Search

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]

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]

Medium-chain fatty alcohols (MCFOHs, C6 to C12) are potential substitutes for fossil fuels, such as diesel and jet fuels, and have wide applications in various manufacturing processes. While today MCFOHs are mainly sourced from petrochemicals or plant oils, microbial biosynthesis represents a scalable, reliable, and sustainable alternative. Here, we aim to establish a Saccharomyces cerevisiae platform capable of selectively producing MCFOHs. This was enabled by tailoring the properties of a bacterial carboxylic acid reductase from Mycobacterium marinum (MmCAR). Extensive protein engineering, including directed evolution, structure-guided semirational design, and rational design, was implemented. MmCAR variants with enhanced activity were identified using a growth-coupled high-throughput screening assay relying on the detoxification of the enzyme's substrate, medium-chain fatty acids (MCFAs). Detailed characterization demonstrated that both the specificity and catalytic activity of MmCAR was successfully improved and a yeast strain harboring the best MmCAR variant generated 2.8-fold more MCFOHs than the strain expressing the unmodified enzyme. Through deletion of the native MCFA exporter gene TPO1, MCFOH productionwas further improved, resulting in a titer of 252mg/L for the final strain, which represents a significant improvement in MCFOH production in minimal medium by S. cerevisiae. [ABSTRACT FROM AUTHOR]

Mycobacterium marinum CAR (Mm CAR) is one of the most widely used CARs as the key enzyme for the synthesis of aldehydes, alcohols and further products from the respective carboxylic acids. Herein, we describe the first functionally secreted 131 kDa CAR and its isolated A-domain using Komagataella phaffii and a methanol-free constitutive expression strategy. Precipitated and lyophilized Mm CAR (500 µg) was isolated from the culture supernatant and showed no decrease in activity for piperonylic acid (80% conversion), even when stored for up to 3 weeks at 4°C. Lyophilized Mm CAR precipitate gave 48% yield of E/Z -nonanal-4-nitrobenzoyloxime from the reduction of nonanoic acid and in-situ derivatization with O -4-nitrobenzoyl-hydroxylamine. Furthermore, K. phaffii could successfully secrete the Mm CAR adenylation domain. Its activity was confirmed by the amidation of benzoic acid with n -hexylamine. Neither enzyme variant was glycosylated by the yeast. In summary, functional CAR can be secreted by K. phaffii and used for cell free conversion of carboxylic acids to various products. [Display omitted] • Active full-length Mm CAR and Mm CAR A-domain were secreted by Komagataella phaffii for the first time. • Mm CAR A-domain was active for amidation of benzoic acid with n-hexylamine. • Precipitated, lyophilized full-length Mm CAR can be stored without activity loss at 4°C for at least 3 weeks. [ABSTRACT FROM AUTHOR]

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]

• The Pycnoporus cinnabarinus genome encodes for a repertoire of carboxylic acid reductase (CAR) enzymes. • Type IV CARs can be classified into more than one subclass (or subtype). • A new functionally-distinct member of the CAR enzyme family was identified. Carboxylic acid reductases (CARs) are attracting burgeoning attention as biocatalysts for organic synthesis of aldehydes and their follow-up products from economic carboxylic acid precursors. The CAR enzyme class as a whole, however, is still poorly understood. To date, relatively few CAR sequences have been reported, especially from fungal sources. Here, we sought to increase the diversity of the CAR enzyme class. Six new CAR sequences from the white-rot fungus Pycnoporus cinnabarinus were identified from genome-wide mining. Genome and gene clustering analysis suggests that these Pc CAR enzymes play different natural roles in Basidiomycete systems, compared to their type II Ascomycete counterparts. The cDNA sequences of all six Pccar genes were deduced and analysis of their corresponding amino acid sequence showed that they encode for proteins of similar properties that possess a conserved modular functional tri-domain arrangement. Phylogenetic analyses showed that all Pc CAR enzymes cluster together with the other type IV CARs. One candidate, Pc CAR4, was cloned and over-expressed recombinantly in Escherichia coli. Subsequent biotransformation-based screening with a panel of structurally-diverse carboxylic acid substrates suggest that Pc CAR4 possessed a more pronounced substrate specificity compared to previously reported CARs, preferring to reduce sterically-rigid carboxylic acids such as benzoic acid. These findings thus present a new functionally-distinct member of the CAR enzyme class. [ABSTRACT FROM AUTHOR]

• Identification and expression of novel carboxylate reductase (CAR) from the thermophilic fungus Thermothelomyces thermophila. • Characterization of the second subtype III CAR focusing on stability attributes. • Thermothelomyces thermophila CAR is slightly more thermotolerant than Neurospora crassa CAR and has a different pH optimum. • Successful in vitro application of the Tt CAR reducing vanillic acid to vanillin with cell-free regeneration of ATP and NADPH. A novel type III fungal CAR was identified from the organism Thermothelomyces thermophila. High expression levels were observed in E. coli using the pETDuet-1 plasmid system in combination with an autoinduction protocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested and Tt CAR showed activity for all substrates. High specific activities for aromatic substrates and short chain aliphatic substrates were observed, comparable to those of Nc CAR, the first type III fungal CAR. Tt CAR's pH and temperature optima were at 6.5 and 30 °C, respectively. Up to 20% (v/v) cosolvents did not show a decrease in specific activity of Tt CAR using (E)-cinnamic acid as a substrate. Its half-life at 40 °C was determined to be 8.25 h and its melting temperature (T m) was 56 °C. In vitro reactions with Tt CAR reduced 95.2% of 10 mM vanillic acid, which correlated to a titer of 1.4 g L−1 of vanillin. The space time yield of 0.029 g L-1 h-1 indicates that further improvements would be necessary for an industrially relevant application. This would be especially important when competing against de novo synthesis of bio vanillin by microbial strains producing >30 g L-1. In de novo and in vivo biosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe less than 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-step bioconversion from eugenol to vanillin very attractive. [ABSTRACT FROM AUTHOR]