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Rerouting plant terpene biosynthesis enables momilactone pathway elucidation

Nature Chemical Biology volume 17, pages 205–212 (2021)Cite this article

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Abstract

Momilactones from rice have allelopathic activity, the ability to inhibit growth of competing plants. Transferring momilactone production to other crops is a potential approach to combat weeds, yet a complete momilactone biosynthetic pathway remains elusive. Here, we address this challenge through rapid gene screening in Nicotiana benthamiana, a heterologous plant host. This required us to solve a central problem: diminishing intermediate and product yields remain a bottleneck for multistep diterpene pathways. We increased intermediate and product titers by rerouting diterpene biosynthesis from the chloroplast to the cytosolic, high-flux mevalonate pathway. This enabled the discovery and reconstitution of a complete route to momilactones (>10-fold yield improvement in production versus rice). Pure momilactone B isolated from N. benthamiana inhibited germination and root growth in Arabidopsis thaliana, validating allelopathic activity. We demonstrated the broad utility of this approach by applying it to forskolin, a Hedgehog inhibitor, and taxadiene, an intermediate in taxol biosynthesis (~10-fold improvement in production versus chloroplast expression).

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Fig. 1: Proposed momilactone biosynthetic pathway including previously characterized steps.
Fig. 2: High-titer production of diterpenoid-derived products in N. benthamiana through cytosolic diterpenoid engineering.
Fig. 3: Biosynthesis of tailored diterpenoids is enhanced via cytosolic expression of the momilactone and forskolin pathways.
Fig. 4: Complete reconstitution of momilactone biosynthesis in N. benthamiana.
Fig. 5: Momilactone B inhibits root growth of A. thaliana seedlings.

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Data availability

Data supporting the findings of this study are presented in the published article (including its Supplementary Information) or are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by National Institutes of Health (NIH) U01 grant GM110699 and NIH R01 grant GM121527. We thank C. Liou and K. Smith for discussions and feedback on the manuscript. We thank E. Holmes, Y. Chen and J. Kim for help in developing Arabidopsis inhibition assays; H. Cartwright at the Advanced Imaging Facility in the Carnegie Institution for Science, Department of Plant Biology, for training and assistance with confocal microscopy imaging; C. Miller for help with confocal imagining analysis; and K. Smith and S. Kim for help with flash column purification and NMR analysis.

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Authors and Affiliations

  1. Department of Chemical Engineering, Stanford University, Stanford, CA, USA

    Ricardo De La Peña & Elizabeth S. Sattely

  2. Howard Hughes Medical Institute, Stanford, CA, USA

    Elizabeth S. Sattely

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  1. Ricardo De La Peña
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Contributions

R.D.L.P. and E.S.S. designed the experiments. R.D.L.P. performed experiments. R.D.L.P. and E.S.S. analyzed the data and wrote the paper.

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Correspondence to Elizabeth S. Sattely.

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Extended data

Extended Data Fig. 1 Characterization of pathway intermediates from overexpression of cytOsCPS4 + cytOsKSL4 via MEV engineering.

GC-MS total ion chromatogram (TIC) and MS spectra of major and minor pathway products from overexpression of HMGR, cytGGPPS, cytOsCPS4, and cytOsKSL4 in N. benthamiana. Representative TICs are shown for the experimental sample and GFP control. Three total products with m/z corresponding to diterpene derived scaffolds were observed only upon expression of pathway genes. The major product identified corresponded to 1 and putative structures for two minor products were hypothesized. For m/z corresponding to syn-copalyl alcohol see Supplemental Fig. 9. Putative ion structures for the major product were assigned as reference. Putative structures are preliminary and based on predicted chemical formulas combined with analysis of the fragment ions observed in the MS spectra.

Extended Data Fig. 2 Characterization of pathway intermediates from overexpression of cytOsCPS4 + cytOsKSL4 + CYP99A3 via MEV engineering.

GC-MS total ion chromatogram (TIC) and MS spectra of products from overexpression of HMGR, cytGGPPS, cytOsCPS4, cytOsKSL4, and CYP99A3 in N. benthamiana. Representative TICs are shown for the experimental sample and controls. Three products with m/z corresponding to diterpene derived scaffolds were observed only upon expression of pathway genes. The major product identified corresponded to 2 and the two minor products corresponded to syn-pimaradien-19-al and syn-pimaradien-19-ol29. For m/z corresponding to syn-copalyl alcohol see Supplementary Fig. 9. Putative ion structures for 2 were assigned as reference. Putative structures are preliminary and based on predicted chemical formulas combined with analysis of the fragment ions observed in the MS spectra. An endogenous metabolite co-eluting with 2 was identified (* in the TIC). b, MS spectra of endogenous metabolite. c, GC-MS extracted ion chromatogram (EIC) using m/z = 241.2, corresponding to an abundant fragment found in the MS of 2, confirmed that 2 is only present upon expression of the requisite diTPSs and CYP99A3.

Extended Data Fig. 3 Characterization of pathway intermediates from overexpression of cytOsCPS4 + cytOsKSL4 + CYP76M8 via MEV engineering.

