Krzysztof Zienkiewicz

Stress-induced neutral lipid biosynthesis in microalgae - Molecular, cellular and physiological insights.

Photosynthetic microalgae have promise as biofuel feedstock. Under certain conditions, they produce substantial amounts of neutral lipids, mainly in the form of triacylglycerols (TAGs), which can be converted to fuels. Much of our current knowledge on the genetic and molecular basis of algal neutral lipid metabolism derives mainly from studies of plants, i.e. seed tissues, and to a lesser extent from direct studies of algal lipid metabolism. Thus, the knowledge of TAG synthesis and the cellular trafficking of TAG precursors in algal cells is to a large extent based on genome predictions, and most aspects of TAG metabolism have yet to be experimentally verified. The biofuel prospects of microalgae have raised the interest in mechanistic studies of algal TAG biosynthesis in recent years and resulted in an increasing number of publications on lipid metabolism in microalgae. In this review we summarize the current findings on genetic, molecular and physiological studies of TAG accumulation in microalgae. Special emphasis is on the functional analysis of key genes involved in TAG synthesis, molecular mechanisms of regulation of TAG biosynthesis, as well as on possible mechanisms of lipid droplet formation in microalgal cells. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii

In plants, neutral lipids are frequently synthesized and stored in seed tissues, where the assembly of lipid droplets (LDs) coincides with the accumulation of triacylglycerols (TAGs). In addition, photosynthetic, vegetative cells can form cytosolic LDs and much less information is known about the makeup and biogenesis of these LDs. Here we focus on Chlamydomonas reinhardtii as a reference model for LDs in a photosynthetic cell, because in this unicellular green alga LD dynamics can be readily manipulated by nitrogen availability. Nitrogen deprivation leads to cellular quiescence during which cell divisions cease and TAGs accumulate. The major lipid droplet protein (MLDP) forms a proteinaceous coat surrounding mature LDs. Reducing the amount of MLDP affects LD size and number, TAG breakdown and timely progression out of cellular quiescence following nitrogen resupply. Depending on nitrogen availability, MLDP recruits different proteins to LDs, tubulins in particular. Conversely, depolymerization of microtubules drastically alters the association of MLDP with LDs. LDs also contain select chloroplast envelope membrane proteins hinting at an origin of LDs, at least in part, from chloroplast membranes. Moreover, LD surface lipids are rich in de novo synthesized fatty acids, and are mainly composed of galactolipids which are typical components of chloroplast membranes. The composition of the LD membrane is altered in the absence of MLDP. Collectively, our results suggest a mechanism for LD formation in C.xa0reinhardtii involving chloroplast envelope membranes by which specific proteins are recruited to LDs and a specialized polar lipid monolayer surrounding the LD is formed.

Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens

Endosymbiosis of bacteria by eukaryotes is a defining feature of cellular evolution. In addition to well-known bacterial origins for mitochondria and chloroplasts, multiple origins of bacterial endosymbiosis are known within the cells of diverse animals, plants and fungi. Early-diverging lineages of terrestrial fungi harbor endosymbiotic bacteria belonging to the Burkholderiaceae. We sequenced the metagenome of the soil-inhabiting fungus Mortierella elongata and assembled the complete circular chromosome of its endosymbiont, Mycoavidus cysteinexigens, which we place within a lineage of endofungal symbionts that are sister clade to Burkholderia. The genome of M. elongata strain AG77 features a core set of primary metabolic pathways for degradation of simple carbohydrates and lipid biosynthesis, while the M. cysteinexigens (AG77) genome is reduced in size and function. Experiments using antibiotics to cure the endobacterium from the host demonstrate that the fungal host metabolism is highly modulated by presence/absence of M. cysteinexigens. Independent comparative phylogenomic analyses of fungal and bacterial genomes are consistent with an ancient origin for M. elongata - M. cysteinexigens symbiosis, most likely over 350 million years ago and concomitant with the terrestrialization of Earth and diversification of land fungi and plants.

Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts

BackgroundPhotosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail.ResultsBased on their expression profile, six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6) were chosen for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds.ConclusionsTaken together, our results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.

