McMasterU

Why Algae?

Green microalgae are photosynthetic, unicellular eukaryotes that can be used as a sustainable biomanufacturing platform. They have been used in industry for more than a century, with early cultivation centered around producing biomass for aquaculture feed and dietary supplements (Allen & Nelson, 1910). At the turn of the 21st century, microalgae became the primary research for biofuels due to rising oil prices and the demand for energy security, however, these products proved too low-value to be competitive (Mahmood et al., 2023). At present, microalgae are being employed to produce antibodies, recombinant proteins, antimicrobial peptides, pigments, and other bioactive compounds in the laboratory (Gong et al., 2011; Niraula et al., 2025a; Rajput et al., 2024; Velasquez, 2025; Wang et al., 2025).

Compared to microbial systems that depend on sugar-based feedstocks, green microalgae directly capture CO2 and sunlight to fix carbon and produce biomass, while other needed materials can be supplied from wastewater (Abdur Razzak et al., 2024). This approach is both cost-effective and environmentally sustainable. Moreover, engineered algae can be cultivated in photobioreactors (Figure 1), which can be installed near high-emission facilities, like industrial plants and breweries, creating a carbon recycling system (Silkina et al., 2024).

Algae Bioreactors

Figure 1: Microalgae cultivation using different bioreactor systems (A) Open race-way pond (B) Closed tubular bioreactors (C) Flat-panel bioreactors (Lewandowski et al., 2018)

Beyond these applications, microalgae share many biological traits with plants, including chloroplasts for photosynthesis, carbon fixation mechanisms, and sterol and isoprenoid biosynthesis pathways (Lohr et al., 2012). The commonalities allow them to produce pigments, antioxidants, and other metabolites traditionally sourced from crops, but with less demand for nutrients or competition for farmland (Peter et al., 2022; Yan et al., 2016). Moreover, as eukaryotes, they are capable of post-translational modifications and thus are a more suitable system for producing complex proteins and metabolites that cannot be synthesized in bacteria (Abdur Razzak et al., 2024).

Product Selection

Our team aimed to select a product that was locally relevant, high value, and challenging to source by existing means. Many compounds and materials were initially considered, summarized in the table below:


Sterol Backbone

Figure 2: Basic sterol structure (Created in MolDraw).

Ultimately, our team decided to focus on producing sterols, which are a subgroup of steroids that are naturally produced by eukaryotes and some prokaryotes (Hoshino & Gaucher, 2021). Sterols are a family of organic compounds known as 3-hydroxy steroids, meaning they have four carbon rings, a hydroxyl group attached to the third carbon of the first ring, and a varying functional group attached to carbon 17 (Figure 2) (CheBI, 2017).

Sterols have applications in numerous pharmaceutical, cosmetic, and food products. Low-cost plant sterols, known as phytosterols, can be used to lower cholesterol, in turn lowering risk for cardiovascular disease (Randhir et al., 2020). More interestingly, they can also be converted into valuable steroid compounds for medical hormonal treatment (Donova, 2023). Microbial fermentation of phytosterols to produce compounds such as progesterone and hydrocortisone is more economical, scalable, and sustainable compared to the current alternative, chemical synthesis. Bacterial fermentation using Mycobacterium and Rhodococcus have been shown to allow for high-yield targeted conversion of phytosterols precursors into steroid drugs, and currently, a mix of microbiological and chemical processes is used by the pharmaceutical industry to produce these compounds (Fernández-Cabezón et al., 2018).

Currently, progesterone is commercially synthesized from diosgenin, a steroid compound from Dioscorea yams (Xu et al., 2022). However, this method is not sustainable due to their extensive use of agricultural land, fresh water, and time required for cultivation. Microalgae serves as a promising alternative to current agricultural methods due to their fast growth rate, ability to utilize non-arable land and water unsuitable for human consumption, and diverse range of sterol and sterol precursors that they are able to produce (Randhir et al., 2020). Additionally, progesterone can be synthesized from ergosterol (Hu et al., 2017), a compound produced by microalgae (Voshall et al., 2021), which is why our team decided to overexpress ergosterol in microalgae.

Why Ergosterol?

