Microbial Synthesis of the Forskolin Precursor Manoyl Oxide in an Enantiomerically Pure Form

Abstract

Forskolin is a promising medicinal compound belonging to a plethora of specialized plant metabolites that constitute a rich source of bioactive high-value compounds. A major obstacle for exploitation of plant metabolites is that they often are produced in small amounts and in plants difficult to cultivate. This may result in insufficient and unreliable supply leading to fluctuating and high sales prices. Hence, substantial efforts and resources have been invested in developing sustainable and reliable supply routes based on microbial cell factories. Here, we report microbial synthesis of (13R)-manoyl oxide, a proposed intermediate in the biosynthesis of forskolin and other medically important labdane-type terpenoids. Process optimization enabled synthesis of enantiomerically pure (13R)-manoyl oxide as the sole metabolite, providing a pure compound in just two steps with a yield of 10 mg/liter. The work presented here demonstrates the value of a standardized bioengineering pipeline and the large potential of microbial cell factories as sources for sustainable synthesis of complex biochemicals.

FIG 1

Schematic overview of the gene assembly procedure. (A) Open reading frames are cloned individually into standardized entry expression vectors. (B and C) Assembly of pGGPP (B) and pMO (C). Transcriptional units consisting of a T7 promoter, a consensus Shine-Dalgarno sequence, and the open reading frame are PCR amplified with generic oligonucleotides that anneal outside the different ORFs in the flanking regions common to all entry plasmids. The generic oligonucleotides facilitate specific assembly through defined linker regions (regions A to D, Eu, and Ed). Transcriptional units are assembled in a single cloning reaction by USER Fusion. Start and stop codons of open reading frames are indicated by green and red bars, respectively. The T7 promoter and terminator are denoted pT7 and T7t, respectively.

FIG 2

In vivo synthesis of two manoyl oxide isomers in E. coli. (A) Total ion GC-MS chromatograms (TIC) of hexane extract from E. coli transformed with either pGGPP and pMO (top) or empty vectors (bottom). IS, internal standard (1 mg/liter 1-eicosene). (B) Mass spectra of metabolites 1 and 2 identified as 13R- and 13S-MO, respectively, based on comparison to the Wiley Registry of Mass Spectral Data (see Fig. S2 in the supplemental material). (C) Relative quantification of 13R- and 13S-MO synthesis in E. coli. Yields are normalized to a 1-mg/liter 1-eicosene internal standard. n.d., neither 13R- nor 13S-MO was detectable. Numbers on the x axis refer to the combination of plasmids present in the analyzed strain, as follows: 1, pET-Duet/pCDF-Duet; 2, pMO/pCDF-Duet; 3, pMO/pSyn; 4, pMO/pGGPP; 5, pET-Duet/pGGPP; 4+mev, pMO/pGGPP supplemented with 9 mM mevalonolactone. Data represent the averages for four independent replicates with standard deviations. (D) Biosynthetic route to 13R-MO. Solid arrows indicate enzymatic reactions. The enzyme catalyzing each step is depicted next to the reaction arrow. The dashed arrows indicate the intrinsic degradation of labda-13-en-8-ol diphosphate into equimolar amounts of 13R- and 13S-MO occurring in the absence of CfTPS3. Phosphate groups in the chemical structures are abbreviated as “P”.

FIG 3

Impact of iterative improvements of culture conditions and extraction time on manoyl oxide yields. E. coli transformed with pGGPP and pMO was cultivated at either 25°C for 16 h postinduction or at 16°C for 112 h postinduction. Cultures were extracted with hexane for either 1 or 16 h. Graphs depict the average yields of the two manoyl oxide isomers from two independent colonies each analyzed in four independent replicates. Error bars denote standard deviations.

FIG 4

Identification and elimination of indole in E. coli extracts. (A) Total ion chromatograms from GC-MS analysis of extracts from E. coli. The plasmid combination in question is listed in the top left corner of each chromatogram along with the initial concentration of tryptophan (Trp or trp) in the media. The metabolites observed were identified as 13R-MO (1), indole (3), and impurities from the instrument (4 and 5). IS, internal standard (1 mg/liter 1-eicosene). (B) Total ion chromatogram of authentic indole standard (top) as well as mass spectra of compound 3 (middle) and indole standard (bottom). Based on a comparison of retention time and mass spectra with authentic standard data, compound 3 was identified as indole.