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Large-scale, sustainable production of sesquiterpenes from cyanobacteria

Funded by: BMBF (Bundesministerium für Bildung und Forschung), 2024-2026

Cyanobacteria, photoautotrophic prokaryotes that inhabit vast niches of the Earth, are quickly gaining traction in applied research. With more knowledge and more tools becoming available, research is progressing to a point in which they are being considered more and more as a biotechnological chassis. In recent years, they have been engineered to biosynthesize various relevant bulk and fine chemicals.

Terpenes are a large group of chemical compounds, comprising more than 20.000 structurally unique molecules. They can be subdivided by the amount of isoprene units they consist of, and their precursors are formed by repeated elongation of IPP and DMAPP to GPP, FPP and GGPP. These backbones are then further processed by terpene synthases, often forming bioactive compounds. Terpenes occur in all organisms and have many different functions.

Sesquiterpenes are derived from FPP and consist of 3 isoprene units. They are often cyclic and volatile, and many can be found in plants, serving as insect repellants, antimicrobial compounds or phytohormones. In this project, we are interested in valencene, which is found naturally in many citrus fruits, but also many other plants. It is used in industry as an aroma ingredient, but it also serves as the precursor for nootkatone (Figure 1), another sesquiterpene which is also used as a flavor ingredient due to its characteristic grapefruit smell, but has shown promise as an insect repellant.

Why cyanobacteria?

As mentioned, the biosynthetic precursors for terpenes can be found produced in practically all organisms, as their derivatives are involved in essential cellular processes. Cyanobacteria, along with other heterotrophic bacteria, derive their terpene backbones from the MEP-Pathway. Since cyanobacteria also perform oxygenic photosynthesis, and photosynthetic pigments such as carotenoids and chlorophyll are derived from the MEP pathway as well (Figure 2), they already have a naturally high metabolic flux towards terpene precursors. In fact, it was shown previously in this group that carbon could be redirected from pigment synthesis towards valencene via inducible expression systems1. Other photosynthetic organisms such as Rhodobacter capsulatus have shown promise in this field as well2,3.

Cyanobacteria share a common ancestor with plant chloroplasts. Many terpenes, especially sesquiterpenes, are synthesized in the plant chloroplast; however, the yield of terpenes from plants is extremely low, and the extraction is costly. Cyanobacteria are not only capable of supplying the necessary carbon backbones and reducing equivalents from photosynthesis, but are able to be cultivated more easily and efficiently without competing for agricultural land, while still reaping the rewards from photosynthesis.

Metabolic engineering

In previous works, we were able to show that combining strategic knockouts of the squalene synthesis pathway, a non-essential side-reaction, with chemically inducible expression of the terpene synthase improved product yield1. The cyanobacterial metabolism offers vast potential for improvement, with many possibilities of redirecting the metabolic flux. One popular target which was previously studied as well by our group4 is the MEP-Pathway (Fig. 2). In a collaborative effort with another project which focuses on the production of synthetic triterpenes, we are studying combinatorial overexpression, as well as regulatory bottlenecks, including cofactor supply.

High-density cultivation

Often, the productivities reported in cyanobacteria are quite low compared with their long-standing heterotroph competitors. However, when comparing biomass-specific productivities directly, the numbers are in the same range. Biotechnological production from microorganisms not only relies on a robust production strain, but also on scaling up the process and optimizing along the way. While this is comparatively straight-forward for heterotrophic chassis organisms on the lab-scale, cyanobacterial growth is often quickly limited by insufficient light and CO2 in a standard shake-flask setup. To maximize growth and production rates and reach high cell densities, we aim to study our production strains in a highly controlled environment that enables such high cell densities in collaboration with CellDEG, a company from Berlin specializing in highly controlled photobioreactors.

Practical Bachelor or Master thesis

If you are interested in working on this topic for your thesis, please contact me via E-Mail with your CV and a short introduction.

Key publications:

  1. Dietsch, M.; Behle, A.; Westhoff, P.; Axmann, I. M. Metabolic Engineering of Synechocystis Sp. PCC 6803 for the Photoproduction of the Sesquiterpene Valencene. Metab. Eng. Commun. 2021, 13, e00178. https://doi.org/10.1016/j.mec.2021.e00178.
  2. Troost, K.; Loeschcke, A.; Hilgers, F.; Özgür, A. Y.; Weber, T. M.; Santiago-Schübel, B.; Svensson, V.; Hage-Hülsmann, J.; Habash, S. S.; Grundler, F. M. W.; Schleker, A. S. S.; Jaeger, K.-E.; Drepper, T. Engineered Rhodobacter Capsulatus as a Phototrophic Platform Organism for the Synthesis of Plant Sesquiterpenoids. Front. Microbiol. 2019, 10, 1998. https://doi.org/10.3389/fmicb.2019.01998.
  3. Loeschcke, A.; Dienst, D.; Wewer, V.; Hage-Hülsmann, J.; Dietsch, M.; Kranz-Finger, S.; Hüren, V.; Metzger, S.; Urlacher, V. B.; Gigolashvili, T.; Kopriva, S.; Axmann, I. M.; Drepper, T.; Jaeger, K.-E. The Photosynthetic Bacteria Rhodobacter Capsulatus and Synechocystis Sp. PCC 6803 as New Hosts for Cyclic Plant Triterpene Biosynthesis. PLoS ONE 2017, 12 (12), e0189816. https://doi.org/10.1371/journal.pone.0189816.
  4. Germann, A. T.; Nakielski, A.; Dietsch, M.; Petzel, T.; Moser, D.; Triesch, S.; Westhoff, P.; Axmann, I. M. A Systematic Overexpression Approach Reveals Native Targets to Increase Squalene Production in Synechocystis Sp. PCC 6803. Front. Plant Sci. 2023, 14, 1024981. https://doi.org/10.3389/fpls.2023.1024981.


Research Associate

Dr. Anna Behle
Universitätsstr. 1 Building: 22.07
Floor/Room: 00.042

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