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Broadening applications of Euglena gracilis by stable nuclear transformation and targeted proteomic studies

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posted on 2022-07-27, 06:01 authored by Bishal Khatiwada

Euglena gracilis is a unicellular microalga with multiple biotechnological applications in areas such as the production of nutraceuticals, cosmeceuticals and biofuels. Also, Euglena has shown the capacity to survive in heavy metal contaminated water. However, little is known about the mechanisms of this adaptation. The transcriptome of E. gracilis 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 Euglena 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 Euglena gracilis.

From the three different transformation approaches explored, transformants from the biolistic bombardment and electroporation were able to transiently express the hygromycin (hptII) gene. In contrast, Agrobacterium-mediated transformation produced stable nuclear transformants suggesting its potential as the preferred method to transform Euglena. The availability of a stable transformation platform will pave the way for further improvement of E. gracilis strains for the production of valuable compounds.

In the second study, the ability of E. gracilis to tolerate and accumulate heavy metals was investigated. The wild type (WT) E. gracilis (Z-strain) and E. gracilis var. saccharophila (B-strain) were exposed to the heavy metals cadmium (Cd), lead (Pb) and mercury (Hg). A comprehensive proteomic profile of E. gracilis 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 E. gracilis Z-strain is presented.

The role of chloroplasts in the accumulation of heavy metals by E. gracilis was studied by creating a variant Euglena 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. E. gracilis 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 (LHCB) 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 E. gracilis 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.


Table of Contents

Chapter 1 Molecular tools and applications of Euglena gracilis - from biorefineries to bioremediation -- Chapter 2 Nuclear transformation of the versatile microalga Euglena gracilis -- Chapter 3 Proteomic response of Euglena gracilis to heavy metal exposure - identification of key proteins involved in heavy metal tolerance and accumulation -- Chapter 4 Probing the role of the chloroplasts in heavy metal tolerance and accumulation in Euglena gracilis -- Chapter 5 Summary and future perspectives -- Appendix I -- Appendix II


A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy

Awarding Institution

Macquarie University

Degree Type

Thesis PhD


Thesis (PhD), Macquarie University, Department of Molecular Sciences, 2020

Department, Centre or School

Department of Molecular Sciences

Year of Award


Principal Supervisor

Helena Nevalainen

Additional Supervisor 1

Anwar Sunna

Additional Supervisor 2

Angela Sun


Copyright disclaimer: Copyright Bishal Khatiwada 2020




xiii, 14-184 pages

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