Ergosterol (ergosta-5,7,22-trien-3β-ol) is a type of sterol found in green algae and fungi (Rangsinth et al., 2023). In C. reinhardtii, ergosterol makes up 50% of the sterol profile and is the most commercially relevant, with the other 50% of sterols produced being 7-dehydroporiferasterol. In the cell, ergosterol serves important roles in membrane maintenance (fluidity, permeability, integrity). Ergosterol can also act as a precursor for vitamin D12 and progesterone synthesis from a biomanufacturing perspective (Hu et al., 2017; Rangsinth et al., 2023). There is also evidence that ergosterol can play bioactive roles, acting as an antioxidant, anti-inflammatory, and anti-cancer compound (Dupont et al., 2021; Rangsinth et al., 2023).

Chassis Selection

It was important to select a chassis that naturally produced the target compound in well-documented pathways to mitigate issues in design and development.

Algae strains

Microscopy images (magnification 1:1000) of (A) Chlamydomonas reinhardtii (B) Chlorella sorokinana (C) Phaeodactylum tricornutum (Hickman, 2016).

Chlamydomonas reinhardtii is a unicellular green algae (~5–10 µm) known widely as the model organism for green microalgae (Neupert et al., 2009). The species has a well-characterized genome and, in bioproduction, C. reinhardtii has been increasingly used to express recombinant proteins in the nucleus via methods like glass bead and gene gun transformation (D.-H. Zhang et al., 2017). C. reinhardtii serves as a versatile platform for recombinant protein production through its chloroplast, nuclear, and mitochondrial genomes, which enable post-translational modifications typical of eukaryotes, not found in prokaryotic hosts. It is designated as Generally Recognized As Safe (GRAS) by the FDA, further supporting its use in research and biotechnology (Niraula et al., 2025b). Additionally, recent research suggests microalgae are a promising alternative sterol source to plants due to their well-documented sterol content and wide range of sterols that can be produced (Randhir et al., 2020). C. reinhardtii was ultimately chosen to conduct our proof of concept as it has a well-characterized ergosterol biosynthesis pathway (Miller et al., 2012).

Chlamydomonas reinhardtii.

Figure 3: Chlamydomonas reinhardtii. (A) Light microscope image (B) Illustration of cell structure and organelles (Dupuis & Merchant, 2023).

Optimizing for ergosterol production in C. reinhardtii

C. reinhardtii synthesizes terpenoids, the class of chemicals are sterols categorized under, through the methylerythritol phosphate (MEP) pathway. The MEP pathway is a chloroplast-localized pathway in most photosynthetic organisms that uses pyruvate and glyceraldehyde 3-phosphate (G3P) as precursors. The MEP pathway produces key intermediates like isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). These serve as building blocks for many terpenoids, including sterols, carotenoids, and quinones (Chatzivasileiou et al., 2019; Yahya et al., 2023). In contrast to fungi, which use the mevalonate (MVA) pathway to produce IPP and DMAPP precursors and converts squalene to lanosterol, C. reinhardtii produces squalene precursors exclusively via the MEP pathway and similarly to plants, converts squalene to cycloartenol instead (Brumfield et al. 2010a). This also differs from higher plants and some bacteria, which possess both the MEP and MVA pathways (Kuzuyama & Seto, 2012).

MEP and MVA

Figure 4: Metabolic pathway for ergosterol synthesis in C. reinhardtii. Enzymes of interest are detonated in pink and transcription factors are teal (Anwar et al., 2024; Brumfield et al., 2017; Voshall et al., 2021b; Wichmann et al., 2022). Created in BioRender.


After evaluating potential targets, a few key strategies were chosen to optimize ergosterol production in C. reinhardtii:

1. Introduction to Alternative SQS

Squalene synthase (SQS) catalyzes the committed step in sterol synthesis, in which two molecules of farnesyl pyrophosphate (FPP) condense to form squalene (Jaramillo-Madrid et al., 2020). This is a key step directing flux of the isoprenoid metabolic pathway towards sterol products, as FPP can also be processed into other products (Wichmann et al., 2022). Evidence suggests that overexpression of native squalene synthase in C. reinhardtii does not result in an increase in the amount of squalene produced (Kajikawa et al., 2015). Native SQS may be subject to tight regulation within C. reinhardtii, resulting in limited flux through the pathway despite increased transcript levels. An alternative approach is heterologous expression of highly active SQSs from other organisms (Cagney & O’Neill, 2024). Additionally, alternative enzymes with low genetic similarity to the C. reinhardtii genes were selected to avoid the chance of regulation by endogenous systems.

