Microalgal lipid biosynthesis: phylogeny of acetyl-CoA carboxylase and gene expression patterns of key enzymes

Lipids from microalgae have become a valuable commodity in the last 20 years. Two of the potential industrial applications are the production of biodiesel from short-chain saturated and monounsaturated fatty acids (FAs) and the use of very long-chain polyunsaturated fatty acids (VLC-PUFAs) to supplement human / animal diets. However, high production costs are still prohibitive and an increase in yield is desired. Understanding the cellular mechanisms by which microalgae produce these FAs and how these mechanisms can be manipulated to potentially increase yield is of immediate interest to the industry. Generally, the lipid content of algae increases under nitrogen starvation, whereas FA length and desaturation decreases. While the general biochemical processes are known, research investigating the expression of the different enzymes involved under different growth conditions and growth phases is still lacking. This kind of knowledge will be invaluable for targeted manipulations of environmental conditions with the aim to increase yields of desired types of FAs for different applications. In algae, de novo synthesis of FAs up to C18 occurs in the plastids, where the key enzyme acetyl-CoA carboxylase (ACCase) catalyses the first committing step. Further elongation and desaturation of FAs to VLC-PUFAs is associated with the endoplasmic reticulum (ER). At this step, cytosolic ACCase provides malonyl-CoA for the elongation of fatty acids in the ER. In addition to the two locations, there are two types of ACCase: the bacterial, multi-subunit heteromeric ACCase and the eukaryotic multi-domain homomeric ACCase. While cytosolic ACCase in eukaryotes is always homomeric, plastidial ACCase, if present, can be either heteromeric or homomeric. The multidomain nature of homomeric ACCase makes it easier to study and manipulate and shows great potential for improving FA synthesis and elongation. It is therefore important to identify the type of ACCase present in the plastids of algae, to enable gene expression studies to be conducted. The two main aims of this thesis were: 1) to increase the general knowledge of ACCase in algae and provide a phylogenetic analysis of the relationships between algae in terms of ACCase; 2) to correlate the gene expression of plastidial and cytosolic ACCase and selected desaturases under nitrogen-deplete and nitrogen-replete growth conditions during different growth phases with FA content and composition data. Despite the recognition of the existence of heteromeric and homomeric ACCase forms, generalised published statements still list the heteromeric form as the one present in algal plastids. In the present research, heteromeric and homomeric ACCase amino acid sequences obtained from the National Center for Biotechnology Information (NCBI) and the Joint Genomic Institute (JGI) were synthesized with taxonomic data. This elucidated that the presence of heteromeric or homomeric ACCase is dependent on the origin of the plastid. Plastids derived from a primary endosymbiotic event (Chlorophyta, with the exception of the green algal class Prasinophyceae, and Rhodophyta) contain heteromeric ACCase. In contrast, plastids derived through secondary or tertiary endosymbiosis (Chlorarachniophyta, Cryptophyta, Stramenopiles, Haptophyta, and apicoplast (rudimentary plastid) containing Apicomplexa), contain homomeric ACCase. This highlights that the majority of algae actually contain homomeric plastidial ACCase. To reconstruct a Bayesian inference tree, five novel sequences of homomeric ACCase from Isochrysis aff. galbana (TISO), Nannochloropsis oculata and Chromera velia were sequenced and used together with all other publicly available amino acid sequences of algal ACCases to date. The cytosolic and plastidial ACCase sequences of the Stramenopiles, Alveolates and Rhizaria (SAR) formed two strongly supported clades. These clades agree with the recently proposed new phylogeny from the literature, where the Rhizaria are related to the Stramenopiles and Alveolates. In addition, the plastidial ACCase clade also included the Prasinophyte sequences, while the cytosolic ACCase clade containing the SAR species excluded the Prasinophytes. This shows that, while the ancestor hosts of the SAR and Prasinophytes are not closely related, they acquired the plastidial homomeric ACCase from the same source and not through a duplication event from either host. To investigate the expression of selected genes at different growth phases, I. aff. galbana (TISO) and Chromera velia were cultivated over 12 days in nitrogen-replete and nitrogen-deplete conditions. Cell density and nutrient status were measured daily, while gene expression, dry weight and lipid content were measured during the logarithmic, late logarithmic and stationary growth phases. The cell density of C. velia of cultures grown with and without nitrogen did not differ notably during the first four days of culture. In contrast, nitrogen-replete cultures of I. aff. galbana (TISO) exhibited higher cell densities from the first day of culturing, compared to cultures grown in nitrogen-deplete conditions. During active growth, FA content is generally considered to be low, since the FA content is shared by the daughter cells at cell division. In terms of gene expression patterns of plastidial ACCase and lipid content, distinct differences were observed between I. aff. galbana (TISO) and C. velia. The expression of plastidial ACCase of C. velia was significantly upregulated during the late logarithmic and stationary phases in both types of cultures, and nitrogen-deplete cultures showed a more pronounced response to nutrient starvation (N+: 2-fold; N-: 4-fold). The upregulation of plastidial ACCase expression strongly correlated with an increase in FA content. Conversely, I. aff. galbana (TISO) showed a significant 8-fold upregulation of plastidial ACCase during the logarithmic growth phase of nutrient-replete cultures only, consistent with higher growth rates. Since the FA content of I. aff. galbana (TISO) did not vary significantly between treatments and growth phases, the upregulation of plastidial ACCase can be explained by the algae maintaining a stable FA content during active growth. Finally, the expression of cytosolic ACCase in C. velia decreased by approximately half with culture age, independent of nutrient status. This decrease correlated well with an average increase in short-chain fatty acids (≤C18) of approximately 240%, showing that cytosolic ACCase plays an important part in supplying the elongases located in the ER with malonyl-CoA for the elongation of FAs beyond C18. Isochrysis aff. galbana (TISO) has been used extensively in aquaculture because of its high content of docosahexaenoic acid (DHA, C22:6n-3), a nutritional supplement important for the health of most aquaculture animals. Therefore, the expression of several desaturases (d6FAD, d8FAD, d5FAD and d4FAD) involved in the desaturation and elongation pathway were measured. Similar to the total FA content, the average DHA content of 12.2 mg g⁻¹ ash-free dry weight did not vary significantly between treatments and growth phases. The expression of d6FAD was not significantly upregulated at any time. In contrast, d8FAD, which provides a bypass of the normal d6FAD pathway, was upregulated 7-fold during logarithmic growth of nutrient-replete cultures. Correspondingly, the expression of d5FAD and d4FAD were upregulated 8.5- fold during logarithmic growth. None of the intermediate FAs in the DHA synthesis pathway were present at high concentrations, suggesting an efficient conversion at each step of the pathway. Even though there was no increase in total DHA content analogous to the increase in gene expression, it is important to keep in mind that the cells were actively growing during this phase. The upregulation of these desaturases therefore allowed the microalgae to maintain adequate levels of the membrane FA DHA during active growth. In summary, the present thesis provides the first comprehensive review of the plastidial ACCase type in microalgal phyla and classes. This is of great importance in terms of algal phylogeny, but also for further investigations of algal lipid synthesis. Moreover, it was shown that growing algae in nitrogen-deplete conditions does not always result in an accumulation of FAs, although there is a strong correlation between the expression of plastidial ACCase and FA production. Similarly, even thought the DHA content in I. aff. galbana (TISO) did not differ between growth phases, the involved desaturases were highly upregulated during logarithmic growth to maintain membrane FA DHA levels. Additional research should focus on microalgal strains with differences in their VLC-PUFA content between growth phases, to further investigate the differences in gene expression between different algal species.