Reductive Amination of Biobased Levulinic Acid to Unnatural Chiral γ-Amino Acid Using an Engineered Amine Dehydrogenase

Abstract

Optically pure (S)-4-aminopentanoic acid is a pivotal precursor in the synthesis of therapeutic molecules and pyrrolidinone derivatives. Enantioselective reductive amination of levulinic acid catalyzed by amine dehydrogenases, a readily sustainable material from biobased lignocellulosic waste, represents an attractive approach for the synthesis of (S)-4-aminopentanoic acid. However, the natural amine dehydrogenases reported so far showed insufficient activity toward levulinic acid. Herein, we engineered a naturally occurring amine dehydrogenase from a thermophilic bacterium Petrotoga mobilis (PmAmDH) by directed evolution. The catalytic efficiency of the most active mutant PmAmDH I80T/P224S/E296G was elevated by 18 folds in comparison to the wild-type enzyme. Using PmAmDH I80T/P224S/E296G coupled with formate dehydrogenase for reduced nicotinamide adenine dinucleotide regeneration, 0.5 M of levulinic acid was reductively aminated in more than 97% conversion at 40 °C, generating the corresponding product (S)-4-aminopentanoic acid with >99% ee and 90% yield. Furthermore, we also successfully developed a chemoenzymatic cascade route for the synthesis of (S)-4-aminopentanoic acid from renewable starch. These results indicated that the engineered amine dehydrogenase PmAmDH I80T/P224S/E296G can serve as an efficient biocatalyst for the manufacture of highly valued chiral unnatural amino acids using renewable feedstocks.

A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase

“…With the column temperature maintained at 30°C, mobile phase H 2 SO 4 (5 mM) ran at a flow rate of 0.6 ml/min. ( Cai et al, 2020 ). ( R )-4-aminopentanoic acid in the reaction mixtures were labeled using 1-fluoro-2,4-dinitrophenyl-5-L-alanineamide (FDAA) and then analyzed on a Develosil ODS-UG-5 column (150 × 4.6 mm).…”

“… Mayol et al (2016) identified a wild-type amine dehydrogenase from Petrotoga mobilis ( Pm AmDH), which is capable of reductive amination of LA to ( S )-4-aminopentanoic acid. Subsequently, Cai et al (2020) engineered Pm AmDH through directed evolution and obtained mutants with increased activity, thereby achieving efficient synthesis of ( S )-4-aminopentanoic acid. However, there are still no relevant reports on the dehydrogenase that converts LA into ( R )-4-aminopentanoic acid.…”

Reprogramming natural proteins using unnatural amino acids

“…20,23 Even naturally occurring amine dehydrogenases have been engineered to improve their substrate specicity and catalytic efficiency. Cai et al 24 have synthesized g-aminobutyric acid from biobased lignocellulosic waste using an engineered amine dehydrogenase.…”

“…A new generation of chemical and drug discovery tools has emerged from conformationally constrained peptides. Asymmetric alkylation of a uorine-modied Ni(II) Schiff base complex (23,24) yielded an unnatural alkenyl amino acid (25,26) required for peptide 'stapling' (Fig. 2c).…”

Improving the Thermostability and Activity of Transaminase From Aspergillus terreus by Charge-Charge Interaction

“…Chiral amines are important components of many significant bioactive compounds, pharmaceutical intermediates and agrochemical industry products (Bornscheuer et al, 2012 ; Mathew and Yun, 2012a ; Ghislieri and Turner, 2014 ; Park et al, 2014 ; Fuchs et al, 2015 ; Ferrandi and Monti, 2017 ; Dawood et al, 2018 ; Cai et al, 2020 ). In addition to optical rotation, enantiomers of chiral drugs have the same physical properties, but they are absorbed, activated or degraded by the metabolic system of the human body in different ways during the pharmacological action, resulting in different efficacies and toxicities (Burke and Henderson, 2002 ).…”