A trait-based ecological perspective on the soil microbial antibiotic-related genetic machinery.

Antibiotic resistance crisis dictates the need for resistance monitoring and the search for new antibiotics. The development of monitoring protocols is hindered by the great diversity of resistance factors, while the "streetlight effect" denies the possibility of discovering novel drugs based on existing databases. In this study, we address these challenges using high-throughput environmental screening viewed from a trait-based ecological perspective. Through an in-depth analysis of the metagenomes of 658 topsoil samples spanning Europe, we explored the distribution of 241 prokaryotic and fungal genes responsible for producing metabolites with antibiotic properties and 485 antibiotic resistance genes. We analyzed the diversity of these gene collections at different levels and modeled the distribution of each gene across environmental gradients. Our analyses revealed several nonparallel distribution patterns of the genes encoding sequential steps of enzymatic pathways synthesizing large antibiotic groups, pointing to gaps in existing databases and suggesting potential for discovering new analogues of known antibiotics. We show that agricultural activity caused a continental-scale homogenization of microbial antibiotic-related machinery, emphasizing the importance of maintaining indigenous ecosystems within the landscape mosaic. Based on the relationships between the proportion of the genes in the metagenomes with the main predictors (soil pH, land cover type, climate temperature and humidity), we illustrate how the properties of chemical structures dictate the distribution of the genes responsible for their synthesis across environments. With this understanding, we propose general principles to facilitate the discovery of antibiotics, including principally new ones, establish abundance baselines for antibiotic resistance genes, and predict their dissemination.

Trapping the substrate radical of heme synthase AhbD.

The heme synthase AhbD catalyzes the last step of the siroheme-dependent heme biosynthesis pathway, which is operative in archaea and sulfate-reducing bacteria. The AhbD-catalyzed reaction consists of the oxidative decarboxylation of two propionate side chains of iron-coproporphyrin III to the corresponding vinyl groups of heme b. AhbD is a Radical SAM enzyme employing radical chemistry to achieve the decarboxylation reaction. Previously, it was proposed that the central iron ion of the substrate iron-coproporphyrin III participates in the reaction by enabling electron transfer from the initially formed substrate radical to an iron-sulfur cluster in AhbD. In this study, we investigated the substrate radical that is formed during AhbD catalysis. While the iron-coproporphyrinyl radical was not detected by electron paramagnetic resonance (EPR) spectroscopy, trapping and visualization of the substrate radical was successful by employing substrate analogs such as coproporphyrin III and zinc-coproporphyrin III. The radical signals detected by EPR were analyzed by simulations based on density functional theory (DFT) calculations. The observed radical species on the substrate analogs indicate that hydrogen atom abstraction takes place at the ß-position of the propionate side chain and that an electron donating ligand is located in proximity to the central metal ion of the porphyrin.

Aspartate in tumor microenvironment and beyond: Metabolic interactions and therapeutic perspectives.

Aspartate is a proteinogenic non-essential amino acid with several essential functions in proliferating cells. It is mostly produced in a cell autonomous manner from oxalacetate via glutamate oxalacetate transaminases 1 or 2 (GOT1 or GOT2), but in some cases it can also be salvaged from the microenvironment via transporters such as SLC1A3 or by macropinocytosis. In this review we provide an overview of biosynthetic pathways that produce aspartate endogenously during proliferation. We discuss conditions that favor aspartate uptake as well as possible sources of exogenous aspartate in the microenvironment of tumors and bone marrow, where most available data have been generated. We highlight metabolic fates of aspartate, its various functions, and possible approaches to target aspartate metabolism for cancer therapy.

Integrated transcriptome and metabolism unravel critical roles of carbon metabolism and oxidoreductase in mushroom with Korshinsk peashrub substrates.

