Continuous process for manoyl-oxide production and in-situ extraction from microalgae cultures

Eukaryotic green microalgae hold potential as green-cell factories for the production of heterologous compounds such diterpenes. Previously, a C. reinhardtii UVM4 strain was genetically modified to express heterologous diterpene synthases (diTPSs) and the enzymes participating in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. In this work, bench-scale continuous production of manoyl oxide (MO) was performed to prove the technical feasibility of the production system and to estimate the MO production rates. Furthermore, the influence of the operational conditions like biomass concentration, light availability, and the extraction capacity on the MO productivity was assessed. In this work, we showed that it is possible to continuously produce/extract of manoyl oxide from a genetically engineered C. reinhardtii cultures in bench-scale photobioreactors, producing up to 6.7 mg l⁻¹ d⁻¹. Next, we analyze the influence of the culture conditions on the biomass and manoyl oxide production rates. We found that increasing the light intensity enhances manoyl oxide production but, if diterpenes are not efficiently extracted from the culture, the MO production is significantly hindered, decreasing 7.5 times in relation to the maximum production rate. From our experimental results, we conclude that the extraction capacity is one of the most important factors affecting the efficiency of MO production in microalgal cultures and, if the extraction process is not efficient, increasing the light availability in the culture has a minor impact on the MO productivity.