Production of the sesquiterpenoid (+ )-nootkatone by metabolic engineering of Pichia pastoris

摘要

The sesquiterpenoid ( + )-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of ( + )-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnolo-gical approaches, using cell extracts or whole-cell systems for the production of ( + )-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+ )-nootkatone from ( + )-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+ )-valencene. Intracellular production of (+ )-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+ )-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+ )-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the ( + )-nootkatone yield to 208mgL~(-1) cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of ( + )-nootkatone from simple carbon sources.