Acyl-CoA:diacylglycerol acyltransferase: Properties, physiological roles, metabolic engineering and intentional control

Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the last reaction in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG). DGAT activity resides mainly in DGAT1 and DGAT2 in eukaryotes and bifunctional wax ester synthase-diacylglycerol acyltransferase (WSD) in bacteria, which are all membrane-bound proteins but exhibit no sequence homology to each other. Recent studies also identified other DGAT enzymes such as the soluble DGAT3 and diacylglycerol acetyltransferase (EaDAcT), as well as enzymes with DGAT activities including defective in cuticular ridges (DCR) and steryl and phytyl ester synthases (PESs). This review comprehensively discusses research advances on DGATs in prokaryotes and eukaryotes with a focus on their biochemical properties, physiological roles, and biotechnological and therapeutic applications. The review begins with a discussion of DGAT assay methods, followed by a systematic discussion of TAG biosynthesis and the properties and physiological role of DGATs. Thereafter, the review discusses the three-dimensional structure and insights into mechanism of action of human DGAT1, and the modeled DGAT1 from Brassica napus. The review then examines metabolic engineering strategies involving manipulation of DGAT, followed by a discussion of its therapeutic applications. DGAT in relation to improvement of traits of farmed animals is also discussed along with DGATs in various other eukaryotic organisms

Microalgal enzymes with biotechnological applications

Enzymes are essential components of biological reactions and play important roles in the scaling and optimization of many industrial processes. Due to the growing commercial demand for new and more efficient enzymes to help further optimize these processes, many studies are now focusing their attention on more renewable and environmentally sustainable sources for the production of these enzymes. Microalgae are very promising from this perspective since they can be cultivated in photobioreactors, allowing the production of high biomass levels in a cost-efficient manner. This is reflected in the increased number of publications in this area, especially in the use of microalgae as a source of novel enzymes. In particular, various microalgal enzymes with different industrial applications (e.g., lipids and biofuel production, healthcare, and bioremediation) have been studied to date, and the modification of enzymatic sequences involved in lipid and carotenoid production has resulted in promising results. However, the entire biosynthetic pathways/systems leading to synthesis of potentially important bioactive compounds have in many cases yet to be fully characterized (e.g., for the synthesis of polyketides). Nonetheless, with recent advances in microalgal genomics and transcriptomic approaches, it is becoming easier to identify sequences encoding targeted enzymes, increasing the likelihood of the identification, heterologous expression, and characterization of these enzymes of interest. This review provides an overview of the state of the art in marine and freshwater microalgal enzymes with potential biotechnological applications and provides future perspectives for this field

Lipid Droplets: Packing Hydrophobic Molecules Within the Aqueous Cytoplasm

Article discusses how lipid droplets, also known as oil bodies or lipid bodies, are plant organelles that compartmentalize neutral lipids as a hydrophobic matrix covered by proteins embedded in a phospholipid monolayer. The authors review the growing body of knowledge about lipid droplets in plant cells, describe the evolutionary similarity and divergence in their associated subcellular machinery, and point to gaps that deserve future attention

Caleosin/Peroxygenases:multifunctional proteins in plants

BACKGROUND: Caleosin/peroxygenases (CLO/PXGs) are a family of multifunctional proteins that are ubiquitous in land plants and also found in some fungi and green algae. CLO/PXGs were initially described as a class of plant lipid-associated proteins with some similarities to the oleosins that stabilize lipid droplets (LDs) in storage tissues such as seeds. However, we now know that CLO/PXGs have more complex structure, distribution and functions than oleosins. Structurally, CLO/PXGs share conserved domains that confer specific biochemical features with diverse localizations and functions.SCOPE: This review surveys the structural properties of CLO/PXGs and their biochemical roles. In addition to their highly conserved structures, CLO/PXGs have peroxygenase activities and are involved in several aspects of oxylipin metabolism in plants. The enzymatic activities and the spatiotemporal expression of CLO/PXGs are described and linked with their wider involvement in plant physiology. Plant CLO/PXGs have many roles in both biotic and abiotic stress responses in plants and in their responses to environmental toxins. Finally, some intriguing developments in the biotechnological uses of CLO/PXGs are addressed.CONCLUSIONS: It is now two decades since caleosin/peroxygenases (CLO/PXGs) were first recognized as a new class of lipid-associated proteins, and only 15 years since their additional enzymatic functions as a novel class of peroxygenases was discovered. There are many interesting research questions that remain to be addressed in future physiological studies of plant CLO/PXGs and also their recently discovered roles in the sequestration and possibly detoxification of a wide variety of lipidic xenobiotics that can challenge plant welfare.</p

