In vivo plug-and-play: a modular multi-enzyme single-cell catalyst for the asymmetric amination of ketoacids and ketones
| Autor: | Elisabeth Lorenz, Wolfgang Kroutil, Nina Richter, Judith E. Farnberger, Volker F. Wendisch |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
Ketone
lcsh:QR1-502 Bioengineering Stereoisomerism 010402 general chemistry 01 natural sciences Applied Microbiology and Biotechnology Reductive amination α-Amino acids lcsh:Microbiology Substrate Specificity Catalysis Bacterial Proteins Leucine Escherichia coli Amines Isoleucine Chiral amines Amination Transaminases Modular concept chemistry.chemical_classification 010405 organic chemistry Research Ketones NAD Keto Acids Combinatorial chemistry 0104 chemical sciences Amino acid Biochemistry chemistry Biocatalysis Single-cell biotransformation Amine gas treating Asymmetric reductive amination Flexibility Plasmids Biotechnology |
| Zdroj: | Microbial Cell Factories, Vol 16, Iss 1, Pp 1-17 (2017) Microbial Cell Factories |
| ISSN: | 1475-2859 |
| DOI: | 10.1186/s12934-017-0750-5 |
| Popis: | Background Transaminases 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. Results By 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%). Conclusions The 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. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0750-5) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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