Efficient encapsulation of Hinoki essential oil with ß-cyclodextrin using an ultrasound-aided co-precipitation technique for dual anti-Listeria monocytogenes and anti-Staphylococcus aureus activities.

Listeria monocytogenes (L. monocytogenes) and Staphylococcus aureus (S. aureus) are widely acknowledged as two of the most dangerous foodborne pathogens. Nevertheless, reports on the development of non-toxic food preservatives that specifically target these two bacterial strains are scarce. Here, we present an inclusion complex (IC) of Hinoki essential oil with ß-cyclodextrin, which exhibited dual anti-L. monocytogenes and anti-S. aureus activities. For the first time, an innovative ultrasound-aided co-precipitation technique was utilized for the preparation of IC. Compared with the traditional co-precipitation method, this new technique demonstrated superior encapsulation and time efficiencies, making it well-suited for large-scale production. X-ray diffraction and scanning electron microscopy analyses revealed a transition in the morphological and crystal structures after formation of the IC. Fourier transform infrared spectroscopy and Raman spectroscopy analyses indicated that Hinoki essential oil was effectively encapsulated by ß-cyclodextrin. The differential scanning calorimetry and thermogravimetric thermograms indicated that the formed IC was more thermally stable than the free Hinoki essential oil. Importantly, 100 % antibacterial ratios for both L. monocytogenes and S. aureus were determined, indicating that the IC prepared in this study is a promising food preservative.

The Metabolic Regulation of Amino Acid Synthesis Counteracts Reactive Nitrogen Stress via Aspergillus nidulans Cross-Pathway Control.

Nitric oxide (NO) is a natural reactive nitrogen species (RNS) that alters proteins, DNA, and lipids and damages biological activities. Although microorganisms respond to and detoxify NO, the regulation of the cellular metabolic mechanisms that cause cells to tolerate RNS toxicity is not completely understood. We found that the proline and arginine auxotrophic proA5 and argB2 mutants of the fungus Aspergillus nidulans require more arginine and proline for normal growth under RNS stress that starves cells by accumulating fewer amino acids. Fungal transcriptomes indicated that RNS stress upregulates the expression of the biosynthetic genes required for global amino acids, including proline and arginine. A mutant of the gene disruptant, cpcA, which encodes the transcriptional regulation of the cross-pathway control of general amino acid synthesis, did not induce these genes, and cells accumulated fewer amino acids under RNS stress. These results indicated a novel function of CpcA in the cellular response to RNS stress, which is mediated through amino acid starvation and induces the transcription of genes for general amino acid synthesis. Since CpcA also controls organic acid biosynthesis, impaired intermediates of such biosynthesis might starve cells of amino acids. These findings revealed the importance of the mechanism regulating amino acid homeostasis for fungal responses to and survival under RNS stress.

Protocatechuate hydroxylase is a novel group A flavoprotein monooxygenase with a unique substrate recognition mechanism.

Para-hydroxybenzoate hydroxylase (PHBH) is a group A flavoprotein monooxygenase that hydroxylates p-hydroxybenzoate to protocatechuate (PCA). Despite intensive studies of Pseudomonas aeruginosa p-hydroxybenzoate hydroxylase (PaPobA), the catalytic reactions of extremely diverse putative PHBH isozymes remain unresolved. We analyzed the phylogenetic relationships of known and predicted PHBHs and identified eight divergent clades. Clade F contains a protein that lacks the critical amino acid residues required for PaPobA to generate PHBH activity. Among proteins in this clade, Xylophilus ampelinus PobA (XaPobA) preferred PCA as a substrate and is the first known natural PCA 5-hydroxylase (PCAH). Crystal structures and kinetic properties revealed similar mechanisms of substrate carboxy group recognition between XaPobA and PaPobA. The unique Ile75, Met72, Val199, Trp201, and Phe385 residues of XaPobA form the bottom of a hydrophobic cavity with a shape that complements the 3-and 4-hydroxy groups of PCA and its binding site configuration. An interaction between the δ-sulfur atom of Met210 and the aromatic ring of PCA is likely to stabilize XaPobA-PCA complexes. The 4-hydroxy group of PCA forms a hydrogen bond with the main chain carbonyl of Thr294. These modes of binding constitute a novel substrate recognition mechanism that PaPobA lacks. This mechanism characterizes XaPobA and sheds light on the diversity of catalytic mechanisms of PobA-type PHBHs and group A flavoprotein monooxygenases.

