Broadening applications of <i>Euglena gracilis</i> by stable nuclear transformation and targeted proteomic studies
Creators: name: Khatiwada, Bishal
Publisher: name: Macquarie University
Year: 2022
Subjects: Other education not elsewhere classified

description: <em>Euglena gracilis </em>is a unicellular microalga with multiple biotechnological applications in areas such as the production of nutraceuticals, cosmeceuticals and biofuels. Also, <em>Euglena </em>has shown the capacity to survive in heavy metal contaminated water. However, little is known about the mechanisms of this adaptation. The transcriptome of <em>E. gracilis </em>was published recently but a complete genome sequence is not available yet. This lack of genome sequence information impedes the process of strain engineering. Moreover, the rather complex nature of the <em>Euglena </em>genome and its cell wall-like structure made up of a proteinaceous pellicle have hampered the development of genetic engineering tools, including a method for obtaining genetically stable transformants. In this study, a method for achieving stable nuclear transformation was developed and studies into the mechanism for heavy metal tolerance were conducted by targeted proteomics with the view of broadening potential applications of <em>Euglena gracilis</em>. From the three different transformation approaches explored, transformants from the biolistic bombardment and electroporation were able to transiently express the hygromycin (<em>hptII</em>) gene. In contrast, <em>Agrobacterium-</em>mediated transformation produced stable nuclear transformants suggesting its potential as the preferred method to transform <em>Euglena</em>. The availability of a stable transformation platform will pave the way for further improvement of <em>E. gracilis </em>strains for the production of valuable compounds. In the second study, the ability of <em>E. gracilis </em>to tolerate and accumulate heavy metals was investigated. The wild type (WT) <em>E. gracilis </em>(Z-strain) and <em>E. gracilis </em>var. <em>saccharophila </em>(B-strain) were exposed to the heavy metals cadmium (Cd), lead (Pb) and mercury (Hg). A comprehensive proteomic profile of <em>E. gracilis </em>after exposure to these heavy metals, obtained using sequential window acquisition of all theoretical mass spectra (SWATH‐MS) is presented for the first time. The key proteins involved in heavy metal tolerance and accumulation were identified and a possible mechanism for heavy metal accumulation in the <em>E. gracilis </em>Z-strain is presented. The role of chloroplasts in the accumulation of heavy metals by <em>E. gracilis </em>was studied by creating a variant <em>Euglena </em>Zm-strain lacking mature chloroplasts and compared to chloroplast-forming Z-strain. Transmission electron microscopy (TEM) was used to locate the deposition of heavy metals in intracellular organelles and a proteomic study was conducted to identify key proteins involved in heavy metal transportation and accumulation. <em>E. gracilis </em>chloroplasts were found to have a role in the accumulation of Cd, whereas Hg and Pb were mostly deposited in the cytosol of both strains. The Zm-strain lacking chloroplasts was able to accumulate a higher amount of Hg suggesting that the chloroplasts in the WT Z-strain were affected by the Hg toxicity resulting in decreased cellular ability to accumulate Hg. Comparative proteomics study of the two strains revealed that the heavy metal transporter MTP2 may have a role in Cd transportation to the chloroplasts. A multidrug resistance-associated protein identified by proteomics could be a potential Hg transporter to either cytosol or mitochondria. From the results of the proteomic study, a gene encoding the light-harvesting chlorophyll a-b binding protein (<em>LHCB</em>) and a phytochelatin synthase (PCS) gene were selected to investigate their role in Cd tolerance in the Z-strain. The result indicated that the strains transformed with both genes showed higher tolerance to Cd compared to the WT Z-strain. This study provides new leads into the mechanism of heavy metal tolerance and accumulation in <em>E. gracilis </em>and guidance for the future development of strains for phycoremediation of Cd and Hg utilizing the information gathered from proteins involved in heavy metal tolerance and the nuclear transformation platform established in this work.