Delineation of the CYP505E subfamily of fungal self-sufficient in-chain hydroxylating cytochrome P450 monooxygenases.

Cytochrome P450 monooxygenases (CYP450s) are abundant in eukaryotes, specifically in plants and fungi where they play important roles in the synthesis and degradation of secondary metabolites. In eukaryotes, the best studied "self-sufficient" CYP450s, with a fused redox partner, belong to the CYP505 family. Members of the CYP505 family are generally considered sub-terminal fatty acid hydroxylases. CYP505E3 from Aspergillus terreus, however, gives remarkable in-chain hydroxylation at the ω-7 position of C10 to C16 alkanes and C12 and C14 fatty alcohols. Because CYP505E3 is a promising catalyst for the synthesis of δ-dodecalactone, we set out to delineate the unique ω-7 hydroxylase activity of CYP505E3. CYP505E3 and six additional CYP505Es as well as four closely related CYP505s from four different subfamilies were expressed in Pichia pastoris. Only the CYP505Es, sharing more than 70% amino acid identity, displayed significant ω-7 hydroxylase activity toward 1-dodecanol, dodecanoic acid, and tetradecanoic acid giving products that can readily be converted to δ-dodecalactone. Concentrations of δ-dodecalactone, directly extracted from dodecanoic acid biotransformations, were higher than previously obtained with E. coli. Searches of the UniProt and NCBI databases yielded a total of only 23 unique CYP505Es, all from the Aspergillaceae. Given that CYP505Es with this remarkable activity occur in only a few Aspergillus and Penicillium spp., we further explored the genetic environments in which they occur. These were found to be very distinct environments which include a specific ABC transporter but could not be linked to apparent secondary metabolite gene clusters. KEY POINTS: • Identified CYP505Es share > 70% amino acid identity. • CYP505Es hydroxylate 1-dodecanol, dodecanoic, and tetradecanoic acid at ω-7 position. • CYP505E genes occur in Aspergillus and Penicillium spp. near an ABC transporter.

CYP505E3: A Novel Self-Sufficient ω-7 In-Chain Hydroxylase.

The self-sufficient cytochrome P450 monooxygenase CYP505E3 from Aspergillus terreus catalyzes the regioselective in-chain hydroxylation of alkanes, fatty alcohols, and fatty acids at the ω-7 position. It is the first reported P450 to give regioselective in-chain ω-7 hydroxylation of C10-C16 n-alkanes, thereby enabling the one step biocatalytic synthesis of rare alcohols such as 5-dodecanol and 7-tetradecanol. It shows more than 70 % regioselectivity for the eighth carbon from one methyl terminus, and displays remarkably high activity towards decane (TTN≈8000) and dodecane (TTN≈2000). CYP505E3 can be used to synthesize the high-value flavour compound δ-dodecalactone via two routes: 1) conversion of dodecanoic acid into 5-hydroxydodecanoic acid (24 % regioselectivity), which at low pH lactonises to δ-dodecalactone, and 2) conversion of 1-dodecanol into 1,5-dodecanediol (55 % regioselectivity), which can be converted into δ-dodecalactone by horse liver alcohol dehydrogenase.

Alkyl Formate Ester Synthesis by a Fungal Baeyer-Villiger Monooxygenase.

We investigated Baeyer-Villiger monooxygenase (BVMO)-mediated synthesis of alkyl formate esters, which are important flavor and fragrance products. A recombinant fungal BVMO from Aspergillus flavus was found to transform a selection of aliphatic aldehydes into alkyl formates with high regioselectivity. Near complete conversion of 10 mm octanal was achieved within 8 h with a regiomeric excess of ∼80 %. Substrate concentration was found to affect specific activity and regioselectivity of the BVMO, as well as the rate of product autohydrolysis to the primary alcohol. More than 80 % conversion of 50 mm octanal was reached after 72 h (TTN nearly 20 000). Biotransformation on a 200 mL scale under unoptimized conditions gave a space-time yield (STY) of 4.2 g L-1 d-1 (3.4 g L-1 d-1 extracted product).

Cytochrome P450 reductase from Candida apicola: versatile redox partner for bacterial P450s.

Candida apicola belongs to a group of yeasts producing surface-active glycolipids consisting of sophorose and long-chain (ω)- or (ω-1)-hydroxy fatty acids. Hydroxylation of the fatty acids in this strain is likely catalyzed by cytochrome P450 monooxygenases (P450), which require reducing equivalents delivered via a cytochrome P450-diflavin reductase (CPR). We herein report cloning and characterization of the cpr gene from C. apicola ATCC 96134. The gene encoding a protein of 687 amino acids was cloned in Escherichia coli and the enzyme was expressed in functional form after truncation of its N-terminal putative membrane anchor. The truncated recombinant protein showed cytochrome c reducing activity (K (M) of 13.8 µM and k (cat) of 1,915 per minute). Furthermore, we herein demonstrate to our best knowledge for the first time the use of a eukaryotic CPR to transfer electrons to bacterial P450s (namely CYP109B1 and CYP154E1). Cloning and characterization of this CPR therefore is not only an important step in the study of the P450 systems of C. apicola, but also provides a versatile redox partner for the characterization of other bacterial P450s with appealing biotechnological potential. The GenBank accession number of the sequence described in this article is JQ015264.