Genes encoding a novel thermostable bacteriocin in the thermophilic bacterium Aeribacillus pallidus PI8.

AIM: Aeribacillus pallidus PI8 is a Gram-positive thermophilic bacterium that produces thermostable antimicrobial substances against several bacterial species, including Geobacillus kaustophilus HTA426. In the present study, we sought to identify genes of PI8 with antibacterial activity. METHODS AND RESULTS: We isolated, cloned, and characterized a thermostable bacteriocin from A. pallidus PI8 and named it pallidocyclin. Mass spectrometric analyses of pallidocyclin revealed that it had a circular peptide structure, and its precursor was encoded by pcynA in the PI8 genome. pcynA is the second gene within the pcynBACDEF operon. Expression of the full-length pcynBACDEF operon in Bacillus subtilis produced intact pallidocyclin, whereas expression of pcynF in G. kaustophilus HTA426 conferred resistance to pallidocyclin. CONCLUSION: Aeribacillus pallidus PI8 possesses the pcynBACDEF operon to produce pallidocyclin. pcynA encodes the pallidocyclin precursor, and pcynF acts as an antagonist of pallidocyclin.

Gallic acid fermentation by metabolically engineered Escherichia coli producing p-hydroxybenzoate hydroxylase from Hylemonella gracilis NS1.

Plant-derived phenolic gallic acid (GA) is an important raw material for antioxidants and food additives. Efforts to ferment GA using microbial processes have aimed at minimizing production costs and environmental load using enzymes that hydroxylate p-hydroxybenzoate and protocatechuate (PCA). Here, we found a p-hydroxybenzoate hydroxylase (PobA) in the bacterium Hylemonella gracilis NS1 (HgPobA) with 1.5-fold more hydroxylation activity than that from Pseudomonas aeruginosa PAO1 and thus converted PCA to GA more efficiently. The PCA hydroxylation activity of HgPobA was improved by introducing the amino acid substitutions L207V/Y393F or T302A/Y393F. These mutants had 2.9- and 3.7-fold lower Kmapp for PCA than wild-type HgPobA. An Escherichia coli strain that reinforces shikimate pathway metabolism and produces HgPobA when cultured for 60 h generated 0.27 g L-1 of GA. This is the first report of fermenting glucose to generate GA using a natural enzyme from the PobA family. The E. coli strain harboring the HgPobA L207V/Y393F mutant increased GA production to 0.56 g L-1. During the early stages of culture, GA was fermented at a 10-fold higher rate by a strain producing either HgPobA L207V/Y393F or T302A/Y393F compared with wild-type HgPobA, which agreed with the high kcatapp/Kmapp PCA values of this mutant. We enhanced a PobA isozyme and its PCA hydroxylating function to efficiently and cost-effectively ferment GA.

Preparation of Green Anti-Staphylococcus aureus Inclusion Complexes Containing Hinoki Essential Oil.

This study aimed to prepare anti-Staphylococcus aureus inclusion complexes (ICs) of Hinoki essential oil (HEO) with ß-cyclodextrin (ß-CD) and 2-hydroxypropyl-ß-cyclodextrin (2-HP-ß-CD). An ultrasound-assisted kneading method was applied for the complexation for the first time. The recovery yield, embedding fraction and loading capacity of the HEO/ß-CD ICs were 92.5%, 78.0% and 11.9%, respectively, while the corresponding values were 80.8%, 73.7% and 12.9% for the HEO/2-HP-ß-CD ICs. As well, a comparative study confirmed the efficiency of the ultrasound-assisted kneading method was higher than the traditional kneading method. The results of SEM, XRD, GC-MS and FT-IR suggested the successful formation of ICs. A significant anti-Staphylococcus aureus activity of the fabricated ICs was demonstrated using a colony counting method. Notably, when the dose in liquid culture medium was 20 g L-1, inhibitory rates of 99.8% for HEO/ß-CD ICs and 100% for HEO/2-HP-ß-CD ICs were achieved. Furthermore, the hydrophilic property of the ICs was proved by water contact angle measurements, implying they have the potential to act as anti-Staphylococcus aureus agents for blending with hydrophilic biodegradable materials for diverse food packaging utilizations.

