Two Distinct Enzymes Have Both Phytoene Desaturase and 3,4-Desaturase Activities Involved in Carotenoid Biosynthesis by the Extremely Halophilic Archaeon Haloarcula japonica.

The extremely halophilic archaeon Haloarcula japonica accumulates the C50 carotenoid, bacterioruberin (BR). To reveal the BR biosynthetic pathway, unidentified phytoene desaturase candidates were functionally characterized in the present study. Two genes encoding the potential phytoene desaturases, c0507 and d1086, were found from the Ha. japonica genome sequence by a homology search using the Basic Local Align Search Tool. Disruption mutants of c0507 and d1086 and their complemented strains transformed with expression plasmids for c0507 and d1086 were subsequently constructed. High-performance liquid chromatography (HPLC) ana-lyses of carotenoids produced by these strains revealed that C0507 and D1086 were both bifunctional enzymes with the same activities as both phytoene desaturase (CrtI) and 3,4-desaturase (CrtD). C0507 and D1086 complemented each other during BR biosynthesis in Ha. japonica. This is the first study to identify two distinct enzymes with both CrtI and CrtD activities in an extremely halophilic archaeon.

Nitrous oxide respiration in acidophilic methanotrophs.

Aerobic methanotrophic bacteria are considered strict aerobes but are often highly abundant in hypoxic and even anoxic environments. Despite possessing denitrification genes, it remains to be verified whether denitrification contributes to their growth. Here, we show that acidophilic methanotrophs can respire nitrous oxide (N2O) and grow anaerobically on diverse non-methane substrates, including methanol, C-C substrates, and hydrogen. We study two strains that possess N2O reductase genes: Methylocella tundrae T4 and Methylacidiphilum caldifontis IT6. We show that N2O respiration supports growth of Methylacidiphilum caldifontis at an extremely acidic pH of 2.0, exceeding the known physiological pH limits for microbial N2O consumption. Methylocella tundrae simultaneously consumes N2O and CH4 in suboxic conditions, indicating robustness of its N2O reductase activity in the presence of O2. Furthermore, in O2-limiting conditions, the amount of CH4 oxidized per O2 reduced increases when N2O is added, indicating that Methylocella tundrae can direct more O2 towards methane monooxygenase. Thus, our results demonstrate that some methanotrophs can respire N2O independently or simultaneously with O2, which may facilitate their growth and survival in dynamic environments. Such metabolic capability enables these bacteria to simultaneously reduce the release of the key greenhouse gases CO2, CH4, and N2O.

Rhizobium hidalgonense and Rhizobium redzepovicii as faba bean (Vicia faba L.) microsymbionts in Mexican soils.

As a legume crop widely cultured in the world, faba bean (Vicia faba L.) forms root nodules with diverse Rhizobium species in different regions. However, the symbionts associated with this plant in Mexico have not been studied. To investigate the diversity and species/symbiovar affiliations of rhizobia associated with faba bean in Mexico, rhizobia were isolated from this plant grown in two Mexican sites in the present study. Based upon the analysis of recA gene phylogeny, two genotypes were distinguished among a total of 35 isolates, and they were identified as Rhizobium hidalgonense and Rhizobium redzepovicii, respectively, by the whole genomic sequence analysis. Both the species harbored identical nod gene cluster and the same phylogenetic positions of nodC and nifH. So, all of them were identified into the symbiovar viciae. As a minor group, R. hidalgonense was only isolated from slightly acid soil and R. redzepovicii was the dominant group in both the acid and neutral soils. In addition, several genes related to resistance to metals (zinc, copper etc.) and metalloids (arsenic) were detected in genomes of the reference isolates, which might offer them some adaptation benefits. As conclusion, the community composition of faba bean rhizobia in Mexico was different from those reported in other regions. Furthermore, our study identified sv. viciae as the second symbiovar in the species R. redzepovicii. These results added novel evidence about the co-evolution, diversification and biogeographic patterns of rhizobia in association with their host legumes in distinct geographic regions.

Safflower CtFLS1-Induced Drought Tolerance by Stimulating the Accumulation of Flavonols and Anthocyanins in Arabidopsis thaliana.

