Research into the cause of brackish marsh recession in the Fraser River estuary

At least 160 ha (30%) of the brackish marsh on Sturgeon Bank in the Fraser River estuary have disappeared since 1989. A collaborative effort between the provincial and federal governments and industry aims to determine the cause(s) of the recession to inform future restoration efforts. Three adjacent low-elevation brackish marshes along the delta front have also receded to varying degrees. River training structures and regular dredging of the Fraser River divert sediments and fresh water, and thus alter sediment and salinity patterns along the delta front. Lesser snow geese (Anser c. caerulescens) preferentially grub bulrush, and the Fraser-Skagit population has increased 2-3 fold over the last three decades. Investigation of the marsh recession to date includes (1) describing historical rates and patterns of recession, (2) assessing the present environment (e.g., sediment accretion rates and salinity regimes), and (3) conducting inferential experiments to identify factors that contribute to marsh loss and prevent recovery. No single recession hypothesis we tested singularly explains the recession. The anticipated effects of climate change, especially sea-level rise, pose additional threats to all tidal marshes in the Fraser River estuary

Protein Engineering of Baeyer-Villiger Monooxygenases

In dieser Arbeit wurde die Regioselektivität verschiedener Baeyer-Villiger Monooxygenasen für die Umsetzung ausgewählter Substrate mittels Protein-Engineering umgekehrt oder verbessert. Anhand der Ergebnisse konnten Einblicke in die Regulation der Regioselektivität dieser Enzyme gewonnen werden und es wurden hot-spots identifiziert, die für die Regioselektivität der verschiedenen Baeyer-Villiger Monooxygenasen bedeutend waren. In Nebenprojekten konnte sowohl die Sulfoxidationsaktivität wie auch die Stabilität von Baeyer-Villiger Monooxygenasen mittels Protein-Engineering verbessert werden.In this work, the regioselectivity of different Baeyer-Villiger monooxygenases (BVMOs) for the conversion of selected substrates was reversed or improved by protein engineering. These studies highlight the importance of substrate positioning for the regioselectivity and that the position of the substrate can be efficiently influenced by introducing proper mutations. It was shown that the beneficial mutations for all BVMOs were partly in corresponding positions. Additionally, the sulfoxidation activity and the stability of BVMOs were targeted and improved by applying protein engineering

Functional assembly of camphor converting two-component Baeyer-Villiger monooxygenases with a flavin reductase from E-coli

The major limitation in the synthetic application of two-component Baeyer-Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.AuthorCount:5;</p

LuxAB-based microbial cell factories for the sensing, manufacturing and transformation of industrial aldehydes

The application of genetically encoded biosensors enables the detection of small molecules in living cells and has facilitated the characterization of enzymes, their directed evolution and the engineering of (natural) metabolic pathways. In this work, the LuxAB biosensor system from Photorhabdus luminescens was implemented in Escherichia coli to monitor the enzymatic production of aldehydes from primary alcohols and carboxylic acid substrates. A simple high-throughput assay utilized the bacterial luciferase—previously reported to only accept aliphatic long-chain aldehydes—to detect structurally diverse aldehydes, including aromatic and monoterpene aldehydes. LuxAB was used to screen the substrate scopes of three prokaryotic oxidoreductases: an alcohol dehydrogenase (Pseudomonas putida), a choline oxidase variant (Arthrobacter chlorophenolicus) and a carboxylic acid reductase (Mycobacterium marinum). Consequently, high-value aldehydes such as cinnamaldehyde, citral and citronellal could be produced in vivo in up to 80% yield. Furthermore, the dual role of LuxAB as sensor and monooxygenase, emitting bioluminescence through the oxidation of aldehydes to the corresponding carboxylates, promises implementation in artificial enzyme cascades for the synthesis of carboxylic acids. These findings advance the bio-based detection, preparation and transformation of industrially important aldehydes in living cells.publishedVersionPeer reviewe

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: The first crystal structure of a type II Baeyer-Villiger monooxygenase

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9\uc5 resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a \u3b2-bulge at the C-terminus of \u3b2-strand 3, which is a feature observed in many proteins of this superfamily.Peer reviewed: YesNRC publication: Ye

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: The first crystal structure of a type II Baeyer-Villiger monooxygenase

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9\uc5 resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a \u3b2-bulge at the C-terminus of \u3b2-strand 3, which is a feature observed in many proteins of this superfamily.Peer reviewed: YesNRC publication: Ye

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase. Corrigendum

: A statement is amended in the article by Isupov et al. [(2015). Acta Cryst. D71, 2344-2353]