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Widespread biosynthesis of 16-carbon terpenoids in bacteria

Nature Chemical Biology volume 19, pages 1532–1539 (2023)Cite this article

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Abstract

Terpenoids are the most diverse group of specialized metabolites with numerous applications. Their biosynthesis is based on the five-carbon isoprene building block and, as a result, almost all terpenoids isolated to date are based on backbones that contain multiples of five carbon atoms. Intrigued by the discovery of an unusual bacterial terpenoid with a 16-carbon skeleton, here we investigate whether the biosynthesis of 16-carbon terpenoids is more widespread than this single example. We mine bacterial genomic information and identify potential C16 biosynthetic clusters in more than 700 sequenced genomes. We study selected clusters using a yeast synthetic biology platform and reveal that the encoded synthases produce at least 47 different noncanonical terpenoids. By thorough chemical analysis, we explain the structures of 13 C16 metabolites, most of which possess intricate highly strained bi- and tricyclic backbones. Our results unveil the existence of an extensive class of terpenoids in bacteria.

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Fig. 1: The identified gene clusters can be classified into four distinct types.
Fig. 2: Heatmap showing the product profile of the analyzed terpene synthases.
Fig. 3: Terpene synthases belonging to type 1 clusters produce intricate bi- and tricyclic terpene scaffolds.
Fig. 4: Characteristic examples and products of terpene synthases present in type 2 clusters.
Fig. 5: Characteristic examples of type 3 clusters and products of corresponding terpene synthases with PSPP as substrate.

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Data availability

The data that support the findings of this study are available within the main text and its Supplementary Information file. Source data are provided with this paper. The NCBI RefSeq Genome Database (https://www.ncbi.nlm.nih.gov/refseq/) was used for biosynthetic gene cluster analysis and the identified clusters are provided in the source data for Fig. 1 file. Data are also available from the corresponding author upon request.

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Acknowledgements

We are grateful to F. Geu-Flores (University of Copenhagen) and K. Miettinen (University of Copenhagen) for critical manuscript reading. We thank J. Olsen and M. Ramirez (University of Copenhagen) for their assistance in running analytical instruments. This work was financially supported by the Novo Nordisk Foundation grant nos. NNF19OC0055204 and NNF22OC0080100 (to S.C.K.). Work at the National and Kapodistrian University of Athens was supported by the research project BioNP (grant no. 70/3/14685 to E.I.). Y.-T.D. acknowledges the Chinese Scholarship Council for a PhD scholarship (CSC grant no. 201904910446).

Author information

Author notes

  1. Codruta Ignea

    Present address: Department of Bioengineering, McGill University, Montreal, Quebec, Canada

Authors and Affiliations

  1. Biochemical Engineering Group, Plant Biochemistry Section, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark

    Yao-Tao Duan, Codruta Ignea, Yong Zhao & Sotirios C. Kampranis

  2. Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, Athens, Greece

    Aikaterini Koutsaviti, Maria Harizani, Vassilios Roussis & Efstathia Ioannou

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  1. Yao-Tao Duan
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Contributions

S.C.K. conceived the study. S.C.K. and Y.-T.D. performed bioinformatics analysis. Y.-T.D., C.I., Y.Z., V.R., E.I. and S.C.K. designed experiments. Y.-T.D., C.I. and Y.Z. conducted all cloning, engineering of S. cerevisiae and chromatographic data analysis. Y.-T.D. and Y.Z. performed large-scale yeast cultivation and extraction. A.K., M.H. and E.I. conducted the isolation of C16 terpenoids from yeast cultures and analyzed NMR data. Y.-T.D., Y.Z., V.R., E.I. and S.C.K. wrote the paper. All authors read and commented on the final version of the paper.

Corresponding authors

Correspondence to Yong Zhao, Efstathia Ioannou or Sotirios C. Kampranis.

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Nature Chemical Biology thanks Yang Hai, Kui Hong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phylogenetic analysis of the different methyltransferase proteins encoded in the 40 investigated clusters.

Enzymes selected for further characterization are indicated in bold. The 2-methyl-GPP synthase from Pseudoanabaena limnetica (GPPMTase) is included in the analysis for comparison. Phylogenetic tree constructed with MEGA v11.0.13 (www.megasoftware.net). Source data are provided as a Source Data file.

Extended Data Fig. 2 Functional characterization of selected methyltransferases.

GC–MS chromatograms of the extracts of yeast cells expressing the different selected methyltransferase genes. All methyltransferases belonging to clade α analyzed in this experiment produced predominantly PSPP. This was confirmed by the detection of its hydrolysis products presodorifelool, presodorifenol, and 2,3-dihydro-presodorifenol. The same products were also synthesized by the prototypical PSPP synthase, SpSodMT. Enzymes belonging to the MTβ group did not produce any new product. List of methyltransferases analyzed and abbreviations used: Burkholderia singularis strain TSV85 MTα (BsMTα), Burkholderia territorii strain SCHI0024 MTα (BtMTα), Pseudomonas marginalis pv. marginalis strain ICMP 14937 MTα (PmMTα), Mesorhizobium sp. strain IRAMC:0171 MTα (Ms1MTα), Morganella morganii strain AR_0133 MTα (MmMTα), Burkholderia multivorans MTα (BmMTα), Pseudomonas chlororaphis strain PA23 MTα (PcMTα), Pseudomonas fluorescens strain FW300-N2E3MTα (PfMTα), Burkholderia pyrrocinia strain DSM 10685 MTα (BpMTα), Pseudomonas lini strain DSM 16768 MTα (PlMTα), P. chlororaphis strain Lzh-T5 MTβ (PcT5MTβ), B. territorii strain SCHI0024 MTβ (BtTPS-MTβ), Burkholderia cepacia strain MTβ (BcTPS-MTβ). Source data is provided as a Source Data file.