Edible fungi cultivation serves as an efficient biological approach to transforming agroforestry byproducts, particularly Korshinsk peashrub (KP) branches into valuable mushroom (Lentinus edodes) products. Despite the widespread use of KP, the molecular mechanisms underlying its regulation of mushroom development remain largely unknown. In this study, we conducted a combined analysis of transcriptome and metabolism of mushroom fruiting bodies cultivated on KP substrates compared to those on apple wood sawdust (AWS) substrate. Our aim was to identify key metabolic pathways and genes that respond to the effects of KP substrates on mushrooms. The results revealed that KP induced at least a 1.5-fold increase in protein and fat content relative to AWS, with 15% increase in polysaccharide and total sugar content in mushroom fruiting bodies. There are 1196 differentially expressed genes (DEGs) between mushrooms treated with KP relative to AWS. Bioinformatic analysis show significant enrichments in amino acid metabolic process, oxidase activity, malic enzyme activity and carbon metabolism among the 698 up-regulated DEGs induced by KP against AWS. Additionally, pathways associated with organic acid transport and methane metabolism were significantly enriched among the 498 down-regulated DEGs. Metabolomic analysis identified 439 differentially abundant metabolites (DAMs) in mushrooms treated with KP compared to AWS. Consistent with the transcriptome data, KEGG analysis on metabolomic dataset suggested significant enrichments in carbon metabolism, alanine, aspartate and glutamate metabolism among the up-regulated DAMs by KP. In particular, some DAMs were enhanced by 1.5-fold, including D-glutamine, L-glutamate, glucose and pyruvate in mushroom samples treated with KP relative to AWS. Targeted metabolomic analysis confirmed the contents of DAMs related to glutamate metabolism and energy metabolism. In conclusion, our findings suggest that reprogrammed carbon metabolism and oxidoreductase pathways act critical roles in the enhanced response of mushroom to KP substrates.

Harnessing the power of Neobacillus niacini AUMC-B524 for silver oxide nanoparticle synthesis: optimization, characterization, and bioactivity exploration.

BACKGROUND: Biotechnology provides a cost-effective way to produce nanomaterials such as silver oxide nanoparticles (Ag2ONPs), which have emerged as versatile entities with diverse applications. This study investigated the ability of endophytic bacteria to biosynthesize Ag2ONPs. RESULTS: A novel endophytic bacterial strain, Neobacillus niacini AUMC-B524, was isolated from Lycium shawii Roem. & Schult leaves and used to synthesize Ag2ONPS extracellularly. Plackett-Burman design and response surface approach was carried out to optimize the biosynthesis of Ag2ONPs (Bio-Ag2ONPs). Comprehensive characterization techniques, including UV-vis spectral analysis, Fourier transform infrared spectroscopy, transmission electron microscopy, X-ray diffraction, dynamic light scattering analysis, Raman microscopy, and energy dispersive X-ray analysis, confirmed the precise composition of the Ag2ONPS. Bio-Ag2ONPs were effective against multidrug-resistant wound pathogens, with minimum inhibitory concentrations (1-25 µg mL-1). Notably, Bio-Ag2ONPs demonstrated no cytotoxic effects on human skin fibroblasts (HSF) in vitro, while effectively suppressing the proliferation of human epidermoid skin carcinoma (A-431) cells, inducing apoptosis and modulating the key apoptotic genes including Bcl-2 associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), Caspase-3 (Cas-3), and guardian of the genome (P53). CONCLUSIONS: These findings highlight the therapeutic potential of Bio-Ag2ONPs synthesized by endophytic N. niacini AUMC-B524, underscoring their antibacterial efficacy, anticancer activity, and biocompatibility, paving the way for novel therapeutic strategies.

Biosynthetic Strategies of Berberine Bridge Enzyme-like Flavoprotein Oxidases toward Structural Diversification in Natural Product Biosynthesis.

