## 3 Results
The highest product levels could be detected after three days of cultivation, where cells have typically reached the beginning of the stationary growth phase. The elongation of the cultivation time did not show increased product accumulation so that all further production experiments were carried out for three days. Under standard phototrophic conditions, conventional light bulbs are used for the illumination of _R. capsulatus_ cells [39; 49]. This conventional light source offers a broad emission spectrum with a relatively high proportion in the infrared (IR) light range (>750 nm; 3.6 mW cm\({}^{-2}\) at 850 nm) suitable for excitation of bacteriochlorophyll \(a\) (BChl _a_) exhibiting excitation maxima at 800 and 860 nm (Figure S6, Supplementary Materials, Reference [91]). To improve the illumination conditions for sesquiterpene production under phototrophic conditions, we subsequently analyzed if the use of (i) alternative cultivation vessels offering a better light penetration of cell cultures by a more favorable surface-area-to-volume ratio (Table S2, Supplementary Materials) or (ii) a customized IR-LED array (5.6 mW cm\({}^{-2}\) at 850 nm) suitable for specific excitation of the photopigment BChl \(a\) with high light intensities can help to increase product formation. To investigate the influence of illumination conditions on the heterologous production of \(\beta\)-caryophyllene, the strain SB1003-MVA carrying the expression vector pRhon5 Hi-2-QHS1-ispA was cultivated over three days under photoheterotrophic conditions and constant illumination with bulb light or IR light in an ammonium-depleted medium in screw neck vials. As shown in Figure 2, the change of cultivation vessel geometry resulted only in a slight increase of \(\beta\)-caryophyllene production of _R. capsulatus_ SB1003-MVA (pRhon5 Hi-2-QHS1-ispA), whereas high irradiation with IR light led to a 1.9-fold increase of the final product accumulation. These results indicate that the applied illumination conditions should be taken into account to reach high product yields when _R. capsulatus_ is used as an alternative terpene production host. This assumption is further supported by the observation that product levels were much lower in _R. capsulatus_ SB1003-MVA (pRhon5 Hi-2-QHS1-ispA) cultures that have been grown under non-phototrophic, i.e., microaerobic conditions (Figure 2, green bars). For non-phototrophic cultivation, _R. capsulatus_ SB1003-MVA (pRhon5 Hi-2-QHS1-ispA) was grown in 100 mL, unbaffled shake flasks containing different volumes of medium in the dark to implement different aeration conditions  
Figure 1: Heterologous \(\beta\)-caryophyllene production in the _R. capsulatus_ strains SB1003 and SB1003-MVA. The \(\beta\)-caryophyllene synthase gene _QHS1_ from _A. ammonia_ was expressed in _R. capsulatus_ SB1003 wild type (grey bars) and SB1003-MVA (blue bars), which additionally carries the MVA gene cluster from _P. zazvanthinifaciens_ to enable a second isopenoid biosynthesis route. Moreover, the _ispA_ gene encoding the _R. capsulatus_ FPP synthase was co-expressed on the same plasmid to further enhance sesquiterpene production titers. Product formation was determined in cell cultures after three days of photoheterotrophic cultivation (gas-tight hungate tubes, 30 \({}^{\circ}\)C) under ammonium depletion and constant illumination with bulb light (3.6 mW cm\({}^{-2}\) at 850 nm). The produced \(\beta\)-caryophyllene was sampled in overlaid _n_-dodecane phases for GC-FID analysis. The increase of \(\beta\)-caryophyllene production in engineered _R. capsulatus_ strains is shown as relative values. To this end, the _R. capsulatus_ SB1003 carrying the plasmid-encoded _QHS1_ gene was used as a reference strain. Values are means of three independent biological replicates (_n_ = 3) and error bars indicate the respective standard deviations. (green bars). Those conditions could lead to the formation of \(\beta\)-caryophyllene oxide, the oxygenated derivative of \(\beta\)-caryophyllene.