AUTHOR=Troost Katrin , Loeschcke Anita , Hilgers Fabienne , Özgür Armagan Yakup , Weber Tim Moritz , Santiago-Schübel Beatrix , Svensson Vera , Hage-Hülsmann Jennifer , Habash Samer S. , Grundler Florian M. W. , Schleker A. Sylvia S. , Jaeger Karl-Erich , Drepper Thomas 

TITLE=Engineered Rhodobacter capsulatus as a Phototrophic Platform Organism for the Synthesis of Plant Sesquiterpenoids

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.01998

DOI=10.3389/fmicb.2019.01998

ISSN=1664-302X

ABSTRACT=<p>Sesquiterpenoids are a large class of natural compounds offering manifold properties valuable for food, cosmetics, agriculture, and pharma industry. Production in microorganisms is a sustainable approach to provide sesquiterpenoids for research and industrial use independent of their natural sources. This requires the functional transfer of the respective biocatalytic pathways in an adequate host microorganism offering a sufficient supply of precursors that is ideally adjusted to the individual demand of the recombinant biosynthesis route. The phototrophic purple bacterium <italic>Rhodobacter capsulatus</italic> offers unique physiological properties that are favorable for biosynthesis of hydrophobic terpenes. Under phototrophic conditions, it develops a large intracytoplasmic membrane suitable for hosting membrane-bound enzymes and metabolites of respective biosynthetic pathways. In addition, <italic>Rhodobacter</italic> harbors an intrinsic carotenoid biosynthesis that can be engineered toward the production of foreign terpenes. Here, we evaluate <italic>R. capsulatus</italic> as host for the production of plant sesquiterpenoids under phototrophic conditions using patchoulol and valencene as a proof of concept. The heterologous expression of patchoulol synthase PcPS from <italic>Pogostemon cablin</italic> as well as the valencene synthases CsVS from <italic>Citrus sinensis</italic> and CnVS from <italic>Callitropsis nootkatensis</italic> led to the production of the respective sesquiterpenoids in <italic>R. capsulatus</italic>. To analyze, if gradually adjustable formation of the key precursor farnesylpyrophosphate (FPP) is beneficial for sesquiterpene synthesis under phototrophic conditions, the intrinsic 1-deoxy-<sc>D</sc>-xylulose 5-phosphate (DXP) pathway genes as well as the heterologous mevalonate pathway genes were modularly expressed in various combinations. To this end, different plasmids and chromosomally integrated expression tools were developed harboring the strong and tightly controlled P<italic><sub><italic>nif</italic></sub></italic> promoter for heterologous gene expression. Notably, comparative studies identified a distinct combination of precursor biosynthetic genes as best-performing setup for each of the tested sesquiterpene synthases. In summary, we could demonstrate that <italic>R. capsulatus</italic> is a promising alternative platform organism that is suited for sustainable sesquiterpenoid formation under phototrophic cultivation conditions. A modular engineering of <italic>R. capsulatus</italic> strains via tailored co-expression of FPP biosynthetic genes further allowed adaptation of sesquiterpene precursor formation to its catalytic conversion by different plant terpene synthases.</p>