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

Summary Nannochloropsis oceanica is an oleaginous microalga rich in ω3 long‐chain polyunsaturated fatty acids (LC‐PUFAs) content, in the form of eicosapentaenoic acid (EPA). We identified the enzymes involved in LC‐PUFA biosynthesis in N. oceanica CCMP1779 and generated multigene expression vectors aiming at increasing LC‐PUFA content in vivo. We isolated the cDNAs encoding four fatty acid desaturases (FAD) and determined their function by heterologous expression in S. cerevisiae. To increase the expression of multiple fatty acid desaturases in N. oceanica CCMP1779, we developed a genetic engineering toolkit that includes an endogenous bidirectional promoter and optimized peptide bond skipping 2A peptides. The toolkit also includes multiple epitopes for tagged fusion protein production and two antibiotic resistance genes. We applied this toolkit, towards building a gene stacking system for N. oceanica that consists of two vector series, pNOC‐OX and pNOC‐stacked. These tools for genetic engineering were employed to test the effects of the overproduction of one, two or three desaturase‐encoding cDNAs in N. oceanica CCMP1779 and prove the feasibility of gene stacking in this genetically tractable oleaginous microalga. All FAD overexpressing lines had considerable increases in the proportion of LC‐PUFAs, with the overexpression of Δ12 and Δ5 FAD encoding sequences leading to an increase in the final ω3 product, EPA.

Heterologous co-expression of a yeast diacylglycerol acyltransferase (ScDGA1) and a plant oleosin (AtOLEO3) as an efficient tool for enhancing triacylglycerol accumulation in the marine diatom Phaeodactylum tricornutum

BackgroundMicroalgae are promising alternate and renewable sources for producing valuable products such as biofuel and essential fatty acids. Although this is the case, there are still challenges impeding on the effective commercial production of microalgal products. For instance, their product yield is still too low. Therefore, this study was oriented towards enhancing triacylglycerol (TAG) accumulation in the diatom Phaeodactylum tricornutum (strain Pt4). To achieve this, a type 2 acyl-CoA:diacylglycerol acyltransferase from yeast (ScDGA1) and the lipid droplet (LD) stabilizing oleosin protein 3 from Arabidopsis thaliana (AtOLEO3) were expressed in Pt4.ResultsThe individual expression of ScDGA1 and AtOLEO3 in Pt4 resulted in a 2.3- and 1.4-fold increase in TAG levels, respectively, in comparison to the wild type. The co-expression of both, ScDGA1 and AtOLEO3, was accompanied by a 3.6-fold increase in TAG content. On the cellular level, the lines co-expressing ScDGA1 and AtOLEO3 showed the presence of the larger and increased numbers of lipid droplets when compared to transformants expressing single genes and an empty vector. Under nitrogen stress, TAG productivity was further increased twofold in comparison to nitrogen-replete conditions. While TAG accumulation was enhanced in the analyzed transformants, the fatty acid composition remained unchanged neither in the total lipid nor in the TAG profile.ConclusionsThe co-expression of two genes was shown to be a more effective strategy for enhancing TAG accumulation in P. tricornutum strain Pt4 than a single gene strategy. For the first time in a diatom, a LD protein from a vascular plant, oleosin, was shown to have an impact on TAG accumulation and on LD organization.

Galactoglycerolipid Lipase PGD1 Is Involved in Thylakoid Membrane Remodeling in Response to Adverse Environmental Conditions in Chlamydomonas

The Chlamydomonas lipase PGD1 plays important roles in thylakoid lipid remodeling, thylakoid architecture, and tolerance to adverse environments. Photosynthesis occurs in the thylakoid membrane, where the predominant lipid is monogalactosyldiacylglycerol (MGDG). As environmental conditions change, photosynthetic membranes have to adjust. In this study, we used a loss-of-function Chlamydomonas reinhardtii mutant deficient in the MGDG-specific lipase PGD1 (PLASTID GALACTOGLYCEROLIPID DEGRADATION1) to investigate the link between MGDG turnover, chloroplast ultrastructure, and the production of reactive oxygen species (ROS) in response to different adverse environmental conditions. The pgd1 mutant showed altered MGDG abundance and acyl composition and altered abundance of photosynthesis complexes, with an increased PSII/PSI ratio. Transmission electron microscopy showed hyperstacking of the thylakoid grana in the pgd1 mutant. The mutant also exhibited increased ROS production during N deprivation and high light exposure. Supplementation with bicarbonate or treatment with the photosynthetic electron transport blocker DCMU protected the cells against oxidative stress in the light and reverted chlorosis of pgd1 cells during N deprivation. Furthermore, exposure to stress conditions such as cold and high osmolarity induced the expression of PGD1, and loss of PGD1 in the mutant led to increased ROS production and inhibited cell growth. These findings suggest that PGD1 plays essential roles in maintaining appropriate thylakoid membrane composition and structure, thereby affecting growth and stress tolerance when cells are challenged under adverse conditions.

