Chemoenzymatic Synthesis with Plant Oxidases and Metabolic Engineering Enable Rapid Access to Rare Gibberellins

Gibberellins are structurally complex diterpenoid plant hormones with widespread agricultural applications. However, of the 136 known congeners found in nature, very few are easily accessible, preventing further research regarding their biological function. In biosynthesis, oxidases from plants generate the diverse oxidation patterns of natural gibberellins. Plant oxidases, in contrast to microbial oxidases, are very challenging to use for chemoenzymatic synthesis. Here, we develop a chemoenzymatic and metabolic engineering platform using plant oxidases that allows the production and full characterization of eight rare gibberellins as well as 16 ent-kaurene derivatives with various oxidation patterns. The filamentous fungus Aspergillus oryzae was engineered to produce common gibberellins and pathway intermediates in high titers, resulting in a panel of 20 diterpenoid substrates. These were screened using leaf disks from the model plant Nicotiana benthamiana producing ten different plant oxidases from diterpenoid metabolism. From the 200 substrate-enzyme pairs a total of 65 compounds were identified. We scaled up five reactions to produce milligram quantities of oxidized diterpenoids. Finally, by adding one of the plant oxidase genes to A. oryzae, we also accessed 15β-hydroxylated gibberellins in a single step by metabolic engineering. In summary, our work enables the flexible and sustainable synthesis of rare gibberellins and other highly oxidized diterpenoids. More importantly, our work demonstrates how plant oxidases can be used in chemoenzymatic synthesis and total biosynthesis campaigns, which will help to better utilize the catalytic potential of these previously neglected enzymes in the future.