Machine learning-assisted medium optimization revealed the discriminated strategies for improved production of the foreign and native metabolites.

The composition of medium components is crucial for achieving the best performance of synthetic construction in genetically engineered cells. Which and how medium components determine the performance, e.g., productivity, remain poorly investigated. To address the questions, a comparative survey with two genetically engineered Escherichia coli strains was performed. As a case study, the strains carried the synthetic pathways for producing the aromatic compounds of 4-aminophenylalanine (4APhe) or tyrosine (Tyr), common in the upstream but differentiated in the downstream metabolism. Bacterial growth and compound production were examined in hundreds of medium combinations that comprised 48 pure chemicals. The resultant data sets linking the medium composition to bacterial growth and production were subjected to machine learning for improved production. Intriguingly, the primary medium components determining the production of 4PheA and Tyr were differentiated, which were the initial resource (glucose) of the synthetic pathway and the inducer (IPTG) of the synthetic construction, respectively. Fine-tuning of the primary component significantly increased the yields of 4APhe and Tyr, indicating that a single component could be crucial for the performance of synthetic construction. Transcriptome analysis observed the local and global changes in gene expression for improved production of 4APhe and Tyr, respectively, revealing divergent metabolic strategies for producing the foreign and native metabolites. The study demonstrated that ML-assisted medium optimization could provide a novel point of view on how to make the synthetic construction meet the designed working principle and achieve the expected biological function.

Local calcium signal transmission in mycelial network exhibits decentralized stress responses.

Many fungi live as mycelia, which are networks of hyphae. Mycelial networks are suited for the widespread distribution of nutrients and water. The logistical capabilities are critical for the extension of fungal survival areas, nutrient cycling in ecosystems, mycorrhizal symbioses, and virulence. In addition, signal transduction in mycelial networks is predicted to be vital for mycelial function and robustness. A lot of cell biological studies have elucidated protein and membrane trafficking and signal transduction in fungal hyphae; however, there are no reports visualizing signal transduction in mycelia. This paper, by using the fluorescent Ca2+ biosensor, visualized for the first time how calcium signaling is conducted inside the mycelial network in response to localized stimuli in the model fungus Aspergillus nidulans. The wavy propagation of the calcium signal inside the mycelium or the signal blinking in the hyphae varies depending on the type of stress and proximity to the stress. The signals, however, only extended around 1,500 µm, suggesting that the mycelium has a localized response. The mycelium showed growth delay only in the stressed areas. Local stress caused arrest and resumption of mycelial growth through reorganization of the actin cytoskeleton and membrane trafficking. To elucidate the downstream of calcium signaling, calmodulin, and calmodulin-dependent protein kinases, the principal intracellular Ca2+ receptors were immunoprecipitated and their downstream targets were identified by mass spectrometry analyses. Our data provide evidence that the mycelial network, which lacks a brain or nervous system, exhibits decentralized response through locally activated calcium signaling in response to local stress.

Microbial pyrazine diamine is a novel electrolyte additive that shields high-voltage LiNi1/3Co1/3Mn1/3O2 cathodes.

