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- Title
From Cell-Free Protein Synthesis to Whole-Cell Biotransformation: Screening and Identification of Novel α-Ketoglutarate-Dependent Dioxygenases for Preparative-Scale Synthesis of Hydroxy-l-Lysine.
- Authors
Rolf, Jascha; Nerke, Philipp; Britner, Annette; Krick, Sebastian; Lütz, Stephan; Rosenthal, Katrin
- Abstract
The selective hydroxylation of non-activated C-H bonds is still a challenging reaction in chemistry. Non-heme Fe2+/α-ketoglutarate-dependent dioxygenases are remarkable biocatalysts for the activation of C-H-bonds, catalyzing mainly hydroxylations. The discovery of new Fe2+/α-ketoglutarate-dependent dioxygenases with suitable reactivity for biotechnological applications is therefore highly relevant to expand the limited range of enzymes described so far. In this study, we performed a protein BLAST to identify homologous enzymes to already described lysine dioxygenases (KDOs). Six novel and yet uncharacterized proteins were selected and synthesized by cell-free protein synthesis (CFPS). The subsequent in vitro screening of the selected homologs revealed activity towards the hydroxylation of l-lysine (Lys) into hydroxy-l-lysine (Hyl), which is a versatile chiral building block. With respect to biotechnological application, Escherichia coli whole-cell biocatalysts were developed and characterized in small-scale biotransformations. As the whole-cell biocatalyst expressing the gene coding for the KDO from Photorhabdus luminescens showed the highest specific activity of 8.6 ± 0.6 U gCDW−1, it was selected for the preparative synthesis of Hyl. Multi-gram scale product concentrations were achieved providing a good starting point for further bioprocess development for Hyl production. A systematic approach was established to screen and identify novel Fe2+/α-ketoglutarate-dependent dioxygenases, covering the entire pathway from gene to product, which contributes to accelerating the development of bioprocesses for the production of value-added chemicals.
- Subjects
DIOXYGENASES; PROTEIN synthesis; BIOCONVERSION; CHEMICAL reactions; PHOTORHABDUS luminescens; ENZYMES
- Publication
Catalysts (2073-4344), 2021, Vol 11, Issue 9, p1038
- ISSN
2073-4344
- Publication type
Article
- DOI
10.3390/catal11091038