Production of C20, C30 and C40 terpenes in the engineered phototrophic bacterium Rhodobacter capsulatus
Graphical abstract
Introduction
Terpenes are one of the largest groups of secondary metabolites that are ubiquitously present in organisms of all kingdoms of life (Chen et al., 2011; Dickschat, 2016; Schmidt-Dannert, 2014). A multitude of plant terpenes are applicable as ingredients in drugs, cosmetics or food products because they exhibit diverse relevant properties including antibiotic and antioxidant activities, a pleasant color, fragrance or flavor (Efferth, 2017; Mahizan et al., 2019; Schempp et al., 2018; Wołosik et al., 2013). Since the chemical synthesis of many terpenes is economically unfeasible and the extraction from their natural sources cannot be realized in a sustainable way, biotechnological approaches represent a promising strategy for accessing these compounds (Marienhagen and Bott, 2013; Schempp et al., 2018).
Terpenes are biosynthesized from the C5 isoprene building blocks isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), and classified as hemi- (C5), mono- (C10), sesqui- (C15), di- (C20), tri-(C30) and tetraterpenes (C40). The isoprene precursor scaffolds IPP and DMAPP are generated via the mevalonate (MVA) pathway, which uses acetyl-CoA as substrate, or the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway (also referred to as DXP pathway), which uses glyceraldehylde-3-phosphate (GAP) and pyruvate. While the MVA pathway occurs in all eukaryotes and is less abundant in prokaryotes, the MEP pathway is predominant in bacteria as well as in plastids of green algae and plants (Frank and Groll, 2017; Lombard and Moreira, 2011). IPP and DMAPP are used as substrates for a series of condensation steps to yield geranyl pyrophosphate GPP (C10), farnesyl pyrophosphate FPP (C15) and geranylgeranyl pyrophosphate GGPP (C20). GPP can be converted to monoterpenoids, FPP to sesqui- and triterpenoids, and GGPP to di- and tetraterpenoids by respective terpene synthase enzymes.
Key factors for successful heterologous production of terpenes in bacteria are a suitable host organism and the employed terpene synthase enzyme. Specifically, the bacterial host should provide a sufficient precursor supply and an appropriate storage capacity for potentially toxic compounds (Cravens et al., 2019; Das et al., 2007; Schempp et al., 2018). To further optimize terpene production via metabolic engineering, molecular genetic tools need to be established for the applied host system. In addition, terpene synthase enzymes from diverse origin were reported to often differ significantly in their performance for a recombinant biosynthesis approach and thus represent a critical determinant of success (Beekwilder et al., 2014; Qiao et al., 2019; Troost et al., 2019). Efforts in microbial terpene production have long focused on the use of established workhorses such as E. coli and yeast, but more recently, also alternative host organisms of the class of phototrophic bacteria are receiving increased attention (Moser and Pichler, 2019; Schempp et al., 2018). An established representative of this group is the Gram-negative facultative phototrophic non-sulfur purple α-proteobacterium Rhodobacter capsulatus. This bacterium possesses the MEP pathway to yield IPP and DMAPP, which are converted into each other by the IPP isomerase Idi. The FPP synthase IspA catalyzes the elongation via GPP to FPP, which is then further elongated by GGPP synthase CrtE, providing the C20 scaffold GGPP as substrate for the phytoene synthase and the following biosynthetic steps to form the Rhodobacter-specific carotenoids spheroidene and spheroidenone (see Fig. 1). Under phototrophic growth conditions, R. capsulatus generates an intracytoplasmic membrane system (ICM) from invaginations of the cytoplasmic membrane (Chory et al., 1984). Besides its function of housing the photosynthesis apparatus including the photopigments bacteriochlorophyll a and the carotenoids, the ICM offers a storage space for heterologously produced enzymes and compounds, thus possibly enabling the bacterium to sustain increased accumulation of hydrophobic products. The applicability of Rhodobacter species for heterologous terpene production has been shown for several examples including sesqui-, tri- and tetraterpenes (Beekwilder et al., 2014; Hage-Hülsmann et al., 2019; Khan et al., 2015; Loeschcke et al., 2017, 2013; Orsi et al., 2019; Troost et al., 2019).
For sesquiterpenes, it was shown that the production capacity of Rhodobacter can be increased by strengthening the elongation of prenyl phosphates by co-expression of FPP synthase along with a sesquiterpene synthase. Further, terpene production can be enhanced by increased synthesis of isoprene units via the intrinsic MEP pathway by co-expression of DxS (1-deoxy-d-xylulose 5-phosphate synthase) and Idi. Moreover, the introduction of the MVA pathway, which does not naturally occur in this host, was demonstrated to enhance terpene production. Such measures have enabled successful sesquiterpene production in Rhodobacter (Beekwilder et al., 2014; Orsi et al., 2019; Troost et al., 2019). Two Rhodobacter species, namely R. capsulatus and R. sphaeroides have raised particular interest as microbial cell factories (Heck and Drepper, 2017; Orsi et al., 2021) and the latter is industrially applied for the production of the sesquiterpenes valencene and nootkatone marketed by Isobionics (now BASF). The heterologous biosynthesis of other classes of terpenes in Rhodobacter species is, however, less explored.
