Ammonia-lyases and aminomutases are mechanistically and structurally diverse enzymes which catalyze the deamination and/or isomerization of amino acids in nature by cleaving or shifting a C–N bond. ...Of the many protein families in which these enzyme activities are found, only a subset have been employed in the synthesis of optically pure fine chemicals or in medical applications. This review covers the natural diversity of these enzymes, highlighting particular enzyme classes that are used within industrial and medical biotechnology. These highlights detail the discovery and mechanistic investigations of these commercially relevant enzymes, along with comparisons of their various applications as stand-alone catalysts, components of artificial biosynthetic pathways and biocatalytic or chemoenzymatic cascades, and therapeutic tools for the potential treatment of various pathologies.
The synthesis of substituted D‐phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one‐pot approach by coupling ...phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high‐throughput solid‐phase screening method has also been developed to identify PALs with higher rates of formation of non‐natural D‐phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the D‐configured product. Furthermore, the system was extended to the preparation of those L‐phenylalanines which are obtained with a low ee value using PAL amination.
PAL around: The cascade coupling of phenylalanine ammonia lyase (PAL) with L‐amino acid deaminase (LAAD) or D‐amino acid oxidase (DAAO), led to the conversion of cheap and easily accessible cinnamic acids into both non‐natural D‐ and L‐phenylalanines by a chemoenzymatic process. PAL variants possessing higher D‐activity were selected by using a novel solid‐phase screening assay and employed to increase yield and ee values.
Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chemical processes. Such products range from pharmaceutical and ...agrochemical building blocks to fine and bulk chemicals and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technology. Directed evolution involves repeated rounds of (i) random gene library generation, (ii) expression of genes in a suitable host and (iii) screening of libraries of variant enzymes for the property of interest. Both in vitro screening-based methods and in vivo selection-based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodology, applications in synthetic transformations and strategies for the generation of new enzyme function.
Enzymes of the class I lyase-like family catalyze the asymmetric addition of ammonia to arylacrylates, yielding high value amino acids as products. Recent examples include the use of phenylalanine ...ammonia lyases (PALs), either alone or as a gateway to deracemization cascades (giving (S)- or (R)-α-phenylalanine derivatives, respectively), and also eukaryotic phenylalanine aminomutases (PAMs) for the synthesis of the (R)-β-products. Herein, we present the investigation of another family member, EncP from Streptomyces maritimus, thereby expanding the biocatalytic toolbox and enabling the production of the missing (S)-β-isomer. EncP was found to convert a range of arylacrylates to a mixture of (S)-α- and (S)-β-arylalanines, with regioselectivity correlating to the strength of electron-withdrawing/-donating groups on the ring of each substrate. The low regioselectivity of the wild-type enzyme was addressed via structure-based rational design to generate three variants with altered preference for either α- or β-products. By examining various biocatalyst/substrate combinations, it was demonstrated that the amination pattern of the reaction could be tuned to achieve selectivities between 99:1 and 1:99 for β:α-product ratios as desired.
Recent developments of stereoselective biocatalytic and chemocatalytic methods are discussed. The review provides a guide to the use of biocatalytic methods in the area of chemical synthesis with ...focused attention on retrosynthetic considerations and analysis. The transformations presented are organized according to bond disconnections and attendant synthetic methods. The review is expected to lead to better understanding of the characteristics and distinctions of the two complementary approaches. It depicts for researchers in bio‐ and chemocatalysis a road map of challenges and opportunities for the evolution (and at times revolution) in chemical synthesis.
Partners in synthesis: This Review provides a guide to the use of biocatalytic methods in the area of chemical synthesis with focus on retrosynthetic considerations and analysis. It is expected to lead to better understanding of the characteristics and distinctions of the two complementary approaches by depicting a road map of challenges and opportunities for the evolution in chemical synthesis.
Enantiomerically pure chiral amines are valuable building blocks for the synthesis of pharmaceutical drugs and agrochemicals. Indeed it is estimated that currently 40 % of pharmaceuticals contain a ...chiral amine component in their structure. Chiral amines are also widely used as resolving agents for diastereomeric salt crystallization. One of the challenges of preparing chiral amines in enantiomerically pure form is the development of cost-effective and sustainable catalytic methods that are able to address the requirement for the entire range of primary, secondary and tertiary amines. In this review we highlight various biocatalytic strategies that have been developed, particularly those based upon asymmetric synthesis or their equivalent therefore (i.e. dynamic kinetic resolution, deracemisation) in which yields and enantiomeric excesses approaching 100 % can be attained. Particular attention is given to the use of monoamine oxidase (MAO-N) from
Aspergillus niger
which has been engineered by directed evolution to provide a tool-box of variants which can generate enantiomerically pure primary, secondary and tertiary amines. These MAO-N variants are combined with non-selective chemical reducing agents in deracemisation processes.
Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, ...and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site‐directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles.
CARpe diem: Carboxylic acid reductases can be used as biocatalysts for the activation and subsequent amidation of a range of carboxylic acids with primary and secondary amines, as well as ammonia. The enzymes were used to synthesize the anticonvulsant ilepcimide. Key: adenosine triphosphate (ATP), nicotinamide adenine dinucleotide phosphate (NADPH).
Biocatalytic retrosynthesis Turner, Nicholas J; O'Reilly, Elaine
Nature chemical biology,
05/2013, Letnik:
9, Številka:
5
Journal Article
Recenzirano
The recent development of a broad range of biocatalysts that can be applied in organic synthesis has brought into focus the need to rethink the way in which organic target molecules might be ...constructed in the future. To aid synthetic chemists in identifying where biocatalysts might be usefully applied Turner and O'Reilly propose that guidelines and rules for biocatalytic retrosynthesis be developed and that this new approach be embedded in the future training and education of organic chemists.
The microbial production of fine chemicals provides a promising biosustainable manufacturing solution that has led to the successful production of a growing catalog of natural products and high-value ...chemicals. However, development at industrial levels has been hindered by the large resource investments required. Here we present an integrated Design-Build-Test-Learn (DBTL) pipeline for the discovery and optimization of biosynthetic pathways, which is designed to be compound agnostic and automated throughout. We initially applied the pipeline for the production of the flavonoid (2
)-pinocembrin in
, to demonstrate rapid iterative DBTL cycling with automation at every stage. In this case, application of two DBTL cycles successfully established a production pathway improved by 500-fold, with competitive titers up to 88 mg L
. The further application of the pipeline to optimize an alkaloids pathway demonstrates how it could facilitate the rapid optimization of microbial strains for production of any chemical compound of interest.
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