Microalgae being photosynthetic and having quick growth cycles can prove to be excellent candidates as biofactories for the production of aromatic compounds like para-hydroxybenzoic acid (pHBA) that act as a monomer in liquid crystal polymers. We developed transgenic lines of the model alga Chlamydomonas reinhardtii by performing nuclear transformation using electroporation. The transgenic cell lines expressed the ubiC gene that utilized chorismate from the shikimate pathway as a substrate to produce pHBA. The maximum yield of pHBA measured in these lines was 80 mg/L.
Accruing pHBA can be toxic to the cells and the mechanism by which C. reinhardtii could detoxify pHBA is not known. C. reinhardtii genome was thus scanned for sequences similar to UDP-glucosyltransferase (UGT) that can transfer the glucose moiety to pHBA, rendering it non-toxic to the cell lines. Our analysis suggested the absence of any potential UGTs that could glycosylate pHBA and detoxify it. We further performed feeding experiments to test the ability of wt-type C. reinhardtii cells to detoxify pHBA and understand its fate. C. reinhardtii cells were fed with varying concentrations of pHBA and harvested at different time intervals. The HPLC chromatograms indicated a majority of the pHBA was catabolized. Based on these results, literature was reviewed to find a suitable UGT candidate to enable the engineering of the glycosylation mechanism in the alga. A transgenic algal line with gene encoding UGT89B1 was created and fed with varying concentrations of pHBA. HPLC chromatograms from the extracts revealed the presence of phenolic glucoside. Following this, C. reinhardtii was co-transformed with ubiC and gene encoding UGT89B1; this led to the production of pHBA and further glycosylation to pHBA glucoside (phenolic glucoside). The maximum yield of pHBA yields in these cell lines was 180 mg/L. Growing C. reinhardtii lines producing pHBA on a large scale can lead to problems like contamination with bacteria and other algal species, a decline in pH, and a rise in temperature. To circumvent these problems, we explored the plausibility of using other algal strains. We analyzed the genome of Galdieria sulphuraria, a photosynthetic thermophile, that can use more than fifty different carbon sources for its growth and metabolism. We hypothesized several of these traits to have arisen by acquiring genes through horizontal gene transfer. We used a segmentation and clustering algorithm to identify regions of the genome that have atypical nucleotide composition. The atypical segments identified by the method were further analyzed using phylogenetic methods to further support claims of their alien origin. Our method identified 69 genes that were not previously reported as alien genes, some of which could be implicated in imparting resistance to environmental stress factors. Our experiments also revealed G. sulphuraria has a greater extent of alien genes compared to C. reinhardtii.
| Identifer | oai:union.ndltd.org:unt.edu/info:ark/67531/metadc1873837 |
| Date | 12 1900 |
| Creators | Saxena, Garima Girish |
| Contributors | Azad, Rajeev, Henard, Calvin, Allen, Michael, Manning, Schonna, Dixon, Richard A. |
| Publisher | University of North Texas |
| Source Sets | University of North Texas |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Format | xiii, 143 pages, Text |
| Rights | Public, Saxena, Garima Girish, Copyright, Copyright is held by the author, unless otherwise noted. All rights Reserved. |
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