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The scarcity of practical methods for aldehyde synthesis in chemistry necessitates the development of mild, selective procedures. Carboxylic acid reductases catalyze aldehyde formation from stable carboxylic acid precursors in an aqueous solution. Carboxylic acid reductases were employed to catalyze aldehyde formation in a cell-free system with activation energy and reducing equivalents provided through auxiliary proteins for ATP and NADPH recycling. In situ product removal was used to suppress over-reduction due to background enzyme activities, and an N-protected 4-formyl-piperidine pharma synthon was prepared in 61% isolated yield. This is the first report of preparative aldehyde synthesis with carboxylic acid reductases employing crude, commercially available enzyme preparations., (© 2021 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Addressing the challenges associated with the development of in vitro biocatalytic carboxylate reductions for potential applications, important aspects of the co-factor regeneration systems and strategies for minimizing over-reduction were investigated. The ATP recycling can be performed with similarly high efficiency exploiting the polyphosphate source by combining Meiothermus ruber polyphosphate kinase and adenylate kinase or with Sinorhizobium meliloti polyphosphate kinase instead of the latter. Carboxylate reductions with the enzyme candidates used in this work allow operating at co-factor concentrations of adenosine 5'-triphosphate and β-nicotinamide adenine dinucleotide 2'-phosphate of 100 μM and, thereby, reducing the amounts of alcohols formed by side activities in the enzyme preparations. This study confirmed the expected benefits of carboxylic acid reductases in chemoselectively reducing the carboxylates to the corresponding aldehydes while leaving reductively-sensitive nitro, ester and cyano groups intact., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Increasing demand for chemicals from renewable resources calls for the development of new biotechnological methods for the reduction of oxidized bio-based compounds. Enzymatic carboxylate reduction is highly selective, both in terms of chemo- and product selectivity, but not many carboxylate reductase enzymes (CARs) have been identified on the sequence level to date. Thus far, their phylogeny is unexplored and very little is known about their structure-function-relationship. CARs minimally contain an adenylation domain, a phosphopantetheinylation domain and a reductase domain. We have recently identified new enzymes of fungal origin, using similarity searches against genomic sequences from organisms in which aldehydes were detected upon incubation with carboxylic acids. Analysis of sequences with known CAR functionality and CAR enzymes recently identified in our laboratory suggests that the three-domain architecture mentioned above is modular. The construction of a distance tree with a subsequent 1000-replicate bootstrap analysis showed that the CAR sequences included in our study fall into four distinct subgroups (one of bacterial origin and three of fungal origin, respectively), each with a bootstrap value of 100%. The multiple sequence alignment of all experimentally confirmed CAR protein sequences revealed fingerprint sequences of residues which are likely to be involved in substrate and co-substrate binding and one of the three catalytic substeps, respectively. The fingerprint sequences broaden our understanding of the amino acids that might be essential for the reduction of organic acids to the corresponding aldehydes in CAR proteins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Novel synthetic strategies for the production of high-value chemicals based on the 12 principles of green chemistry are highly desired. Herein, we present a proof of concept for two novel chemo-enzymatic one-pot cascades allowing for the production of valuable fragrance and flavor aldehydes. We utilized renewable phenylpropenes, such as eugenol from cloves or estragole from estragon, as starting materials. For the first strategy, Pd-catalyzed isomerization of the allylic double bond and subsequent enzyme-mediated (aromatic dioxygenase, ADO) alkene cleavage were performed to obtain the desired aldehydes. In the second route, the double bond was oxidized to the corresponding ketone via a copper-free Wacker oxidation protocol followed by enzymatic Baeyer–Villiger oxidation (phenylacetone monooxygenase from Thermobifida fusca), esterase-mediated (esterase from Pseudomonas fluorescens, PfeI) hydrolysis and subsequent oxidation of the primary alcohol (alcohol dehydrogenase from Pseudomonas putida, AlkJ) to the respective aldehyde products. Eight different phenylpropene derivatives were subjected to these reaction sequences, allowing for the synthesis of seven aldehydes in up to 55% yield after 4 reaction steps (86% for each step). [ABSTRACT FROM AUTHOR]