Berberine bridge enzyme-like oxidases are often involved in natural product biosynthesis and are seen as essential enzymes for the generation of intricate pharmacophores. These oxidases have the ability to transfer a hydride atom to the FAD cofactor, which enables complex substrate modifications and rearrangements including (intramolecular) cyclizations, carbon-carbon bond formations, and nucleophilic additions. Despite the diverse range of activities, the mechanistic details of these reactions often remain incompletely understood. In this Review, we delve into the complexity that BBE-like oxidases from bacteria, fungal, and plant origins exhibit by providing an overview of the shared catalytic features and emphasizing the different reactivities. We propose four generalized modes of action by which BBE-like oxidases enable the synthesis of natural products, ranging from the classic alcohol oxidation reactions to less common amine and amide oxidation reactions. Exploring the mechanisms utilized by nature to produce its vast array of natural products is a subject of considerable interest and can lead to the discovery of unique biochemical activities.

The total biosynthesis route of rosmarinic acid in Sarcandra glabra based on transcriptome sequencing.

Sarcandra glabra is a widely distributed and valuable plant in food and daily chemical industries, and is also a common-used medicinal plant for treating inflammatory diseases and tumors. Rosmarinic acid (RA) with significant pharmacological activity is an abundant and important constituent in S. glabra, however, little information about key enzymes involving the biosynthesis of RA in S. glabra is available and the underlying biosynthesis mechanisms of RA in S. glabra remain undeciphered. Therefore, in this study, by full-length transcriptome sequencing analyses of S. glabra, we screened the RA biosynthesis candidate genes based on sequence similarity and conducted enzymatic function characterization in vitro and in vivo. As a result, a complete set of 7 kinds of enzymes (SgPALs, SgC4H, Sg4CL, SgTATs, SgHPPRs, SgRAS and SgC3H) involving the biosynthesis route of RA from phenylalanine and tyrosine, were identified and fully characterized. This research systematically revealed the complete biosynthesis route of RA in S. glabra, which helps us better understand the process of RA synthesis and accumulation, especially the substrate promiscuities of SgRAS and SgC3H provide the molecular biological basis for the efficient biosynthesis of specific and abundant RA in S. glabra. The 7 kinds of key enzymes revealed in this study can be utilized as tool enzymes for production of RA by synthetic biology methods.

Sucrose Phosphate Synthase Genes in Plants: Its Role and Practice for Crop Improvement.

Plants convert solar energy and carbon dioxide into organic compounds through photosynthesis. Sucrose is the primary carbonate produced during photosynthesis. Sucrose phosphate synthase (SPS) is the key enzyme controlling sucrose biosynthesis in plants. There are at least three SPS gene families in higher plants, named A, B, and C. However, in monocotyledonous plants from Poaceae, there are at least five SPS gene families, named A, B, C, DIII, and DIV. Each family of SPS genes in different plants shows a divergent expression pattern. So different families of SPS genes participate in diverse biological functions, including sucrose accumulation, plant growth and production, and abiotic stress tolerance. SPS activity in plants is regulated by exogenous factors through gene expression and reversible protein phosphorylation. It is a practicable way to improve crop traits through SPS gene transformation. This work analyzes the cloning, phylogeny, and regulatory mechanism of the SPS gene in plants, reviews its biological function as well as its role in crop improvement, and discusses the challenges and future perspectives. This paper can serve as a reference for further study on plant SPS genes and eventually for crop improvement.

Recent progresses in the cyclization and oxidation of polyketide biosynthesis.

Polyketides represent an important class of natural products, renowned for their intricate structures and diverse biological activities. In contrast to common fatty acids, polyketides possess relatively more rigid carbon skeletons, more complex ring systems, and chiral centers. These structural features are primarily achieved through distinctive enzymatic cyclizations and oxidations as tailoring steps. In this opinion, we discuss the recent progress in deciphering the mechanisms of cyclization and oxidation within polyketide biosynthesis. By shedding light on these enzymatic processes, this article seeks to motivate the community to unravel the remaining mysteries surrounding cyclase and oxidase functionalities and to explore novel polyketide natural products through genome mining.

A transcription factor of SHI family AaSHI1 activates artemisinin biosynthesis genes in Artemisia annua.