2. Introduction to Alternative SQE

Squalene epoxidase (also called squalene monooxygenase) catalyzes the conversion of squalene to squalene epoxide (Kajikawa et al., 2015). In eukaryotes, this is generally the rate-limiting step of sterol synthesis, and so was chosen as a target for overexpression to increase flux through the sterol pathway (Jaramillo-Madrid et al., 2020). However, overexpression of native SQE may also face similar issues with tight regulation by endogenous systems (Jaramillo-Madrid et al., 2020). Heterologous expression of SQE genes from other organisms has shown to successfully increase sterol intermediate production in Phaedactylum tricornutum and sterols in Nicotiana tabacum (Jaramillo-Madrid et al., 2020; Kaushal et al., 2023).

Alternative Enzymes Selected for Introduction

EnzymeSource OrganismAmino Acid % SimilarityRationale
SQSSaccharomyces cerevisiae34%S. cerevisiae produces more sterols than C. reinhardtii, indicating that it may have higher SQS activity (Commault et al., 2021; He et al., 2000).
SQSThermosynechococcus elongatus BP-135%Possesses a SQS with a kcat/KmFPP of 1.8 µM-1s-1 (compared to 0.21 µM-1s-1 for S. cerevisiae SQS or 0.51 µM-1s-1 for human SQS) (Lee & Poulter, 2008). This kinetic parameter is indicative of the efficiency of an enzyme.
SQES. cerevisiae34%S. cerevisiae produces more sterols than C. reinhardtii, indicating that it may have higher SQE activity (Commault et al., 2021; He et al., 2000).
SQEBotrycoccus braunii51%B. braunii is a green algae well-known for producing large amounts of triterpenes, and so may have a more active squalene epoxidase. Complementation of an S. cerevisiae erg1 (SQE) mutant with B. braunii SQE restored ergosterol auxotrophy (Uchida et al., 2015). SQE from another green algae may also be better adapted to the cellular environment of C. reinhardtii.

3. Introduction to MVA Pathway

To increase upstream flux towards the production of precursors of sterols, we also aimed to introduce a synthetic MVA pathway, which produces the IPP and DMAPP like the MEP pathway, but is not naturally present in green algae. The MVA pathway also draws from acetyl-CoA, a different initial precursor as the MEP pathway, preventing pyruvate and G3P reserves from being depleted (J. Wang et al., 2025). This will be done using an upstream and downstream module as summarized in Figure 5.

MVA

Figure 5: Proposed synthetic MVA modules for heterologous expression in C. reinhardtii. Gene names for enzymes that will be introduced are italicized. Upstream module (pink) genes are sourced from Enterococcus faecalis. Remaining genes are part of the downstream module. mvK is sourced from Methanosarcina mazei (blue), pmk and mvaD from S. cerevisiae (purple) and Idi from Escherichia coli (green) (Gomes et al., 2022; J. Wang et al., 2025). Created in BioRender.

Heterologous Enzyme Localization

To ensure SQS from the alternative enzymes function in C. reinhardtii, the protein needs to localize to its correct site of action, which may be different in other organisms. SQS operates in the endoplasmic reticulum (ER) so the addition of an N-terminal signal sequence and C-terminal anchor was required to guide the enzymes (Nakayama et al., 2007). Binding immunoglobulin Protein (BiP) was chosen as the N-terminal signal sequence as it is an ER-resident chaperone that facilitates nascent protein translocation and folding within the ER lumen (Srivastava et al., 2013). Its signal sequence has been successfully employed in C. reinhardtii to drive heterologous proteins into the secretory pathway (Molino et al., 2018). A KDEL retention motif may be considered further as well for future localization as it is a sequence that redirects proteins back to the ER when misguided (Chung et al., 2003).

For anchoring the expressed protein within the ER membrane, C-terminal transmembrane domain (TMD) of Protein Tyrosine Phosphatase 1B (PTP1B) was chosen. PTP1B’s hydrophobic C-terminal sequence functions as a strong ER anchor, maintaining the protein’s catalytic domain on the cytosolic face of the ER which is necessary for SQS and SQE in C. reinhardtii (Anderie et al., 2007).

Considerations for Future Work

Though not in the scope of the wet lab work for this project, future work should consider that the overaccumulation of ergosterol could cause potential toxic effects or membrane destabilization (Hu et al., 2017b). These effects may be mediated by introducing efflux pumps to facilitate secretion of ergosterol outside of the cell, using an inducible promoter to prevent overexpression of ergosterol in early growth stages, or increasing expression of lipid droplet proteins to encourage storage of more sterols (Hu et al., 2017b; Qin et al., 2024; Sun et al., 2021).

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