title: Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli.
abstract: Cytochrome P450s are interesting biocatalysts due to their ability to hydroxylate non-activated hydrocarbons in a selective manner. However, to date only a few P450-catalyzed processes have been implemented in industry due to the difficulty of developing economically feasible processes. In this study, we have used the CYP153A heme domain from Marinobacter aquaeolei fused to the reductase domain of CYP102A1 from Bacillus megaterium (BM3) expressed in Escherichia coli. This self-sufficient protein chimera CYP153A-CPRBM3 G307A mutant is able to selectively hydroxylate medium and long chain length fatty acids at the terminal position. ω-Hydroxylated fatty acids can be used in the field of high-end polymers and in the cosmetic and fragrance industry. Here, we have identified the limitations for implementation of a whole-cell P450-catalyzed reaction by characterizing the chosen biocatalyst as well as the reaction system. Despite a well-studied whole-cell P450 catalyst, low activity and poor stability of the artificial fusion construct are the main identified limitations to reach sufficient biocatalyst yield (mass of product/mass of biocatalyst) and space-time yield (volumetric productivity) essential for an economically feasible process. Substrate and product inhibition are also challenges that need to be addressed, and the application of solid substrate is shown to be a promising option to improve the process.
authors.affiliation: Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, DK-2800, Kgs. Lyngby, Denmark.
authors.forename: M T
authors.initials: MT
authors.lastname: Lundemo
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Notonier
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: G
authors.initials: G
authors.lastname: Striedner
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Hauer
authors.affiliation: Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, DK-2800, Kgs. Lyngby, Denmark. jw@kt.dtu.dk.
authors.forename: J M
authors.initials: JM
authors.lastname: Woodley
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D053520:Marinobacter / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System
keywords: Bottleneck identification; Cytochrome P450 monooxygenases; Inhibition; Process development; Whole-cell catalysis