Synthetic DNA Libraries for Protein Engineering Toward Process Improvement in Drug Synthesis.

Speeding-up enzyme engineering by directed evolution is a primary target to be achieved for a wider uptake of biocatalysis in pharmaceutical process development. The capability to rapidly generate the designed sequence diversity has profound implications in the overall optimization of protein function. Drawbacks associated with traditional PCR methods for sequence diversification interfere with the generation of all the variants that have been designed. On the contrary, the enhanced quality of synthetic DNA libraries makes the exploration of sequence space more efficient. Here, methods for the effective utilization of synthetic DNA libraries are described. The overall procedure allows the generation of ready-to-screen libraries within two weeks from synthetic DNA acquisition.

Regio- and Enantio-selective Chemo-enzymatic C-H-Lactonization of Decanoic Acid to (S)-δ-Decalactone.

The conversion of saturated fatty acids to high value chiral hydroxy-acids and lactones poses a number of synthetic challenges: the activation of unreactive C-H bonds and the need for regio- and stereoselectivity. Here the first example of a wild-type cytochrome P450 monooxygenase (CYP116B46 from Tepidiphilus thermophilus) capable of enantio- and regioselective C5 hydroxylation of decanoic acid 1 to (S)-5-hydroxydecanoic acid 2 is reported. Subsequent lactonization yields (S)-δ-decalactone 3, a high value fragrance compound, with greater than 90 % ee. Docking studies provide a rationale for the high regio- and enantioselectivity of the reaction.

The crystal structure of P450-TT heme-domain provides the first structural insights into the versatile class VII P450s.

The first crystal structure of a class VII P450, CYP116B46 from Tepidiphilus thermophilus, has been solved at 1.9 Šresolution. The structure reveals overall conservation of the P450-fold and a water conduit around the I-helix. Active site residues have been identified and sequence comparisons have been made with other class VII enzymes. A structure similarity search demonstrated that the P450-TT structure is similar to enzymes capable of oxy-functionalization of fatty acids, terpenes, macrolides, steroids and statins. The insight gained from solving this structure will provide a guideline for future engineering and modelling studies on this catalytically promiscuous class of enzymes.

Cloning, expression and characterisation of P450-Hal1 (CYP116B62) from Halomonas sp. NCIMB 172: A self-sufficient P450 with high expression and diverse substrate scope.

Cytochrome P450 monooxygenases are able to catalyse a range of synthetically challenging reactions ranging from hydroxylation and demethylation to sulfoxidation and epoxidation. As such they have great potential for biocatalytic applications but are underutilised due to often-poor expression, stability and solubility in recombinant bacterial hosts. The use of self-sufficient P450 s with fused haem and reductase domains has already contributed heavily to improving catalytic efficiency and simplifying an otherwise more complex multi-component system of P450 and redox partners. Herein, we present a new addition to the class VII family with the cloning, sequencing and characterisation of the self-sufficient CYP116B62 Hal1 from Halomonas sp. NCIMB 172, the genome of which has not yet been sequenced. Hal1 exhibits high levels of expression in a recombinant E. coli host and can be utilised from cell lysate or used in purified form. Hal1 favours NADPH as electron donor and displays a diverse range of activities including hydroxylation, demethylation and sulfoxidation. These properties make Hal1 suitable for future biocatalytic applications or as a template for optimisation through engineering.

Characterisation of CYP102A25 from Bacillus marmarensis and CYP102A26 from Pontibacillus halophilus: P450 Homologues of BM3 with Preference towards Hydroxylation of Medium-Chain Fatty Acids.

Cytochrome P450 monooxygenases are highly desired biocatalysts owing to their ability to catalyse a wide variety of chemically challenging C-H activation reactions. The CYP102A subfamily of enzymes are natural catalytically self-sufficient proteins consisting of a haem and FMN-FAD reductase domain fused in a single-component system. They catalyse the oxygenation of saturated and unsaturated fatty acids to produce primarily ω-1, ω-2 and ω-3 hydroxy acids. These monooxygenases have potential applications in biotechnology; however, their substrate range is still limited and there is a continued need to add diversity to this class of biocatalysts. Herein, we present the characterisation of two new members of this class of enzymes, CYP102A25 (BMar) from Bacillus marmarensis and CYP102A26 (PHal) from Pontibacillus halophilus, both of which express readily in a recombinant bacterial host. BMar exhibits the highest activity toward myristic acid and shows moderate activity towards unsaturated fatty acids. PHal exhibits broader activity towards mid-chain-saturated (C14 -C18 ) and unsaturated fatty acids. Furthermore, PHal shows good regioselectivity for the hydroxylation of myristic acid, targeting the ω-2 position for C-H activation.

A biocatalytic cascade for the amination of unfunctionalised cycloalkanes.

Here we describe a one-pot, three-enzyme, cascade involving a cytochrome P450 monooxygenase, an alcohol dehydrogenase and a reductive aminase for the synthesis of secondary amines from cycloalkanes. Amine product concentrations of up to 19.6 mM were achieved. The preparative scale amination of cyclohexane was also demonstrated with a space-time yield of 2 g L-1 d-1.

The self-sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5-hydroxylation of diclofenac.

P450 monooxygenases are able to catalyze the highly regio- and stereoselective oxidations of many organic molecules. However, the scale-up of such bio-oxidations remains challenging due to the often-low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti-inflammatory drug that is persistent in the environment along with the 4'- and 5-hydroxy metabolites. Here we have used the self-sufficient P450 RhF (CYP116B2) from Rhodococcus sp. in a whole cell system to reproducibly catalyze the highly regioselective oxidation of diclofenac to 5-hydroxydiclofenac. The product is a human metabolite and as such is an important standard for environmental and toxicological analysis. Furthermore, access to significant quantities of 5-hydroxydiclofenac has allowed us to demonstrate further oxidative degradation to the toxic quinoneimine product. Our studies demonstrate the potential for gram-scale production of human drug metabolites through recombinant whole cell biocatalysis.