Flavonol synthase gene (FLS) is a member of the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily and plays an important role in plant flavonoids biosynthetic pathways. Safflower (Carthamus tinctorius L.), a key source of traditional Chinese medicine, is widely cultivated in China. Although the flavonoid biosynthetic pathway has been studied in several model species, it still remains to be explored in safflower. In this study, we aimed to elucidate the role of CtFLS1 gene in flavonoid biosynthesis and drought stress responses. The bioinformatics analysis on the CtFLS1 gene showed that it contains two FLS-specific motifs (PxxxIRxxxEQP and SxxTxLVP), suggesting its independent evolution. Further, the expression level of CtFLS1 in safflower showed a positive correlation with the accumulation level of total flavonoid content in four different flowering stages. In addition, CtFLS1-overexpression (OE) Arabidopsis plants significantly induced the expression levels of key genes involved in flavonol pathway. On the contrary, the expression of anthocyanin pathway-related genes and MYB transcription factors showed down-regulation. Furthermore, CtFLS1-OE plants promoted seed germination, as well as resistance to osmotic pressure and drought, and reduced sensitivity to ABA compared to mutant and wild-type plants. Moreover, CtFLS1 and CtANS1 were both subcellularly located at the cell membrane and nucleus; the yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assay showed that they interacted with each other at the cell membrane. Altogether, these findings suggest the positive role of CtFLS1 in alleviating drought stress by stimulating flavonols and anthocyanin accumulation in safflower.

A functional study reveals CsNAC086 regulated the biosynthesis of flavonols in Camellia sinensis.

MAIN CONCLUSION: CsNAC086 was found to promote the expression of CsFLS, thus promoting the accumulation of flavonols in Camellia sinensis. Flavonols, the main flavonoids in tea plants, play an important role in the taste and quality of tea. In this study, a NAC TF gene CsNAC086 was isolated from tea plants and confirmed its regulatory role in the expression of flavonol synthase which is a key gene involved in the biosynthesis of flavonols in tea plant. Yeast transcription-activity assays showed that CsNAC086 has self-activation activity. The transcriptional activator domain of CsNAC086 is located in the non-conserved C-terminal region (positions 171-550), while the conserved NAC domain (positions 1-170) does not have self-activation activity. Silencing the CsNAC086 gene using antisense oligonucleotides significantly decreased the expression of CsFLS. As a result, the concentration of flavonols decreased significantly. In overexpressing CsNAC086 tobacco leaves, the expression of NtFLS was significantly increased. Compared with wild-type tobacco, the flavonols concentration increased. Yeast one-hybrid assays showed CsNAC086 did not directly regulate the gene expression of CsFLS. These findings indicate that CsNAC086 plays a role in regulating flavonols biosynthesis in tea plants, which has important implications for selecting and breeding of high-flavonols-concentration containing tea-plant cultivars.

Structure, mechanism, and evolution of the last step in vitamin C biosynthesis.

Photosynthetic organisms, fungi, and animals comprise distinct pathways for vitamin C biosynthesis. Besides this diversity, the final biosynthetic step consistently involves an oxidation reaction carried out by the aldonolactone oxidoreductases. Here, we study the origin and evolution of the diversified activities and substrate preferences featured by these flavoenzymes using molecular phylogeny, kinetics, mutagenesis, and crystallographic experiments. We find clear evidence that they share a common ancestor. A flavin-interacting amino acid modulates the reactivity with the electron acceptors, including oxygen, and determines whether an enzyme functions as an oxidase or a dehydrogenase. We show that a few side chains in the catalytic cavity impart the reaction stereoselectivity. Ancestral sequence reconstruction outlines how these critical positions were affixed to specific amino acids along the evolution of the major eukaryotic clades. During Eukarya evolution, the aldonolactone oxidoreductases adapted to the varying metabolic demands while retaining their overarching vitamin C-generating function.

Sustained bacterial N2O reduction at acidic pH.

