Browsing by Author "Honda Malca, Sumire"

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Open Access
Substrate characterization and protein engineering of bacterial cytochrome P450 monooxygenases for the bio-based synthesis of omega-hydroxylated aliphatic compounds
(2013) Honda Malca, Sumire; Hauer, Bernhard (Prof. Dr.)
The selective oxyfunctionalization of alkanes and fatty acids is a challenging task in basic and applied chemistry. Biocatalysts belonging to the superfamily of cytochrome P450 monooxygenases (CYPs) can introduce oxygen into a wide variety of molecules in a very regio- and stereospecific manner, which can be used for the synthesis of fine and bulk chemicals. CYPs from the bacterial CYP153A subfamily have been described as alkane hydroxylases with high terminal regioselectivity. In the present work, CYP153A monooxygenases were screened for the synthesis of industrially relevant omega-hydroxylated aliphatic compounds, such as primary alcohols, alpha,omega-diols, omega-hydroxyfatty acids (omega-OHFAs) and alpha,omega-dicarboxylic acids (alpha,omega-DCAs). One enzyme candidate was tailored by rational design and applied in whole cell biotransformations with recombinant E. coli or Pseudomonas strains. The biocatalytic systems were further improved by utilizing a fusion enzyme construct for increased coupling efficiency. In summary, this work constitutes the first example of rational engineering of a CYP153A enzyme, which allowed the identification of key residues for activity and substrate specificity within the enzyme subfamily. CYP153A enzymes have also been applied for the first time in the omega-hydroxylation of fatty acids. Comparative in vivo studies with recombinant E. coli and P. putida cells provided information on the effects of host strains on product yields, eventually leading to the generation of an efficient bacterial whole cell biocatalyst for the synthesis of selected omega-hydroxylated aliphatic compounds.