Mitochondria and chloroplasts function in microalgae energy production

Mitochondrial function, microalgae productivity and energy production

Mitochondria are double-membrane-bound organelles present in eukaryotic organisms. Depending on their physiological requirements, some cells may have about 10 to 1,000 mitochondria whit a proteome composed of more than 1,200 proteins. This organelle plays a critical role in different cellular processes such as energetic metabolism, carbon and nitrogen metabolism, thermoregulation, calcium and iron homeostasis, regulation of apoptosis, and in the synthesis of Fe-S and heme groups (Kotiadis, Duchen & Osellame, 2014; Ryan & Hoogenraad, 2007).

Mitochondrial biogenesis requires the correct function of the electron transport chain (mETC), which is composed by four multiprotein complexes (complexes I–IV) and an ATP synthase (Complex V) (Moller, Rasmusson & Van Aken, 2021), many of them formed by ferrosulfoproteins and hemoproteins (Gray, 1999; Gross & Bhattacharya, 2009; Poyton & McEwen, 1996). Fe-S proteins and hemoproteins participate in a wide variety of cellular processes such as many enzymatic reactions, respiration, cofactor biosynthesis, ribosome biogenesis, regulation of gene expression, and DNA and RNA metabolism in almost all organisms (Busi & Gomez-Casati, 2012; Gomez-Casati et al., 2021; Lill & Muhlenhoff, 2008).

Among other functions, microalgae are biocatalysts for the production of different compounds such as lipids, starch, cellulose, proteins and hydrogen. The ability of these organisms to convert light energy in many compounds of economic importance places them within the organisms with the greatest potential to produce clean energy (Elman et al., 2022; Nath et al., 2015). Thus, many efforts have been made to understand in more detail some processes and metabolic pathways that allow these photosynthetic organisms to supply different energy demands (Nath et al., 2015).

Particularly, bio-hydrogen production has attracted attention as a secondary energy carrier, because this compound is the lightest carbon-neutral fuel, has a high energy capacity per mass unit, and it is easy to store. In addition, it is a renewable energy source, and eco-friendly. Although various technologies are currently being developed for H₂ production, at present none of them have been used as a replacement for traditional fuels (Nath et al., 2015). In this sense, the identification of more robust microalgae or the development of genetically modified strains for various biotechnological applications would be of great scientific interest.

There are other examples of genetic manipulation of microalgae in order to modify metabolic pathways and/or mitochondrial functions to improve their productivity. One of the approaches used was the alteration of lipid metabolism using a multiplexed CRISPR/Cas9 to modify enzymes belonging to the long chain acyl-CoA synthetase (LACS) family (Hao et al., 2022). Although the general role of LACS in other organisms is well known, little is known about how these enzymes regulate lipid metabolism in algae. The study of Phaeodactylum tricornutum knockout/knockdown strains of LACS, particularly the mutants deficient in LACS3, which has a mitochondrial localization, showed a deficiency in the accumulation of triacylglycerol (TAG), as well as an alteration in the fatty acid profile (Hao et al., 2022). This study demonstrated the importance of the proper function of mitochondria in the production of lipids in oleaginous microalgae.

It has been recently described in other organisms that mitochondrial stress would also lead to an increase in lipid accumulation (Jennings et al., 2021; Lee et al., 2013; Turchi et al., 2020). In addition, it was also reported that photoautotrophically or photoheterotrophically cultured stressed microalgae cells can accumulate large quantities of TAG in lipid bodies, reaching about 46–65% of dry weight (Banerjee, Dubey & Shukla, 2016; Du & Benning, 2016; Goold et al., 2015, 2016; Kwak et al., 2016; Li et al., 2010; Vitova et al., 2015). Furthermore, it was shown that the combination of two stress conditions also increased lipid production; however, the use of more than two different conditions (i.e., nutrient limitation, temperature variation, pH, etc.) resulted in a lower growth rate of the algae cells. Based on these data, we propose that the induction of mitochondrial stress, caused, for example, by the deficiency of some enzyme of a key metabolic pathway in the organelle, in conjunction with the application of another type of stress (i.e., nitrogen or phosphorus limitation) could substantially improve the accumulation of lipids, with an increase of the biomass and the productivity of microalgae.

