Carboxylic acid reductases in metabolic engineering

Butler, Neil D.; Kunjapur, Aditya M.

doi:10.1016/j.jbiotec.2019.10.002

Cited by 34 publications

(35 citation statements)

References 119 publications

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“…Additional activation of the carboxylic acid group and protection of other reactive groups to minimize offtarget reductions are in most cases required. [6] All these factors are cost contributors and cause an immense burden onto the environment.…”

Section: Introductionmentioning

confidence: 99%

“…The reactive nature of aldehydes poses a challenge for their production: under oxidative conditions from alcohols, toxic catalysts are being employed, and reductive routes from carboxylic acids suffer from poor atom economy and are prone to overreduction. Additional activation of the carboxylic acid group and protection of other reactive groups to minimize off‐target reductions are in most cases required . All these factors are cost contributors and cause an immense burden onto the environment.…”

Section: Introductionmentioning

confidence: 99%

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In Vivo Reduction of Medium‐ to Long‐Chain Fatty Acids by Carboxylic Acid Reductase (CAR) Enzymes: Limitations and Solutions

Horvat

Winkler

2020

ChemCatChem

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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 À 1 of octanal (STY 0.458 g L À 1 h À 1) with less than 1.5 % of 1-octanol.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

In Vivo Reduction of Medium‐ to Long‐Chain Fatty Acids by Carboxylic Acid Reductase (CAR) Enzymes: Limitations and Solutions

Horvat

Winkler

2020

ChemCatChem

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“…Aldehydes as chemical targets are widely produced by the flavor and fragrance industry [1,2] and used by the pharmaceutical [3] and biofuel industry [4] as reactive intermediates. Industrial scale aldehyde production often resort to ecologically unfriendly, energy‐draining and unselective methods [5] . In recent years, the enzyme class of carboxylic acid reductases (CARs, E.C.1.2.1.30) has been explored as milder and more sustainable alternative to the harsh chemical approaches used by industry [6] .…”

Section: Methodsmentioning

confidence: 99%

“…Industrial scale aldehyde production often resort to ecologically unfriendly, energy-draining and unselective methods. [5] In recent years, the enzyme class of carboxylic acid reductases (CARs, E.C.1.2.1.30) has been explored as milder and more sustainable alternative to the harsh chemical approaches used by industry. [6] CAR enzymes enable the reduction of a broad range of carboxylic acids (a) to the respective aldehydes (b) in nicotinamide adenine dinucleotide phosphate (NADPH)-and adenosine triphosphate (ATP)-dependent catalytic steps (Scheme 1).…”

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confidence: 99%

Amino Benzamidoxime (ABAO)‐Based Assay to Identify Efficient Aldehyde‐Producing Pichia pastoris Clones

et al. 2020

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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.

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“…CARs have a broad substrate scope and have been employed for production of numerous industrially valuable aldehydes and alcohols (Butler and Kunjapur, 2020). They are relatively large, multi-domain enzymes possessing an amino-terminal adenylation domain, a carboxyl-terminal thioester reductase domain, and a central phosphopantetheine-binding domain ( Figure 2B; Finnigan et al, 2017).…”

Section: Carboxylic Acid Reductases (Cars)mentioning

confidence: 99%

Biosynthesis of Fatty Alcohols in Engineered Microbial Cell Factories: Advances and Limitations

Krishnan

McNeil

Stuart

2020

Front. Bioeng. Biotechnol.

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Concerns about climate change and environmental destruction have led to interest in technologies that can replace fossil fuels and petrochemicals with compounds derived from sustainable sources that have lower environmental impact. Fatty alcohols produced by chemical synthesis from ethylene or by chemical conversion of plant oils have a large range of industrial applications. These chemicals can be synthesized through biological routes but their free forms are produced in trace amounts naturally. This review focuses on how genetic engineering of endogenous fatty acid metabolism and heterologous expression of fatty alcohol producing enzymes have come together resulting in the current state of the field for production of fatty alcohols by microbial cell factories. We provide an overview of endogenous fatty acid synthesis, enzymatic methods of conversion to fatty alcohols and review the research to date on microbial fatty alcohol production. The primary focus is on work performed in the model microorganisms, Escherichia coli and Saccharomyces cerevisiae but advances made with cyanobacteria and oleaginous yeasts are also considered. The limitations to production of fatty alcohols by microbial cell factories are detailed along with consideration to potential research directions that may aid in achieving viable commercial scale production of fatty alcohols from renewable feedstock.

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Carboxylic acid reductases in metabolic engineering

Cited by 34 publications

References 119 publications

In Vivo Reduction of Medium‐ to Long‐Chain Fatty Acids by Carboxylic Acid Reductase (CAR) Enzymes: Limitations and Solutions

In Vivo Reduction of Medium‐ to Long‐Chain Fatty Acids by Carboxylic Acid Reductase (CAR) Enzymes: Limitations and Solutions

Amino Benzamidoxime (ABAO)‐Based Assay to Identify Efficient Aldehyde‐Producing Pichia pastoris Clones

Biosynthesis of Fatty Alcohols in Engineered Microbial Cell Factories: Advances and Limitations

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