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Abstract
BackgroundTransaminases have become a key tool in biocatalysis to introduce the amine functionality into a range of molecules like prochiral α-ketoacids and ketones. However, due to the necessity of shifting the equilibrium towards the product side (depending on the amine donor) an efficient amination system may require three enzymes. So far, this well-established transformation has mainly been performed in vitro by assembling all biocatalysts individually, which comes along with elaborate and costly preparation steps. We present the design and characterization of a flexible approach enabling a quick set-up of single-cell biocatalysts producing the desired enzymes. By choosing an appropriate co-expression strategy, a modular system was obtained, allowing for flexible plug-and-play combination of enzymes chosen from the toolbox of available transaminases and/or recycling enzymes tailored for the desired application.ResultsBy using a two-plasmid strategy for the recycling enzyme and the transaminase together with chromosomal integration of an amino acid dehydrogenase, two enzyme modules could individually be selected and combined with specifically tailored E. coli strains. Various plug-and-play combinations of the enzymes led to the construction of a series of single-cell catalysts suitable for the amination of various types of substrates. On the one hand the fermentative amination of α-ketoacids coupled both with metabolic and non-metabolic cofactor regeneration was studied, giving access to the corresponding α-amino acids in up to 96% conversion. On the other hand, biocatalysts were employed in a non-metabolic, “in vitro-type” asymmetric reductive amination of the prochiral ketone 4-phenyl-2-butanone, yielding the amine in good conversion (77%) and excellent stereoselectivity (ee = 98%).ConclusionsThe described modularized concept enables the construction of tailored single-cell catalysts which provide all required enzymes for asymmetric reductive amination in a flexible fashion, representing a more efficient approach for the production of chiral amines and amino acids.
Highlights
Transaminases have become a key tool in biocatalysis to introduce the amine functionality into a range of molecules like prochiral α-ketoacids and ketones
A range of well-working techniques has been developed like the coupling of l-alanine dependent TAs with an alanine dehydrogenase (AlaDH) and an enzyme for nicotinamide cofactor regeneration, which can be for instance formate dehydrogenase or glucose dehydrogenase [20, 21]
Design and construction of modular plug‐and‐play single‐cell biocatalysts The aim was the development of a recombinant E. coli single-cell system providing all required enzymes for the asymmetric reductive amination of prochiral α-ketoacids and ketones in a flexible plug-and-play fashion
Summary
Transaminases have become a key tool in biocatalysis to introduce the amine functionality into a range of molecules like prochiral α-ketoacids and ketones. A range of well-working techniques has been developed like the coupling of l-alanine dependent TAs with an alanine dehydrogenase (AlaDH) and an enzyme for nicotinamide cofactor regeneration, which can be for instance formate dehydrogenase or glucose dehydrogenase [20, 21] This orthogonal three-enzyme cascade has successfully demonstrated its usefulness in a range of TA-catalyzed transformations and has occupied a permanent spot in cell-free biocatalysis involving TAs. the assembly of multiple biocatalysts in vitro, which is generally termed “systems biocatalysis” [22,23,24], implies the individual production of each required enzyme, which is time-consuming and lacks elegance. The obtained toolbox of single-cell biocatalysts was successfully employed for the asymmetric reductive amination of α-ketoacids and prochiral ketones, investigating both fermentative transformations coupled to the host’s metabolism and non-metabolic “in vitro-type” transformations
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