Nicholas J. Weise

Synthesis of D‐ and L‐Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process

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.

The Bacterial Ammonia Lyase EncP: A Tunable Biocatalyst for the Synthesis of Unnatural Amino Acids

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.

Biocatalytic transamination with near-stoichiometric inexpensive amine donors mediated by bifunctional mono- and di-amine transaminases

The discovery and characterisation of enzymes with both monoamine and diamine transaminase activity is reported, allowing conversion of a wide range of target ketone substrates with just a small excess of amine donor. The diamine co-substrates (putrescine, cadaverine or spermidine) are bio-derived and the enzyme system results in very little waste, making it a greener strategy for the production of valuable amine fine chemicals and pharmaceuticals.

Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases

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.

Putrescine Transaminases for the Synthesis of Saturated Nitrogen Heterocycles from Polyamines

Putrescine transaminase (pATA; EC 2.6.1.82) catalyzes the transfer of an amino group from terminal diamine donor molecules to keto acid acceptors by using pyridoxal‐5′‐phosphate as a cofactor. The ygjG genes from Escherichia coli K12, Bacillus megaterium, and Bacillus mycoides were successfully cloned and expressed in E. coli BL21(DE3) cells. The three putrescine transaminases were all shown to prefer diaminoalkanes as substrates and thereby generated cyclic imines from the ω‐amino aldehyde intermediates. The addition of a mild chemical reducing agent rapidly reduced the imine intermediate in situ to furnish a range of N‐heterocycle products. We applied pATA in a biomimetic synthesis of 2,3‐dihydro‐1H‐indolizinium‐containing targets, notably the bioactive alkaloid ficuseptine.

Intensified biocatalytic production of enantiomerically pure halophenylalanines from acrylic acids using ammonium carbamate as the ammonia source

An intensified, industrially-relevant strategy for the production of enantiopure halophenylalanines has been developed using the novel combination of a cyanobacterial phenylalanine ammonia lyase (PAL) and ammonium carbamate reaction buffer. The process boasts STYs up to >200 g L−1 d−1, ees ≥ 98% and simplified catalyst/reaction buffer preparation and work up.

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

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.

Identification of Novel Bacterial Members of the Imine Reductase Enzyme Family that Perform Reductive Amination

Reductive amination of carbonyl compounds constitutes one of the most efficient ways to rapidly construct chiral and achiral amine frameworks. Imine reductase (IRED) biocatalysts represent a versatile family of enzymes for amine synthesis through NADPH‐mediated imine reduction. The reductive aminases (RedAms) are a subfamily of IREDs that were recently shown to catalyze imine formation as well as imine reduction. Herein, a diverse library of novel enzymes were expressed and screened as cell‐free lysates for their ability to facilitate reductive amination to expand the known suite of biocatalysts for this transformation and to identify more enzymes with potential industrial applications. A range of ketones and amines were examined, and enzymes were identified that were capable of accepting benzylamine, pyrrolidine, ammonia, and aniline. Amine equivalents as low as 2.5 were employed to afford up to >99 % conversion, and for chiral products, up to >98 % ee could be achieved. Preparative‐scale reactions were conducted with low amine equivalents (1.5 or 2.0) of methylamine, allylamine, and pyrrolidine, achieving up to >99 % conversion and 76 % yield.

Bio-derived production of cinnamyl alcohol via a three step biocatalytic cascade and metabolic engineering

The construction of biocatalytic cascades for the production of chemical precursors is fast becoming one of the most efficient approaches to multi-step synthesis in modern chemistry. However, despite the use of low solvent systems and renewably-resourced catalysts in reported examples, many cascades are still dependent on petrochemical starting materials, which as of yet cannot be accessed in a sustainable fashion. Herein we report the production of the versatile chemical building block cinnamyl alcohol from the primary metabolite and fermentation product L-phenylalanine. Through the combination of three biocatalyst classes (phenylalanine ammonia lyase, carboxylic acid reductase and alcohol dehydrogenase) the target compound could be reached in high purity, demonstrable at 100 mg scale achieving 53 % yield using ambient temperature and pressure in aqueous solution. This system represents a synthetic strategy in which all components present at time zero are biogenic and thus minimising damage to the environment. Further we extend this biocatalytic cascade by its inclusion in a L-phenylalanine overproducing strain of Escherichia coli. This metabolically engineered strain produces cinnamyl alcohol in mineral media using a glycerol and glucose as carbon source. This study demonstrates the potential to establish green routes to the synthesis of cinnamyl alcohol from a waste stream such as glycerol derived, for example, from lipase treated biodiesel.

Zymophore identification enables the discovery of novel phenylalanine ammonia lyase enzymes

The suite of biological catalysts found in Nature has the potential to contribute immensely to scientific advancements, ranging from industrial biotechnology to innovations in bioenergy and medical intervention. The endeavour to obtain a catalyst of choice is, however, wrought with challenges. Herein we report the design of a structure-based annotation system for the identification of functionally similar enzymes from diverse sequence backgrounds. Focusing on an enzymatic activity with demonstrated synthetic and therapeutic relevance, five new phenylalanine ammonia lyase (PAL) enzymes were discovered and characterised with respect to their potential applications. The variation and novelty of various desirable traits seen in these previously uncharacterised enzymes demonstrates the importance of effective sequence annotation in unlocking the potential diversity that Nature provides in the search for tailored biological tools. This new method has commercial relevance as a strategy for assaying the ‘evolvability’ of certain enzyme features, thus streamlining and informing protein engineering efforts.