The photosynthetic bacteria Rhodobacter capsulatus and Synechocystis sp. PCC 6803 as new hosts for cyclic plant triterpene biosynthesis

Abstract

Cyclic triterpenes constitute one of the most diverse groups of plant natural products. Besides the intriguing biochemistry of their biosynthetic pathways, plant triterpenes exhibit versatile bioactivities, including antimicrobial effects against plant and human pathogens. While prokaryotes have been extensively used for the heterologous production of other classes of terpenes, the synthesis of cyclic triterpenes, which inherently includes the two-step catalytic formation of the universal linear precursor 2,3-oxidosqualene, is still a major challenge. We thus explored the suitability of the metabolically versatile photosynthetic α-proteobacterium Rhodobacter capsulatus SB1003 and cyanobacterium Synechocystis sp. PCC 6803 as alternative hosts for biosynthesis of cyclic plant triterpenes. Therefore, 2,3-oxidosqualene production was implemented and subsequently combined with different cyclization reactions catalyzed by the representative oxidosqualene cyclases CAS1 (cycloartenol synthase), LUP1 (lupeol synthase), THAS1 (thalianol synthase) and MRN1 (marneral synthase) derived from model plant Arabidopsis thaliana. While successful accumulation of 2,3-oxidosqualene could be detected by LC-MS analysis in both hosts, cyclase expression resulted in differential production profiles. CAS1 catalyzed conversion to only cycloartenol, but expression of LUP1 yielded lupeol and a triterpenoid matching an oxidation product of lupeol, in both hosts. In contrast, THAS1 expression did not lead to cyclic product formation in either host, whereas MRN1-dependent production of marnerol and hydroxymarnerol was observed in Synechocystis but not in R. capsulatus. Our findings thus indicate that 2,3-oxidosqualene cyclization in heterologous phototrophic bacteria is basically feasible but efficient conversion depends on both the respective cyclase enzyme and individual host properties. Therefore, photosynthetic α-proteo- and cyanobacteria are promising alternative candidates for providing new bacterial access to the broad class of triterpenes for biotechnological applications.

Comparative analysis of inducible promoters in cyanobacteria

“…Since the most efficient and commonly used Ni 2+ -responsive promoter, P nrsB , has already been investigated in detail elsewhere 13 , we chose P coaT . This promoter was successfully used towards small-scale biotechnological production of plant terpenes 38 and ethylene 39 in Synechocystis , as well as for mimicking a null mutant in the filamentous cyanobacterium Anabaena sp . by selectively removing Co 2+ and Zn 2+ from the media 40 .…”

Identifying and engineering the ideal microbial terpenoid production host

“…Rhodobacter capsulatus produced 110 mg L −1 of botryococcene in an autotrophic cultivation setup supplying only CO 2 , H 2 , and O 2 that yielded almost threefold more in titer compared with a glucose-based fed batch (Khan et al 2015). The first synthesis of triterpenoids derived from 2,3-oxidosqualene in cyanobacteria was described by Loeschcke et al (2017) who engineered Synechocystis sp. PCC 6803 for the production of cycloartenol, lupeol, and marneral.…”

Marine Proteobacteria as a source of natural products: advances in molecular tools and strategies

“…the lasso peptide brevunsin by Sphingomonas subterranea 155 or a triterpene in Rhodococcus capsulatus. 156 Terrestrial Beta-, Gamma-and Deltaproteobacteria including the genera Burkholderia, 157,158 Pseudomonas, 68,120,159,160 and Myxococcus 161 have been shown to be promising cell factories. Therefore, marine representatives of Proteobacteria could also be the candidates for future marine cell factories.…”