Nitrous oxide (N2O) is a climate-active gas with emissions predicted to increase due to agricultural intensification. Microbial reduction of N2O to dinitrogen (N2) is the major consumption process but microbial N2O reduction under acidic conditions is considered negligible, albeit strongly acidic soils harbor nosZ genes encoding N2O reductase. Here, we study a co-culture derived from acidic tropical forest soil that reduces N2O at pH 4.5. The co-culture exhibits bimodal growth with a Serratia sp. fermenting pyruvate followed by hydrogenotrophic N2O reduction by a Desulfosporosinus sp. Integrated omics and physiological characterization revealed interspecies nutritional interactions, with the pyruvate fermenting Serratia sp. supplying amino acids as essential growth factors to the N2O-reducing Desulfosporosinus sp. Thus, we demonstrate growth-linked N2O reduction between pH 4.5 and 6, highlighting microbial N2O reduction potential in acidic soils.

An unexpected role of EasDaf: catalyzing the conversion of chanoclavine aldehyde to chanoclavine acid.

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2), an important intermediate produced by the enzyme EasDaf or its counterpart FgaDH from chanoclavine-I (CC, 1). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasDaf not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. KEY POINTS: • EAs possess complicated skeletons and are widely used in several clinical diseases • EasDaf belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasDaf for dehydrogenation was analyzed by molecular docking and site mutations.

Structural basis for the intracellular regulation of ferritin degradation.

The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy-a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4-FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.

FTH1 overexpression using a dCasRx translation enhancement system protects the kidney from calcium oxalate crystal-induced injury.

The engineered clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is currently widely applied in genetic editing and transcriptional regulation. The catalytically inactivated CasRx (dCasRx) has the ability to selectively focus on the mRNA coding region without disrupting transcription and translation, opening up new avenues for research on RNA modification and protein translation control. This research utilized dCasRx to create a translation-enhancement system for mammals called dCasRx-eIF4GI, which combined eukaryotic translation initiation factor 4G (eIF4GI) to boost translation levels of the target gene by recruiting ribosomes, without affecting mRNA levels, ultimately increasing translation levels of different endogenous proteins. Due to the small size of dCasRx, the dCasRx-eIF4GI translation enhancement system was integrated into a single viral vector, thus optimizing the delivery and transfection efficiency in subsequent applications. Previous studies reported that ferroptosis, mediated by calcium oxalate (CaOx) crystals, significantly promotes stone formation. In order to further validate its developmental potential, it was applied to a kidney stone model in vitro and in vivo. The manipulation of the ferroptosis regulatory gene FTH1 through single-guide RNA (sgRNA) resulted in a notable increase in FTH1 protein levels without affecting its mRNA levels. This ultimately prevented intracellular ferroptosis and protected against cell damage and renal impairment caused by CaOx crystals. Taken together, this study preliminarily validated the effectiveness and application prospects of the dCasRx-eIF4GI translation enhancement system in mammalian cell-based disease models, providing novel insights and a universal tool platform for protein translation research and future therapeutic approaches for nephrolithiasis.

Dissecting the role of soybean rhizosphere-enriched bacterial taxa in modulating nitrogen-cycling functions.

Crop roots selectively recruit certain microbial taxa that are essential for supporting their growth. Within the recruited microbes, some taxa are consistently enriched in the rhizosphere across various locations and crop genotypes, while others are unique to specific planting sites or genotypes. Whether these differentially enriched taxa are different in community composition and how they interact with nutrient cycling need further investigation. Here, we sampled bulk soil and the rhizosphere soil of five soybean varieties grown in Shijiazhuang and Xuzhou, categorized the rhizosphere-enriched microbes into shared, site-specific, and variety-specific taxa, and analyzed their correlation with the diazotrophic communities and microbial genes involved in nitrogen (N) cycling. The shared taxa were dominated by Actinobacteria and Thaumarchaeota, the site-specific taxa were dominated by Actinobacteria in Shijiazhuang and by Nitrospirae in Xuzhou, while the variety-specific taxa were more evenly distributed in several phyla and contained many rare operational taxonomic units (OTUs). The rhizosphere-enriched taxa correlated with most diazotroph orders negatively but with eight orders including Rhizobiales positively. Each group within the shared, site-specific, and variety-specific taxa negatively correlated with bacterial amoA and narG in Shijiazhuang and positively correlated with archaeal amoA in Xuzhou. These results revealed that the shared, site-specific, and variety-specific taxa are distinct in community compositions but similar in associations with rhizosphere N-cycling functions. They exhibited potential in regulating the soybean roots' selection for high-efficiency diazotrophs and the ammonia-oxidizing and denitrification processes. This study provides new insights into soybean rhizosphere-enriched microbes and their association with N cycling. KEY POINTS: • Soybean rhizosphere affected diazotroph community and enriched nifH, amoA, and nosZ. • Shared and site- and variety-specific taxa were dominated by different phyla. • Rhizosphere-enriched taxa were similarly associated with N-cycle functions.

