Photobiocatalytic Oxyfunctionalization with High Reaction
Rate using a Baeyer–Villiger Monooxygenase from Burkholderia xenovorans in Metabolically Engineered
Cyanobacteria
posted on 2021-12-10, 13:03authored byElif Erdem, Lenny Malihan-Yap, Leen Assil-Companioni, Hanna Grimm, Giovanni Davide Barone, Carole Serveau-Avesque, Agnes Amouric, Katia Duquesne, Véronique de Berardinis, Yagut Allahverdiyeva, Véronique Alphand, Robert Kourist
Baeyer–Villiger
monooxygenases (BVMOs) catalyze the oxidation
of ketones to lactones under very mild reaction conditions. This enzymatic
route is hindered by the requirement of a stoichiometric supply of
auxiliary substrates for cofactor recycling and difficulties with
supplying the necessary oxygen. The recombinant production of BVMO
in cyanobacteria allows the substitution of auxiliary organic cosubstrates
with water as an electron donor and the utilization of oxygen generated
by photosynthetic water splitting. Herein, we report the identification
of a BVMO from Burkholderia xenovorans (BVMOXeno) that exhibits higher reaction
rates in comparison to currently identified BVMOs. We report a 10-fold
increase in specific activity in comparison to cyclohexanone monooxygenase
(CHMOAcineto) in Synechocystis sp. PCC 6803 (25 vs 2.3 U gDCW–1 at
an optical density of OD750 = 10) and an initial rate of
3.7 ± 0.2 mM h–1. While the cells containing
CHMOAcineto showed a considerable reduction
of cyclohexanone to cyclohexanol, this unwanted side reaction was
almost completely suppressed for BVMOXeno, which was attributed to the much faster lactone formation and a
10-fold lower KM value of BVMOXeno toward cyclohexanone. Furthermore, the whole-cell
catalyst showed outstanding stereoselectivity. These results show
that, despite the self-shading of the cells, high specific activities
can be obtained at elevated cell densities and even further increased
through manipulation of the photosynthetic electron transport chain
(PETC). The obtained rates of up to 3.7 mM h–1 underline
the usefulness of oxygenic cyanobacteria as a chassis for enzymatic
oxidation reactions. The photosynthetic oxygen evolution can contribute
to alleviating the highly problematic oxygen mass-transfer limitation
of oxygen-dependent enzymatic processes.