GC-MS total ion chromatogram (TIC) and MS spectra of major and minor pathway products from overexpression of HMGR, cytGGPPS, cytOsCPS4, cytOsKSL4, and CYP76M8 in N. benthamiana. Representative TICs are shown for the experimental sample and the no diterpene synthase control. Three total products with m/z corresponding to diterpene derived scaffolds were observed only upon expression of pathway genes. The major product identified corresponded to syn-pimaradien-6-ol and putative structures for two minor products were hypothesized (For syn-copalyl alcohol see Supplemental Fig. 9, for *, corresponding to an endogenous metabolite, see Extended Data Fig. 2). Putative ion structures for the major product were assigned as reference. Putative structures are preliminary and based on predicted chemical formulas combined with analysis of the fragment ions observed in the MS spectra.

Extended Data Fig. 4 Characterization of pathway intermediates from overexpression of cytOsCPS4 + cytOsKSL4 + CYP99A3 + CYP76M8 via MEV engineering.

GC-MS total ion chromatogram (TIC) and MS spectra of products from overexpression of HMGR, cytGGPPS, cytOsCPS4, cytOsKSL4, CYP99A3, and CYP76M8 in N. benthamiana. Representative TICs are shown for the experimental sample and controls. Products with m/z corresponding to diterpene derived scaffolds were observed only upon expression of pathway genes. Putative ion structures for 4, 5, and 2G were assigned as reference. Putative structures are preliminary and based on predicted formulas combined with analysis of the fragment ions observed in the MS spectra.

Extended Data Fig. 5 Oxidation of syn-pimaradiene scaffold via overexpression of various tailoring oxidases.

a, Oxidation of syn-pimaradiene can proceed via overexpression of either CYP99A3 or CYP76M8. n=3 biological independent leaf samples examined over three independent experiments. b, Overexpression of OsMAS, CYP99A3 and CYP76M8 is required for biosynthesis of the lactone ring in 6 and 8. Average GC-MS ion abundances of oxidized syn-pimaradiene scaffolds after overexpression of indicated genes via cytosolic engineering. Values and error bars represent the mean and the standard deviation of biological triplicates. n=3 biological independent leaf samples examined over three independent experiments.

Extended Data Fig. 6 Characterization of pathway intermediates from overexpression of cytOsCPS4 + cytOsKSL4 + CYP99A3 + CYP76M8 + OsMAS via MEV engineering.

GC-MS total ion chromatogram (TIC) and MS spectra of major and minor pathway products from overexpression of HMGR, cytGGPPS, cytOsCPS4, cytOsKSL4, CYP99A3, CYP76M8, and OsMAS in N. benthamiana. Representative TICs are shown for the experimental sample and the no diterpene synthase control. Four total products with m/z corresponding to diterpene derived scaffolds were observed only upon expression of pathway genes. Two major products identified corresponded to 6 and 8, and putative structures for two minor products were hypothesized. Putative ion structures for the major product were assigned as reference. Putative structures are preliminary and based on predicted chemical formulas combined with analysis of the fragment ions observed in the MS spectra.

Extended Data Fig. 7 Expression of OsMAS results in the reduction of levels of several oxidized syn-pimaradiene scaffolds.

Average GC-MS ion abundances of oxidized syn-pimaradiene intermediates after overexpression of indicated genes via cytosolic engineering. Co-expression with OsMAS results in reduction of levels of 4, 5, and increase in levels of 6. Values and error bars represent the mean and the standard deviation of biological triplicates. n=3 biological independent leaf samples examined over three independent experiments. P values were calculated using unpaired, two-tailed t-tests.

Extended Data Fig. 8 Characterization of pathway intermediates from overexpression of cytOsCPS4 + cytOsKSL4 + CYP99A3 + CYP76M8 + OsMAS + CYP76M14 + CYP701A8 via MEV engineering.

GC-MS total ion chromatogram (TIC) and MS spectra of products from overexpression of HMGR, cytGGPPS, cytOsCPS4, cytOsKSL4, CYP99A3, CYP76M8, OsMAS, CYP76M14, and CYP701A8 in N. benthamiana. Representative TICs are shown for the experimental sample and control. a, Two products with m/z corresponding to diterpene derived scaffolds were observed only upon expression of pathway genes. The products identified corresponded to momilactone B and 7. Putative ion structures were assigned as reference. Structures of 7 is preliminary and based on a predicted chemical formula combined with analysis of the fragment ions observed in the MS spectra. b, The major product identified corresponded to momilactone B (See Supplementary Figs. 11 and 12 for structural characterization).

Extended Data Fig. 9 Momilactone B inhibits A. thaliana seed germination.

a, Phenotype of A. thaliana seeds after 10 days of incubation in MS medium plates with different momilactone B concentrations. SA, salicylic acid.

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De La Peña, R., Sattely, E.S. Rerouting plant terpene biosynthesis enables momilactone pathway elucidation. Nat Chem Biol 17, 205–212 (2021). https://doi.org/10.1038/s41589-020-00669-3

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