Enantioselective transformation of phytoplankton-derived dihydroxypropanesulfonate by marine bacteria.

Chirality, a fundamental property of matter, is often overlooked in the studies of marine organic matter cycles. Dihydroxypropanesulfonate (DHPS), a globally abundant organosulfur compound, serves as an ecologically important currency for nutrient and energy transfer from phytoplankton to bacteria in the ocean. However, the chirality of DHPS in nature and its transformation remain unclear. Here, we developed a novel approach using chiral phosphorus-reagent labeling to separate DHPS enantiomers. Our findings demonstrated that at least one enantiomer of DHPS is present in marine diatoms and coccolithophores, and that both enantiomers are widespread in marine environments. A novel chiral-selective DHPS catabolic pathway was identified in marine Roseobacteraceae strains, where HpsO and HpsP dehydrogenases at the gateway to DHPS catabolism act specifically on R-DHPS and S-DHPS, respectively. R-DHPS is also a substrate for the dehydrogenase HpsN. All three dehydrogenases generate stable hydrogen bonds between the chirality-center hydroxyls of DHPS and highly conserved residues, and HpsP also form coordinate-covalent bonds between the chirality-center hydroxyls and Zn2+, which determines the mechanistic basis of strict stereoselectivity. We further illustrated the role of enzymatic promiscuity in the evolution of DHPS metabolism in Roseobacteraceae and SAR11. This study provides the first evidence of chirality's involvement in phytoplankton-bacteria metabolic currencies, opening a new avenue for understanding the ocean organosulfur cycle.

Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin.

Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light accumulated diatoxanthin to diadinoxanthin in low-light in diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin-diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.

Radical fluorine transfer catalysed by an engineered nonheme iron enzyme.

Nonheme iron enzymes stand out as one of the most versatile biocatalysts for molecular functionalization. They facilitate a wide array of chemical transformations within biological processes, including hydroxylation, chlorination, epimerization, desaturation, cyclization, and more. Beyond their native biological functions, these enzymes possess substantial potential as powerful biocatalytic platforms for achieving abiological metal-catalyzed reactions, owing to their functional and structural diversity and high evolvability. To this end, our group has recently engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for abiological radical transformations not previously known in biology. Notably, we have demonstrated that a nonheme iron enzyme, (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE), can be repurposed into an efficient and selective biocatalyst for radical fluorine transfer reactions. This marks the first known instance of a redox enzymatic process for C(sp3)F bond formation. This chapter outlines the detailed experimental protocol for engineering SvHPPE for fluorination reactions. Furthermore, the provided protocol could serve as a general guideline that might facilitate other engineering endeavors targeting nonheme iron enzymes for novel catalytic functions.

Clinical application value of pre-pregnancy carrier screening in Chinese Han childbearing population.