BACKGROUND: Transcription factors (TFs) of plant-specific SHORT INTERNODES (SHI) family play a significant role in regulating development and metabolism in plants. In Artemisia annua, various TFs from different families have been discovered to regulate the accumulation of artemisinin. However, specific members of the SHI family in A. annua (AaSHIs) have not been identified to regulate the biosynthesis of artemisinin. RESULTS: We found five AaSHI genes (AaSHI1 to AaSHI5) in the A. annua genome. The expression levels of AaSHI1, AaSHI2, AaSHI3 and AaSHI4 genes were higher in trichomes and young leaves, also induced by light and decreased when the plants were subjected to dark treatment. The expression pattern of these four AaSHI genes was consistent with the expression pattern of four structural genes of artemisinin biosynthesis and their specific regulatory factors. Dual-luciferase reporter assays, yeast one-hybrid assays, and transient transformation in A. annua provided the evidence that AaSHI1 could directly bind to the promoters of structural genes AaADS and AaCYP71AV1, and positively regulate their expressions. This study has presented candidate genes, with AaSHI1 in particular, that can be considered for the metabolic engineering of artemisinin biosynthesis in A. annua. CONCLUSIONS: Overall, a genome-wide analysis of the AaSHI TF family of A. annua was conducted. Five AaSHIs were identified in A. annua genome. Among the identified AaSHIs, AaSHI1 was found to be localized to the nucleus and activate the expression of structural genes of artemisinin biosynthesis including AaADS and AaCYP71AV1. These results indicated that AaSHI1 had positive roles in modulating artemisinin biosynthesis, providing candidate genes for obtaining high-quality new A. annua germplasms.

Integrative Transcriptomic and Metabolic Analyses Reveal That Flavonoid Biosynthesis Is the Key Pathway Regulating Pigment Deposition in Naturally Brown Cotton Fibers.

Brown cotton is a major cultivar of naturally colored cotton, and brown cotton fibers (BCFs) are widely utilized as raw materials for textile industry production due to their advantages of being green and dyeing-pollution-free. However, the mechanisms underlying the pigmentation in fibers are still poorly understood, which significantly limits their extensive applications in related fields. In this study, we conducted a multidimensional comparative analysis of the transcriptomes and metabolomes between brown and white fibers at different developmental periods to identify the key genes and pathways regulating the pigment deposition. The transcriptomic results indicated that the pathways of flavonoid biosynthesis and phenylpropanoid biosynthesis were significantly enriched regulatory pathways, especially in the late development periods of fiber pigmentation; furthermore, the genes distributed in the pathways of PAL, CHS, F3H, DFR, ANR, and UFGT were identified as significantly up-regulated genes. The metabolic results showed that six metabolites, namely (-)-Epigallocatechin, Apiin, Cyanidin-3-O-glucoside, Gallocatechin, Myricetin, and Poncirin, were significantly accumulated in brown fibers but not in white fibers. Integrative analysis of the transcriptomic and metabolomic data demonstrated a possible regulatory network potentially regulating the pigment deposition, in which three MYB transcription factors promote the expression levels of flavonoid biosynthesis genes, thereby inducing the content increase in (-)-Epigallocatechin, Cyanidin-3-O-glucoside, Gallocatechin, and Myricetin in BCFs. Our findings provide new insights into the pigment deposition mechanism in BCFs and offer references for genetic engineering and breeding of colored cotton materials.

Unlocking the Transcriptional Reprogramming Repertoire between Variety-Dependent Responses of Grapevine Berries to Infection by Aspergillus carbonarius.

