumigatiaffinis (Accession No. PP235788.1). Molecular identification confirmed its novelty, representing the first report of PTX production by A. fumigatiaffinis, an endophyte of Artemisia judaica. Optimization through full factorial design of experiments (DOE) and response surface methodology (RSM) significantly enhanced PTX production to 110.23 µg L-1 from 1 g of dry weight of the fungal culture under optimal conditions of pH 8.0, 150 µg L-1 becozyme supplementation, and 18 days of fermentation in potato dextrose broth. The presence of paclitaxel was confirmed using thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry. These findings maximize the role of endophytic fungus to produce a secondary metabolite that might be able to replace the chemically produced PTX and gives an opportunity to provide a sustainable source of PTX eco-friendly at high concentrations. KEY POINTS: • Endophytic fungi, like A. fumigatiaffinis, show promise for eco-friendly paclitaxel production • Optimization strategies boost paclitaxel yield significantly, reaching 110.23 µg L -1 • Molecular identification confirms novelty, offering a sustainable PTX source.

Dysregulated proteasome activity and steroid hormone biosynthesis are associated with mortality among patients with acute COVID-19.

The persistence of coronavirus disease 2019 (COVID-19)-related hospitalization severely threatens medical systems worldwide and has increased the need for reliable detection of acute status and prediction of mortality. We applied a systems biology approach to discover acute-stage biomarkers that could predict mortality. A total 247 plasma samples were collected from 103 COVID-19 (52 surviving COVID-19 patients and 51 COVID-19 patients with mortality), 51 patients with other infectious diseases (IDCs) and 41 healthy controls (HCs). Paired plasma samples were obtained from survival COVID-19 patients within 1 day after hospital admission and 1-3 days before discharge. There were clear differences between COVID-19 patients and controls, as well as substantial differences between the acute and recovery phases of COVID-19. Samples from patients in the acute phase showed suppressed immunity and decreased steroid hormone biosynthesis, as well as elevated inflammation and proteasome activation. These findings were validated by enzyme-linked immunosorbent assays and metabolomic analyses in a larger cohort. Moreover, excessive proteasome activity was a prominent signature in the acute phase among patients with mortality, indicating that it may be a key cause of poor prognosis. Based on these features, we constructed a machine learning panel, including four proteins [C-reactive protein (CRP), proteasome subunit alpha type (PSMA)1, PSMA7, and proteasome subunit beta type (PSMB)1)] and one metabolite (urocortisone), to predict mortality among COVID-19 patients (area under the receiver operating characteristic curve: 0.976) on the first day of hospitalization. Our systematic analysis provides a novel method for the early prediction of mortality in hospitalized COVID-19 patients.

Identification of tomato F-box proteins functioning in phenylpropanoid metabolism.

Phenylpropanoids, a class of specialized metabolites, play crucial roles in plant growth and stress adaptation and include diverse phenolic compounds such as flavonoids. Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are essential enzymes functioning at the entry points of general phenylpropanoid biosynthesis and flavonoid biosynthesis, respectively. In Arabidopsis, PAL and CHS are turned over through ubiquitination-dependent proteasomal degradation. Specific kelch domain-containing F-Box (KFB) proteins as components of ubiquitin E3 ligase directly interact with PAL or CHS, leading to polyubiquitinated PAL and CHS, which in turn influences phenylpropanoid and flavonoid production. Although phenylpropanoids are vital for tomato nutritional value and stress responses, the post-translational regulation of PAL and CHS in tomato remains unknown. We identified 31 putative KFB-encoding genes in the tomato genome. Our homology analysis and phylogenetic study predicted four PAL-interacting SlKFBs, while SlKFB18 was identified as the sole candidate for the CHS-interacting KFB. Consistent with their homolog function, the predicted four PAL-interacting SlKFBs function in PAL degradation. Surprisingly, SlKFB18 did not interact with tomato CHS and the overexpression or knocking out of SlKFB18 did not affect phenylpropanoid contents in tomato transgenic lines, suggesting its irreverence with flavonoid metabolism. Our study successfully discovered the post-translational regulatory machinery of PALs in tomato while highlighting the limitation of relying solely on a homology-based approach to predict interacting partners of F-box proteins.

Mapping a sustainable approach: biosynthesis of lactobacilli-silver nanocomposites using whey-based medium for antimicrobial and bioactivity applications.

This study explores a sustainable approach for synthesizing silver nanocomposites (AgNCs) with enhanced antimicrobial and bioactivity using safe Lactobacillus strains and a whey-based medium (WBM). WBM effectively supported the growth of Lactobacillus delbrueckii and Lactobacillus acidophilus, triggering a stress response that led to AgNCs formation. The synthesized AgNCs were characterized using advanced spectroscopic and imaging techniques such as UV‒visible, Fourier transform infrared (FT-IR) spectroscopy, transmission electron (TEM), and scanning electron microscopy with energy dispersive X-ray analysis (SEM-Edx). Lb acidophilus-synthesized AgNCs in WBM (had DLS size average 817.2-974.3 ± PDI = 0.441 nm with an average of metal core size 13.32 ± 3.55 nm) exhibited significant antimicrobial activity against a broad spectrum of pathogens, including bacteria such as Escherichia coli (16.47 ± 2.19 nm), Bacillus cereus (15.31 ± 0.43 nm), Clostridium perfringens (25.95 ± 0.03 mm), Enterococcus faecalis (32.34 ± 0.07 mm), Listeria monocytogenes (23.33 ± 0.05 mm), methicillin-resistant Staphylococcus aureus (MRSA) (13.20 ± 1.76 mm), and filamentous fungi such as Aspergillus brasiliensis (33.46 ± 0.01 mm). In addition, Lb acidophilus-synthesized AgNCs in WBM exhibit remarkable free radical scavenging abilities, suggesting their potential as bioavailable antioxidants. These findings highlight the dual functionality of these biogenic AgNCs, making them promising candidates for applications in both medicine and nutrition.

A RsrC-RsrA-RsrB transcriptional circuit positively regulates polysaccharide-degrading enzyme biosynthesis and development in Penicillium oxalicum.

Filamentous fungi produce polysaccharide-degrading enzymes, which is controlled by poorly understood transcriptional circuits. Here we show that a circuit comprising RsrC-RsrA-RsrB (Rsr: production of raw-starch-degrading enzyme regulator) that positively regulates production of raw starch-degrading enzymes in Penicillium oxalicum. Transcription factor (TF) RsrA is essential for biosynthesis of raw starch-degrading enzymes. RsrB and RsrC containing Zn2Cys6- and C2H2-zinc finger domains, act downstream and upstream of RsrA, respectively. RsrA activates rsrB transcription, and three nucleotides (G-286, G-287 and G-292) of rsrB promoter region are required for RsrA, in terms of TF, for binding. RsrB165-271 binds to DNA sequence 5'-TCGATCAGGCACGCC-3' in the promoter region of the gene encoding key raw-starch-degrading enzyme PoxGA15A. RsrC specifically binds rsrA promoter, but not amylase genes, to positively regulate the expression of rsrA and the production of raw starch-degrading enzymes. These findings expand complex regulatory network of fungal raw starch-degrading enzyme biosynthesis.