## 3 Results
As previously described, a filling volume of 60 mL is most suitable for the induction of intrinsic terpene formation and P\({}_{nif}\)-mediated target gene expression in _R. capsulatus_[39], which is corroborated by the observed \(\beta\)-caryophyllene production levels. Nevertheless, only a quarter of the product yield could be achieved under microaerobic, non-phototrophic growth conditions when compared to the corresponding values of phototrophically grown cells (_R. capsulatus_ SB1003-MVA, pRhon5 Hi-2-QHS1-ispA, 3 days, bulb light), and only traces of the oxygenated derivative were detectable (data not shown). However, to fully understand the effects of varying cultivation conditions on \(\beta\)-caryophyllene production, further experiments have to be performed in future studies. To accurately determine the final product titers, we analyzed (i) the individual transfer efficiency of \(\beta\)-caryophyllene from intact cells into the \(n\)-dodecane phase, (ii) the effect of the ICM, which is formed by _R. capsulatus_ cells under phototrophic conditions, on sesquiterpenoid extraction, (iii) the differences in terpene transfer efficiencies when comparing single and repeated \(n\)-dodecane extraction, and finally (iv) the effect of the presence and absence  
Figure 2: Heterologous \(\beta\)-caryophyllene production in the _R. capsulatus_ strain SB1003-MVA with dependence on the cultivation time and illumination conditions. The \(\beta\)-caryophyllene accumulation was determined in _R. capsulatus QHS1_ expression strains SB1003-MVA (pRhon5 Hi-2-QHS1) and SB1003-MVA (pRhon5 Hi-2-QHS1-ispA). First, product formation was determined in cell cultures after three, four, and five days of photoheterotrophic cultivation in 15 mL humgate tubes using standard illumination conditions (bulb lights, 3.6 mW cm\({}^{-2}\) at 850 nm) and RCV medium supplemented with 0.1% serine. Blue bars represent the results of this experiment. Second, illumination conditions were changed by cultivating _R. capsulatus_ strain SB1003-MVA (pRhon5 Hi-2-QHS1-ispA) for three days under photoheterotrophic conditions using either constant illumination with bulb lights (BL; 3.6 mW cm\({}^{-2}\) at 850 nm, yellow bar) or IR-emitting diodes (IR; 5.6 mW cm\({}^{-2}\) at 850 nm, red bar). Here, 4.5-mL screw neck vials were used to improve light penetration due to a more favorable surface-area-to-volume ratio of this cultivation vessel. For non-phototrophic cultivation, the same strain was grown in 100-mL, unbaffled shake flasks containing different volumes of serine-supplemented RCV medium (shake flask, green bars). In all cultures, the produced \(\beta\)-caryophyllene was sampled in overlaid \(n\)-dodecane phases for GC-FID analysis. The increase of \(\beta\)-caryophyllene production is shown as relative values using _R. capsulatus_ SB1003-MVA carrying the QHS1 expression plasmid pRhon5 Hi-2-QHS1 as a reference strain. Values are the means of three independent biological replicates (\(n=3\)) and the error bars indicate the respective standard deviations. of organic solvent on the final product titers (Supplementary Method section \"Analysis of \(n\)-dodecane-mediated \(\beta\)-caryophyllene extraction from phototrophically grown _R. capsulatus_\"). Finally, we were able to determine a product titer of 90 \(\pm\) 19 mg L\({}^{-1}\)\(\beta\)-caryophyllene for _R. capsulatus_ SB1003-MVA with pRhon5 Hi-2-QHS1-ispA after 3 days of cultivation in humgate tubes under bulb light. This titer could be further increased by using IR light and screw neck vials for cultivation, reaching a final product titer of 139 \(\pm\) 31 mg L\({}^{-1}\). Based on these values and the reached cell densities, the respective productivities were further calculated (Table S3, Supplementary Materials). In summary, we showed that _R. capsulatus_ can efficiently synthesize the sesquiterpene \(\beta\)-caryophyllene.