Project

Converting isoprenoid biosynthesis into an energy-generating process for sustainable production

We aim to implement a metabolic network to increase the energetic efficiency of isoprenoid biosynthesis in Rhodobacter sphaeroides. If the energy demand of the bioconversion is decreased sufficiently or even becomes energy generating, the process may become suitable for sustainable production of bulk chemicals.

Background

Considering accelerated climate change due to fossil-based CO2 emissions, biotechnology can contribute to enabling a green, circular bioeconomy and help mitigate the effects of the environmental crisis.

Isoprenoids (also called terpenes) are a class of very versatile natural compounds. Currently, they are mostly used in high value applications (flavour ingredients, pharmaceuticals, pigments). Traditionally, these isoprenoids are extracted from plants, but due to low yields and/or seasonal supply, there is a strong shift towards biotechnological processes (Ro et al., 2006).

In an earlier project, we engineered the ability of Rhodobacter sphaeroides to overproduce isoprenoids. To this end, heterotrophic growth was studied, metabolic flux analysis was performed and novel genome editing tools have been developed (Mougiakos et al., 2019; Orsi, et al., 2020; Orsi et al., 2019). Furthermore, the native isoprenoid-producing pathway (MEP) was coupled with a heterologous pathway (MVA), which significantly increased the isoprenoid titres (Orsi, et al., 2020). This previous research transitioned R. sphaeroides into a mature microbial cell factory and serves as an exciting starting point for future research (Orsi et al., 2021).

Isoprenoids can also be used as bulk chemicals for low-value applications – e.g. as biofuels or building blocks – and as such contribute even stronger to the reduction of our fossil resource dependency (Keasling et al., 2021). However, currently employed technologies are not yet efficient enough. One of the main bottlenecks is that isoprenoid biosynthesis requires the input of a lot of metabolic energy.

Aim

This project aims to increase the energetic efficiency of isoprenoid biosynthesis in R. sphaeroides. If this energy demand can be decreased significantly, or even becomes negative, this may make the process suitable for the production of bulk chemicals as well.

We will approach this by:

  • Replacing energy-requiring metabolic steps with solutions that do not require energy input, or are even energy generating.
  • The strains will be assessed in terms of TRY (titre, rate and yield), but also using C13 labelling, growth profiles and bioreactor cultivations.
  • Uncoupling product formation from growth.

Technologies

  • Strain engineering, metabolic engineering, CRISPR-Cas9 mediated genome engineering.
  • Strain cultivation in batch, continuous and retentostat modes.
  • A variety of analytical methods (C13 labelling, growth profiling, gas chromatography, mass spectrometry, etc.)

Contact

Thesis projects are available for enthusiastic Bachelor’s and Master’s students. If you are interested in this project, feel free to reach out to Matic Kostanjsek.

References

  1. Keasling, J., Garcia Martin, H., Lee, T. S., Mukhopadhyay, A., Singer, S. W., & Sundstrom, E. (2021). Microbial production of advanced biofuels. Nature Reviews Microbiology, 0123456789. https://doi.org/10.1038/s41579-021-00577-w
  2. Mougiakos, I., Orsi, E., Ghiffary, M. R., Post, W., de Maria, A., Adiego-Perez, B., Kengen, S. W. M., Weusthuis, R. A., & van der Oost, J. (2019). Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering. Microbial Cell Factories, 18(1), 204. https://doi.org/10.1186/s12934-019-1255-1
  3. Orsi, E., Beekwilder, J., Eggink, G., Kengen, S. W. M., & Weusthuis, R. A. (2021). The transition of Rhodobacter sphaeroides into a microbial cell factory. Biotechnology and Bioengineering, 118(2), 531–541. https://doi.org/10.1002/bit.27593
  4. Orsi, E., Beekwilder, J., Gelder, D., Houwelingen, A., Eggink, G., Kengen, S. W. M., & Weusthuis, R. A. (2020). Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides. Microbial Biotechnology, 13(4), 1082–1093. https://doi.org/10.1111/1751-7915.13562
  5. Orsi, E., Beekwilder, J., Peek, S., Eggink, G., Kengen, S. W. M., & Weusthuis, R. A. (2020). Metabolic flux ratio analysis by parallel 13C labeling of isoprenoid biosynthesis in Rhodobacter sphaeroides. Metabolic Engineering, 57, 228–238. https://doi.org/10.1016/j.ymben.2019.12.004
  6. Orsi, E., Folch, P. L., Monje-López, V. T., Fernhout, B. M., Turcato, A., Kengen, S. W. M., Eggink, G., & Weusthuis, R. A. (2019). Characterization of heterotrophic growth and sesquiterpene production by Rhodobacter sphaeroides on a defined medium. Journal of Industrial Microbiology and Biotechnology, 46(8), 1179–1190. https://doi.org/10.1007/s10295-019-02201-6
  7. Ro, D.-K., Paradise, E. M., Ouellet, M., Fisher, K. J., Newman, K. L., Ndungu, J. M., Ho, K. A., Eachus, R. A., Ham, T. S., Kirby, J., Chang, M. C. Y., Withers, S. T., Shiba, Y., Sarpong, R., & Keasling, J. D. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature, 440(7086), 940–943. https://doi.org/10.1038/nature04640