Molecular Biotechnology
Our groups’ research contributes to the field of applied microbiology for industrial biotechnology. We use rational metabolic engineering (including synthetic biology) and non-targeted strategies to develop yeast strains for the conversion of renewable resources into fuels, bulk and fine chemicals. While past research focused on plant-based raw materials such as glycerol (by-product of biodiesel industry) and D-galacturonic acid (an ingredient in pectin-rich agro-industrial side streams), we currently aim at establishing yeast with the ability to convert CO2-based sources of carbon and energy to grow yeast and produce valuable target molecules. Among the products, the economically relevant platform chemical succinic acid received particular attention since the exploited formation pathway fixes CO2. Our group is experienced in targeted yeast strain construction including methods such as
• Gibson Assembly
• Plasmid- and genome-based expression
• Homologous recombination
• CRISPR/Cas9
Non-targeted approaches such as adaptive laboratory evolution and transcript factor engineering have also been successfully applied in past research projects. We collaborate with several academic and industrial partners.
The Nevoigt Laboratory is an international leader in the field of glycerol utilization by baker’s yeast for the production of chemicals, particularly succinic acid. Our lab developed the first S. cerevisiae strains able to efficiently utilize glycerol and they formed the basis of interesting novel applications such as the production of various chemicals, yeast biomass and proteins. We have successfully established the CRISPR/Cas9 system as a tool for yeast engineering. Our current projects are listed below.
A) Projects related to alternative feedstocks for yeast:
- Glycerol - Glycerol is an attractive source of carbon and energy due to several reasons. We successfully overcame the limitations of the popular yeast cell factory Saccharomyces cerevisiae (baker’s yeast) to utilize glycerol. Our currently best strain is able to grow with a maximum specific rate (µmax) of 0.31 h-1 (Perpelea et al., submitted manuscript). We are working on demonstrating high cell density biomass production from pure and crude glycerol.
- D-galacturonic acid (D-GalUA) – One advantage of glycerol among the available plant-based sources of carbon and energy is its relatively high degree of reduction. This can be used to produce chemicals whose production pathway requires reducing power/electrons. However, co-feeding of glycerol can also support the utilization of renewable substrates that are more oxidized than sugars such as D-GalUA. We successfully engineered a strain that is able consume D-GalUA with the highest reported maximum specific rate of 0.23 g gCDW-1 h-1 in synthetic minimal medium when glycerol was added.
- Methanol and methanol-derived compounds – Green methanol can be produced from CO2 and renewable energy. Our goal is to use methanol as a substrate for fermentative processes with the yeast Saccharomyces cerevisiae. Alternatively, we work on equipping the yeast with pathways for the use of organic molecules with multiple carbon atoms that can be generated by in-vitro enzyme cascades from methanol in the future. The particular target of this project is to produce compounds with antioxidant properties. These are of utmost importance, especially for the animal feed industry.
B) Projects related to particular target products produced by engineered yeast:
- Succinic acid (SA) - Among the target products from glycerol, succinic acid production was mainly promoted (Malubhoy et al., Rendulic et al. 2024). To our knowledge, we achieved the highest product yield obtained in engineered yeast so far (0.66 gSA/gGlycerol), and we are currently exploring routes to further improve titer, yield and rate.
In 2024, the Nevoigt Laboratory consists of one Humboldt fellow, one postdoctoral fellow, four PhD students, and one technician. The funding of the current projects originate from the German Research Foundation (DFG) and the Federal Ministry of Education and Research (BMBF).
The group leader is author on 56 peer-reviewed publications, two book chapters, seven patent applications, and co-editor of one book and two special issues.
Rendulić, T., Perpelea, A., Ragas Ortiz, J.P., Casal, M., Nevoigt, E., (2024) Mitochondrial membrane transporters as attractive targets for the fermentative production of succinic acid from glycerol in Saccharomyces cerevisiae. FEMS Yeast Res. 24.
Perpelea, A., Martins L.C., Wijaya, A.W., Rippert, D., Klein, M., Angelov, A., Peltonen, K., Teleki, A. , Liebl, W., Richard, P., Thevelein, J.M., Takors, R., Sá-Correia, I., Nevoigt, E. (2021) Towards valorization of pectin-rich agro-industrial residues: engineering of Saccharomyces cerevisiae for co-fermentation of D-galacturonic acid and glycerol. Metab. Eng. 69, 1-14.
Xiberras, J., Klein, M., Nevoigt, E. Engineering Saccharomyces cerevisiae for succinic acid production from glycerol and carbon dioxide. Front. Bioeng. Biotechnol. 8, 566.
Xiberras, J., Klein, M., Nevoigt, E. (2019) Glycerol as a substrate for Saccharomyces cerevisiae based bioprocesses - Knowledge gaps regarding the central carbon catabolism of this 'non-fermentable' carbon source. Biotechnol Adv. 37, 107378
Islam, Z.U., Klein, M., Ødum, A.S.R., Nevoigt, E. (2017) A modular metabolic engineering approach for the production of 1,2-propanediol from glycerol by Saccharomyces cerevisiae. Metab. Eng. 44, 223-235.
Klein, M., Swinnen, S., Thevelein, J.M., Nevoigt, E. (2016) Glycerol metabolism and transport in yeast and fungi: established knowledge and ambiguities. Invited minireview for Environ. Microbiol. 19, 878-893.
Klein, M., Carrillo, M., Xiberras, J., Islam, Z.-U., Swinnen, S., Nevoigt, E. (2016) Towards the exploitation of glycerol’s high reducing power in Saccharomyces cerevisiae-based bioprocesses. Metab. Eng. 38, 464-472.
Swinnen, S., Ho, P.-W., Klein, M., Nevoigt, E. (2016) Genetic determinants for enhanced glycerol growth of Saccharomyces cerevisiae. Metab. Eng. 36, 68-79.
Swinnen, S., Klein, M., Carrillo, M., McInnes, J., Nguyen, H.T.T., and Nevoigt, E. (2013) Re-evaluation of glycerol utilization in the species Saccharomyces cerevisiae: characterization of an isolate which grows on glycerol without supporting supplements. Biotechnol. Biofuels 6, 157.
Nevoigt, E. (2008) Progress in metabolic engineering of the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 72, 379-412.
Alper, H., Moxley, J., Nevoigt, E., Fink, G. R., and Stephanopoulos, G. (2006) Engineering yeast transcription machinery for improved bioethanol tolerance and production. Science 314, 1565-1568.