The uncontrolled oxidative decomposition of electrolyte while operating at high potential (> 4.2 V vs Li/Li+) severely affects the performance of high-energy density transition metal oxide-based materials as cathodes in Li-ion batteries. To restrict this degradative response of electrolyte species, the need for functional molecules as electrolyte additives that can restrict the electrolytic decomposition is imminent. In this regard, bio-derived molecules are cost-effective, environment friendly, and non-toxic alternatives to their synthetic counter parts. Here, we report the application of microbially synthesized 2,5-dimethyl-3,6-bis(4-aminobenzyl)pyrazine (DMBAP) as an electrolyte additive that stabilizes high-voltage (4.5 V vs Li/Li+) LiNi1/3Mn1/3Co1/3O2 cathodes. The high-lying highest occupied molecular orbital of bio-additive (DMBAP) inspires its sacrificial in situ oxidative decomposition to form an organic passivation layer on the cathode surface. This restricts the excessive electrolyte decomposition to form a tailored cathode electrolyte interface to administer cyclic stability and enhance the capacity retention of the cathode.

Carboxypeptidase G and pterin deaminase metabolic pathways degrade folic acid in Variovorax sp. F1.

BACKGROUND: Folic acid (FA) is a synthetic vitamin (B9) and the oxidized form of a metabolic cofactor that is essential for life. Although the biosynthetic mechanisms of FA are established, its environmental degradation mechanism has not been fully elucidated. The present study aimed to identify bacteria in soil that degrade FA and the mechanisms involved. RESULTS: We isolated the soil bacterium Variovorax sp. F1 from sampled weed rhizospheres in a grassland and investigated its FA degradation mechanism. Cultured Variovorax sp. F1 rapidly degraded FA to pteroic acid (PA), indicating that FA hydrolysis to PA and glutamate. We cloned the carboxypeptidase G (CPG) gene and found widely distributed paralogs within the Variovorax genus. Recombinant CPG preferred FA and deaminofolic acid as substrates, indicating its involvement in FA degradation by Variovorax. Prolonged culture of Variovorax sp. F1 resulted in decreased rates of deaminofolic acid (DFA) and deaminopteroic acid (DPA) accumulation. This indicated that the deamination reaction also comprised a route of FA degradation. We also identified an F1 gene that was orthologous to the pterin deaminase gene (Arad3529) of Agrobacterium radiobacter. The encoded protein deaminated FA and PA to DFA and DPA, which was consistent with the deamination activity of FA and PA in bacterial cell-free extracts. CONCLUSION: We discovered that the two enzymes required for FA degradation pathways in isolates of Variovorax sp. F1 comprise CPG and pterin deaminase, and that DFA and PA are intermediates in the generation of DPA.

Pseudomonas cannabina pv. alisalensis Virulence Factors Are Involved in Resistance to Plant-Derived Antimicrobials during Infection.

Bacteria are exposed to and tolerate diverse and potentially toxic compounds in the natural environment. While efflux transporters are generally thought to involve bacterial antibiotic resistance in vitro, their contributions to plant bacterial virulence have so far been poorly understood. Pseudomonas cannabina pv. alisalensis (Pcal) is a causal agent of bacterial blight of Brassicaceae. We here demonstrated that NU19, which is mutated in the resistance-nodulation-cell division (RND) transporter encoded gene, showed reduced virulence on cabbage compared to WT, indicating that the RND transporter contributes to Pcal virulence on cabbage. We also demonstrated that brassinin biosynthesis was induced after Pcal infection. Additionally, the RND transporter was involved in resistance to plant-derived antimicrobials and antibiotics, including the cabbage phytoalexin brassinin. These results suggest that the RND transporter extrudes plant-derived antimicrobials and contributes to Pcal virulence. We also found that the RND transporter contributes to Pcal virulence on Brassicaceae and tomato, but not on oat. These results suggest that the RND transporter contributes to Pcal virulence differentially depending on the host-plant species. Lastly, our expression-profile analysis indicated that the type-three secretion system (TTSS), which is essential for pathogenesis, is also involved in suppressing brassinin biosynthesis. Taken together, our results suggest that several Pcal virulence factors are involved in resistance to plant-derived antimicrobials and bacterial survival during infection.

Fermentative production of 2-(4-aminophenyl)ethylamine to synthesize a novel heat-resistant biopolyurea.

