Exploring Linalool (De)hydratase-Isomerase for Enzymatic Hydration of Alkenes.

Acyclic monoterpenes constitute a large and highly abundant class of secondary plant metabolites and are, therefore, attractive low-cost raw materials for the chemical industry. To date, numerous biocatalysts for their transformation are known, giving access to highly sought-after monoterpenoids. In view of the high selectivity associated with many of these reactions, the demand for enzymes generating commercially important target molecules is unabated.

Recombinant expression, purification and biochemical characterization of kievitone hydratase from Nectria haematococca.

Kievitone hydratase catalyzes the addition of water to the double bond of the prenyl moiety of plant isoflavonoid kievitone and, thereby, forms the tertiary alcohol hydroxy-kievitone. In nature, this conversion is associated with a defense mechanism of fungal pathogens against phytoalexins generated by host plants after infection. As of today, a gene sequence coding for kievitone hydratase activity has only been identified and characterized in Fusarium solani f.

Process development for oxidations of hydrophobic compounds applying cytochrome P450 monooxygenases in-vitro.

Cytochrome P450 monooxygenases are a unique family of enzymes that are able to catalyze regio- and stereospecific oxidations for a broad substrate range. However, due to limited enzyme activities and stabilities, hydrophobicity of substrates, as well as the necessity of a continuous electron and oxygen supply the implementation of P450s for industrial processes remains challenging. Aim of this study was to point out key aspects for the development of an efficient synthesis concept for cytochrome P450 catalyzed oxidations.

Semi-rational engineering of cytochrome CYP153A from Marinobacter aquaeolei for improved ω-hydroxylation activity towards oleic acid.

ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket.

Enhancing cytochrome P450-mediated conversions in P. pastoris through RAD52 over-expression and optimizing the cultivation conditions.

Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts.

Structure-Based Mechanism of Oleate Hydratase from Elizabethkingia meningoseptica.

Hydratases provide access to secondary and tertiary alcohols by regio- and/or stereospecifically adding water to carbon-carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD).

Over-expression of ICE2 stabilizes cytochrome P450 reductase in Saccharomyces cerevisiae and Pichia pastoris.

Membrane-anchored cytochrome P450 enzymes (CYPs) are a versatile and interesting class of enzymes for industrial applications, as they are capable of regio- and stereoselectively hydroxylating hydrophobic molecules. However, CYP activity requires sufficient levels of suitable cytochrome P450 reductases (CPRs) for regeneration of catalytic capacity, which is a bottleneck in many industrial applications. Searching for positive effectors of membrane-anchored CYP/CPR function, we transformed and screened selected strains from a Saccharomyces cerevisiae knockout collection for Hyoscyamus muticus premnaspirodiene oxygenase (HPO; CYP) and Arabidopsis thaliana CPR (AtCPR) expression levels, as well as for activity towards (+)-valencene.

Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris.

The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone.

Enantiodivergent, biocatalytic routes to both taxol side chain antipodes.

[Reaction: see text]. Two enantiocomplementary bakers' yeast enzymes reduced an alpha-chloro-beta-keto ester to yield precursors for both enantiomers of the N-benzoyl phenylisoserine Taxol side chain. After base-mediated ring closure of the chlorohydrin enantiomers, the epoxides were converted directly to the oxazoline form of the target molecules using a Ritter reaction with benzonitrile.

Stereoselective, biocatalytic reductions of alpha-chloro-beta-keto esters.

Eighteen known and putative reductases from baker's yeast (Saccharomyces cerevisiae) were tested for the ability to reduce a series of alpha-chloro-beta-keto esters. In nearly all cases, it was possible to produce at least two of the four possible alpha-chloro-beta-hydroxy ester diastereomers with high optical purities. The utility of this approach was demonstrated by reducing ethyl 2-chloroacetoacetate to the corresponding syn-(2R,3S)-alcohol on a multigram scale using whole cells of an Escherichia coli strain overexpressing a single yeast reductase identified from the screening studies.

Systematic investigation of Saccharomyces cerevisiae enzymes catalyzing carbonyl reductions.

Eighteen key reductases from baker's yeast (Saccharomyces cerevisiae) have been overproduced in Escherichia coli as glutathione S-transferase fusion proteins. A representative set of alpha- and beta-keto esters was tested as substrates (11 total) for each purified fusion protein. The stereoselectivities of beta-keto ester reductions depended both on the identity of the enzyme and the substrate structure, and some reductases yielded both L- and D-alcohols with high stereoselectivities.