Source data

Extended Data Fig. 3 Product profiles of yeast cells expressing selected terpene synthases and SpSodMT.

GC-APCI-qToF-HRMS (coupled gas chromatography/atmospheric pressure chemical ionization/quadrupole-time-of-flight/high resolution mass spectrometry) ion-extracted chromatograms (EIC, [M + H]+ = 219.2107 ± 0.001 for the putative molecular formula C16H26) showing the product profile of yeast cells co-expressing SpSodMT and different terpene synthases (blue) versus three control strains: yeast cells expressing only SpSodMT (black), yeast cells expressing only the terpene synthase gene (red), and yeast cells carrying two empty vectors (yellow). a. Yeast cells expressing BsmTPS produced five C16 compounds (1, 14-17). b. Yeast cells expressing PfTPS produced two C16 compounds, compounds 2 and 18. c. Yeast cells expressing PbTPS produced seven C16 compounds (3, 19-24). d. Yeast cells expressing BpTPS produced two C16 compounds (3 and 4). e. Yeast cells expressing PcTPS produced six C16 compounds (5, 25-29). f. Yeast cells expressing SsTPS produced one C16 compound (25). g. Yeast cells expressing Vs01TPS2 produced one C16 compound (23). h. Yeast cells expressing Ms1TPS2 produced two C16 compounds (30 and 31). i. Yeast cells expressing PmaTPS2 produced compounds 32, 33, and 34. j. Yeast cells expressing Pc66TPS1 produced compound 35. k. Yeast cells expressing PczmTPS2 produced thirteen C16 compounds (6-8, 15, 36-44). l. Yeast cells expressing PcT5TPS1 produced eight C16 compounds (9-13, 45-47). m. Yeast cells expressing PlTPS produced compounds 3, 19-24. n. Yeast cells expressing PccTPS produced compounds 3, 19-24. o. Yeast cells expressing BsTPS3 produced compounds 3 and 4. p. Yeast cells expressing MmTPS produced compounds 3 and 4. q. Yeast cells expressing PmaTPS1 produced compound 5. r. Yeast cells expressing BuTPS produced two C16 compounds 5 and 25. Characterization of compounds 1-47 as putative C16 terpenoids was based on their high-resolution APCI-MS (HR-APCI-MS) spectra shown in Supplementary Note 1. The structures of compounds 1-13 were subsequently elucidated by analysis of their NMR data after their isolation in pure form. Compounds 1-13 are indicated in bold. Figure composed using MS PowerPoint.

Extended Data Fig. 4 Product profiles of selected terpene synthases determined using in vitro activity assays.

GC-APCI-QqToF ion-extracted chromatograms (EIC, [M + H]+ = 219.2107 ± 0.001 for the putative molecular formula C16H26) for in vitro activity assays using extracts from yeast cells expressing SpSodMT and different terpene synthases as enzyme source (a-r). Yeast cells expressing only SpSodMT (black) and yeast cells carrying two empty vectors (yellow) were used as controls. SAM and FPP were used as substrates in all the experiments (details in Methods section). Figure composed using MS PowerPoint.

Extended Data Fig. 5 Metabolic engineering to improve the titer of heraclitene.

When both the BsmTPS and SpSodMT genes were co-expressed from high-copy number plasmids in AM109 yeast cells, the titer of compound 1 was 4.2 mg/L. Assuming that this could be due to limited substrate availability, we set out to improve the supply of PSPP. We constructed a fusion of the yeast FPP synthase Erg20p with SpSodMT, in order to increase the levels of FPP and at the same time improve its channeling to SpSodMT. Expression of the gene fusion from a high-copy number plasmid led to only marginal improvement in the titer of compound 1, suggesting that PSPP availability was not the limiting factor at this stage. Thus, we considered that conversion of PSPP by the terpene synthase may be the limiting step. To address this, we constructed a fusion between BsmTPS and Erg20p. This led to a 2-fold increase in the titer of 1, reaching 8.2 mg/L, when the ERG20-BsmTPS gene fusion was expressed together with SpSodMT. When both ERG20-SpSodMT and ERG20-BsmTPS fusions were co-expressed, no further improvement was obtained, confirming the earlier observation that PSPP availability was not limiting. To further improve production, we then proceeded to construct a yeast strain (AM109-97) where the SpSodMT and ERG20-BsmTPS genes were integrated into the genome because this leads to stable and robust production. Genomic integration resulted in a remarkable 56-fold increase in the titer of compound 1, reaching 231.7 mg/L. Error bars show standard error of the mean of three independent biological replicates (n = 3). Analysis was carried our using Microsoft Office Professional Plus 2016 (Microsoft Excel). Source data is provided as a Source Data file.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–3, Figs. 1–3 and Notes 1 and 2.

Reporting Summary

Supplementary Data

Lists of primers, yeast strains, yeast plasmids, synthetic gene sequences and source data for Supplementary Figs. 2 and 3.

Source data

Source Data Fig. 1

Spreadsheet with the bacterial gene cluster information used in the analysis described in Fig. 1.

Source Data Fig. 2

Statistical source data for the heatmap in Fig. 2.

Source Data Extended Data Fig. 2

Methyltransferase protein accession numbers and gene sequences used in the phylogenetic analysis.

Source Data Extended Data Fig. 5

Statistical source data.

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Duan, YT., Koutsaviti, A., Harizani, M. et al. Widespread biosynthesis of 16-carbon terpenoids in bacteria. Nat Chem Biol 19, 1532–1539 (2023). https://doi.org/10.1038/s41589-023-01445-9

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