Identification and characterization of 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates of CYP102A1.

CYP102A1 is an efficient medium- to long-chain fatty acid hydroxylase that is able to accept a wide range of non-natural substrates which bear no resemblance to the natural ones. 4-Hexylbenzoic acid (HBA) and 4-nonyloxybenzoic acid (NOBA) were identified as CYP102A1 substrates via screening studies using the BD Oxygen Biosensor System. Spectroscopic binding studies showed that these two substrates bind in the active site of CYP102A1 with K(d) values of 2.6 ± 0.1 µM for HBA and 1.9 ± 0.2 µM for NOBA. NADPH consumption rates in the presence of HBA and NOBA were 45 ± 1 min(-1) and 61 ± 1 min(-1), respectively. The coupling efficiency for NADPH was 57% for NOBA, while it was 77% for HBA. During whole-cell biotransformations, HBA was converted into ω-1- and ω-2-hydroxyhexylbenzoic acid, whereas NOBA was oxidized to ω-2-hydroxynonyloxybenzoic acid and ω-2,ω-4-dihydroxynonyloxybenzoic acid. HBA was used as a fatty acid mimic to compare whole-cell biotransformations with cell-free extracts. Whole-cell biotransformations carried out in a biphasic system resulted in 86% conversion of 5 mM HBA, producing 3.8 mM ω-2- and 0.5 mM ω-1-hydroxyhexylbenzoic acid in 4 h with a turnover number of 4.1 min(-1), whereas 100% conversion of 5 mM HBA was obtained in 1 h with crude cell extracts and a cofactor regeneration system, giving a turnover number of 10.5 min(-1).

Fungal BVMOs as alternatives to cyclohexanone monooxygenase.

FAD-dependent Baeyer-Villiger monooxygenases (BVMOs) have proven to be useful biocatalysts in the selective and specific oxygenation of various ketones. Despite the cloning, heterologous expression and characterization of close to 80 members of this enzyme family, some sub-groups of BVMOs still remain underrepresented and their evolutionary relationship uncertain. Until recently, very few fungal BVMOs have been described. Our previous investigations into BVMOs from the fungus Aspergillus flavus, yielded very little activity on simple cyclic ketones. Here we report on another four BVMOs from A. flavus that are more closely related to cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. Evolutionary analysis with other characterized BVMOs show their closest relationship to be with either cycloalkanone monooxygenase (CAMO) or 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase (OTEMO). The OTEMO-related BVMOAFL706 and BVMOAFL334 were heterologously expressed in E. coli, purified and shown to be able to convert a range of cyclic and substituted cyclic ketones. Of the unsubstituted cyclic ketones, cyclohexanone showed the highest conversion with maximum turnover frequencies reaching 4.3s-1 for BVMOAFL706. Unlike CHMOacinet, and many of the closely related BVMOs, no substrate inhibition was observed with cyclohexanone to a concentration of up to 30mM, creating the possibility for applications requiring high substrate loading. Aliphatic ketones were also readily converted with excellent regioselectivity. Similar to CHMOacinet, acetophenones were not converted and the oxidation of rac-cis-bicyclo[3.2.0]hept-2-en-6-one occurs enantiodivergently, with the (1R,5S) isomer converted to the "normal" lactone and the (1S,5R) isomer to the "abnormal" lactone.

Structural and Catalytic Characterization of a Fungal Baeyer-Villiger Monooxygenase.

Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that convert ketones to esters. Due to their high regio-, stereo- and enantioselectivity and ability to catalyse these reactions under mild conditions, they have gained interest as alternatives to chemical Baeyer-Villiger catalysts. Despite their widespread occurrence within the fungal kingdom, most of the currently characterized BVMOs are from bacterial origin. Here we report the catalytic and structural characterization of BVMOAFL838 from Aspergillus flavus. BVMOAFL838 converts linear and aryl ketones with high regioselectivity. Steady-state kinetics revealed BVMOAFL838 to show significant substrate inhibition with phenylacetone, which was more pronounced at low pH, enzyme and buffer concentrations. Para substitutions on the phenyl group significantly improved substrate affinity and increased turnover frequencies. Steady-state kinetics revealed BVMOAFL838 to preferentially oxidize aliphatic ketones and aryl ketones when the phenyl group are separated by at least two carbons from the carbonyl group. The X-ray crystal structure, the first of a fungal BVMO, was determined at 1.9 Å and revealed the typical overall fold seen in type I bacterial BVMOs. The active site Arg and Asp are conserved, with the Arg found in the "in" position. Similar to phenylacetone monooxygenase (PAMO), a two residue insert relative to cyclohexanone monooxygenase (CHMO) forms a bulge within the active site. Approximately half of the "variable" loop is folded into a short α-helix and covers part of the active site entry channel in the non-NADPH bound structure. This study adds to the current efforts to rationalize the substrate scope of BVMOs through comparative catalytic and structural investigation of different BVMOs.