Recently, a novel mechanism was also described in C. reinhardtii, which showed that it has the ability to modify the location of its mitochondria inside the cells. The positioning of cell organelles was associated with various metabolic and signaling functions in unicellular organisms. Under normal conditions, it was observed that mitochondria are randomly distributed in this microalga, and relocate to the cell periphery under low inorganic carbon levels (Harmon et al., 2022). This mechanism is not known in detail; however, it is believed to be related to a carbon concentration mechanism. It is of particular importance to identify some mutant strains where this organelle relocation does not occur in order to understand the role of mitochondria in this process and its relationship with energy production (Harmon et al., 2022). Thus, based on these data, it was proposed that mitochondria would have a fundamental but also a little-known role in the accumulation of lipids in microalgae and also in the production of H₂, which would have important biotechnological implications.

Crosstalk between mitochondria and chloroplasts in microalgae productivity

Various works have shown that the mitochondria have a key role in the provision of cellular energy, as well as in the participation in retrograde regulation mechanisms involved in the biogenesis of other organelles (i.e., chloroplasts) in plants and algae (Busi et al., 2011; Liu & Butow, 2006; Liu et al., 2018). Although mitochondria and chloroplasts were traditionally considered as autonomous compartments, there is much evidence that the crosstalk between these organelles directly impacts on cell physiology (Raghavendra & Padmasree, 2003). For example, mitochondrial metabolism, particularly the bioenergetic reactions of oxidative electron transport and phosphorylation are still active in the light and are essential for maintain photosynthetic carbon assimilation (Busi et al., 2011; Raghavendra & Padmasree, 2003).

To achieve this goal, the correct function of the mETC and the photosynthetic machinery are essential. Numerous studies reported that mETC plays a crucial role in the retrograde regulation of the expression of different genes related to the photosynthetic process in different organisms (Liu et al., 2018; Matsuo & Obokata, 2006). Particularly, in C. reinhardtii, it was showed that the induction of photosynthetic genes responds to different signals from the mitochondrial and chloroplast electron transport chains (Matsuo & Obokata, 2006).

According to these results, Li et al. (2008) demonstrated that Chlorella zonfingiensis strains are able to grow and accumulate astaxanthin under dark conditions using glucose as the only source of carbon and energy. It is important to note that astaxanthin, a carotenoid from the terpene family, is a powerful antioxidant and colorant that has numerous applications in the nutraceutical, cosmetic and food industries (Guerin, Huntley & Olaizola, 2003). In this study, an increase in the levels of several chloroplastic enzymes such as β-carotenoid ketolase (BKT) and beta-carotenoid hydroxylase (CHYb) was observed in the presence of glucose analogs and glucosamine (hexokinase inhibitor) that led to an increase in astaxanthin amounts (Li et al., 2008). In contrast, the impairment of the mETC using some inhibitors such as antimycin A or rotenone partially decreased the transcription of those genes, while in the presence of salicyl hydroxamic acid (SHAM), an inhibitor of the alternative mitochondrial pathway, the transcription of such genes was completely inhibited, leading to lower astaxanthin levels. These data are in agreement with the involvement of mitochondria in the induction of the metabolic pathway that leads to the production of astaxanthin (Li et al., 2008). On the other hand, when a similar strategy was used involving mitochondrial respiration inhibitors, and a subsequent proteomic analysis using two-dimensional electrophoresis coupled to matrix assisted laser desorption-time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), it was shown that in Chlorella pyrenoidosa cells, the production of some compounds such as lutein and chlorophyll also depends on the correct function of the mETC (Liu et al., 2018).