BACKGROUND: To explore the clinical application value of pre-conception expanded carrier screening (PECS) in the Chinese Han ethnicity population of childbearing age. METHODS: The results of genetic testing of infertile parents who underwent PECS in the Reproductive Medicine Center of the Second Affiliated Hospital of Zhengzhou University, China, from September 2019 to December 2021, were retrospectively analyzed. The carrier rate of single gene disease, the detection rate of high-risk parents, and the clinical outcome of high-risk parents were statistically analyzed. RESULTS: A total of 1372 Chinese Han ethnicity patients underwent PECS, among which 458 patients underwent the extended 108-gene test, their overall carrier rate was 31.7%, and the detection rate of high-risk parents was 0.3%. The highest carrier rates were SLC22A (2.4%), ATP7B (2.4%), MMACHC (2.2%), PAH (1.8%), GALC (1.8%), MLC1 (1.3%), UNC13D (1.1%), CAPN3 (1.1%), and PKHD1 (1.1%). There were 488 women with fragile X syndrome-FMR1 gene detection, and 6 patients (1.2%) had FMR1 gene mutation. A total of 426 patients were screened for spinal muscular atrophy-SMN1, and the carrier rate was 3.5%, and the detection rate of parents' co-carrier was 0.5%. CONCLUSION: Monogenic recessive hereditary diseases had a high carrier rate in the population. Pre-pregnancy screening could provide good prenatal and postnatal care guidance for patients and preimplantation genetic testing for monogenic/single gene disorders (PGT-M) and prenatal diagnosis could provide more precise reproductive choices for high-risk parents.

Tri-enzyme fusion of tryptophan halogenase achieves a concise strategy for coenzyme self-sufficiency and the continuous halogenation of L-tryptophan.

The halogenase-based catalysis is one of the most environmentally friendly methods for the synthesis of halogenated products, among which flavin-dependent halogenases (FDHs) have attracted great interest as one of the most promising biocatalysts due to the remarkable site-selectivity and wide substrate range. However, the complexity of constructing the NAD+-NADH-FAD-FADH2 bicoenzyme cycle system has affected the engineering applications of FDHs. In this work, a coenzyme self-sufficient tri-enzyme fusion was constructed and successfully applied to the continuous halogenation of L-tryptophan. SpFDH was firstly identified derived from Streptomyces pratensis, a highly selective halogenase capable of generating 6-chloro-tryptophan from tryptophan. Then, using gene fusion technology, SpFDH was fused with glucose dehydrogenase (GDH) and flavin reductase (FR) to form a tri-enzyme fusion, which increased the yield by 1.46-fold and making the coenzymes self-sufficient. For more efficient halogenation of L-tryptophan, a continuous halogenation bioprocess of L-tryptophan was developed by immobilizing the tri-enzyme fusion and attaching it to a continuous catalytic device, which resulted in a reaction yield of 97.6% after 12 h reaction. An FDH from S. pratensis was successfully applied in the halogenation and our study provides a concise strategy for the preparation of halogenated tryptophan mediated by multienzyme cascade catalysis.

Systems Metabolic Engineering for Efficient Violaxanthin Production in Yeast.

Violaxanthin is a plant-derived orange xanthophyll with remarkable antioxidant activity that has wide applications in various industries, such as food, agriculture, and cosmetics. In addition, it is the key precursor of important substances such as abscisic acid and fucoxanthin. Saccharomyces cerevisiae, as a GRAS (generally regarded as safe) chassis, provides a good platform for producing violaxanthin production with a yield of 7.3 mg/g DCW, which is far away from commercialization. Herein, an integrated strategy involving zeaxanthin epoxidase (ZEP) source screening, cytosol redox state engineering, and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration was implemented to enhance violaxanthin production in S. cerevisiae. 58aa-truncated ZEP from Vitis vinifera exhibited optimal efficiency in an efficient zeaxanthin-producing strain. The titer of violaxanthin gradually increased by 17.9-fold (up to 119.2 mg/L, 15.19 mg/g DCW) via cytosol redox state engineering and NADPH supplementation. Furthermore, balancing redox homeostasis considerably improved the zeaxanthin concentration by 139.3% (up to 143.9 mg/L, 22.06 mg/g DCW). Thus, the highest reported titers of violaxanthin and zeaxanthin in S. cerevisiae were eventually achieved. This study not only builds an efficient platform for violaxanthin biosynthesis but also serves as a useful reference for the microbial production of xanthophylls.

