Development of Genetic Transformation and Gene Delivery Methods in the Marine Microalga Micromonas commoda

Li, Yixuan (2026) Development of Genetic Transformation and Gene Delivery Methods in the Marine Microalga Micromonas commoda. Doctoral thesis, University of East Anglia.

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Abstract

Nitrogen availability in the oceans is a key factor influencing the productivity and diversity of microalgae. To overcome nitrogen limitation and competition, marine microalgae have evolved diverse nitrogen metabolic strategies. The green alga Micromonas commoda and the diatom Thalassiosira pseudonana are globally distributed marine microalgae that exhibit distinct nitrogen utilization strategies. Cyanate serves as an alternative nitrogen source in marine environments, and the key enzyme involved in cyanate metabolism is cyanase (CynS), which is present in both M. commoda and T. pseudonana. However, they display different capacities for cyanate utilization, and the role of CynS in nitrogen metabolism is not clear.

In M. commoda, functional genomic studies have been constrained by the lack of efficient and stable genome editing tools. To investigate the function of the CynS gene in M. commoda, the development of efficient and cost-effective transformation methods is essential. In this study, a robust and highly efficient polyethylene glycol (PEG)-mediated transformation method was established for DNA delivery into M. commoda. Transformed cell lines were able to grow on 0.3% seawater agarose plates containing zeocin and expressed enhanced yellow fluorescent protein (eYFP). Furthermore, a CRISPR/Cas9 construct was assembled using Golden Gate cloning, resulting in a 76-nt deletion within the coding region of the CynS gene and generating a frameshift mutation in M. commoda.

The PEG-mediated transformation of T. pseudonana was optimized. Prior to the transformation, Alcalase was employed as the sole enzyme for efficient cell wall removal. Single-cell colonies were isolated on selective plates and the expression of enhanced green fluorescent protein (eGFP) was confirmed.

In addition, the abilities of M. commoda and T. pseudonana to utilize different nitrogen sources were further investigated. M. commoda was able to utilize nitrate, ammonium, and cyanate as sole nitrogen sources, but exhibited growth retardation when grown on urea. In contrast, T. pseudonana was capable of utilizing nitrate, nitrite, ammonium, and urea as sole nitrogen sources, but showed no growth on cyanate. Stable isotope probing (SIP) results revealed low nitrogen uptake rates under cyanate treatment, indicating negligible direct assimilation of cyanate.

This thesis presents an efficient transformation and genome editing system for M. commoda, together with an optimized gene delivery method for T. pseudonana. This provides a foundation for functional gene characterization in marine microalgae.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Environmental Sciences
Depositing User:	Chris White
Date Deposited:	14 Oct 2026 10:02
Last Modified:	14 Oct 2026 10:02
URI:	https://ueaeprints.uea.ac.uk/id/eprint/104774
DOI:

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