Sequential Enzymatic Conversion of α‐Angelica Lactone to γ‐Valerolactone through Hydride‐Independent C=C Bond Isomerization

Abstract A case of hydride‐independent reaction catalyzed by flavin‐dependent ene‐reductases from the Old Yellow Enzyme (OYE) family was identified. α‐Angelica lactone was isomerized to the conjugated β‐isomer in a nicotinamide‐free and hydride‐independent process. The catalytic cycle of C=C bond isomerization appears to be flavin‐independent and to rely solely on a deprotonation–reprotonation sequence through acid–base catalysis. Key residues in the enzyme active site were mutated and provided insight on important mechanistic features. The isomerization of α‐angelica lactone by OYE2 in aqueous buffer furnished 6.3 mm β‐isomer in 15 min at 30 °C. In presence of nicotinamide adenine dinucleotide (NADH), the latter could be further reduced to γ‐valerolactone. This enzymatic tool was successfully applied on semi‐preparative scale and constitutes a sustainable process for the valorization of platform chemicals from renewable resources.


Scale-up
The scale up was performed as described in the experimental section. Upon evaporation of the solvent, GC-MS analysis of the recovered product (18.1 mg) indicated a purity of 98% for -isomer 1b.

Determination of enantiomeric excess
Optical purity of the products was determined by GC-FID measurements by comparison with authentic reference material. A Chirasil Chiraldex DEX-CB column (25 m, 0.32 mm ID, 0.25 µm film thickness) was used. Temperature program: 60 °C, 5 °C/min. to 85 °C, hold for 5 min., 20 °C/min. to 180 °C.

Cloning of OYE2
The synthetic gene for OYE2 was ordered (Thermo Fisher Scientific) and codon-optimized for E. coli.

Site-directed Mutagenesis
Primers that included the desired mutations were designed using the sequence of codon-optimized (E. coli) OYE2-gene (Table S1). All mutants were generated using an Agilent QuikChange II XL Site-Directed Mutagenesis Kit. The obtained plasmids were transformed into XL10-Gold Ultracompetent Cells according to the manufacturer's manual. The correct introduction on mutations was confirmed by sequencing.

Transformation of plasmids
To 100 µL of cell suspension in 1.5 mL Eppendorf tubes, 10 µL of plasmid were added and incubated on ice for 30 min. The samples were then placed in a thermoshaker 42 °C for 10 s [BL21(DE3)] and 30 s (NEB5α) respectively. Afterwards, 250 µL of LB-medium were added and the cells were incubated in the thermomixer for 1 h at 37 °C and 300 rpm. 100 µL of the resulting product were plated on kanamycincontaining agar plates.

Overexpression of OYE2 and variants
Colonies obtained from the transformation step were used to prepare overnight cultures (ONCs; 10 mL LB-medium, 50 µg/mL kanamycin) of all mutants. They were placed in the shaker overnight (37 °C, 120 rpm) and used on the next day to inoculate the main cultures (330 mL LB-medium per shaking flask; 3.3 mL of the ONCs, 50 µg/mL kanamycin). The main cultures were placed in the shaker at 37 °C and 140 rpm until an OD600 of 0.6 was reached. Then, IPTG (0.2 mM) was used for induction. The cultures were again placed in the shaker overnight at 37 °C and 140 rpm. On the next day, the cells were harvested by centrifugation (4000 rpm, 20 min, 4 °C) and the supernatant was discarded. The pellet was resuspended with cold 0.9% NaCl solution and centrifuged again (4000 rpm, 20 min, 4 °C). The supernatant was discarded and the pellets were frozen until the purification was performed.