The aromatic diamine 2-(4-aminophenyl)ethylamine (4APEA) is a potential monomer for polymers and advanced materials. Here, 4APEA was produced by fermentation using genetically engineered Escherichia coli (Masuo et al.2016). Optimizing fed-batch cultures of this strain produced the highest reported yield to date of 4APEA (7.2%; 3.5 g/L versus glucose) within 72 h. Appropriate aeration was important to maximize production and avoid unfavorable 4APEA degradation. Fermented 4APEA was purified from culture medium and polymerized with methylene diphenyldiisocyanate and hexamethylene diisocyanate to produce polyureas PU-1 and PU-2, respectively. The decomposition temperatures for 10% weight loss (Td10) of PU-1 and PU-2 were 276 °C and 302 °C, respectively, and were comparable with that of other thermostable aromatic polyureas. This study is the first to synthesize polyureas from the microbial aromatic diamine. Their excellent thermostability will be useful for the industrial production of heat-resistant polymer materials.

Glucose-Derived Raspberry Ketone Produced via Engineered Escherichia coli Metabolism.

The demand for raspberry ketone (RK) as a plant-based natural flavoring agent is high, but natural RK is one of the most expensive flavor compounds due to its limited content in plants. Here, we produced RK de novo from simple carbon sources in Escherichia coli. We genetically engineered E. coli metabolism to overproduce the metabolic precursors tyrosine and p-coumaric acid and increase RK production. The engineered E. coli produced 19.3- and 1.9 g/L of tyrosine and p-coumaric acid from glucose, respectively. The p-coumaric acid CoA ligase from Agrobacterium tumefaciens and amino acid substituted benzalacetone synthase of Rhemu palmatum (Chinese rhubarb) were overexpressed in E. coli overproducing p-coumaric acid. The overexpression of fabF, encoding ß-ketoacyl-acyl carrier protein synthetase II increased intracellular malonyl-CoA, the precursor of benzalacetone synthase for RK biosynthesis, and improved RK production. Fed-batch cultures given glucose as a carbon source produced 62 mg/L of RK under optimized conditions. Our production system is inexpensive and does not rely on plant extraction; thus, it should significantly contribute to the flavor and fragrance industries.

Coronatine Contributes to Pseudomonas cannabina pv. alisalensis Virulence by Overcoming Both Stomatal and Apoplastic Defenses in Dicot and Monocot Plants.

Pseudomonas cannabina pv. alisalensis is a causative agent of bacterial blight of crucifers including cabbage, radish, and broccoli. Importantly, P. cannabina pv. alisalensis can infect not only a wide range of Brassicaceae spp. but, also, green manure crops such as oat. However, P. cannabina pv. alisalensis virulence mechanisms have not been investigated and are not fully understood. We focused on coronatine (COR) function, which is one of the well-known P. syringae pv. tomato DC3000 virulence factors, in P. cannabina pv. alisalensis infection processes on both dicot and monocot plants. Cabbage and oat plants dip-inoculated with a P. cannabina pv. alisalensis KB211 COR mutant (ΔcmaA) exhibited reduced virulence compared with P. cannabina pv. alisalensis wild type (WT). Moreover, ΔcmaA failed to reopen stomata on both cabbage and oat, suggesting that COR facilitates P. cannabina pv. alisalensis entry through stomata into both plants. Furthermore, cabbage and oat plants syringe-infiltrated with ΔcmaA also showed reduced virulence, suggesting that COR is involved in overcoming not only stomatal-based defense but also apoplastic defense. Indeed, defense-related genes, including PR1 and PR2, were highly expressed in plants inoculated with ΔcmaA compared with WT, indicating that COR suppresses defense-related genes of both cabbage and oat. Additionally, salicylic acid accumulation increases after ΔcmaA inoculation compared with WT. Taken together, COR contributes to causing disease by suppressing stomatal-based defense and apoplastic defense in both dicot and monocot plants. Here, we investigated COR functions in the interaction of P. cannabina pv. alisalensis and different host plants (dicot and monocot plants), using genetically and biochemically defined COR deletion mutants.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2021.