Many aldehydes are volatile compounds with distinct and characteristic olfactory properties. The aldehydic functional group is reactive and, as such, an invaluable chemical multi‐tool to make all sorts of products. Owing to the reactivity, the selective synthesis of aldehydic is a challenging task. Nature has evolved a number of enzymatic reactions to produce aldehydes, and this review provides an overview of aldehyde‐forming reactions in biological systems and beyond. Whereas some of these biotransformations are still in their infancy in terms of synthetic applicability, others are developed to an extent that allows their implementation as industrial biocatalysts. [ABSTRACT FROM AUTHOR]

Carboxylate reductases (CARs) are valuable catalysts for the selective one‐step reduction of carboxylic acids to their corresponding aldehydes. In recent years, numerous new CARs have been made available, studied and applied in the context of biocatalytic syntheses. The preparation of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates represent the two major fields of applications. This review gives a comprehensive overview of the current toolbox of recombinant CARs and their numerous carboxylate substrates. Non‐natural functions of CARs are highlighted and recent insight into the structure‐function relationship of CARs are summarized. [ABSTRACT FROM AUTHOR]

The chemoselective synthesis of aldehydes is a challenging task. Nature provides carboxylic acid reductases (CARs) as elegant tools for the direct reduction of carboxylic acids to their respective aldehydes. The discovery of new CARs and strains that efficiently produce these enzymes necessitates a robust high‐throughput assay with selectivity for aldehydes. We recently reported a simple assay that allows the substrate independent and chemoselective quantification of aldehydes (irrespective of their chemical structure). The assay utilized amino benzamidoxime (ABAO), which forms UV‐active and fluorescent dihydroquinazolines. In this study, we adapted the ABAO‐assay for the identification and comparison of Pichia pastoris clones with the ability to produce aldehydes from carboxylic acids. Specifically, CAR and PPTase from Mycobacterium marinum (MmCAR and MmPPTase) were co‐expressed using different bidirectional promoters (BDPs). A library of 598 clones was screened for piperonal production with the ABAO assay and the results were validated by HPLC quantification. 1 OD unit of the best Pichia pastoris clone 2.A7, regulating MmCAR and MmPPTase expression by two strong constitutive promoters, fully converted 5 mM of piperonylic acid within 2 h. [ABSTRACT FROM AUTHOR]

Fatty aldehyde production by chemical synthesis causes an immense burden to the environment. Within this study, we explored a sustainable, aldehyde‐selective and mild alternative approach by utilizing carboxylic acid reductases (CARs). CARs from Neurospora crassa (NcCAR), Thermothelomyces thermophila (TtCAR), Nocardia iowensis (NiCAR), Mycobacterium marinum (MmCAR) and Trametes versicolor (TvCAR) were overexpressed in E. coli K‐12 MG1655 RARE (DE3) and screened for medium‐ to long‐chain fatty acid (C6–C18) reduction. MmCAR showed the broadest tolerance towards all carbon‐chain lengths and was selected for further investigations of fatty aldehyde synthesis in whole cells. To yield relevant product concentrations, different limitations of CAR whole‐cell conversions were elucidated and compensated. We coupled an in vitro cofactor recycling system to a whole‐cell biocatalyst to support cofactor supply and achieved 12.36 g L−1 of octanal (STY 0.458 g L−1 h−1) with less than 1.5 % of 1‐octanol. [ABSTRACT FROM AUTHOR]

