Polyunsaturated Fatty Acid Metabolism, Immunity And Genetic Engineering

Introduction
Polyunsaturated fatty acids (PUFAs) are 18-22 carbon, straight chained fatty acids that have two or more double bonds and are critical functional components of the cell membrane, lipid storage and have even functional roles in signal transduction pathways (Liu, Green, John Mann, Rapoport, & Sublette, 2015; Weylandt et al., 2015). Generally, they are categorized into three main groups, according to the number of carbon atoms in between the terminal methyl (which is also called omega group) end and the first double bond. Main n-3 in food sources are α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) while main n-6 in food sources are linolenic acid (LA) and arachidonic acid (AA) (Liu et al., 2015; Weylandt et al., 2015). By numerous studies, polyunsaturated acids have been shown to habe beneficial functional potentials againts a number of disorders and diseases such as obesity, auto-inflammation, cardiovascular disorders, hypertriglyceridemia and certain types of cancers (cororectal, mammary…) (Abedi & Sahari, 2014). Even though they take part in many extensive biological and physiological systems in human body, PUFAs are generally known for promoting the brain development. Lipids in general are found in high concentrations in the central nervous system and almost half of the dry weight of the human brain is composed of lipids. Furthermore, polyunsaturated fatty acids make up 35% of the lipids and since they cannot be synthesized from 2 carbon molecules in mammals and also in vertebrates, their uptake from other sources are essential for the development of the human brain. Arachidonic acid and docosahexaenoic acid in particular make up 50 and 40% of the human brain PUFAs respectively. DHA is critical for developing neurons and also neurotransmission (Liu et al., 2015). Additionally, arachidonic acid aids in the growth, repair, maintenance and protection of neurons. Moreover, since PUFAs have important immune regulatory roles, their presence is also required for the inhibition of possible neuroinflammatory conditions (Abedi & Sahari, 2014).

Sources of Polyunsaturated Fatty Acids
Since n-3 and n-6 PUFAs are not synthesized by vertebrates de novo, they are acquired by dietary means. LA, as one of the n-6 PUFAs, is abundant in nature and found in seeds of many plants (soybean, canola, sunflower…) while arachidonic acid is mostly found in meat products and from animals that are fed corn based diets. ALA as the precursor of n-3 PUFAs are found in green leafy vegetables while seafood are the main source for EPA and DHA, even though cattles fed diets in high n-3 PUFA can also provide source for EPA and DHA. Nevertheless seafood as the main source for many PUFAs also present challenges. Carcinogen and non-carcinogen contaminants (methly mercury, heavy metals such as Pb, Cr, Hg…) can result in high risks, especially for children(Komprda, Zelenka, Fajmonova, Fialova, & Kladroba, 2005).

Nutritional Functions of PUFAs
PUFAs have many functional roles in most of the biological systems in the human body. It includes the regulation of the immune system, the cardiovascular system, blood clotting, neurotransmitters, cholesterol and lipid metabolism and structure of membrane lipids in retina and brain. PUFAs have important inhibitory effects on the synthesis of low density lipoprotein (LDL) and they take part in the elimination of the very same molecule, thus have a positive effect on prevention of cardiovascular disorders. Moreover, PUFAs also have functional roles in shaping the endocrine and cardiovascular systems, PUFAs reduce the blood pressure(Abedi & Sahari, 2014).

Figure 1: Molecular Structure and Categorization of Polyunsaturated Fatty Acids (Abedi & Sahari, 2014)

It has also been reported that PUFAs have beneficial effects on various diseases such as skin diseases, asthma, lupus erythematosus and multiple sclerosis. They are also important components of the membrane phospholipids and play important roles in the structure and function of the cell membranes, they can regulate membrane bound enzymes. Furthermore, PUFAs generally have anti-inflammatory effects on the immune system and inflammation, which makes them important regulators of immunity and key inhibitors of auto-immune and auto-inflammatory disorders (Abedi & Sahari, 2014).

Figure 2: Arachidonic Acid (Tallima & El Ridi, 2018)

Figure 3: The Extensive Functional Roles of PUFAs (Stewart, Feinle-Bisset, & Keast, 2011)

Polyunsaturated Fatty Acid Metabolism in Microalgae
As in other major ecosystems, primary producers (such as unicellular phytoplanktons) as the main sources of nutrients, hold a key importance in regulating the transfer of nutrients and essential molecules throughout the food web (Jonasdottir, 2019). Carbon fixed in the form of glucose via photosynthesis and the metabolism of phosphorus and nitrogen, therefore carbohydrates, lipids and proteins are not only the building blocks of the microalgae, but also they provide the essential molecules to the upper trophic levels in the marine ecosystems. In the phytoplankton cells, carbon is found in all macromolecules such as carbohydrates, lipids and proteins. Additionally, nitrogen is mostly involved in the structure of the proteins, and, since it is essential for phytoplankton growth, the nitrogen content has been used to indicate the quality of the cell. Apart from its structural function in proteins, nitrogen is also an essential part of vitamins, enzymes and some lipid complexes (Jonasdottir, 2019).

