Structural and functional insights into asymmetric enzymatic dehydration of alkenols.

The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of ß-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5-C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD. The three-dimensional structure of LinD from Castellaniella defragrans revealed a pentamer with active sites at the protomer interfaces. Furthermore, the structure of LinD in complex with the product geraniol provides initial mechanistic insights into this bifunctional enzyme. Site-directed mutagenesis confirmed active site amino acid residues essential for its dehydration and isomerization activity. These structural and mechanistic insights facilitate the development of hydrating catalysts, enriching the toolbox for novel bond-forming biocatalysis.

Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol.

1,4-Butanediol (BDO) is an important commodity chemical used to manufacture over 2.5 million tons annually of valuable polymers, and it is currently produced exclusively through feedstocks derived from oil and natural gas. Herein we report what are to our knowledge the first direct biocatalytic routes to BDO from renewable carbohydrate feedstocks, leading to a strain of Escherichia coli capable of producing 18 g l(-1) of this highly reduced, non-natural chemical. A pathway-identification algorithm elucidated multiple pathways for the biosynthesis of BDO from common metabolic intermediates. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic operation of the oxidative tricarboxylic acid cycle, thereby generating reducing power to drive the BDO pathway. The organism produced BDO from glucose, xylose, sucrose and biomass-derived mixed sugar streams. This work demonstrates a systems-based metabolic engineering approach to strain design and development that can enable new bioprocesses for commodity chemicals that are not naturally produced by living cells.

Remarkably favorable hydration of carbonyl substituents in dicationic bis(arene) ruthenium complexes.

The addition of water and methanol to the carbonyl substituents in the dicationic bis(arene)ruthenium complexes [(p-cymene)Ru(C6H5CHO)][OTf]2 (1) and [(p-cymene)Ru(C6H5C(O)CH3)][OTf]2 (2) is remarkably favorable-the equilibrium constants for these reactions are 1 million times greater than those for uncomplexed benzaldehyde and acetophenone. The addition of methanol to the carbonyl group in the o-anisaldehyde complex, [(p-cymene)Ru(C6H4{OCH3}CHO)][OTf]2 (6), occurs with a high degree of kinetic selectivity (94% de).