Table 1.
Strain constructs used in this study and their genetic features.
Fig 1.
(A) Pathways for targeted biosynthesis of triterpenes in R. capsulatus and Synechocystis by implementation of A. thaliana biosynthesis modules. In both organisms, the common triterpene precursor FPP is provided by the native 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. To establish the first step of the triterpene-specific precursor module in Synechocystis, a knock-out mutant of the shc gene encoding the squalene-hopene cyclase of the intrinsic hopanoid biosynthesis was employed [37]. In R. capsulatus, heterologous expression of SQS1 was implemented to form squalene. For monooxygenation of squalene to the central precursor 2,3-oxidosqualene, SQE1 was heterologously expressed in both host strains. Subsequently, the cyclization module was designed to convert the linear precursor 2,3-oxidosqualene into plant sterols and further cyclic triterpenes. To cover different triterpene scaffolds, the OSC enzymes CAS1, LUP1, THAS1, and MRN1 from A. thaliana were expressed in each host to synthesize cycloartenol (sterol), lupeol (exhibiting the often occurring pentacyclic scaffold) as well as thalianol and marneral (representing more unusual tri- or monocyclic structures). Respective substrate folding is indicated (CBC, chair-boat-chair; CCC, chair-chair-chair; CB, chair-boat). (B) Schematic representation of expression constructs used for triterpenoid biosynthesis in R. capsulatus and Synechocystis. The respective genes were arranged as synthetic operons, using native ammonium (Pnif) and cobalt (PcoaT) regulated promoters for transcription activation. FPP, farnesyl pyrophosphate; SQS, squalene synthase; SQE, squalene epoxidase; OSC, oxidosqualene cyclase.
Fig 2.
LC-MS detection of triterpenoids produced in engineered R. capsulatus SB1003 (Rc) and Synechocystis sp. PCC 6803 (Syn) strains.
Cells of the respective strains (indicated in chromatograms and color-coded with red for Rc-, green for Syn-strains) were subjected to extraction and analysis after expression of A. thaliana triterpenoid biosynthesis genes (SQS1, squalene synthase; SQE1, squalene epoxidase; CAS1, cycloartenol synthase; LUP1, lupeol synthase; MRN1, marneral synthase). Triterpenoid identity was verified by comparison of retention times, as well as MS and MS/MS spectra to commercial or biological references (S2 Fig). (A) Signals in EICs of m/z 411.399 at RT 14.9 min correspond to squalene (C30H50), signals in EICs of m/z 427.393 at RT 11.8 min correspond to 2,3-oxidosqualene (C30H50O). (B) Signals in EICs of m/z 427.393 at RT 10.9 min correspond to cycloartenol (C30H50O). (C) Signals in EICs of m/z 409.383 at RT 9.9 min correspond to lupeol (C30H50O-H2O). Signals in EICs of m/z 427.393 (and 409.383) at RT 6.7 min correspond to lupX (tentatively identified as lupanediol (C30H52O2-H2O)). In both EICs, signals at RT 11.7 min correspond to 2,3-oxidosqualene (C30H50O). (D) Signals in EICs of m/z 429.409 at RT 10.7 min correspond to marnerol (C30H52O), signals in EICs of m/z 445.404 at RT 7.1 min correspond to hydroxymarnerol (C30H52O2). Signals in EICs of m/z 429.409 at RT 11.8 min correspond to the (M+2) isotopic peak of 2,3-oxidosqualene (C30H52O). Peaks in EICs of m/z 445.404 also detected in Syn_Δshc-SQE1 (control) also correspond to 2,3-oxidosqualene-derived signals. As a reference, chromatograms of samples from S. cerevisiae GIL77 (Sc), carrying pYES/DEST-52 with MRN1 or as empty vector control (EVC), are shown. Shown chromatograms are representative for replicate measurements from at least three independent cultivations. The corresponding quantitative data are summarized in Table 2.
Table 2.
Triterpene levels in engineered R. capsulatus SB1003 (Rc) and Synechocystis sp. PCC 6803 (Syn) strains.
Fig 3.
Comparative physiological characterization of triterpene producing strains of R. capsulatus and Synechocystis.
(A) Growth of R. capsulatus (left) and Synechocystis (right) expressing A. thaliana triterpene biosynthesis genes. Cell densities were determined by turbidity measurements at 660 nm (R. capsulatus) or 750 nm (Synechocystis) to record growth curves. Strains carrying the precursor biosynthetic modules for the generation of squalene and 2,3-oxidosqualene were compared to strains that additionally harbored different cyclization modules or an empty vector as control. Data represent mean values from three independent cultivations, as well as the respective standard deviations. The time point of cobalt (Co2+)-induction in Synechocystis is indicated. The y-axes are scaled logarithmically (base e). (B) Whole cell absorbance spectra of R. capsulatus (upper panel) and Synechocystis (lower panel) expressing the precursor synthesis modules. Pigment profiles were recorded at time point 52 h (R. capsulatus) and 164 h (Synechocystis) of the respective cultivation period (cf. A), and are representative for replicate data obtained from at least three independent cultivations. Typical pigment absorption is indicated. All values were normalized to OD660nm (R. capsulatus) and OD750nm (Synechocystis), respectively. BChl, bacteriochlorophyll a; Car, carotenoids; Chl, chlorophyll a; PC, phycocyanin. (C) Carotenoid content of R. capsulatus (upper panel) and Synechocystis (lower panel). Normalized values at 500 nm were plotted as a measure of carotenoid content in the host cells. R. capsulatus was analyzed at time points 24 h and 52 h during the cultivation. Pigments of Synechocystis were measured before and after cobalt-induction at time points 48 h and 164 h, respectively. Data represent mean results from three independent experiments, as well as the respective standard deviations. EVC, empty vector control; WT, wild type and Δshc both harbor the empty vector.