Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biochemistry

Supervisor

Edgell, David R.

Abstract

Phosphorus (P) is essential for all life. The bioavailability of phosphorus in oceans impacts diatoms like Phaeodactylum tricornutum (P. tricornutum). P. tricornutum is extensively used in transcriptomic studies to understand pathways involved in P-acquisition. However, activation mechanisms and roles of regulatory elements in P-acquisition responses remains unclear. Here, I deleted predicted phosphate regulatory sites in P.tricornutum’s HASP1 promoter to create different HASP1-eGFP constructs. Under P-depletion, two constructs showed increased eGFP secretion. Additionally, a HASP1 knockout strain was grown under different phosphorus sources to determine if HASP1 is a phytase. Cells grown in full phosphorus utilized organic sources of phosphorus and cells grown under P-depletion used inorganic sources. HASP1 knockout cells were also used to understand transcriptional responses under phosphorus depletion using RNAseq. This revealed the loss of HASP1 was not essential to cell survival. Understanding diatom responses under P-starvation will contribute to improving P. tricornutum for synthetic biology applications.

Summary for Lay Audience

Phosphorus (P) is an essential element for all life forms. The amount of phosphorus in marine environments often fluctuates which impacts the ecology and physiology of marine phytoplankton living in these environments, specifically diatoms. Scientists are curious to understand how certain species living in these environments like the diatom, Phaeodactylum tricornutum (P. tricornutum), can regulate internal systems to cope with adverse environmental conditions. One method is called a phosphate starvation response (or PSR). The process involves activating genes responsible for phosphate acquisition. Much of what we know about this phenomenon is through studies that look at what genes are turned up or down in response to phosphorus depletion. The downfall is we do not have a comprehensive understanding of how P-acquisition responses are activated in response to phosphate depletion, as well as localization and function of proteins involved. Thankfully, due to recent technological advances, we have the tools required to genetically manipulate regions within sections of DNA responsible for turning genes on or off to understand how genes are expressed.

In this study, I use a portion of the P. tricornutum genetic code, called the Highly Abundant Secreted Protein 1 (HASP1) promoter, which contains 4 regions predicted to be responsible for the activation of a phosphate starvation response. I create a series of deletions in the predicted sites and design novel P. tricornutum strains that are grown in various low- phosphorus media. 2 out of 3 mutant constructs showed an upregulation in promoter activity compared to normal P. tricornutum strains. As a result, there was an increase of protein secretion. These results introduce novel expression systems for the production of new proteins. Furthermore, the growth of the HASP1 knockout strain was assessed under different phosphorus sources to observe phytase-like potential. Results showed the phosphorus source utilized was dependent on phosphorus concentration. Lastly, responses in the HASP1 knockout revealed many genes were activated in response to nutrient depletion over time. Using new strategies to assess phosphate regulation in diatoms holds promising results in developing novel expression systems that may be used for biotechnological purposes.

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