Figure 4: The Building Blocks of Phytoplanktons (Jonasdottir, 2019)

Protein is the main organic group, making up the 40–60% of the organic mass, with carbohydrates contributing approximately 17–40% and lipids about 16–26%. Nevertheless, as shown in figure 5, different species of phytoplanktons in different growth conditions vary in this proportion. Lipids have a wide range of functions in these organisms, ranging from energy storage, membrane structure, digestion to photosensitivity. Triacylglycerol, galactolipids and phospholipids are the main lipid structures and fatty acids are the primary building units of these lipids (Jonasdottir, 2019).

Figure 5: Content of Organic Matter in Phytoplankton Species (Jonasdottir, 2019)

Fatty Acid Synthesis in Phytoplanktons
Fatty acid synthesis in microalgae takes place in aerobic pathways in the chloroplast and endoplasmic reticulum. The glucose fixed during the photosynthesis is converted to pyruvate, then to acetyl CoA which can follow through the Krebs cycle or form Malonly CoA. From there on, for fatty acid synthesis, the phytoplankton cell undergoes 4 major steps. In step 1, fatty acid synthesis starts in the chloroplast where Malonyl-CoA and Acetyl-CoA contribute 2 carbons each to form the first fatty acid chain. In step 2, fatty acid elongation takes place, where the 4:0-ACP is elongated with the help of fatty acid synthesase 2 carbons. The cycle ends up with 14–18 carbon length ACP-chains. In step 3, 16 or 18:0-ACP with the help of Δ9 desaturase puts the first double bond on the 9th carbon from the ACP end of the chain. In step 4, The desaturation and elongation process takes place in the endoplasmic reticulum (Jonasdottir, 2019).

Figure 6: Fatty Acid Synthesis Pathways in Phytoplanktons (Jonasdottir, 2019)

Polyunsaturated Fatty Acids and Immunity
The immune system is a complex and dynamic mechanism that protects the body against pathogens and maintain homeostatic balance in the presence of an internal disruption. Even though its fundamental goal is generally thought to be defence against infection and tumor formation, the immune system is intertwined with large-scale systems such as the nervous system and various metabolic systems, and constitutes sophisticated multi-dimensional mechanisms ranging from antibody production to tissue remodeling (Ciraci, Janczy, Sutterwala, & Cassel, 2012; Shaw, McDermott, & Kanneganti, 2011; Wilmanski, Petnicki-Ocwieja, & Kobayashi, 2008).

The immune system is divided into two distinct categories which are also interrelated: innate immunity and adaptive immunity. Compared to adaptive immunity, innate immunity reacts much more rapidly and nonspecifically upon encountering a pathogen or a danger signal whereas adaptive immunity is slower since it responds to invading pathogens in a specific manner and builds up a memory mechanism upon recognition(Ciraci et al., 2012; Shaw et al., 2011).

Macrophages are important components of the innate immunity and have functional roles such as pathogen recognition, pathogen clearance by phagocytosis, cytokine secretion and antigen presentation. M1 subtype of macrophages are generally pro-inflammatory whereas M2 macrophages can present antagonistic functionalities together with their roles against extracellular parasites. It has been reported that inflammation related genes upregulated by lipopolysaccharide (LPS) stimulation of Toll-like Receptors (pro-IL-1B, IL-6, TNF-α) were attenuated by DHA treatment. Moreover, oxidized LDL treated macrophages secreted higher levels of IL-6 and TNFα and omega 3 fatty acid administration diminished the secretion. This anti-inflammatory effect of PUFAs were confirmed by other studies as well. Jin et al. demonstrated that omega 3 fatty acid administration only increased IL-10 secretion in macrophages, while another study demonstrated the suppression of NLRP3 by EPA and DHA treatment. Additionally, Shoeniger et al. showed decreased TLR4 mRNA expression after DHA treatment. PUFA treatment can also lead to macrophage polarization to antagonistic subtypes. ALA and DHA have been shown to polarize M2 macrophages by increasing the expression of Arg1, IL-10 and TGFB genes (IL-10 and TGFB promotes Treg activation while Arginase 1 activation leads to L-arginine depletion which causes inhibiton of nitric oxide synthesis) (Dimitriades, Rodriguez, Zabaleta, & Ochoa, 2014). Even though PUFAs mainly present an anti-inlammatory impact on macrophage phenotypes, Omega 3 fatty acids have also been shown to increase the phagocytic properties of macrophages, which can also be seen as an anti-inflammatory response since pathogen clearance can attenuate pro-inflammatory responses (Gutierrez, Svahn, & Johansson, 2019).