Current trends to comprehend lipid metabolism in diatoms

Diatoms are the most dominant phytoplankton species in oceans and they continue to receive a great deal of attention because of their significant contributions in ecosystems and the environment. Due to triacylglycerol (TAG) profiles that are abundant in medium-chain fatty acids, diatoms have emerged to be better feed stocks for biofuel production, in comparison to the commonly studied green microalgal species (chlorophytes). Importantly, diatoms are also known for their high levels of the essential ω3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and are considered to be a promising alternative source of these components. The two most commonly exploited diatoms include Thalassiosira pseudonana and Phaeodactylum tricornutum. Although obvious similarities between diatoms and chlorophytes exist, there are some substantial differences in their lipid metabolism. This review provides an overview on lipid metabolism in diatoms, with P. tricornutum as the most explored model. Special emphasis is placed on the synthesis and incorporation of very long chain ω3 fatty acids into lipids. Furthermore, current approaches including genetic engineering and biotechnological methods aimed at improving and maximizing lipid production in P. tricornutum are also discussed.

Plant Endocytosis Requires the ER Membrane-Anchored Proteins VAP27-1 and VAP27-3

Through yet-undefined mechanisms, the plant endoplasmic reticulum (ER) has a critical role in endocytosis. The plant ER establishes a close association with endosomes and contacts the plasma membrane (PM) at ER-PM contact sites (EPCSs) demarcated byxa0the ER membrane-associated VAMP-associated-proteins (VAP). Here, we investigated two plant VAPs, VAP27-1 and VAP27-3, and found an interaction with clathrin and a requirement for the homeostasis of clathrin dynamics at endocytic membranes and endocytosis. We also demonstrated direct interaction of VAP27-proteins with phosphatidylinositol-phosphate lipids (PIPs) that populate endocytic membranes. These results support that, through interaction with PIPs, VAP27-proteins bridge the ER with endocytic membranes and maintain endocytic traffic, likely through their interaction with clathrin.

Enhancing oil production and harvest by combining the marine alga Nannochloropsis oceanica and the oleaginous fungus Mortierella elongata

BackgroundAlthough microalgal biofuels have potential advantages over conventional fossil fuels, high production costs limit their application in the market. We developed bio-flocculation and incubation methods for the marine alga, Nannochloropsis oceanica CCMP1779, and the oleaginous fungus, Mortierella elongata AG77, resulting in increased oil productivity.ResultsBy growing separately and then combining the cells, the M. elongata mycelium could efficiently capture N. oceanica due to an intricate cellular interaction between the two species leading to bio-flocculation. Use of a high-salt culture medium induced accumulation of triacylglycerol (TAG) and enhanced the contents of polyunsaturated fatty acids (PUFAs) including arachidonic acid and docosahexaenoic acid in M. elongata. To increase TAG productivity in the alga, we developed an effective, reduced nitrogen-supply regime based on ammonium in environmental photobioreactors. Under optimized conditions, N. oceanica produced high levels of TAG that could be indirectly monitored by following chlorophyll content. Combining N. oceanica and M. elongata to initiate bio-flocculation yielded high levels of TAG and total fatty acids, with ~u200915 and 22% of total dry weight (DW), respectively, as well as high levels of PUFAs. Genetic engineering of N. oceanica for higher TAG content in nutrient-replete medium was accomplished by overexpressing DGTT5, a gene encoding the type II acyl-CoA:diacylglycerol acyltransferase 5. Combined with bio-flocculation, this approach led to increased production of TAG under nutrient-replete conditions (~u200910% of DW) compared to the wild type (~u20096% of DW).ConclusionsThe combined use of M. elongata and N. oceanica with available genomes and genetic engineering tools for both species opens up new avenues to improve biofuel productivity and allows for the engineering of polyunsaturated fatty acids.