Fungal mycelia and bacterial thiamine establish a mutualistic growth mechanism.

Exclusivity in physical spaces and nutrients is a prerequisite for survival of organisms, but a few species have been able to develop mutually beneficial strategies that allow them to co-habit. Here, we discovered a mutualistic mechanism between filamentous fungus, Aspergillus nidulans, and bacterium, Bacillus subtilis The bacterial cells co-cultured with the fungus traveled along mycelia using their flagella and dispersed farther with the expansion of fungal colony, indicating that the fungal mycelia supply space for bacteria to migrate, disperse, and proliferate. Transcriptomic, genetic, molecular mass, and imaging analyses demonstrated that the bacteria reached the mycelial edge and supplied thiamine to the growing hyphae, which led to a promotion of hyphal growth. The thiamine transfer from bacteria to the thiamine non-auxotrophic fungus was directly demonstrated by stable isotope labeling. The simultaneous spatial and metabolic interactions demonstrated in this study reveal a mutualism that facilitates the communicating fungal and bacterial species to obtain an environmental niche and nutrient, respectively.

Invasive growth of Aspergillus oryzae in rice koji and increase of nuclear number.

BACKGROUND: 'Rice koji' is a solid culture of Aspergillus oryzae on steamed rice grains. Multiple parallel fermentation, wherein saccharification of rice by A. oryzae and alcohol fermentation by the budding yeast occur simultaneously, leads to the formation of a variety of ingredients of Japanese sake. In sake brewing, the degree of mycelial invasive growth into the steamed rice, called 'haze-komi', highly correlates with the digestibility and quality of rice koji, since the hyphae growing into the rice secrete amylases and digest starch. RESULTS: In this study, we investigated mycelial distribution of GFP-tagged A. oryzae in rice koji made with different types of rice, such as sake rice and eating rice, with 50 or 90% polishing rate to remove abundant proteins and lipids near the surface. In addition, we compared transcriptomes of A. oryzae in the different types of rice koji. Finally, we found that A. oryzae increases the nuclear number and hyphal width in the course of 1-3 days cultivation. CONCLUSIONS: Our imaging analyses indicate that A. oryzae hyphae grew more deeply into 50% polished rice than 90% polished rice. The increases of nuclear number may be a selectively acquired characteristic for the high secretory capacity during the long history of cultivation of this species.

Symbiotic riboflavin degradation by Microbacterium and Nocardioides bacteria.

Unlike its biosynthetic mechanisms and physiological function, current understanding of riboflavin degradation in soil is limited to a few bacteria that decompose it to lumichrome. Here, we isolated six Microbacterium and three Nocardioides strains. These strains utilized riboflavin and lumichrome, respectively, as carbon sources. Among these strains, we identified Microbacterium paraoxydans R16 (R16) and Nocardioides nitrophenolicus L16 (L16), which were isolated form the same enrichment culture. Co-cultured R16 and L16 reconstituted a riboflavin-degrading interspecies consortium, in which the R16 strain degraded riboflavin to lumichrome and ᴅ-ribose. The L16 strain utilized the lumichrome as a carbon source, indicating that R16 is required for L16 to grow in the consortium. Notably, rates of riboflavin degradation and growth were increased in co-cultured, compared with monocultured R16 cells. These results indicated that a beneficial symbiotic interaction between M. paraoxydans R16 and N. nitrophenolicus L16 results in the ability to degrade riboflavin.

Enzymatic Cascade in Pseudomonas that Produces Pyrazine from α-Amino Acids.

