## 3 Results  
In the past, terpenoids were exclusively obtained from natural plant sources, e.g., by extracting them from essential oils, requiring a complex and time-consuming downstream processing. The heterologous production of sesquiterpenes in a suitable microbial host, however, bears many benefits. For example, it offers the possibility to solely produce a desired compound so that it can be rather easily purified without the need of removing closely related constituents [21; 34]. Thus, we here aimed to reconstitute the plant sesquiterpene pathway of \(\beta\)-caryophyllene in _R. capsulatus_ and optimize the production under phototrophic growth conditions. Since many sesquiterpenoids exhibit promising antimicrobial activities, the antifungal efficacy of \(\beta\)-caryophyllene and its oxidized form against phytopathogenic fungi were evaluated. Establishment of \(\beta\)-Caryophyllene Production in R. capsulatus via Overexpression of Isoprenoid Precursor Genes  
Recently, we described the heterologous synthesis of the plant sesquiterpenoids valencene and patchoulol in the phototrophic bacterium _R. capsulatus_ and its modular improvement by engineering the biosynthesis of the central precursor FPP [39]. To evaluate if _R. capsulatus_ and the modular engineering principle can analogously be applied for the synthesis of the plant-derived sesquiterpene \(\beta\)-caryophyllene, we expressed the gene encoding \(\beta\)-caryophyllene synthase QHS1 from _A. annuu_ in the bacterial host. To this end, the expression vectors pRhon5 Hi-2-QHS1 and pRhon5 Hi-2-QHS1-ispA, carrying an additional copy of the intrinsic FPP synthase gene _ispA_, were transferred to the _R. capsulatus_ wild type strain SB1003 and strain SB1003-MVA. The latter strain additionally contains the chromosomally integrated MVA pathway genes derived from _Paracoccus zexaanthininifaciens_ and thus offers a second isoprenoid biosynthesis pathway. To compare the \(\beta\)-caryophyllene production in all _Rhodobacter_ strains grown under phototrophic conditions, cells were incubated in the absence of molecular oxygen and ammonium under constant bulb light illumination. Terpene accumulation was determined in the late stationary growth phase by analyzing _n_-dodecane samples via GC-FID measurements. The increase of \(\beta\)-caryophyllene production in tested _R. capsulatus_ strains is shown in Figure 1 as relative values using _R. capsulatus_ SB1003 solely carrying the plasmid-encoded _QHS1_ gene as reference strain. As shown in Figure 1, the expression of the \(\beta\)-caryophyllene synthase gene _QHS1_ in _R. capsulatus_ strain SB1003 led to a measurable production of \(\beta\)-caryophyllene. Remarkably, the co-expression of _QHS1_ and _ispA_ in the _R. capsulatus_ strain SB1003 as well as _QHS1_ expression in the engineered SB1003-MVA strain did not result in increased \(\beta\)-caryophyllene synthesis. However, concerted expression of _QHS1_ and _ispA_ in _R. capsulatus_ SB1003-MVA led to a considerable increase of sesquiterpenoid production of about 300% in comparison to the reference strain. Optimization of \(\beta\)-Caryophyllene Production in R. capsulatus via Modification of Cultivation Conditions  
In the above-described experiments, we could demonstrate that modular engineering of the isoprenoid biosynthesis can also be applied to improve \(\beta\)-caryophyllene production in _R. capsulatus_. Next, we analyzed whether the modification of cultivation conditions including a prolonged cultivation time or the change of illumination parameters can further improve the product yield in the better-performing strain SB1003-MVA. First, \(\beta\)-caryophyllene accumulation was comparatively analyzed over five days in photheterotrophically-grown cultures of _R. capsulatus_ strain SB1003-MVA carrying pRhon5 Hi-2-QHS1 or pRhon5 Hi-2-QHS1-ispA. Product formation was determined by analyzing the overlaid _n_-dodecane samples via GC-FID measurements (Figure 2, blue bars).