Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii
- University of Copenhagen, Denmark
- University of California, Berkeley, United States
- Evolva, Denmark
- Nofima, Norway
- KTH - Royal Institute of Technology, Sweden
- Michigan State University, United States
Abstract
Forskolin is a unique structurally complex labdane type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.
Article and author information
Author details
Allison Maree Heskes
Funding
Villum Fonden
- Birger Lindberg Møller
Novo Nordisk
- Birger Lindberg Møller
European Commission
- Irini Pateraki
DOE Office of Science (BER DE-FC02-07ER64494)
- Bjoern Hamberger
Strategic Partnership Grant (15-SPG-Full-3101)
- Bjoern Hamberger
MSU Foundation
- Bjoern Hamberger
Michigan State University (startup funding from the Department of Molecular Biology and Biochemistry)
- Bjoern Hamberger
Michigan State University (AgBioResearch - MICL02454)
- Bjoern Hamberger
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Pateraki et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 5,109
- views
-
- 810
- downloads
-
- 98
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii
eLife 6:e23001.
https://doi.org/10.7554/eLife.23001
Further reading
-
- Biochemistry and Chemical Biology
The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins, combined with the exposure of their residues, accounts for this sensitivity. One context in which IDPs play important roles that are concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat-soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results suggest that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet the mechanisms underlying this synergy differ between IDP families.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here, we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry. Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å, which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II complexes.