PUFAs also have anti-inflammatory effects on neutrophils which are the most abundant white blood cells in blood. EPA induces prostaglandin D3 production, which inhibits neutrophil adhesion and migration. Furthermore, DHA has been shown to increase the phagocytic capacities of neutrophils. Additionally, The effect of omega 3 fatty acids on reactive oxygen species production by neutrophils is species dependent, DHA increased ROS production by rat neutrophils whereas decreased ROS production in goat leukocytes (Gutierrez et al., 2019).

Together with their effects on innate immune cells, PUFAs present anti-inflammatory effects on B and T cells. Omega 3 fatty acids blunt the secretion of IFN-γ, IL-17, and IL-2 by human T helper cells and activated CD8 T cells. Moreover, Omega 3 fatty acids inhibit Th17 activation whereas EPA and DHA have been shown to promote regulatory T-cell proliferation. Interestingly, EPA and DHA increase IgM production by B cells by increasing the number of antibody-producing cells in mouse and human (Gutierrez et al., 2019).

Environmental Factors That Impact PUFA Synthesis in Diatoms
Marine diatoms recently have been shown to be sources secondary metabolites with biological activity. They have a high PUFA content (in some species can reach up to 5%–6% of cell dry weight). Since they are single celled, fast growing organisms, they present a new source for the high yield of PUFAs since extraction and purification of PUFAs are relatively easy in these microorganisms. Environmental factors play a key determining role in PUFA synthesis and yield. Light is an important mediator of biomass and fatty acid production (P. tricornutum cultivated in tubular photobioreactors had decreasing biomass productivity under lowered light, but the EPA content showed increased production.) Moreover, temperature is proven to be another detemining factor, it has been reported that TFA, SAFA, and MUFA concentrations increased at 25 °C compared to 10 °C under low P supply in Cyclotella meneghiniana (Li, Lu, Zheng, Yang, & Liu, 2014). Chemical composition of the environment is another fundamental factor, Many microalgae species present a higher lipid content under nitrogen starvation conditions, including diatom P. tricornutum, green algae Chlorella spp., Botryococcus braunii, Chlamydomonas reinhardtii, and Dunaliella salina. Furthermore, urea and NaNO3 are optimum nitrogen sources for growth of C. fusiformis, while NH4Cl is the best for C. Closterium (Li et al., 2014).

Genetic Engineering Applications Targeting Polyunsaturated Fatty Acid Synthesis and Yield
Genetic engineering has been a reliable and productive method for impacting the pathways related to PUFA synthesis. In the study conducted by Ahmad el al., rapeseed diacylglycerol acyltransferase from rapeseed was transfected to C. Reinhardtii by electroporation since attempts of overexpressing the isoforms of DGATs of same species have been unsuccessful (Ahmad, Sharma, Daniell, & Kumar, 2015). For instance, in C. reinhardtii, there are two types of DGAT homologues which have been shown to play no major roles in lipid accumulation (Ahmad et al., 2015).

Figure 7: pAlgaeDGAT-eGFP Vector Transformed to the Chlamydomonas reinhardtii Cells

Nonetheless, the vector containing the DGAT isoform from rapeseed transformed to the Chlamydomonas reinhardtii doubled the synthesis of neutral lipids. The saturated fatty acid synthesis decreased by 7% while PUFA (ALA) synthesis and yield increased by 12% (Ahmad et al., 2015).

In the study conducted by Poliner et al., a different method of approach was utilized. Fatty acid desaturases (FAD) taking place in the synthesis of EPA in Nannochloropsis oceanica CCMP1779 was determined by first observing the FADs coexpressed under light/dark conditions (Poliner et al., 2018). Protein sequences were generated from isolated cDNA sequences and protein structure of the FADs were determined by using computational analysis (Poliner et al., 2018). Since to generate EPA from the endogenous 18:1D9, it is necessary to introduce four additional desaturases and an elongase to S. Cerevisiae, the coexpressed FADs were introduced to S. Cerevisiae to understand and reveal the metabolic pathway of EPA (Poliner et al., 2018). It was observed that coexpression of the D12, D6, D5, x3 FADs and the D6 FAE in the Sc- D12 + D6 + E6 + D5 + x3 strain resulted in the formation of EPA. Furthermore, The overexpression of single D5 or D12 FADs led to an approximate 25% increase in EPA mol ratio in Nannochloropsis oceanica (Poliner et al., 2018).