Study of molecular mechanisms to increase carbon use efficiency in microalgae

In order to better understand alga\u2019s biology and allow to design biotechnological approaches to improve biomass yield, in this PhD thesis we investigated the molecular mechanism involved in the microalgae carbon use efficiency. In the Chapter 1 we studied the model algae C. reinhardtii. In section A Photosystem II assembly were investigated. Indeed, no detailed studies of the assembly factors of PSII have been performed. In this work we focus on a putative assembly factor of the CP43 subunit, called LPA2 (low PSII accumulation 2), previously identified in A. thaliana. A candidate lpa2 gene in C. reinhardtii was identified by homology and its role was studied in vivo thank to a CRISPR-cas9 mutant. The data collected demonstrated that LPA2 protein is involved in both de novo biogenesis and repair of PSII. In the section B the relationship between chloroplast and mitochondrion metabolism was explored studying a mutant of C. reinhardtii knockout for a mitochondrial transcription factor. Previous studies demonstrated that the mutation affect the mitochondrial respiration and resulted in a light-sensitive phenotype. In this work we investigated how a mutation affecting the mitochondrial respiration perturbed light acclimation of the strain. Chapter 2 regards two species of Chlorella genus. In the section A we elucidated the molecular basis of the improved growth and biomass yield in mixotrophic condition, where the cross-talk between chloroplast and mitochondria metabolism is essential for efficient biomass production. C. sorokiniana is able to combine an autotrophic metabolism with the utilization of reduced carbon source (mixotrophic condition). The de novo assembly transcriptome allowed to identify the regulation of several genes involved in control of carbon flux. In section B genetic basis of the highly productive phenotype of C. vulgaris in low light vs. high light condition was examined. Nuclear and organelle genomes were obtained combining short-reads Illumina, long PacBio reads and Bionano optical mapping, allowing to assembly a near-chromosome scale genome of 14 scaffolds and the two complete circular organelle genomes. All the genes encoding for photosynthetic subunit, as well as, genes involved in the key metabolic pathway were identified. In section C the two Chlorella species was compared for their adaptation to high CO2 level. In C. sorokiniana in 3% CO2 were observed several reorganizations of the photosynthetic machinery leading to an improved carbon fixation, while mitochondrial respiration was essentially unaffected. Instead, in C. vulgaris the 3% CO2 induced an improved uptake of reducing power by chloroplast leading to a reduced mitochondrial respiration. Chapter 3 is focused on the marine algae. In the section A was isolated a chemical mutant of N. gaditana with a reduction chlorophyll content per cell combined with increased lipids productivity. The mutant did not show an increased biomass accumulation but induced an increased lipid content, a class of macromolecules with a higher energy content per gram. This is in any case an indication of improved light energy conversion in line with an improved light penetration in the photobioreactor and more homogenous light availability due to the reduced chlorophyll content per cell in the mutant. Moreover, thank to Illumina sequencing, we found putative genes responsible of the observed phenotype. In the section B cells of T. weissflogii were grown together with an artificial cyanine molecular antenna (Cy5) that extends the absorbance range of the photosynthetic apparatus exploiting light energy in the orange spectral region. The dye was incorporate in the algae increasing light dependent growth, oxygen and biomass production. Time-resolved spectroscopy data indicates that a Cy5-chlorophyll a energy transfer mechanism happen, compatible with a FRET process

MORPHOLOGICAL, BIOCHEMICAL AND TRANSCRIPTOMIC CHARACTERISATION OF Chlorella sorokiniana AND Chlorella zofingiensis DURING NORMAL AND STRESS CONDITIONS

