Photobiocatalytic synthesis of chiral secondary fatty alcohols from renewable unsaturated fatty acids

Abstract

En route to a bio-based chemical industry, the conversion of fatty acids into building blocks is of particular interest. Enzymatic routes, occurring under mild conditions and excelling by intrinsic selectivity, are particularly attractive. Here we report photoenzymatic cascade reactions to transform unsaturated fatty acids into enantiomerically pure secondary fatty alcohols. In a first step the C=C-double bond is stereoselectively hydrated using oleate hydratases from Lactobacillus reuteri or Stenotrophomonas maltophilia. Also, dihydroxylation mediated by the 5,8-diol synthase from Aspergillus nidulans is demonstrated. The second step comprises decarboxylation of the intermediate hydroxy acids by the photoactivated decarboxylase from Chlorella variabilis NC64A. A broad range of (poly)unsaturated fatty acids can be transformed into enantiomerically pure fatty alcohols in a simple one-pot approach.

Fig. 1. Natural fatty acids as building blocks. In recent years, biocatalytic methodologies for the transformation of fatty acids have practically exploded.

For example: a hydrolase-catalysed esterification of amidation, b reductase-catalysed reduction of the carboxylate group to the corresponding aldehyde and alcohol^,, c P450-peroxygenase-catalysed oxidative decarboxylation yielding terminal alkenes^–, d photodecarboxylase-catalysed decarboxylation yielding alkanes^,, e hydratase-catalysed water addition to C=C-bonds, f lipoxygenase-catalysed allylic hydroperoxidation, g use of mono-, di- and per-oxygenases for the terminal hydroxylation and further transformation into acids or amines as polymer building blocks^–,, and h multi-enzyme cascades yielding short-chain acids^–.

Fig. 2. Proposed photoenzymatic cascades to transform unsaturated fatty acids into secondary alcohols.

a Cascade 1 comprises the (stereoselective) addition of water to C=C-double bonds catalysed by fatty acid hydratases (FAHs) followed by the decarboxylation mediated by the photoactivated decarboxylase from Chlorella variabilis NC64A (CvFAP) generating secondary long-chain alcohols; b cascade 2 combines 5,8-diol synthase from Aspergillus nidulans (AnDS) with CvFAP yielding diols.

Fig. 3. Proposed photoenzymatic cascade to transform oleic acid into 9-heptadecanol.

a: Recation scheme. b shows a representative time course of the cascade reaction. Reaction conditions: [oleic acid] = 7 mM, [LrOhyA cells] = 15 g L⁻¹, [CvFAP] = 2 µM, Tris-HCl buffer pH 8.0 (100 mM, with 50 mM of NaCl), illumination with blue light (λ = 450 nm; intensity = 13.7 mE L⁻¹ s⁻¹): oleic acid (black squares), 10-hydroxystearic acid (green circles), 9-heptadecanol (blue diamonds). Values represent the average of duplicates (n = 2). Error bars indicate the standard deviation.

Fig. 4. Preliminary product scope of the proposed photoenzymatic reaction system.

Reaction conditions: [substrate] = 5 mM, [LrOhyA-cells] = 20 g L⁻¹, [CvFAP] = 2 µM, Tris-HCl buffer (100 mM, with 50 mM of NaCl), blue light (λ = 450 nm; intensity = 13.7 mE L⁻¹ s⁻¹). The reactions were performed in a two-step fashion: first the LrOhyA-catalysed hydration reaction was performed for 11 h followed by addition of CvFAP and illumination for another 6 h. nd not determined. Conversion = [product]_final × [substrate]_initial⁻¹ × 100%; determined via GC, conversions determined via ¹H NMR are shown in Supplementary Tables 2 and 3. The enantiomeric excess (e.e.) was determined by ¹H NMR analysis after the fatty alcohols were derivatised by (S)-( + )-O-acetylmandelic acid (details see Supplementary Tables 1 and 2).

Fig. 5. Trienzymatic cascade for the transformation of triolein into 9-heptadecanol using a two-liquid-phase approach.

The aqueous reaction medium is supplemented with neat triolein (triglyceride phase) serving as substrate reservoir and product sink. In the reaction sequence, triolein is hydrolysed by the lipase from Candida rugosa (CrLip, located at the interphase) liberating glycerol and oleic acid. The latter is hydrated and decarboxylated (catalysed by LrOHyA and CvFAP) yielding 9-heptadeconol, which partitions back into the hydrophobic phase.

Fig. 6. Photoenzymatic cascade.

a Reaction scheme of the photoenzymatic cascade combining SmOhA and CvFAP in a single expression host. b Time course of the conversion of oleic acid using co-expressed enzymes. Oleic acid (1a, black circles) was converted via 10-hydroxystearic acid (1b, green squares) into 9-heptadecanol (1c, blue diamonds) and the side-product (Z)-heptadec-8-ene (1f, grey empty circles) using the freshly designed, all-inclusive E. coli BL21 (DE3) pACYC-PelBSS-OhyA/pET28a-CvFAP. [oleic acid] = 5 mM, [E. coli co-expressing SmOhyA and CvFAP] = 7 g dry cells L⁻¹, Tris-HCl buffer pH 6.5 (50 mM), illumination with blue light (λ = 450 nm; intensity = 13.7 mE L⁻¹s⁻¹). For the reaction, first the SmOhyA-catalysed hydration reaction was performed for 0.125 h followed by CvFAP-catalysed decarboxylation under illumination for another 1.625 h. Values represent the average of duplicates (n = 2). Error bars indicate the standard deviation.

Fig. 7. Photoenzymatic cascade.

a Reaction scheme of the photoenzymatic cascade transforming oleic acid into Photoenzymatic diol synthesis-decarboxylation of oleic acid. b Typical time course [oleic acid] = 15 mM, [AnDS cells] = 7 g L⁻¹, [CvFAP cells] = 7 g L⁻¹, HEPES buffer pH 7.5 (50 mM, with 10% (v/v) DMSO), illumination with blue light (λ = 450 nm; intensity = 13.7 mE L⁻¹ s⁻¹): oleic acid (black circles), 8-hydroperoxy-9(Z)-octadecenoic acid (1g, green empty triangles), 5,8-dihydroxy-9(Z)-octadecenoic acid (1d, green triangles), (4S,7R,Z)-heptadec-8-ene-4,7-diol (1e, blue squares), (Z)-heptadec-8-ene (1f, grey circles). For the reaction, first the AnDS-catalysed diol synthetic reaction was performed for 2 h followed by addition of CvFAP and illumination for another 7 h. The absolute configuration is based on the enantioselectivity of the synthase as previously established by Oh and coworkers^,.