In the present study, we thus aimed to focus on the production of representatives of C20, C30 and C40 terpenes, namely casbene, squalene and β-carotene, in R. capsulatus SB1003. Hence, we employed the casbene synthase RcCS from the castor bean shrub Ricinus communis (Mau and West, 1994), the squalene synthase SQS1 from the small flowering plant thale cress Arabidopsis thaliana (Busquets et al., 2008), and the phytoene desaturase CrtI together with lycopene cyclase CrtY of the proteobacterium Pantoea ananatis (Misawa et al., 1990). We further evaluated metabolic engineering strategies, which were recently established for sesquiterpene production in R. capsulatus by implementing co-expression of precursor biosynthetic genes ispA, dxs, and idi of the intrinsic isoprenoid metabolism and all genes of the MVA pathway (Troost et al., 2019), for optimizing the production of di-, tri- and tetraterpenes. The here presented data demonstrate that the metabolic engineering approach could successfully be transferred resulting in an enhanced production of casbene and β-carotene. Surprisingly, squalene production could not be improved when the A. thaliana synthase was employed. We therefore tested four alternative squalene synthases from the thermophilic cyanobacterium Thermosynechococcus elongatus, the green colonial microalga Botryococcus braunii, the Gram-negative methanotroph Methylococcus capsulatus, and Homo sapiens, finding highest production by the latter two in an engineered background. Therefore, the modular metabolic engineering approaches applied in this study enabled us to identify genetic setups that resulted in improved product titers of the target terpenes and demonstrate applicability of R. capsulatus as a platform organism for di-, tri-, and tetraterpene production.
Section snippets
Bacterial strains and cultivation conditions
Escherichia coli strains DH5α (Hanahan, 1983) and S17-1 (Simon et al., 1983) were used for cloning and conjugational transfer (Klipp et al., 1988) of DNA into Rhodobacter capsulatus. E. coli cells were cultivated in LB medium (Luria/ Miller, Carl Roth: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) under constant shaking or on LB agar plates containing 1.5 % agar (w/v, Bacto agar, Difco) at 37 °C. Kanamycin was added to a final concentration of 50 μg/mL. R.capsulatus strains
Engineering concepts for the production of C20, C30 and C40 terpenes in R. capsulatus
In order to analyze if the recently developed tools for modular engineering of sesquiterpenoid synthesis in R. capsulatus (Troost et al., 2019) can be transferred to the phototrophic production of C20, C30 and C40 terpenes, we used the diterpene casbene, the triterpene squalene and the tetraterpene β-carotene as showcase targets (Fig. 1). The C20 diterpene casbene is produced in Euphorbiaceae (Dueber et al., 1978). The compound is known to exhibit antifungal activity (Moesta and West, 1985) and
Discussion
This study clearly demonstrates the applicability of R. capsulatus as an alternative phototrophic production host for di-, tri-, and tetraterpenes using casbene, squalene and β-carotene as model compounds. In addition, we show the effects of metabolic engineering and use of different terpene biosynthetic enzymes on terpene production .
The beneficial effects of enhancing the precursor synthesis in the isoprenoid anabolic pathway for terpenoid production are well-known (Moser and Pichler, 2019).
CRediT authorship contribution statement
Jennifer Hage-Hülsmann: Data curation, Writing - original draft. Oliver Klaus: Investigation, Methodology, Data curation. Karl Linke: Methodology. Katrin Troost: Methodology. Lukas Gora: Methodology. Fabienne Hilgers: Methodology. Astrid Wirtz: Methodology. Beatrix Santiago-Schübel: Methodology. Anita Loeschcke: Data curation, Writing - review & editing, Visualization, Supervision. Karl-Erich Jaeger: Conceptualization, Writing - review & editing. Thomas Drepper: Conceptualization, Writing -
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
JHH was funded by the Deutsche Forschungsgemeinschaft viaCEPLAS - Cluster of Excellence on Plant Science (EXC1028). The work of OK was funded by the state of North Rhine Westphalia (NRW) and the European Regional Development Fund (EFRE), Project “CLIB-Kompetenzzentrum Biotechnologie (CKB)”34.EFRE-0300096. KT, FH and AL were funded by the Ministry of Culture and Science of the German State of North Rhine-Westphalia through NRW Strategieprojekt BioSC (No. 313/323‐400‐00213). The authors
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Equally contributed.