Chlorella has been identified as one of the most interesting microalgae species, which has high nutritional values, high growth rate, and is able to produce a wide range of metabolites in response to environmental changes. The objectives of this study are to characterise the morphology and biochemical contents and to identify the genes and miRNAs involved in regulating the production of carotenoids and lipids in Chlorella sorokiniana and Chlorella zofingiensis when cultured under high light intensity combined with glucose supplementation. In this study, stress was introduced to the Chlorella cultures by adding 2% glucose and increasing the light intensity from 10 to 100 µmol photons m-1 s-1. Then, the pigments, total phenolic contents, and antioxidant activities of both Chlorella species were evaluated. The results showed that both strains grew larger when cultured under stress condition. Although the total carotenoid content was increased under stress condition, reduction of the pigment and total phenolic contents associated with lower antioxidant activity were also recorded. Subsequently, the transcriptome of C. sorokiniana was sequenced using Illumina paired-end sequencing, and 198,844,110 raw reads with the length of 100 bp were produced. After pre-processing, ~95% of high quality reads were de novo assembled using Trinity software into 18,310 contigs. Analysis of differential gene expression by DESeq2 package showed that a total of 767 genes were upregulated and 948 genes were downregulated in stress conditions. Then, miRNAs that regulate the genes during normal and stress conditions of both C. sorokiniana and C. zofingiensis were profiled and analysed using CLC Genomic Workbench and OmiRas. From both analysis pipelines, the known and predicted novel miRNAs were identified. Although most of the identified miRNAs were not functionally determined, this study suggests that they were species-specific, which may have roles in regulating genes during stress condition. In conclusion, identifying the genes and the regulation of various metabolite productions under different growth conditions are useful for further strain enhancement of the microalgae