The selective and direct reduction of carboxylic acids into the corresponding aldehydes by chemical methods is still a challenging task in synthesis. Several reductive and oxidative chemical methods are known to produce aldehydes, but most of them require expensive reagents, special reaction conditions, are two‐step procedures and often lack chemoselectivity. Nature provides an elegant tool, so called carboxylic acid reductases (CARs) for the direct reduction of carboxylic acids to aldehydes. Discovery as well as engineering of novel CAR enzymes necessitates a robust, product selective high‐throughput assay (HTA). We report a simple and fast HTA that allows the substrate‐independent and chemoselective quantification of aldehydes (irrespective of their chemical structure) and is sensitive to the nM range. The HTA was validated by NMR and GC analyses and in microbial cells by reexamination of the substrate scope of CAR from Nocardia iowensis (CARNi). The results were fully consistent with reported data. [ABSTRACT FROM AUTHOR]

The direct generation of aldehydes from carboxylic acids is often a challenging synthetic task but undoubtedly attractive in view of abundant supply of such feedstocks from nature. Though long known, biocatalytic carboxylate reductions are at an early stage of development, presumably because of their co‐factor requirement. To establish an alternative to whole‐cell‐based carboxylate reductions which are limited by side reactions, we developed an in vitro multi‐enzyme system that allows for quantitative reductions of various carboxylic acids with full recycling of all cofactors and prevention of undesired over‐reductions. Regeneration of adenosine 5′‐triphosphate is achieved through the simultaneous action of polyphosphate kinases from Meiothermus ruber and Sinorhizobium meliloti and β‐nicotinamide adenine dinucleotide 2′‐phosphate is reduced by a glucose dehydrogenase. Under these conditions and in the presence of the carboxylate reductases from Neurospora crassa or Nocardia iowensis, various aromatic, heterocyclic and aliphatic carboxylic acids were quantitatively reduced to the respective aldehydes. [ABSTRACT FROM AUTHOR]

Carboxylate reductases (CARs) are emerging as valuable catalysts for the selective one-step reduction of carboxylic acids to their corresponding aldehydes. The substrate scope of CARs is exceptionally broad and offers potential for their application in diverse synthetic processes. Two major fields of application are the preparation of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates. The latest applications of CARs are dominated by in vivo cascades and chemo-enzymatic reaction sequences. The challenge to fully exploit product selectivity is discussed. Recent developments in the characterization of CARs are summarized, with a focus on aspects related to the domain architecture and protein sequences of CAR enzymes. [ABSTRACT FROM AUTHOR]

Carboxylate reductases (CARs, E.C. 1.2.1.30) generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and fromthe literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants in vivo and in vitro. Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of NcCAR variants when replaced by alanine. Finally, we showed that NcCAR wild-type and mutants efficiently reduce aliphatic acids. [ABSTRACT FROM AUTHOR]

Synthetic enzyme cascades in living cells often lack efficiency owing to the formation of byproducts by endogenous enzymes or toxicity of the cascade intermediates. Highly reactive aldehyde species can trigger a metabolic stress response, and this leads to undesired side reactions and decreased yields. Owing to the metabolic background of Escherichia coli ( E. coli), aldehydes may be irreversibly oxidized to carboxylic acids or reduced to the corresponding alcohols. Herein, we applied an approach to equilibrate the aldehyde concentration in vivo. We oxidized primary alcohols to the corresponding aldehydes by AlkJ, an alcohol dehydrogenase from Pseudomonas putida. Introduction of a carboxylic acid reductase from Nocardia iowensis allowed the target compound to be retrieved from the carboxylate sink. Further reduction of the aldehydes to alcohols by endogenous E. coli enzymes completed the equilibration between alcohols, aldehydes, and carboxylic acids. Thus, the aldehyde concentrations remained below nonviable concentrations. We demonstrated the concept on several primary alcohols, which reached the redox equilibrium within 6 h and persisted up to 24 h. Subsequent combination with a dihydroxyacetone-dependent aldolase (Fsa1-A129S, E. coli) demonstrated that the reactive aldehyde species were freely available and gave the aldol product, (3 S,4 R)-1,3,4-trihydroxy-5-phenylpentan-2-one, in 70 % yield within short reaction times. [ABSTRACT FROM AUTHOR]