Conclusion
The extensive functional roles and potentials of the polyunsaturated fatty acids prove these fatty acids to be critical in human diet as their roles in brain development, prevention of obesity, auto-inflammation and cardiovascular diseases. Since PUFAs are mostly extracted from seafood, the genetic engineering of the pathways related to PUFA synthesis in fast growing, microorganisms recently is an important factor in augmenting the yield and enrichment of these fatty acids. In future studies, genetic engineering studies will not only gather more data and prove to be useful, but they will also be essential in a world with increasing pollution and decreasing resources.

Reference:

Abedi, E., & Sahari, M. A. (2014). Long-chain polyunsaturated fatty acid sources and evaluation of their nutritional and functional properties. Food Sci Nutr, 2(5), 443-463. doi:10.1002/fsn3.121
Ahmad, I., Sharma, A. K., Daniell, H., & Kumar, S. (2015). Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2. Plant Biotechnol J, 13(4), 540-550. doi:10.1111/pbi.12278
Ciraci, C., Janczy, J. R., Sutterwala, F. S., & Cassel, S. L. (2012). Control of innate and adaptive immunity by the inflammasome. Microbes Infect, 14(14), 1263-1270. doi:10.1016/j.micinf.2012.07.007
Dimitriades, V., Rodriguez, P. C., Zabaleta, J., & Ochoa, A. C. (2014). Arginase I levels are decreased in the plasma of pediatric patients with atopic dermatitis. Ann Allergy Asthma Immunol, 113(3), 271-275. doi:10.1016/j.anai.2014.06.010
Gutierrez, S., Svahn, S. L., & Johansson, M. E. (2019). Effects of Omega-3 Fatty Acids on Immune Cells. Int J Mol Sci, 20(20). doi:10.3390/ijms20205028
Jonasdottir, S. H. (2019). Fatty Acid Profiles and Production in Marine Phytoplankton. Mar Drugs, 17(3). doi:10.3390/md17030151
Komprda, T., Zelenka, J., Fajmonova, E., Fialova, M., & Kladroba, D. (2005). Arachidonic acid and long-chain n-3 polyunsaturated fatty acid contents in meat of selected poultry and fish species in relation to dietary fat sources. J Agric Food Chem, 53(17), 6804-6812. doi:10.1021/jf0504162
Li, H. Y., Lu, Y., Zheng, J. W., Yang, W. D., & Liu, J. S. (2014). Biochemical and genetic engineering of diatoms for polyunsaturated fatty acid biosynthesis. Mar Drugs, 12(1), 153-166. doi:10.3390/md12010153
Liu, J. J., Green, P., John Mann, J., Rapoport, S. I., & Sublette, M. E. (2015). Pathways of polyunsaturated fatty acid utilization: implications for brain function in neuropsychiatric health and disease. Brain Res, 1597, 220-246. doi:10.1016/j.brainres.2014.11.059
Poliner, E., Pulman, J. A., Zienkiewicz, K., Childs, K., Benning, C., & Farre, E. M. (2018). A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production. Plant Biotechnol J, 16(1), 298-309. doi:10.1111/pbi.12772
Shaw, P. J., McDermott, M. F., & Kanneganti, T. D. (2011). Inflammasomes and autoimmunity. Trends Mol Med, 17(2), 57-64. doi:10.1016/j.molmed.2010.11.001
Stewart, J. E., Feinle-Bisset, C., & Keast, R. S. (2011). Fatty acid detection during food consumption and digestion: Associations with ingestive behavior and obesity. Prog Lipid Res, 50(3), 225-233. doi:10.1016/j.plipres.2011.02.002
Tallima, H., & El Ridi, R. (2018). Arachidonic acid: Physiological roles and potential health benefits - A review. J Adv Res, 11, 33-41. doi:10.1016/j.jare.2017.11.004
Weylandt, K. H., Serini, S., Chen, Y. Q., Su, H. M., Lim, K., Cittadini, A., & Calviello, G. (2015). Omega-3 Polyunsaturated Fatty Acids: The Way Forward in Times of Mixed Evidence. Biomed Res Int, 2015, 143109. doi:10.1155/2015/143109
Wilmanski, J. M., Petnicki-Ocwieja, T., & Kobayashi, K. S. (2008). NLR proteins: integral members of innate immunity and mediators of inflammatory diseases. J Leukoc Biol, 83(1), 13-30. doi:10.1189/jlb.0607402