The bio-based chemical industry is seeking sustainable alternatives for producing chemical building blocks. Fatty acids, particularly from agricultural waste or non-edible sources, are attractive starting materials. Traditional chemical methods for fatty acid conversion are limited to biodiesel or cosmetic ester production. However, recent advancements have opened new avenues for valorizing fatty acids, including the discovery of fatty acid decarboxylases (like OleT_5-7 or UndA/B) enabling the synthesis of terminal alkenes, and the use of P450 monooxygenases, peroxygenases, or dioxygenases for hydroxylation. Long-chain secondary alcohols, valuable in cosmetics, oleochemicals, and natural product synthesis, are currently inaccessible from natural fatty acids via efficient methods. Existing synthetic routes involve Grignard reactions, resulting in racemic products and significant waste. This research explores the potential of a photoenzyme, CvFAP from *Chlorella variabilis* NC64A, for the synthesis of functionalized alkanes and its application in creating secondary fatty alcohols from unsaturated fatty acids. CvFAP's high chemoselectivity and functional group tolerance under mild conditions make it a promising candidate. The study proposes two photoenzymatic cascades: one using fatty acid hydratase and the other using diol synthase, both followed by CvFAP-catalyzed decarboxylation to produce the desired alcohols.

The existing literature extensively covers various biocatalytic methodologies for transforming fatty acids. These include hydrolase-catalyzed esterification or amidation, reductase-catalyzed reduction to aldehydes or alcohols, P450-peroxygenase-catalyzed oxidative decarboxylation to terminal alkenes, photodecarboxylase-catalyzed decarboxylation to alkanes, hydratase-catalyzed water addition to C=C bonds, lipoxygenase-catalyzed allylic hydroperoxidation, and the use of mono-, di-, and peroxygenases for hydroxylation. Several studies have focused on multi-enzyme cascades for producing short-chain acids. However, there was a gap in the literature regarding the efficient and selective synthesis of long-chain secondary alcohols from natural fatty acids. The authors highlight the limitations of existing chemical methods and the potential of the newly discovered photoenzyme, CvFAP, as a key enabling technology for this transformation.

The study employed a multi-step enzymatic approach. CvFAP, the photoactivated carboxylic acid decarboxylase, was produced recombinantly in *E. coli*. For the hydration step, oleate hydratase from *Lactobacillus reuteri* (LrOhyA) was also recombinantly expressed in *E. coli*. Initially, a two-step procedure was used, with the hydration step first, followed by the addition of CvFAP and illumination for decarboxylation. Optimization of reaction parameters focused on enhancing product formation, addressing LrOhyA's instability. The substrate scope was investigated using a range of (poly)unsaturated fatty acids. Optical purity was determined via O-acylation with (S)-(+)-O-acetylmandelic acid and NMR analysis. To address the low solubility of fatty acids, a two-liquid-phase system was explored, using triolein as the organic phase. A tri-enzymatic cascade was developed incorporating lipase from *Candida rugosa* (CrLip) for triglyceride hydrolysis. To improve efficiency, the authors constructed a co-expression system in *E. coli* combining SmOhyA (from *Stenotrophomonas maltophilia*) and CvFAP. Finally, a cascade using 5,8-diol synthase from *Aspergillus nidulans* (AnDS) was implemented for dihydroxylation, followed by CvFAP-mediated decarboxylation. Detailed procedures for biocatalyst preparation, cascade reactions (including one-pot, two-step, and tri-enzymatic variations), and preparative-scale synthesis are provided in the supplementary materials. Analytical methods included gas chromatography (GC), GC/MS, and NMR.

The study successfully demonstrated the photoenzymatic cascade synthesis of chiral secondary fatty alcohols from unsaturated fatty acids. Using LrOhyA and CvFAP, a broad range of substrates were converted into the corresponding alcohols with acceptable to good yields (24-74%). The resulting alcohols were largely enantiomerically pure. The two-liquid-phase system using triolein significantly improved yields (up to 87%). The co-expression of SmOhyA and CvFAP in *E. coli* resulted in a much faster hydration step compared to the two-step approach. The use of AnDS allowed for the synthesis of diols followed by decarboxylation. A preparative-scale synthesis of an optically pure alcohol from linoleic acid yielded 82.5 mg (32.5% isolated yield). While the current system showed promise, limitations include poor substrate loadings, low reaction rates (especially for hydration), and the need for multiple catalyst systems. The optimization of the cascade reaction including the use of a two-liquid phase system has shown to be a very promising approach to improve product titres and yields. The data included time-course studies for various transformations (Figs. 3, 6, 7), showing the conversion of substrates to products and intermediates. Substrate scope and product enantiomeric excesses are presented (Fig. 4 and Supplementary Figs. 8-48).

The findings directly address the research question of developing an efficient and sustainable method for synthesizing chiral secondary fatty alcohols from renewable resources. The successful implementation of the photoenzymatic cascades with multiple enzyme systems demonstrates a viable route to these valuable chemicals. The high enantiomeric purity of the products is a significant achievement. The use of two-liquid phase systems and the co-expression of enzymes show considerable potential for improving reaction efficiency and yield. The results are highly relevant to the field of green chemistry and biocatalysis, offering an environmentally friendly and selective alternative to traditional chemical synthesis. Further research into optimization of the different steps and investigation of the properties of the synthesized secondary fatty alcohols will be important for future developments and applications.

This study successfully demonstrated the feasibility of synthesizing enantiomerically pure secondary fatty alcohols from unsaturated fatty acids using photobiocatalytic cascades. The use of multiple enzymes and optimization strategies, such as the two-liquid phase system, considerably improved reaction efficiency. Future work will focus on enhancing product yields, exploring broader substrate scopes, and investigating potential applications of these chiral alcohols, including their biological activities.

The current system suffers from limitations including poor substrate solubility, low reaction rates (particularly in the hydration step), and the requirement for two separate enzyme systems. While the use of two-liquid phase systems and co-expression have helped mitigate some of these issues, further optimizations are needed to improve overall efficiency and cost-effectiveness for large-scale applications. The product titres achieved remain relatively low at this stage.