Aspergillus carbonarius causes severe decays on berries in vineyards and is among the main fungal species responsible for grape contamination by ochratoxin A (OTA), which is the foremost mycotoxin produced by this fungus. The main goal of this study was to investigate at the transcriptome level the comparative profiles between two table grape varieties (Victoria and Fraoula, the white and red variety, respectively) after their inoculation with a virulent OTA-producing A. carbonarius strain. The two varieties revealed quite different transcriptomic signatures and the expression profiles of the differential expressed genes (DEGs) highlighted distinct and variety-specific responses during the infection period. The significant enrichment of pathways related to the modulation of transcriptional dynamics towards the activation of defence responses, the triggering of the metabolic shunt for the biosynthesis of secondary metabolites, mainly phenylpropanoids, and the upregulation of DEGs encoding phytoalexins, transcription factors, and genes involved in plant-pathogen interaction and immune signaling transduction was revealed in an early time point in Fraoula, whereas, in Victoria, any transcriptional reprogramming was observed after a delay. However, both varieties, to some extent, also showed common expression dynamics for specific DEG families, such as those encoding for laccases and stilbene synthases. Jasmonate (JA) may play a critical modulator role in the defence machinery as various JA-biosynthetic DEGs were upregulated. Along with the broader modulation of the transcriptome that was observed in white grape, expression profiles of specific A. carbonarius genes related to pathogenesis, fungal sporulation, and conidiation highlight the higher susceptibility of Victoria. Furthermore, the A. carbonarius transcriptional patterns directly associated with the regulation of the pathogen OTA-biosynthesis gene cluster were more highly induced in Victoria than in Fraoula. The latter was less contaminated by OTA and showed substantially lower sporulation. These findings contribute to uncovering the interplay beyond this plant-microbe interaction.

Abscisic Acid Regulates the Occurrence and Recovery of the Striped Leaf Phenotype in Response to Lacking Light at the Base of Sheath in Rice by Modulating Carbohydrate Metabolism.

Rice B03S mutants with intermittent leaf discoloration were developed from the photoperiod- and thermosensitive genic male sterile (PTGMS) rice line Efeng 1S. After these plants were deeply transplanted, the new leaves manifested typical stripe patterns. In this study, deep and shallow transplantation of B03S was carried out, and aluminum shading was performed directly on the leaf sheath. It was determined that the reason for the appearance of the striped leaf trait was that the base of leaf sheath lacked light, at which time the sheath transformed from the source organ to the sink organ in rice. To elucidate the related metabolic changes in glycometabolism and abscisic acid (ABA) biosynthesis and transcriptional regulation in the leaf sheath, ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) combined with transcriptome and real-time quantitative PCR (qPCR) validation were used for analysis after deep and shallow transplantation. The result indicates that the leaf sheath may need to compete with the new leaves for sucrose produced by the photosynthesis of old leaves in response to lacking light at the base of sheath. Moreover, the ABA content increases in the leaf sheath when the gene expression of ABA2 and AAO1 is upregulated at the same time, enhancing the plant's resistance to the adverse condition of shading at the leaf sheath. Furthermore, exogenous spraying of B03S with ABA solution was carried out to help recovery under shading stress. The result indicates that the synthesis of endogenous ABA in the leaf sheath is reduced by spraying ABA. At the same time, ABA regulates sucrose metabolism by inhibiting the expression of the SUS gene. This allows for more sucrose synthesized by the old leaves to be transported to the new leaves, resulting an obvious recovery effect of the strip leaf character due to the re-balance of sugar supply and demand in B03S. These findings improve the understanding of the physiological function and metabolic mechanism of the rice leaf sheath, provide a theoretical basis for uneven leaf coloration in nature, and provide theoretical guidance for rice production via seedling transplantation or direct seeding.

Proteomic and phosphoproteomic analysis of a Haematococcus pluvialis (Chlorophyceae) mutant with a higher heterotrophic cell division rate reveals altered pathways involved in cell proliferation and nutrient partitioning.

