On Dec 16th, MSc student Ana Luísa Amaro successfully defended her MSc thesis in Biology-Biotechnology on ZOOM.
The title of her project is ‘Uncaging glycolysis in Chlamydomonas reinhardtii to enable growth on external glucose’.
Green microalgae are among the most promising organisms for production of food and high-value biomolecules. But, high production costs due to low yield in phototrophic (light-driven) cultivation prohibit large-scale commercial exploitation. A promising avenue is to develop heterotrophic growth in fermenters using sugar as organic carbon sources. However, many microalgae cannot utilize sugars for growth because they do not have the transport machinery for importing sugar and/or for delivering it to compartmentalized sugar metabolizing pathways.
In her thesis, Ana has sought to understand the inability of the model microalgae Chlamydomonas reinhardtii to grow on sugar. Ana successfully generated a range of expression constructs that required advanced molecular biology skills to overcome the challenges posed by the G/C rich C.reinhardtii genome. Using her constructs in combination with flow cytometry and confocal microscopy, Ana showed that the first two dedicated steps of the glycolytic pathway of C.reinhardtii are compartmentalized within the chloroplast. These findings confirmed the first indications of a compartmentalized glycolytic pathway which were generated 35 years ago. Moreover, Ana correctly predicted and removed the transit peptides in both of these proteins and showed their relocalization to the cytosol. In parallel, Ana used RT-PCR and sequence analyses to show incorrect intron splicing as the probable cause for our inability to express the human GLUT1 transporter in C.reinhardtii. Finally, Ana took many steps to establish fluorescence-ELISA based enzymatic carbohydrate metabolism assays and she also established liquid 2-fluordeoxglucose growth inhibition assays to determine whether 2-deoxyglucose exerts its growth inhibition by targeting the glycolysis or the OPPP. Through her thesis, Ana has established some important prerequisites for decompartmentalizing glycolysis in C.reinhardtii. We believe that this will enable this microalgae to grow on sugar and potentially achieve much higher production yield than what is currently possible