Recombinant human aldehyde oxidase (AO) was expressed in Escherichia coli. Different cell disruption methods and conditions of cell culture in shake flasks and bioreactors and of biotransformation on an analytical scale were tested to optimize the synthesis of oxidized AO drug metabolites. The volumetric productivity was increased 24-fold by optimizing the cell culture conditions. The highest yield was achieved in a 25 L stirred tank bioreactor under non-oxygen-limited conditions and high lactose feed rate. Suspensions of highly concentrated and well-aerated whole cells at neutral pH and relatively low temperatures led to the best conversion. The solvent for the substrate and the buffering agent for the biotransformation had an important effect. In a biotransformation with AO, 210 mg of famciclovir was converted to diacetyl penciclovir a yield of 82 %. The optimized protocol represents a viable method for the preparative synthesis of oxidized AO metabolites of drugs. [ABSTRACT FROM AUTHOR]

In this study, we present the synthesis of chiral fragrance aldehydes, which was tackled by a combination of chemo-catalysis and a multi-enzymatic in vivo cascade reaction and the development of a highly versatile high-throughput assay for the enzymatic reduction of carboxylic acids. We investigated a biocompatible metal-catalyzed synthesis for the preparation of α or β substituted cinnamic acid derivatives which were fed directly into the biocatalytic system. Subsequently, the target molecules were synthesized by an enzymatic cascade consisting of a carboxylate reduction, followed by the selective C-C double bond reduction catalyzed by appropriate enoate reductases. We investigated a biocompatible oxidative Heck protocol and combined it with cells expressing a carboxylic acid reductase from Neurospora crassa (NcCAR) and an ene reductase from Saccharomyces pastorianus for the production fragrance aldehydes. [ABSTRACT FROM AUTHOR]

The Cover shows a biochemical cell factory (Escherichia coli) for the synthesis of chiral polyhydroxylated compounds via an artificial enzyme cascade.In their Communication, T. Bayer et al. presented a rational approach for tuning the concentration of toxic aldehyde intermediates in living cells. By combining an alcohol dehydrogenase and a carboxylic acid reductase, the redox equilibrium between alcohol, aldehyde and carboxylic acid species can be adjusted, thus maintaining the aldehyde concentration in a ‘hidden reservoir’ and therefore below the toxicity level, yet freely available for subsequent aldolase mediated reaction. More information can be found in the Communication by T. Bayer et al. on page 2919 in Issue 15, 2017 (DOI: 10.1002/cctc.201700469). [ABSTRACT FROM AUTHOR]

A correction to the article "Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa" is presented.

Carboxylic acid reductases (CARs) are well-known for their eminent selective one-step synthesis of carboxylic acids to aldehydes. To date, however, few CARs have been identified and characterized, especially from fungal sources. In this study, the CAR from the white rot fungus Pycnoporus cinnabarinus (Pc CAR2) was expressed in Escherichia coli. Pc CAR2′s biochemical properties were explored in vitro after purification, revealing a melting temperature of 53 °C, while the reaction temperature optimum was at 35 °C. In the tested buffers, the enzyme showed a pH optimum of 6.0 and notably, a similar activity up to pH 7.5. Pc CAR2 was immobilized to explore its potential as a recyclable biocatalyst. Pc CAR2 showed no critical loss of activity after six cycles, with an average conversion to benzaldehyde of more than 85% per cycle. Immobilization yield and efficiency were 82% and 76%, respectively, on Ni-sepharose. Overall, our findings contribute to the characterization of a thermotolerant fungal CAR, and established a more sustainable use of the valuable biocatalyst. [Display omitted] • The carboxylic acid reductase (CAR) from Pycnoporus cinnabarinus Pc CAR2 is a pH and thermotolerant enzyme. • Targeted immobilization of Pc CAR2 maintains essential conformational flexibility of the enzyme and enables recycling. • All known CARs from subtype IV are from Basidiomycetes. [ABSTRACT FROM AUTHOR]