Recently, Uwizeye et al. (2021) performed a 3D microscopy morphometric analysis on seven different phytoplankton species. The results allowed to postulate that the phytoplankton subcellular topology is modulated by energy management constraints. In addition, lighting changes (i.e., from low light to high light, a condition that was associated with stress production in microalgae) induced photosynthesis and respiration, increasing the occupation of the cell volume by mitochondria and also increasing the contact points between mitochondria and chloroplasts. In Nannochloropsis sp., for example, these structural changes were also associated with an increased respiratory and photosynthetic responses (Uwizeye et al., 2021). In addition, a recent work reported that the interaction and the energy exchange between chloroplasts and mitochondria increases the growth of microalgae and also the production of hydrogen, and that the molecular mechanism behind this particular phenotype would be related to the biogenesis of mitochondria (Elman et al., 2022).

These data support the hypothesis that the correct function of the mETC, the subcellular architecture, the contact points between mitochondria and chloroplasts, as well as the signals related to mitochondrial retrograde regulation between mitochondria and chloroplasts towards the nucleus are involved not only in the biosynthesis and accumulation of photosynthetic pigments in algae, but also in the biosynthesis of vitamins, bioproducts, starch, different lipids and in the production of hydrogen and biogas. In addition, the coordination of the functioning of the nucleus and the organelles would influence the adaptation to grow under different conditions, modulating the physiological and metabolic responses to maximize survival as well as biomass production (Fig. 1). Further studies are needed to elucidate the signals that regulate the crosstalk between mitochondria and chloroplasts and how these signals ultimately affect biomass generation and microalgae productivity, as well as the key metabolic pathways in the production of metabolites of interest.

Engineering microalgae nuclear and organelle genomes

As we mentioned above, in a few years there will be a major challenge to produce high amounts of sustainable, cheap and environmentally friendly energy. A promising solution to overcome many issues in different areas such as agriculture, industry, medical sciences and energy production would be the development of design organisms (Cochrane et al., 2020). Synthetic biology has expanded the ability to enhance the genotype of an organism by the integration of optimized genes, leading to the selective improvement of different metabolic pathways and cellular phenotypes (Alper et al., 2010). Although there are various reports where the engineering of different microalgae was carried out by modifying the nuclear genome, little is known about the genetic modification of organelle genomes (Kumar et al., 2020).

In this sense, engineering genome organelles by introducing optimized genes would have several advantages over a nuclear transformation. First, genes are organized in polycistronic units, there is an absence of gene silencing and also reduced positional effects in organellar DNA (Alper et al., 2010; Cochrane et al., 2020). On the other hand, organelles allow metabolic pathways to be compartmentalized, which increases their efficiency and metabolic flow, and minimizes the competition of certain metabolites with other metabolic pathways located in other cellular compartments (Cochrane et al., 2020). The main goal of this strategy would be to modify the genomes of mitochondria and chloroplasts to improve them at a biochemical level, which could lead to greater productivity. Although there are numerous works where synthetic biology was applied in the design of other microorganisms of biotechnological interest, few work has been done related to the genetic modification of microalgae genomes. One of the main reasons would be that there is still little information of complete microalgae sequenced genomes compared to other microorganisms such as bacteria (Grama, Liu & Li, 2022; Kumar et al., 2020). However, in the last few years, several techniques have been developed that allowed the modification and editing of these genomes, however, the genetic engineering of algae was proved to be effective improving performance under laboratory conditions, but with limited success in industry.

Recently, a rapid and efficient method for engineering microalgae mitochondrial genomes was described. Cochrane et al. (2020) reported the use of the model organism P. tricornutum, which is a widely characterized diatom due to its ability to store high amounts of lipids for biofuel production. The described strategy includes a methodology to clone the complete chromosomes of microalgae inside S. cerevisiae and E. coli.

