Glucosinolates are complex specialized plant metabolites characteristic of the order of Brassicales, such as e.g. broccoli. Besides being well-known flavor compounds (e.g. mustard and wasabi), glucosinolates have gained recognition due to their anti-cancer properties. This has primed a strong desire to develop a stable, rich source of glucosinolates in a microbial cell factory.
Recent successful synthetic biology applications have demonstrated the incredible potential of microbial cell factories for production of high-value chemicals. The genetic ‘parts’ required for glucosinolate engineering are known and the feasibility of engineering them has been demonstrated in both E. coli and yeast. Our focus is now on unleashing the potential and breaking the yield barrier by increasing the flux through the pathways.
Towards building an efficient yeast cell factory, you will apply the newly developed OrthoRep system (Ravikumar et al. 2018) that allows rapid evolution of a gene of interest by using an error-prone DNA polymerase that specifically targets this gene. You will be using OrthoRep to enable expression of one challenging protein. Additionally, you will apply the CASCADE platform (Strucko et al., 2017) to tune the gene copy number and ‘omics-based approaches to assess the metabolic impact of introducing the glucosinolate pathway on the microbial host. As part of the engineering subgroup in the DynaMo Center of Excellence, we have several other projects in case you finish the proposed project.
- Employing the OrthoRep system to ‘revive’ the LSU1 gene that has proven challenging to express in S. cerevisiae. LSU1 is a key enzyme in the methionine chain elongation pathway that are critical for production of glucoraphanin, associated with the health-promoting properties of broccoli.
- Characterization of two yeast strains containing either a single copy or six copies of the biosynthetic pathway producing benzyl glucosinolate
- Targeted proteomics will be applied to investigate how the gene copy number affects protein levels in the yeast cells.
- An ‘omics approach will be employed to identify and test specific genetic targets for global strain optimization.
In the DynaMo Center of Excellence, we have created optimal opportunities for critical thinking, excellent science and outstanding training of the next generation of young scientists. In return, we expect you to be enthusiastic, dedicated and eager to learn and advance your skills in many disciplines (e.g. research, oral and written presentations etc.).
For further information, do not hesitate to send an email to either Professor Barbara Halkier (firstname.lastname@example.org) or PhD student William Thomas Wajn (email@example.com).
Ravikumar, A., Arzumanyan, G. A., Obadi, M. K. A., Javanpour, A. A., & Liu, C. C. (2018). Scalable, Continuous Evolution of Genes at Mutation Rates above Genomic Error Thresholds. Cell, 175(7), 1946-1957.e13. https://doi.org/10.1016/j.cell.2018.10.021
Strucko, T., Buron, L. D., Jarczynska, Z. D., Nødvig, C. S., Mølgaard, L., Halkier, B. A., & Mortensen, U. H. (2017). CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast. Scientific Reports, 7(January), 1–12. https://doi.org/10.1038/srep41431
Methods used:Heterologous gene expression in yeast, LC-MS on selected metabolites, yeast genotyping, sequencing, protein extraction and digestion, targeted proteomics, OrthoRep, strain optimization and basic molecular biology tools.
Keywords:Synthetic biology, yeast engineering, biotechnology, microbial cell factory, strain optimization
Project home page:https://dynamo.ku.dk/