Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (SMO) (StyA1) and another styrene monooxygenase fused to an NADH-flavin oxidoreductase (StyA2B). StyA1 and StyA2B convert styrene and chemical analogues to the corresponding epoxides at the expense of FADH 2 provided from StyA2B. The StyA1/StyA2B system presents the highest monooxygenase activity in an equimolar ratio of StyA1 and StyA2B, indicating (transient) protein complex formation. StyA1 is also active when FADH 2 is supplied by StyB from Pseudomonas sp. VLB120 or PheA2 from Rhodococcus opacus 1CP. However, in both cases the reductase produces an excess of FADH 2 , resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the FADH 2 -induced activation of StyA1 requires interprotein communication. We conclude that the StyA1/StyA2B system represents a novel type of multifunctional flavoprotein monooxygenase. Its unique mechanism of cofactor utilization provides new opportunities for biotechnological applications and is highly relevant from a structural and evolutionary point of view.The environmentally harmful hydrocarbon styrene is readily biodegradable by various classes of microorganisms covering Gram-negative and Gram-positive bacteria as well as fungi (e.g., ascomycetes). Two major pathways for styrene mineralization have been described (reviewed in references 23, 26, and 33); of these, the most common one is initiated by a monooxygenase-catalyzed epoxidation of the vinyl side chain. Due to their biotechnological potential, the styrene monooxygenases (SMOs) involved in this reaction have received considerable attention. Most SMOs have been described for pseudomonads and were investigated for their biochemical properties (5,13,27,31,46) and their biotechnological applicability in cell-free (16, 17) or whole-cell systems (3,12,28,29,30,32,37). All SMOs investigated thus far convert styrene in a highly enantioselective manner to (S)-styrene oxide, which is a useful precursor for several chiral synthons and pharmaceuticals (2,6,14,26,34). Moreover, the relaxed substrate specificity of SMOs allows an enantioselective conversion of substituted styrene derivatives and structurally related compounds, like indene and dihydronaphthalene, as well as phenylalkylsulfides ( Fig. 1) (17, 40, 45), thus increasing their biocatalytic potential.Typical SMOs of pseudomonads consist of two enzymatically active protein components encoded by genes that are usually clustered adjacent to each other (styA and styB) (Fig. 2a) (26,43,46). The flavin reductase subunit (StyB) reduces flavin adenine dinucleotide (FAD) at the expense of NADH.The monooxygenase subunit (StyA) then utilizes the reduced flavin (FADH 2 ) to activate molecular oxygen for styrene attack (Fig. 2b)....

We propose a joint experimental and theoretical approach to the automated reconstruction of elemental fluxes in microbial communities. While stable isotope probing of proteins (protein-SIP) has been successfully applied to study interactions and elemental carbon and nitrogen fluxes, the volume and complexity of mass spectrometric data in protein-SIP experiments pose new challenges for data analysis. Together with a flexible experimental setup, the novel bioinformatics tool MetaProSIP offers an automated high-throughput solution for a wide range of (13)C or (15)N protein-SIP experiments with special emphasis on the analysis of metaproteomic experiments where differential labeling of organisms can occur. The information calculated in MetaProSIP includes the determination of multiple relative isotopic abundances, the labeling ratio between old and new synthesized proteins, and the shape of the isotopic distribution. These parameters define the metabolic capacities and dynamics within the investigated microbial culture. MetaProSIP features a high degree of reproducibility, reliability, and quality control reporting. The ability to embed into the OpenMS framework allows for flexible construction of custom-tailored workflows. Software and documentation are available under an open-source license at www.openms.de/MetaProSIP.

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Ashes from lignite combustion for power generation contain considerable amounts of strategic metals, metalloids and rare earth elements. Within the presented project bioleaching with different types of microorganisms was investigated to recover valuable metals from lignite ashes. An increased mobilization of several metals ions was observed with the gluconic acid-producing bacteriumAcetobacter methanolicusand the silicate-solubilizing bacteriumBacillus circulans. Most promising results were achieved with sulfuric acid-producing microorganisms and bioleaching could even be increased at higher temperatures or by reductive bioleaching using acidophilic, iron-reducing bacteria.

Ashes from lignite combustion for power generation contain strategic metals, metalloids and rare earth elements and may thus be a potential source of industrially demanded metals. The presented project focuses on the assessment and exploitation of this potential raw material. Lignite ash assessment showed that largest ash amounts for potential exploitation are available in the Lusatia district, Saxony. Mechanical ash pre-treatment in principle provided enriched fractions by different methods but still suffered from low yields of enriched fractions. Thermal ash processing showed multiple significant phase changes compared to original ash. Subsequent chemical leaching using HClaq resulted in high metal extraction. Alternatively, bioleaching was applied using acidophilic Fe (II) and S-oxidizing or Fe (III)-reducing microorganisms (MO) as well as heterotrophic MO. The results indicated likewise high and partly specific metal mobilizations. Industrial ash exploitation was accomplished by direct reaction with acids resulting in Al-Fe-solutions which potentially can be applied in water treatment.