Novel concepts to utilize carbon dioxide are required to reach a circular carbon economy and minimize environmental issues. To achieve these goals, photo‐, electro‐, thermal‐, and biocatalysis are key tools to realize this, preferentially in aqueous solutions. Nevertheless, catalytic systems that operate efficiently in water are scarce. Here, we present a general strategy for the identification of enzymes suitable for CO ₂ reduction based on structural analysis for potential carbon dioxide binding sites and subsequent mutations. We discovered that the phenolic acid decarboxylase from Bacillus subtilis (BsPAD) promotes the aqueous photocatalytic CO ₂ reduction selectively to carbon monoxide in the presence of a ruthenium photosensitizer and sodium ascorbate. With engineered variants of BsPAD, TONs of up to 978 and selectivities of up to 93 % (favoring the desired CO over H ₂ generation) were achieved. Mutating the active site region of BsPAD further improved turnover numbers for CO generation. This also revealed that electron transfer is rate‐limiting and occurs via multistep tunneling. The generality of this approach was proven by using eight other enzymes, all showing the desired activity underlining that a range of proteins is capable of photocatalytic CO ₂ reduction.

The application of genetically encoded biosensors enables the detection of small molecules in living cells and has facilitated the characterization of enzymes, their directed evolution and the engineering of (natural) metabolic pathways. In this work, the LuxAB biosensor system from Photorhabdus luminescens was implemented in Escherichia coli to monitor the enzymatic production of aldehydes from primary alcohols and carboxylic acid substrates. A simple high-throughput assay utilized the bacterial luciferase—previously reported to only accept aliphatic long-chain aldehydes—to detect structurally diverse aldehydes, including aromatic and monoterpene aldehydes. LuxAB was used to screen the substrate scopes of three prokaryotic oxidoreductases: an alcohol dehydrogenase (Pseudomonas putida), a choline oxidase variant (Arthrobacter chlorophenolicus) and a carboxylic acid reductase (Mycobacterium marinum). Consequently, high-value aldehydes such as cinnamaldehyde, citral and citronellal could be produced in vivo in up to 80% yield. Furthermore, the dual role of LuxAB as sensor and monooxygenase, emitting bioluminescence through the oxidation of aldehydes to the corresponding carboxylates, promises implementation in artificial enzyme cascades for the synthesis of carboxylic acids. These findings advance the bio-based detection, preparation and transformation of industrially important aldehydes in living cells.

Olive mill wastewater (OMWW) is produced annually during olive oil extraction and contains most of the health‐promoting 3‐hydroxytyrosol of the olive fruit. To facilitate its recovery, enzymatic transesterification of hydroxytyrosol (HT) was directly performed in an aqueous system in the presence of ethyl acetate, yielding a 3‐hydroxytyrosol acetate rich extract. For this, the promiscuous acyltransferase from Pyrobaculum calidifontis VA1 (PestE) was engineered by rational design. The best mutant for the acetylation of hydroxytyrosol (PestE_I208A_L209F_N288A) was immobilized on EziG² beads, resulting in hydroxytyrosol conversions between 82 and 89 % in one hour, for at least ten reaction cycles in a buffered hydroxytyrosol solution. Due to inhibition by other phenols in OMWW the conversions of hydroxytyrosol from this source were between 51 and 62 %. In a preparative scale reaction, 13.8 mg (57 %) of 3‐hydroxytyrosol acetate was extracted from 60 mL OMWW.

Biocatalytic transesterification is commonly carried out employing lipases in anhydrous organic solvents since hydrolases usually prefer hydrolysis over acyl transfer in bulk water. However, some promiscuous acyltransferases can catalyze acylation in an aqueous solution. In this study, a rational design was performed to enhance the acyltransferase selectivity and substrate scope of the Pyrobaculum calidifontis VA1 esterase (PestE). PestE wild type and variants were applied for the acylation of monoterpene alcohols. The mutant PestE_I208A is selective for (–)-menthyl acetate (E-Value = 55). Highly active acyltransferases were designed, allowing for complete conversion of (–)-citronellol to citronellyl acetate. Additionally, carvacrol was acetylated but with lower conversions. To the best of our knowledge, this is the first example of the biocatalytic acylation of a phenolic alcohol in bulk water. In addition, a high citronellol conversion of 92% was achieved with the more environmentally friendly and inexpensive acyl donor ethyl acetate using PestE_N288F as a catalyst. PestE_N288F exhibits good acyl transfer activity in an aqueous medium and low hydrolysis activity at the same time. Thus, our study demonstrates an alternative synthetic strategy for acylation of compounds without organic solvents.

Combining solid acid catalysts with enzyme reactions in aqueous environments is challenging because either very acidic conditions inactivate the enzymes, or the solid acid catalyst is neutralized. In this study, Amberlyst‐15 encapsulated in polydimethylsiloxane (Amb‐15@PDMS) is used to deprotect the lignin depolymerization product G−C2 dioxolane phenol in a buffered system at pH 6.0. This reaction is directly coupled with the biocatalytic reduction of the released homovanillin to homovanillyl alcohol by recombinant horse liver alcohol dehydrogenase, which is subsequently acylated by the promiscuous acyltransferase/hydrolase PestE_I208A_L209F_N288A in a one‐pot system. The deprotection catalyzed with Amb‐15@PDMS attains up to 97 % conversion. Overall, this cascade enables conversions of up to 57 %.

Fatty aldehydes (FALs) can be derived from fatty acids (FAs) and related compounds and are frequently used as flavors and fragrances. Although chemical methods have been conventionally used, their selective biotechnological production aiming at more efficient and eco‐friendly synthetic routes is in demand. α‐Dioxygenases (α‐DOXs) are heme‐dependent oxidative enzymes biologically involved in the initial step of plant FA α‐oxidation during which molecular oxygen is incorporated into the C_α‐position of a FA (C_n) to generate the intermediate FA hydroperoxide, which is subsequently converted into the shortened corresponding FAL (C_n‐1). α‐DOXs are promising biocatalysts for the flavor and fragrance industries, they do not require NAD(P)H as cofactors or redox partner proteins, and they have a broad substrate scope. Here, we highlight recent advances in the biocatalytic utilization of α‐DOXs with emphasis on newly discovered cyanobacterial α‐DOXs as well as analytical methods to measure α‐DOX activity in vitro and in vivo.