On the other hand, there are several reports where the nuclear transformation of different species of algae was carried out, such as Chlamydomonas, Phaeodactylum, Dunaliella, Nannochloropsis and Haematococcus (Kumar et al., 2020). Some of these reports allowed to improve the photosynthetic efficiency and biomass production, such as the overexpression of the RuBisCo activase (Wei et al., 2017), the fru-1,6-bisphosphate aldolase (Yang et al., 2017) or the engineering of the PSII D1 subunit in Chlamydomonas (Vinyard et al., 2014). Other works focused on the modification of several enzymes and metabolic pathways of lipid metabolism in algae, such as the expression of acetyl-CoA carboxylase, which encodes the enzyme that catalyzes the rate-limiting step in lipid synthesis, together with the malic enzyme, which catalyzes the production of pyruvate from malate. This led to an increase in the levels of accumulated lipids in D. salina (Talebi et al., 2014). A similar result was obtained after the overexpression of chloroplast diacylglycerol acyltransferase 3 in P. tricornutum, which also resulted in increased TAG levels in this algae (Niu et al., 2013) and also in C. reinhardtii (Carro et al., 2022). In addition, the overexpression of RuBisCo activase also increased TAG content in N. oceanica (Wei et al., 2017). Moreover, the possibility to generate knockout organisms by the CRISPR-Cas9 technique allowed the successful modification of different metabolic pathways. This new gene editing technology also drastically increased the possibility for design-driven synthetic biology (Kelterborn et al., 2022). These studies include the modification of phosphoenolpyruvate carboxylase 1 (PEPC1) in C. reinhardtii (Kao & Ng, 2017). PEPC is a cytosolic enzyme that catalyzes the formation of oxaloacetate which is subsequently converted to malate by malate dehydrogenase (MDH). This compound finally enters into the tricarboxylic acid (TCA) cycle to provide substrates for energy production in mitochondria. Thus, the deficiency of a cytosolic enzyme (PEPC1) causes a decrease in the efficiency of the mitochondrial TCA pathway with a redirection of the carbon flow towards lipid accumulation in C. reinhardtii (Deng et al., 2014; Kao & Ng, 2017). Another example of the modification of two enzymes encoded in the nucleus but with chloroplastic localization was the editing of zeaxanthin epoxidase (ZEP), which produce violaxanthin from zeaxanthin, and ADP-glucose pyrophosphorylase (AGP), which is the key enzyme in starch synthesis (Busi et al., 2014; Song et al., 2022). These gene modifications (PEPC1 and ZEP/AGP) produced an increase in lipid content in the C. reinhardtii mutants of about 30% and 81%, respectively.

Recently, it was described another interesting technique based on Agrobacterium tumefaciens transformation to improve lipid and terpenoid synthesis in Schizochytrium sp, a microorganism similar to marine microalgae which has become one of the most effective organisms used for the production of Omega-3 polyunsaturated fatty acids (Huang et al., 2021). The results showed that the overexpression of acetyl-CoA acetyltransferase produced an increase in the accumulation of terpenoid compounds, increasing the production of squalene, β-carotene and astaxanthin as well as the production of saturated fatty acids and polyunsaturated fatty acids. In addition, the deletion of three acyl-CoA oxidase genes also produced an increase in lipid content, demonstrating the usefulness of this strategy mediated by A. tumefaciens for the study of functional genes, as well as for the development of microalgae with higher productivity (Huang et al., 2021) (Table 1).

Another interesting strategy would be to modify the levels of enzymes involved in glycolysis. It was reported that plant glycolytic enzymes are associated with the mitochondrial membrane to quickly direct the final products (pyruvate and/or malate) into mitochondria to continue their oxidation in the TCA cycle. For example, GAPC deficiency in plants showed a decrease in pyruvate levels and a slight increase in malate levels; however, the levels of TCA cycle intermediates decreased, as well as the expression of PEPC, which would lead to an increase in lipid accumulation (Rius et al., 2008). In the same direction, it was observed that the expression of a regulator of GAPC, SINAL7, which belongs from the SINA family proteins of E3 ubiquitin ligases, produced an increase in plant biomass, possibly associated with the decrease in GAPC activity or in GAPC association to the mitochondrial external membrane (Peralta et al., 2016, 2018, 2013). It remains to be evaluated whether the modification of glycolytic enzyme levels or the modification of GAPC regulation by this strategy would be useful to increase lipid accumulation and/or biomass in microalgae.