Blueprints for the rational design of therapeutic mutacin 1140 variants.

Lantibiotics represent a large untapped pipeline of attractive scaffolds for the development of novel antibiotics. Saturation mutagenesis was employed to substitute every amino acid of a lantibiotic called mutacin 1140 (MU1140), creating an unbiased expression library of 418 variants that was used to study the permissiveness to mutagenesis and the "drugability" of several compounds. Contrasting previous reports, the results from this study supported that not all residues involved in lanthionine bridge formation were critical for maintaining optimal activity. While substitutions in lanthionine bridges in Ring A, C, and D invariably lead to inactive variants, permissive substitutions in Abu8 and Ala11 (Ring B) were observed, albeit infrequently. Further, the data generated suggested that the unsaturated bond from Dha5 (Ser5) may not be critically involved in Lipid-II binding but still important for conferring optimal activity. This study identified additional permissive mutations of Ser5, including Ser5His, Ser5Met, Ser5Gln, and Ser5Leu. In contrast, no permissive substitutions were identified for Dhb14, which suggested that this residue may be critical for optimal activity. Novel blueprints are proposed for directing further development of MU1140 variants and other lantibiotics, which may enable the rational design, development, manufacture, and formulation of an entirely new class of anti-infectives.

Baeyer-Villiger Monooxygenase-Mediated Synthesis of Esomeprazole As an Alternative for Kagan Sulfoxidation.

A wild-type Baeyer-Villiger monooxygenase was engineered to overcome numerous liabilities in order to mediate a commercial oxidation of pyrmetazole to esomeprazole, using air as the terminal oxidant in an almost exclusively aqueous reaction matrix. The developed enzyme and process compares favorably to the incumbent Kagan inspired chemocatalytic oxidation, as esomeprazole was isolated in 87% yield, in >99% purity, with an enantiomeric excess of >99%.

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae.

Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.

Efficient, chemoenzymatic process for manufacture of the Boceprevir bicyclic [3.1.0]proline intermediate based on amine oxidase-catalyzed desymmetrization.

The key structural feature in Boceprevir, Merck's new drug treatment for hepatitis C, is the bicyclic [3.1.0]proline moiety "P2". During the discovery and development stages, the P2 fragment was produced by a classical resolution approach. As the drug candidate advanced through clinical trials and approached regulatory approval and commercialization, Codexis and Schering-Plough (now Merck) jointly developed a chemoenzymatic asymmetric synthesis of P2 where the net reaction was an oxidative Strecker reaction. The key part of this reaction sequence is an enzymatic oxidative desymmetrization of the prochiral amine substrate.

Improving catalytic function by ProSAR-driven enzyme evolution.

We describe a directed evolution approach that should find broad application in generating enzymes that meet predefined process-design criteria. It augments recombination-based directed evolution by incorporating a strategy for statistical analysis of protein sequence activity relationships (ProSAR). This combination facilitates mutation-oriented enzyme optimization by permitting the capture of additional information contained in the sequence-activity data. The method thus enables identification of beneficial mutations even in variants with reduced function. We use this hybrid approach to evolve a bacterial halohydrin dehalogenase that improves the volumetric productivity of a cyanation process approximately 4,000-fold. This improvement was required to meet the practical design criteria for a commercially relevant biocatalytic process involved in the synthesis of a cholesterol-lowering drug, atorvastatin (Lipitor), and was obtained by variants that had at least 35 mutations.