PhD Defence Zeinu Mussa Belew
Transport Engineering in Synthetic Biology Identification and characterization of phlorizin transporters enable increased production in yeast
Plants produce a plethora of specialized small molecules (plant natural products) that have pharmaceutical and nutraceutical values. Many of these molecules are usually produced in minute amounts and have complex chemical structures that make production by extraction from native hosts or through chemical synthesis laborious and cost-intensive. To overcome these challenges, microbial engineering has emerged as an alternative for large-scale production of high-value plant natural products. However, plant natural products can accumulate inside the microbial cell factories, which can lead to toxicity or feedback inhibition and thereby low production. Transport engineering can potentially be applied to facilitate final product secretion into the growth medium, thereby improving production and cost-efficient purification. However, the limited knowledge on transporters of plant natural products has hitherto hindered the application of transport engineering technologies.
In this thesis, using phlorizin, the anti-diabetic compound from apple, we provided a proof-of-concept for identification and application of a heterologous transporter for improving heterologous production of plant natural products in yeast. We functionally screened a total of ~600 full-length cDNA transporters library in Xenopus laevis oocytes and identified two Arabidopsis transporters capable of transporting phlorizin. Heterologous expression of one of these phlorizin transporters in phlorizin-producing yeast strain improved production titer by almost 90 %.
The other phlorizin transporter belongs to the Arabidopsis NPF (Nitrate and Peptide transporter Family). Biophysical characterization of the transporter suggested a new transport mechanism for members of the NPF. Here we show that the identified NPF phlorizin transporter appears to co-transport phlorizin together with chloride and proton in the same direction. Furthermore, based on homology searching, we identified and functionally characterized the first native (putative) phlorizin transporters in apple.
Finally, based on homology searching and expression profiling, we identified four SWEET (Sugars Will Eventually be Exported Transporters) family sugar transporters in barley, potentially involved in seed filling. This is the first study to functionally characterize barley SWEET transporters.
Associate Professor Hussam Hassan Nour Eldin Auis, Molecular Plant Biology, PLEN
Professor Dietmar Geiger, University of Würzburg, Germany
Senior Researcher Verena Siewers, Chalmers University of Technology, Sweden
Associate Professor Anja Thoe Fuglsang, PLEN
Reception afterwards in the 117 rooms, Thorvaldsensvej 40, 1st floor