Team:ULaval/Design
Design
Our team intends to use Saccharomyces cerevisiae (S. cerevisiae) to produce adrenaline. We designed a two-plasmid system which harbors synthetic human cDNAs encoding the adrenaline enzymatic pathway. We explored how the insertion of this plasmid system into a strain of S. cerevisiae engineered to overproduce L-tyrosine1 can be used for the biosynthesis of adrenaline. As dopamine and noradrenaline, two of the three metabolic intermediates of the adrenaline enzymatic pathway2 as shown in Figure 1, are also of biomedical interest3, we intend to create three strains of S. cerevisiae producing dopamine, noradrenaline or adrenaline based on plasmid combination.
Figure 1. The adrenaline enzymatic pathway2.
In order to achieve that we will create three plasmids. The first plasmid, pRS31N-TH-LDDC, will carry the genes coding for the first two enzymes of the biosynthetic pathway, tyrosine hydroxylase and aromatic L-amino acid decarboxylase, and will hence allow for the production of dopamine when transformed into the optimised strain of S. cerevisiae. The second plasmid, pRS31H-DBH, will contain the gene coding for dopamine β-hydroxylase in order to produce noradrenaline when combined with the first plasmid. The last construct, pRS31H-DBH-PNMT, will carry both the genes coding for dopamine β-hydroxylase and phenylethanolamine N-methyltransferase. As shown in Figure 2, the combination of the first and the last plasmid will therefore allow for the production of adrenaline. To assure that both plasmids can be maintained within the yeast cells simultaneously, they will be engineered to harbour different resistance genes. The plasmid pRS31N-TH-LDDC will possess a nourseotricin (NatR) resistance gene, while the plasmids pRS31H-DBH and pRS31H-DBH-PNMT will harbour a hygromycin (hPhR) resistance gene.
Finally, all the products will be purified by HPLC using the reverse-phase analytical column Primesep 200 by SIELC Technologies which is made for the efficient separation of catecholamines4.
Figure 2. Workflow of our scientific approach.
Figure 1. The adrenaline enzymatic pathway2.
In order to achieve that we will create three plasmids. The first plasmid, pRS31N-TH-LDDC, will carry the genes coding for the first two enzymes of the biosynthetic pathway, tyrosine hydroxylase and aromatic L-amino acid decarboxylase, and will hence allow for the production of dopamine when transformed into the optimised strain of S. cerevisiae. The second plasmid, pRS31H-DBH, will contain the gene coding for dopamine β-hydroxylase in order to produce noradrenaline when combined with the first plasmid. The last construct, pRS31H-DBH-PNMT, will carry both the genes coding for dopamine β-hydroxylase and phenylethanolamine N-methyltransferase. As shown in Figure 2, the combination of the first and the last plasmid will therefore allow for the production of adrenaline. To assure that both plasmids can be maintained within the yeast cells simultaneously, they will be engineered to harbour different resistance genes. The plasmid pRS31N-TH-LDDC will possess a nourseotricin (NatR) resistance gene, while the plasmids pRS31H-DBH and pRS31H-DBH-PNMT will harbour a hygromycin (hPhR) resistance gene.
Finally, all the products will be purified by HPLC using the reverse-phase analytical column Primesep 200 by SIELC Technologies which is made for the efficient separation of catecholamines4.
Figure 2. Workflow of our scientific approach.
References
1. Gold, N. D. et al., 2015. Metabolic engineering of a tyrosine-overproducing yeast platform using targetedmetabolomics. Microbial Cell Factories, 14(73).
2. Pocock, G., Richards, C. D. & Richards, D. A., 2013. Human Physiology. Fourth ed. Oxford: Oxford University Press.
3. Burchum, J. R. & Rosenthal, L. D., 2016. Lehne's Pharmacology for Nursing Care. 9th ed. USA: Elsevier.
4. SIELC Technologies, 2018. HPLC Separation of Catecholamines. [Online] Available at: http://www.sielc.com/Application-HPLC-Separation-of-Catecholamines.html [Accessed 03 2018].
2. Pocock, G., Richards, C. D. & Richards, D. A., 2013. Human Physiology. Fourth ed. Oxford: Oxford University Press.
3. Burchum, J. R. & Rosenthal, L. D., 2016. Lehne's Pharmacology for Nursing Care. 9th ed. USA: Elsevier.
4. SIELC Technologies, 2018. HPLC Separation of Catecholamines. [Online] Available at: http://www.sielc.com/Application-HPLC-Separation-of-Catecholamines.html [Accessed 03 2018].