A Red Algal Bourbonane Sesquiterpene Synthase Defined by Microgram-Scale NMR-Coupled Crystalline Sponge X‑ray Diffraction Analysis

Sesquiterpene scaffolds
are the core backbones of many medicinally
and industrially important natural products. A plethora of sesquiterpene
synthases, widely present in bacteria, fungi, and plants, catalyze
the formation of these intricate structures often with multiple stereocenters
starting from linear farnesyl diphosphate substrates. Recent advances
in next-generation sequencing and metabolomics technologies have greatly
facilitated gene discovery for sesquiterpene synthases. However, a
major bottleneck limits biochemical characterization of recombinant
sesquiterpene synthases: the absolute structural elucidation of the
derived sesquiterpene products. Here, we report the identification
and biochemical characterization of LphTPS-A, a sesquiterpene synthase
from the red macroalga Laurencia pacifica. Using
the combination of transcriptomics, sesquiterpene synthase expression
in yeast, and microgram-scale nuclear magnetic resonance-coupled crystalline
sponge X-ray diffraction analysis, we resolved the absolute stereochemistry
of prespatane, the major sesquiterpene product of LphTPS-A, and thereby
functionally define LphTPS-A as the first bourbonane-producing sesquiterpene
synthase and the first biochemically characterized sesquiterpene synthase
from red algae. Our study showcases a workflow integrating multiomics
approaches, synthetic biology, and the crystalline sponge method,
which is generally applicable for uncovering new terpene chemistry
and biochemistry from source-limited living organisms.