Harnessing the MEP pathway for heterologous (+)-nootkatone biosynthesis in a green microalga under fine-tuned light and carbon regimes

The high-value sesquiterpenoid (+)-nootkatone has important applications in food, agriculture, and pharmaceutical industries. Extraction from plant material, however, is technically challenging and inefficient due to inherent low concentrations in native sources. Over the last decade, the eukaryotic green microalga Chlamydomonas reinhardtii has emerged as a powerful alternative for heterologous terpenoid production, due to a natively high carbon flux through its MEP pathway. This study describes strategic fusion protein designs of different valencene and farnesyl pyrophosphate (FPP) synthases, which allowed efficient (+)-valencene biosynthesis in the C. reinhardtii cytosol and also found the algal chloroplast to be highly suitable for heterologous production. Successful co-expression of cytochrome P450 monooxygenases resulted in a two-step oxidation towards (+)-nootkatone at a comparably high conversion rate of 76 % and was independent of recombinant reductase activity. In addition, the 1-deoxyxylulose-5-phosphate synthase (DXS) was found to be rate-limiting for increased sesquiterpenoid production. Currently available photobioreactors suffer from limitations in light availability, which can hinder phototrophic growth, especially at higher cell densities. C. reinhardtii harbours the ability to use acetic acid as a carbon source, and fine-tuned cultivation regimes under photo-, mixo-, and heterotrophic conditions were tested to optimize heterologous sesquiterpene production. Customized scale-up cultivations in 2.5 L showed efficient volumetric production of 148 mg/L under phototrophic conditions and a maximal gravimetric production of 76 mg/g_CDW under heterotrophic cultivation regimes, which displays a first industrially relevant (+)-nootkatone production concept in a green cell factory.