Pyrazines are widespread chemical compounds that include pheromones and odors. Herein, a novel mechanism used by Pseudomonas fluorescens SBW25 to biosynthesize monocyclic pyrazines is reported. Heterologous expression of the papABC genes that synthesize the natural α-amino acid 4-aminophenylalanine (4APhe), together with three adjacent papDEF genes of unknown function, in Escherichia coli resulted in the production of 2,5-dimethyl-3,6-bis(4-aminobenzyl)pyrazine (DMBAP), which comprised two symmetrical aminobenzyl moieties derived from 4APhe. It is found that PapD is a novel amino acid C-acetyltransferase, which decarboxylates and transfers acetyl residues to 4APhe, to generate an α-aminoketone, which spontaneously dehydrates and condenses to give dihydro DMBAP. PapF is a novel oxidase in the amine oxidase superfamily that oxidizes dihydro DMBAP to yield the pyrazine ring of DMBAP. These two enzymes constitute a unique mechanism for synthesizing monocyclic pyrazines and might serve as a novel strategy for the enzymatic synthesis of pyrazine derivatives from natural α-amino acids.

5-Methylmellein is a novel inhibitor of fungal sirtuin and modulates fungal secondary metabolite production.

Sirtuin is an NAD+-dependent histone deacetylase that is highly conserved among prokaryotes and eukaryotes. Sirtuin deacetylates histones and non-histone proteins, and it is involved in fungal growth and secondary metabolite production. Here, we screened 579 fungal culture extracts that inhibited the histone deacetylase activity of Sirtuin A (SirA), produced by the fungus Aspergillus nidulans. Eight fungal strains containing three Ascomycota, two Basidiomycota and three Deuteromycetes produced SirA inhibitors. We purified the SirA inhibitor from the culture broth of Didymobotryum rigidum JCM 8837, and identified it as 5-methylmellein-a known polyketide. This polyketide and its structurally-related compound, mellein, inhibited SirA activity with IC50 of 120 and 160 µM, respectively. Adding 5-methylmellein to A. nidulans cultures increased secondary metabolite production in the medium. The metabolite profiles were different from those obtained by adding other sirtuin inhibitors nicotinamide and sirtinol to the culture. These results indicated that 5-methylmellein modulates fungal secondary metabolism, and is a potential tool for screening novel compounds derived from fungi.

Two-Component Flavin-Dependent Riboflavin Monooxygenase Degrades Riboflavin in Devosia riboflavina.

The actinobacterium Microbacterium maritypicum splits riboflavin (vitamin B2) into lumichrome and d-ribose. However, such degradation by other bacteria and the involvement of a two-component flavin-dependent monooxygenase (FMO) in the reaction remain unknown. Here we investigated the mechanism of riboflavin degradation by the riboflavin-assimilating alphaproteobacterium Devosia riboflavina (formerly Pseudomonas riboflavina). We found that adding riboflavin to bacterial cultures induced riboflavin-degrading activity and a protein of the FMO family that had 67% amino acid identity with the predicted riboflavin hydrolase (RcaE) of M. maritypicum MF109. The D. riboflavina genome clustered genes encoding the predicted FMO, flavin reductase (FR), ribokinase, and flavokinase, and riboflavin induced their expression. This finding suggests that these genes constitute a mechanism for utilizing riboflavin as a carbon source. Recombinant FMO (rFMO) protein of D. riboflavina oxidized riboflavin in the presence of reduced flavin mononucleotide (FMN) provided by recombinant FR (rFR), oxidized FMN and NADH, and produced stoichiometric amounts of lumichrome and d-ribose. Further investigation of the enzymatic properties of D. riboflavina rFMO indicated that rFMO-rFR coupling accompanied O2 consumption and the generation of enzyme-bound hydroperoxy-FMN, which are characteristic of two-component FMOs. These results suggest that D. riboflavina FMO is involved in hydroperoxy-FMN-dependent mechanisms to oxygenize riboflavin and a riboflavin monooxygenase is necessary for the initial step of riboflavin degradation.IMPORTANCE Whether bacteria utilize either a monooxygenase or a hydrolase for riboflavin degradation has remained obscure. The present study found that a novel riboflavin monooxygenase, not riboflavin hydrolase, facilitated this process in D. riboflavina The riboflavin monooxygenase gene was clustered with flavin reductase, flavokinase, and ribokinase genes, and riboflavin induced their expression and riboflavin-degrading activity. The gene cluster is uniquely distributed in Devosia species and actinobacteria, which have exploited an environmental niche by developing adaptive mechanisms for riboflavin utilization.