A Cold-Active Flavin-Dependent Monooxygenase from Janthinobacterium svalbardensis Unlocks Applications of Baeyer-Villiger Monooxygenases at Low Temperature.

Cold-active enzymes maintain a large part of their optimal activity at low temperatures. Therefore, they can be used to avoid side reactions and preserve heat-sensitive compounds. Baeyer-Villiger monooxygenases (BVMO) utilize molecular oxygen as a co-substrate to catalyze reactions widely employed for steroid, agrochemical, antibiotic, and pheromone production. Oxygen has been described as the rate-limiting factor for some BVMO applications, thereby hindering their efficient utilization. Considering that oxygen solubility in water increases by 40% when the temperature is decreased from 30 to 10 °C, we set out to identify and characterize a cold-active BVMO. Using genome mining in the Antarctic organism Janthinobacterium svalbardensis, a cold-active type II flavin-dependent monooxygenase (FMO) was discovered. The enzyme shows promiscuity toward NADH and NADPH and high activity between 5 and 25 °C. The enzyme catalyzes the monooxygenation and sulfoxidation of a wide range of ketones and thioesters. The high enantioselectivity in the oxidation of norcamphor (eeS = 56%, eeP > 99%, E > 200) demonstrates that the generally higher flexibility observed in the active sites of cold-active enzymes, which compensates for the lower motion at cold temperatures, does not necessarily reduce the selectivity of these enzymes. To gain a better understanding of the unique mechanistic features of type II FMOs, we determined the structure of the dimeric enzyme at 2.5 Å resolution. While the unusual N-terminal domain has been related to the catalytic properties of type II FMOs, the structure shows a SnoaL-like N-terminal domain that is not interacting directly with the active site. The active site of the enzyme is accessible only through a tunnel, with Tyr-458, Asp-217, and His-216 as catalytic residues, a combination not observed before in FMOs and BVMOs.

Photobiocatalytic Oxyfunctionalization with High Reaction Rate using a Baeyer-Villiger Monooxygenase from Burkholderia xenovorans in Metabolically Engineered Cyanobacteria.

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 (BVMO Xeno ) 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 (CHMO Acineto ) 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 CHMO Acineto showed a considerable reduction of cyclohexanone to cyclohexanol, this unwanted side reaction was almost completely suppressed for BVMO Xeno , which was attributed to the much faster lactone formation and a 10-fold lower K M value of BVMO Xeno 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.

Engineering of NADPH Supply Boosts Photosynthesis-Driven Biotransformations.

Light-driven biocatalysis in recombinant cyanobacteria provides highly atom-efficient cofactor regeneration via photosynthesis, thereby remediating constraints associated with sacrificial cosubstrates. However, despite the remarkable specific activities of photobiocatalysts, self-shading at moderate-high cell densities limits efficient space-time-yields of heterologous enzymatic reactions. Moreover, efficient integration of an artificial electron sink into the tightly regulated network of cyanobacterial electron pathways can be highly challenging. Here, we used C=C bond reduction of 2-methylmaleimide by the NADPH-dependent ene-reductase YqjM as a model reaction for light-dependent biotransformations. Time-resolved NADPH fluorescence spectroscopy allowed direct monitoring of in-cell YqjM activity and revealed differences in NADPH steady-state levels and oxidation kinetics between different genetic constructs. This effect correlates with specific activities of whole-cells, which demonstrated conversions of >99%. Further channelling of electrons toward heterologous YqjM by inactivation of the flavodiiron proteins (Flv1/Flv3) led to a 2-fold improvement in specific activity at moderate cell densities, thereby elucidating the possibility of accelerating light-driven biotransformations by the removal of natural competing electron sinks. In the best case, an initial product formation rate of 18.3 mmol h-1 L-1 was reached, allowing the complete conversion of a 60 mM substrate solution within 4 h.

Hydrogen-Driven Cofactor Regeneration for Stereoselective Whole-Cell C=C Bond Reduction in Cupriavidus necator.

The coupling of recombinantly expressed oxidoreductases to endogenous hydrogenases for cofactor recycling permits the omission of organic cosubstrates as sacrificial electron donors in whole-cell biotransformations. This increases atom efficiency and simplifies the reaction. A recombinant ene-reductase was expressed in the hydrogen-oxidizing proteobacterium Cupriavidus necator H16. In hydrogen-driven biotransformations, whole cells catalyzed asymmetric C=C bond reduction of unsaturated cyclic ketones with stereoselectivities up to >99 % enantiomeric excess. The use of hydrogen as a substrate for growth and cofactor regeneration is particularly attractive because it represents a strategy for improving atom efficiency and reducing side product formation associated with the recycling of organic cofactors.