Haematococcus pluvialis has been used to produce the ketocarotenoid antioxidant, astaxanthin. Currently, heterotrophic cultivation of H. pluvialis is limited by slow growth rates. This work aimed to address this challenge by exploring the mechanisms of acetate metabolism in Haematococcus. Chemical mutagenesis and screening identified H. pluvialis strain KREMS 23D-3 that achieved up to a 34.9% higher cell density than the wild type when grown heterotrophically on acetate. An integrative proteomics and phosphoproteomics approach was employed to quantify 4955 proteins and 5099 phosphorylation sites from 2505 phosphoproteins in the wild-type and mutant strains of H. pluvialis. Among them, 12 proteins were significantly upregulated and 22 significantly downregulated in the mutant while phosphoproteomic analysis identified 143 significantly upregulated phosphorylation sites on 106 proteins and 130 downregulated phosphorylation sites on 114 proteins. Upregulation of anaphase-promoting complex phosphoproteins and downregulation of a putative cell cycle division 20 phosphoprotein in the mutant suggests rapid mitotic progression, coinciding with higher cell division rates. Upregulated coproporphyrinogen oxidase and phosphorylated magnesium chelatase in the mutant demonstrated altered nitrogen partitioning toward chlorophyll biosynthesis. The large proportion of differentially expressed phosphoproteins suggests phosphorylation is a key regulator for protein expression and activity in Haematococcus. Taken together, this study reveals the regulation of interrelated acetate metabolic pathways in H. pluvialis and provides protein targets that may guide future strain engineering work.

Towards a thorough understanding of mammalian glycosylphosphatidylinositol-anchored protein biosynthesis.

Glycosylphosphatidylinositols (GPIs) are glycolipids found ubiquitously in eukaryotes. They consist of a glycan and an inositol phospholipid, and act as membrane anchors of many cell-surface proteins by covalently linking to their C-termini. GPIs also exist as unlinked, free glycolipids on the cell surface. In human cells, at least 160 proteins with various functions are GPI-anchored proteins (GPI-APs). Because the attachment of GPI is required for the cell-surface expression of GPI-APs, a thorough knowledge of the molecular basis of mammalian GPI-AP biosynthesis is important for understanding the basic biochemistry and biology of GPI-APs and their medical significance. In this paper, I review our previous knowledge of the biosynthesis of mammalian GPI-APs and then examine new findings made since 2020.

Reduced seed viability in exchange for transgenerational plant protection: Does the defensive mutualism concept pass the fitness test?

BACKGROUND AND AIMS: Epichloë endophytes are vertically transmitted via grass seeds and chemically defend their hosts against herbivory. Endophyte-conferred plant defence via alkaloid biosynthesis may occur independently of costs for host plant growth. However, fitness consequences of endophyte-conferred defence and transgenerational effects on herbivore resistance of progeny plants, are rarely studied. The aim of this study was to test whether severe defoliation in mother plants affects their seed production, seed germination rate, and the endophyte-conferred resistance of progeny plants. METHODS: In a field study, we tested the effects of defoliation and endophyte symbiosis (Epichloë uncinata) on host plant (Festuca pratensis) performance, loline alkaloid concentrations in leaves and seeds, seed biomass and seed germination rates. In a subsequent greenhouse study, we challenged the progeny of the plants from the field study to aphid herbivory and tested whether defoliation of mother plants affects endophyte-conferred resistance against aphids in progeny plants. KEY RESULTS: Defoliation of the mother plants resulted in a reduction of alkaloid concentrations in leaves and elevated the alkaloid concentrations in seeds when compared with non-defoliated endophyte-symbiotic plants. Viability and germination rate of seeds of defoliated endophyte-symbiotic plants were significantly lower compared to those of non-defoliated endophyte-symbiotic plants and endophyte-free (defoliated and non-defoliated) plants. During six weeks growth, seedlings of defoliated endophyte-symbiotic mother plants had elevated alkaloid concentrations, which negatively correlated with aphid performance. CONCLUSIONS: Endophyte-conferred investment in higher alkaloid levels in seeds -elicited by defoliation- provided herbivore protection in progenies during the first weeks of plant establishment. Better protection of seeds via high alkaloid concentrations negatively correlated with seed germination indicating trade-off between protection and viability.

Five unaddressed questions about cytokinin biosynthesis.

