title: High-cell-density cultivation of recombinant Escherichia coli, purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase.
abstract: We have recently described the biocatalytic characterization of a self-sufficent biosynthetic alkane hydroxylase based on CYP153A13a from Alcanivorax borkumensis SK2 (thereafter A13-Red). Despite remarkable regio- and chemo-selectivity, A13-Red suffers of a difficult-to-reproduce expression and moderate operational stability. In this study, we focused our efforts on the production of A13-Red using high-cell-density cultivation (HCDC) of recombinant Escherichia coli. We achieved 455 mg (5,000 nmol) of functional enzyme per liter of culture. Tight control of cultivation parameters rendered the whole process highly reproducible compared with flask cultivations. We optimized the purification of the biocatalyst that can be performed in either two or three steps depending on the application needed to afford A13-Red up to 95 % homogeneous. We investigated different reaction conditions and found that the total turnover numbers of A13-Red during the in vitro hydroxylation of n-octane could reach up to 3,250 to produce 1-octanol (1.6 mM) over a period of 78 h.
authors.affiliation: Institut Charles Gerhardt Montpellier, UMR 5253 CNRS/ENSCM/UM2/UM1, 8 rue de l'Ecole Normale, 34296, Montpellier, France.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Bordeaux
authors.forename: Diane
authors.initials: D
authors.lastname: de Girval
authors.forename: Robin
authors.initials: R
authors.lastname: Rullaud
authors.forename: Maeva
authors.initials: M
authors.lastname: Subileau
authors.forename: Eric
authors.initials: E
authors.lastname: Dubreucq
authors.forename: Jullien
authors.initials: J
authors.lastname: Drone
mesh_terms: D053521:Alcanivoraceae / Q000201:enzymology* / Q000235:genetics; D042926:Cytochrome P-450 CYP4A / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D009795:Octanes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D009795:Octanes; D011994:Recombinant Proteins; D042926:Cytochrome P-450 CYP4A; C026728:octane