The bacillithiol pathway is required for biofilm formation in Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that can cause infections that range from superficial skin and mucosal infections to life threatening disseminated infections. S. aureus can attach to medical devices and host tissues and form biofilms that allow the bacteria to evade the host immune system and provide protection from antimicrobial agents. To counter host-generated oxidative and nitrosative stress mechanisms that are part of the normal host responses to invading pathogens, S. aureus utilizes low molecular weight (LMW) thiols, such as bacillithiol (BSH). Additionally, S. aureus synthesizes its own nitric oxide (NO), which combined with its downstream metabolites may also protect the bacteria against specific host responses. We have previously shown that LMW thiols are required for biofilm formation in Mycobacterium smegmatis and Pseudomonas aeruginosa. Here, we show that the S. aureus bshC mutant strain, which is defective in the last step of the BSH pathway and lacks BSH, is impaired in biofilm formation. We also identify a possible S-nitrosobacillithiol reductase (BSNOR), similar in sequence to an S-nitrosomycothiol reductase found in M. smegmatis and show that the putative S. aureus bsnoR mutant strain has reduced levels of BSH and decreased biofilm formation. Our studies also show that NO plays an important role in biofilm formation and that acidified sodium nitrite severely reduces biofilm thickness. These studies provide insight into the roles of oxidative and nitrosative stress mechanisms on biofilm formation and indicate that BSH and NO are key players in normal biofilm formation in S. aureus.

Elucidation of Palmarumycin Spirobisnaphthalene Biosynthesis Reveals a Set of Previously Unrecognized Oxidases and Reductases.

Spirobisnaphthalenes (SBNs) are a class of highly oxygenated, fungal bisnaphthalenes containing a unique spiroketal bridge, that displayed diverse bioactivities. Among the reported SBNs, palmarumycins are the major type, which are precursors for the other type of SBNs structurally. However, the biosynthesis of SBNs is unclear. In this study, we elucidated the biosynthesis of palmarumycins, using gene disruption, heterologous expression, and substrate feeding experiments. The biosynthetic gene cluster for palmarumycins was identified to be distant from the polyketide synthase gene cluster, and included two cytochrome P450s (PalA and PalB), and one short chain dehydrogenase/reductase (PalC) encoding genes as key structural genes. PalA is an unusual, multifunctional P450 that catalyzes the oxidative dimerization of 1,8-dihydroxynaphthalene to generate the spiroketal linkage and 2,3-epoxy group. Chemical synthesis of key intermediate and in vitro biochemical assays proved that the oxidative dimerization proceeded via a binaphthyl ether. PalB installs the C-5 hydroxy group, widely found in SBNs. PalC catalyzes 1-keto reduction, the reverse 1-dehydrogenation, and 2,3-epoxide reduction. Moreover, an FAD-dependent oxidoreductase, encoded by palD, which locates outside the cluster, functions as a 1-dehydrogenase. These results provided the first genetic and biochemical evidence for the biosynthesis of palmarumycin SBNs.

Engineering pineapple peel cellulose nanofibrils with oxidase-mimic functionalities for antibacterial and fruit preservation.

In this study, an antibacterial material (CNF@CoMn-NS) with oxidase-like activity was created using ultrathin cobalt­manganese nanosheets (CoMn-NS) with a larger specific surface area grown onto pineapple peel cellulose nanofibrils (CNF). The results showed that the CoMn-NS grew well on the CNF, and the obtained CNF@CoMn-NS exhibited good oxidase-like activity. The imidazole salt framework of the CNF@CoMn-NS contained cobalt and manganese in multiple oxidation states, enabling an active redox cycle and generating active oxygen species (ROS) such as singlet molecular oxygen atoms (1O2) and superoxide radical (·O2-), resulting in the significant inactivation of Staphylococcus aureus (74.14%) and Escherichia coli (54.87%). Importantly, the CNF@CoMn-NS did not exhibit cytotoxicity. The CNF@CoMn-NS further self-assembled into a CNF@CoMn-NS paper with flexibility, stability, and antibacterial properties, which can effectively protect the wound of two varieties of pears from decay caused by microorganisms. This study demonstrated the potential of using renewable and degradable CNF as substrate combined with artificial enzymes as a promising approach to creating antibacterial materials for food preservation and even extending to textiles and biomedical applications.