Cytokinins, a class of phytohormones, play crucial roles in regulating plant growth and stress responses through finely tuned feedback loops involving metabolic and signaling cascades. Cytokinin metabolism modulates the abundance of these biologically active molecules. Over the past 25 years, studies have identified key genes involved in cytokinin biosynthesis and inactivation pathways. Nevertheless, several gaps remain in our understanding, particularly regarding the movement of intermediate metabolites between subcellular compartments and the discrepancy between the product of adenosine phosphate-isopentenyltransferase (IPT) and the substrate preferences of subsequent reactions. In addition, recent gene discoveries related to lonely guy (LOG)-independent pathways suggest a spatial extension of cytokinin biosynthesis into the apoplast. Other intriguing issues remain to be addressed, i.e., elucidating the synthetic pathway for cis-zeatin and unraveling the molecular mechanisms governing selective substrate use by the cytokinin biosynthetic enzyme tumor morphology root (Tmr) derived from the phytopathogen Agrobacterium tumefaciens during crown gall formation. Further studies are needed to reveal a fully comprehensive picture of cytokinin metabolism.

Bioflavonoid Baicalein Modulates Tetracycline Resistance by Inhibiting Efflux Pump in Staphylococcus aureus.

The rise in antibiotic resistance among bacterial pathogens, particularly Staphylococcus aureus, has become a critical global health issue, necessitating the search for novel antimicrobial agents. S. aureus uses various mechanisms to resist antibiotics, including the activation of efflux pumps, biofilm formation, and enzymatic modification of drugs. This study explores the potential of baicalein, a bioflavonoid from Scutellaria baicalensis, in modulating tetracycline resistance in S. aureus by inhibiting efflux pumps. The synergistic action of baicalein and tetracycline was evaluated through various assays. The minimum inhibitory concentration (MIC) of baicalein and tetracycline against S. aureus was 256 and 1.0 µg/mL, respectively. Baicalein at 64 µg/mL reduced the MIC of tetracycline by eightfold, indicating a synergistic effect (fractional inhibitory concentration index: 0.375). Time-kill kinetics demonstrated a 1.0 log CFU/mL reduction in bacterial count after 24 hours with the combination treatment. The ethidium bromide accumulation assay showed that baicalein mediated significant inhibition of efflux pumps, with a dose-dependent increase in fluorescence. In addition, baicalein inhibited DNA synthesis by 73% alone and 92% in combination with tetracycline. It also markedly reduced biofilm formation and the invasiveness of S. aureus into HeLa cells by 52% at 64 µg/mL. These findings suggest that baicalein enhances tetracycline efficacy and could be a promising adjunct therapy to combat multidrug-resistant S. aureus infections.

Step-by-step optimization of a heterologous pathway for de novo naringenin production in Escherichia coli.

Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli. KEY POINTS: • Best enzyme and strain combination were selected for de novo naringenin production. • After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. • This de novo production is the highest reported so far in E. coli.

Total Biosynthesis of Circular Bacteriocins by Merging the Genetic Engineering and Enzymatic Catalysis.

Circular bacteriocins are known for their structural stability and effective antimicrobial properties, positioning them as potential natural food preservatives. However, their widespread application is impeded by restricted availability. This research developed a total biosynthesis platform for circular bacteriocins, with a focus on AS-48 by involving recombinant production of the linear precursor in Escherichia coli, followed by enzymatic cyclization of the precursor into cyclic AS-48 using the ligase butelase-1 in vitro. An important discovery is that, aside from fusion tags, the C-terminal motif LE and LEKKK also could affect the expression yield of the precursor. This biosynthesis platform is both versatile and high-yielding, achieving yields of 10-20 mg/L of AS-48. Importantly, the biosynthetic AS-48 exhibited a secondary structure and antimicrobial activities comparable to those of the native molecules. As such, this work proposes an effective synthetic approach for circular bacteriocins, facilitating their advancement and application in the food industry.