Mediating the performance of anaerobic granular sludge by exogenous flavin supplementation: Flavin binding capacity and behavior, interspecies electron transfer, and methane production.

Although flavins are known as effective electron mediators, the binding capacity of exogenous flavins by anaerobic granular sludge (AGS) and their role in interspecies electron transfer (IET) remains unknown. In this study, AGS was mediated by using three exogenous flavins of riboflavin (RF), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Results showed that the total amounts of flavins associated with extracellular polymeric substance (EPS) of AGS increased by 2.03-2.42 and 3.83-4.94 folds, after exposure to 50 and 200 µM of exogenous flavins, respectively. A large portion of FMN and FAD was transformed into RF by AGS. Exogenous flavin mediation also stimulated the production of EPS and cytochrome c (c-Cyts) as well as cytochrome-bound flavins. The increased abundance of these electron mediators led to a reduced electrochemical impedance of EPS and improved extracellular electron transfer capacity. The methane production of AGS after mediation with exogenous RF, FMN, and FAD increased by 19.03-31.71%, 22.86-26.04%, and 28.51-33.44%, respectively. This study sheds new light on the role of exogenous flavins in promoting the IET process of a complex microbial aggregate of AGS.

A screening system using minimal media identifies a flavin-competing inhibitor of Porphyromonas gingivalis growth.

Chronic periodontitis is caused by dysbiosis of human oral commensals and especially by increase in Porphyromonas gingivalis. Inhibitors of P. gingivalis growth are expected to serve as effective drugs for the periodontal therapy. In the present study, we isolated new growth inhibitors of P. gingivalis using minimal media for P. gingivalis. The minimal media included the previously reported Globulin-Albumin (GA) and the newly developed Lactalbumin-Ferric chloride (LF) and Globulin-Calcium chloride (GC); all supported growth of the wild-type strain of P. gingivalis but did not support the growth of a mutant defective for a type IX secretion system. GC contains CaCl2, indicating that P. gingivalis requires a calcium ion for growth. Using LF and GA, we screened about 100 000 compounds and identified 73 that strongly inhibited the growth of P. gingivalis. More than half of these candidates would not have been obtained if these minimal media had not been used in our screen. One of our candidate inhibitors was diphenyleneiodonium chloride (DPIC), which showed strong bactericidal activity against P. gingivalis. Excess amounts of flavin adenine dinucleotide or flavin mononucleotide suppressed the inhibitory activity of DPIC, suggesting that DPIC would be a novel potent growth inhibitor.

Production of Bioactive 3'-Hydroxystilbene Compounds Using the Flavin-Dependent Monooxygenase Sam5.

The flavin-dependent monooxygenase Sam5 was previously reported to be a bifunctional hydroxylase with a coumarte 3-hydroxylase and a resveratrol 3'-hydroxylase activity. In this article, we showed the Sam5 enzyme has 3'-hydroxylation activities for methylated resveratrol (pinostilbene and pterostilbene), hydroxylated resveratrol (oxyresveratrol) and glycosylated resveratrol (piceid) as substrates. However, the use of piceid, a glycone type stilbene, as a substrate for bioconversion experiments with the Sam5 enzyme expressed in, Escherichia coli does not convert to the hydroxylated compound astringin, but it has converted in vitro enzyme reactions. Finally, we report a novel catalytic activity of Sam5 monooxygenase for the synthesis of piceatannol derivatives, 3'-hydroxylated stilbene compounds. Development of this bioproduction method for the hydroxylation of stilbenes is challenging because of the difficulty in expressing P450-type hydroxylase in E. coli and regionspecific chemical synthesis.

The dynamics of cellular energetics during continuous yeast culture.

A plethora of data is accumulating from high throughput methods on metabolites, coenzymes, proteins, and nucleic acids and their interactions as well as the signalling and regulatory functions and pathways of the cellular network. The frozen moment viewed in a single discrete time sample requires frequent repetition and updating before any appreciation of the dynamics of component interaction becomes possible. Even then in a sample derived from a cell population, time-averaging of processes and events that occur in out-of-phase individuals blur the detailed complexity of single cell organization. Continuously-grown cultures of yeast can become spontaneously self-synchronized, thereby enabling resolution of far more detailed temporal structure. Continuous on-line monitoring by rapidly responding sensors (O2 electrode and membrane-inlet mass spectrometry for O2, CO2 and H2S; direct fluorimetry for NAD(P)H and flavins) gives dynamic information from time-scales of minutes to hours. Supplemented with capillary electophoresis and gas chromatography mass spectrometry and transcriptomics the predominantly oscillatory behaviour of network components becomes evident, with a 40 min cycle between a phase of increased respiration (oxidative phase) and decreased respiration (reductive phase). Highly pervasive, this ultradian clock provides a coordinating function that links mitochondrial energetics and redox balance to transcriptional regulation, mitochondrial structure and organelle remodelling, DNA duplication and cell division events. Ultimately, this leads to a global partitioning of anabolism and catabolism and the enzymes involved, mediated by a relatively simple ATP feedback loop on chromatin architecture.