Design and use of recombinant whole cell systems in organic chemistry: Synthetic applications of cyclohexanone monooxygenase, cytochromes P450 and epoxide hydrolases

The construction of organic compounds through biocatalytic methods has the advantage of offering a more environmentally benign process, compared to modern chemical methodology. The main goal of this work was to develop and apply whole cell biocatalysts that contain specific types of enzymes that are useful in organic chemistry but that present very serious technical problems when they are used as purified enzymes. We focus on two monooxygenases; cyclohexanone monooxygenase (E.C 1.14.13.22) and cytochrome P450 (E.C1.14.14.1) and one hydrolase; mammalian epoxide hydrolase (E.C 3.3.2.3).; After the end of this work, the regio- and stereoselectivity of cyclohexanone monooxygenase utilizing the recombinant microbial strains was examined and a purification scheme was devised using the high levels of protein obtained with an E. coli expression system. The enzyme was also applied to the chemo-enzymatic synthesis of precursors of isoprostane 8-epi -PGF_2α.; The heterologous expression of several mammalian P-450 monooxygenases in yeast and E. coli hosts was done. Members of the families 2B, 2E. 2A and 3A were explored in this research. The co-expression of the electron transport protein needed for catalytic activity of cytochrome P-450 enzymes was done as well. The low catalytic turnover of the P450 enzymes was the main limitation for a complete examination of the substrate specificity in whole cell systems. However, the system devised allowed the production of large amounts of enzyme in a very cheap way.; After the heterologous expression of mouse liver microsomal and cytosolic epoxide hydrolase was explored, two useful bacterial recombinant systems were obtained. These strains represent a superior alternative to the use of isolated enzyme. We could demonstrate the potential utility of recombinant DNA techniques to combat problems associated with the use of enzymes in organic synthesis.