Advancing algal biotechnology for food application

Increasing global temperatures and atmospheric carbon dioxide pose challenges to traditional agriculture, highlighting the need for alternative food production methods. Microalgae, which consume CO2, offer a promising solution for producing food ingredients utilizing non-arable land. Recent advances in synthetic biology have enabled targeted improvements to microalgal strains for this purpose. This thesis focused on engineering Chlamydomonas reinhardtii to achieve three key goals: improving photosynthetic efficiency, accelerating acetate utilization, and optimizing recombinant protein secretion. Chapter 2 focused on overexpressing malate synthase (MS), an enzyme that condenses glyoxylate and acetyl-CoA to form malate, to examine its effect on glycolate secretion, a byproduct of photorespiration. Reducing glycolate secretion is expected to improve photosynthetic efficiency. An expression cassette that placed MS under the control of a constitutive promoter and targeted the MS protein to the chloroplast (CTP-MS) was used to transform the C. reinhardtii cia5 strain, which lacks a carbon-concentrating mechanism and induces photorespiration under ambient CO2 conditions. A time-course experiment measured growth and glycolate production in the transformed lines. However, MS overexpression did not reduce glycolate production and it did not enhance growth compared to the cia5 marker control strains. Chapter 3 examined acetate uptake rate in C. reinhardtii, which can use acetate as its sole source of carbon and energy. Enhanced acetate utilization was achieved using a two-step strategy combining acetyl-CoA synthetase (ACS2) and malate synthase (MS): ACS2 converts acetate to acetyl-CoA, which is then used by MS to produce malate. A dual-gene cassette containing ACS2 and chloroplast-targeted MS, each driven by a constitutive promoter, was assembled using the Chlamydomonas MoClo kit and transformed into the CC-4349 strain. CC-4349 is a cell wall-deficient strain commonly used for high-efficiency nuclear transformation and serves as the parental background of the cia5 knock-out strain described in Chapter 2. Transformants were evaluated by measuring growth (OD750) and acetate consumption. Under mixotrophic conditions (light with acetate and CO2 available), ACS2-MS strains exhibited increased growth relative to the CC-4349 marker controls. When photosynthesis was inhibited by removing the light source, ACS2-MS lines consumed acetate faster than both ACS2-only and marker control across repeated experiments. This fast acetate-consuming strain has potential applications in food production using acetate as a carbon source for biomanufacturing. Chapter 4 explores the feasibility of using C. reinhardtii to secrete thaumatin, a small sweet-tasting protein, as a model for heterologous protein production. Secretion of this heterologous protein may reduce intracellular stress, extend culture longevity, and conserve resources. A secretion construct using a signal peptide (SP) and immune tags (HA and HIS) from the Chlamydomonas MoClo kit was introduced into the CC-400 strain, a cell-wall deficient line frequently used for a high-efficiency nuclear transformation. While immunoblotting of tagged thaumatin transformants revealed faint bands, Green Fluorescent Protein (GFP) constructs, used as a placeholder for thaumatin, showed stronger supernatant signals than controls in spectrofluorometric measurements, verifying the utility of this detection method. Thaumatin transformants were recovered at a lower frequency than the marker-only controls and exhibited a range of culture colors from light yellow to green. GFP transformation efficiency was comparable to the controls. These phenotypes suggest that, while the secretion system was functioning, the thaumatin protein might be a burden on the host. Additional constructs that target thaumatin to the chloroplast, mitochondria and cytosol were built and these lines need further phenotypic characterization. Chapter 5 outlines future directions for engineering of Chlamydomonas reinhardtii for industrial applications, based on the findings in this thesis. While overexpression of CTP-MS altered growth rate and glycolate production in transgenic lines, achieving the improvements in photosynthetic efficiency and biomass reported in tobacco by South (South et al. 2019) may require a dual construct targeting malate synthase and glycolate dehydrogenase to the chloroplast. The rapid acetate consumption observed in the ACS2-MS F5 line represents a promising phenotype for biofactories producing food ingredients. The line may also tolerate high-acetate concentrations, potentially increase production rates, and therefore further characterization of this line is warranted. Thaumatin-producing C. reinhardtii strains were generated, demonstrating the feasibility for a secretion system. However, the observation that the thaumatin-secreting strain showed culture stress, while the GFP strain did not, suggest that more research is needed to better understand the sweet protein characteristics that promote and increase heterologous protein yield in Chlamydomonas reinhardtii.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Food Science & Human Nutrition

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Atkinson, Christine Anne

Contributors dc:contributor

• Jin, Yong-Su
• Miller, Michael J.
• Schmidt, Shelly J.
• Erdman, John W.
• Ort, Donald R.

• Chlamydomonas Reinhardtii
• Thaumatin
• Food Science
• Synthetic Biology

Statement dc:rights

• ©2025 Christine Anne Atkinson All rights reserved.

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/130181

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F130181