Evolution and regulation of photoprotective mechanisms in microalgae

La fotosintesi ossigenica \ue8 un processo per mezzo del quale l\u2019energia solare e l\u2019anidride carbonica (CO2) sono utilizzate per produrre ossigeno (O2) e biomassa. La conversione dell\u2019energia luminosa in energia chimica \ue8 condotta da complessi multiproteici denominati Fotosistema II (PSII) e Fotosistema I (PSI). Il PSII e il PSI mediano la separazione di carica, la raccolta della luce e il trasporto di elettroni dall\u2019acqua, producendo il potere riducente necessario per fissare la CO2 in carboidrati (ATP e NADPH). I Fotosistemi sono composti da due unit\ue0 principali: il centro di reazione, sito in cui avvengono le reazioni biochimiche e la separazione di carica, e il sistema antenna, costituito da complessi proteici di raccolta della luce (LHC), coinvolti principalmente nella raccolta della luce e nel trasferimento dell\u2019energia d\u2019eccitazione al centro di reazione. Gli organismi fotosintetici sfruttano la radiazione fotosinteticamente attiva (PAR) a fini metabolici. Variazioni nell'irradianza, quali l\u2019eccesso di luce, possono determinare condizioni limitanti o di stress, portando alla formazione di specie reattive dell\u2019ossigeno (ROS), le quali, influenzando la crescita delle piante e ne riducono la produttivit\ue0. L'attivazione del processo di dissipazione termica, denominato Non-Photochemical Quenching (NPQ), ha un ruolo fondamentale nella reazione di quenching (smorzamento) degli stati eccitati di singoletto della clorofilla, dissipando l'energia di eccitazione sotto forma di calore, prevenendo quindi lo stress foto-ossidativo. Nelle microalghe fino all'80% dell'energia luminosa assorbita pu\uf2 essere riemessa sotto forma di calore con conseguente riduzione della produttivit\ue0 totale di biomassa. Questa tesi \ue8 incentrata sullo studio della regolazione dell\u2019NPQ in diverse specie di alghe. A tale scopo sono stati applicati diversi approcci quali la trasformazione genetica, la caratterizzazione fenotipica e spettroscopica di cellule intere e di complessi proteici isolati. La regolazione dell\u2019NPQ a livello del PSII e del PSI \ue8 stata ampiamente studiata anche in relazione alle proteine LHC nell\u2019organismo modello per le alghe verdi, Chlamydomonas reinhardtii, in cui \ue8 stata evidenziata un'attivit\ue0 di quenching in entrambi i Fotosistemi. Nella specie di microalga di uso commerciale, Chlorella vulgaris, la regolazione dell\u2019NPQ \ue8 stata studiata in relazione all'accumulo di zeaxantina, evidenziandone una forte dipendenza. Infine, la regolazione fotosintetica \ue8 stata monitorata in cellule intere e in complessi isolati nella microalga Haematococcus pluvialis, in presenza di forti stress indotti dall\u2019eccesso di energia luminosa e dalla carenza di nutrienti.Oxygenic photosynthesis is a process by which sunlight energy and CO2 are used to produce O2 and biomass. The light energy conversion into chemical energy is carried forth by multiproteic complexes called Photosystem II (PSII) and Photosystem I (PSI). PSII and PSI drive charge separation, light harvesting and electron transport from water, producing the reducing power necessary to fix CO2 into carbohydrates (ATP and NADPH). Photosystems are composed by two moieties: a core reaction center, site of biochemical reactions and charge separation, and an antenna system constituted by Light Harvesting Complex (LHC) proteins mainly involved in light harvesting and excitation energy transfer to the reaction center. Photosynthetic organisms use the photosynthetically active radiation (PAR) for their metabolic processes but irradiance undergo changes and light excess becomes a limit or even a stressor leading to the formation of Reactive Oxygen Species (ROS) which influence plant growth and could decrease crop productivity. Photo-oxidative stress can be prevented by activation of thermal dissipation process called Non-Photochemical Quenching (NPQ), which has an important role in quenching chlorophylls singlet excited states dissipating the excitation energy in form of heat. In microalgae, up to 80% of absorbed light energy can be re-emitted as heat with a consequent reduction of total biomass productivity. This thesis was focused into the investigation of NPQ regulation in several algae species. For this purpose, different approaches were applied including genetic transformation, phenotypic and spectroscopic characterization of entire cells and of isolated complexes. The NPQ regulation at the level of both PSII and PSI also in relation with LHC proteins was fully investigated in the model green alga Chlamydomonas reinhardtii evidencing a quenching activity in both Photosystems. In the commercial microalga specie, Chlorella vulgaris, the NPQ regulation was studied in relation with zeaxanthin accumulation evidencing a strong dependency. Finally, in the microalga Haematococcus pluvialis, the photosynthetic regulation was also monitored in entire cells and isolated complexes, in presence of strong stresses induced by light excess and nutrients depletion

Microalgal Enzymes with Biotechnological Application

The aim of this thesis was to address the current state of art of microalgal enzymes with biotechnological application, and to expand the current knowledge of enzymes with the potential to find proper application in industry or novel market niches, via their production and biochemical characterization. Available transcriptomic datasets were used to identify possible enzymes of interest. Specifically, after a review of the available scientific literature (Chapter 1) my experimental activities focused on different enzymes identified in the transcriptome of the marine diatom Cylindrotheca closterium. Studied enzymes were two di-n-butyl phthalate hydrolases (CcDBPH1-2), putatively involved in phthalate degradation (Chapter 2), two platelet activating factor- acetylhydrolase (CcPAF-AH1-2), involved in lipid metabolism and inflammation modulation, which role in diatom is still unknown (Chapter 3), a L-asparaginase (CcASNase), that is an enzyme with a well-supported market niche in cancer treatment and acrylamide mitigation (Chapter 4). The experimental pipeline of CcDBPH1-2, starting from transcript identification till protein functional activity studies, was completed and reached the stage of protein purification and activity assay. In addition, different bioinformatics analyses and cc_dbph1-2 expression by RT-qPCR in C. closterium grown with different concentrations of DBP were also performed confirming evidences of the protein bioremediation activity. The expression of CcPAF-AH1-2 was hampered by the strong toxic effect that recombinant proteins production induced in E. coli cells. However, the mutation of the catalytic serine 243 of CcPAF-AH2 allowed enzyme accumulation in 1L cultures. Finally, the expression of CcASNase was successful in BL21 (DE3) and Rosetta 2 (DE3) E. coli strains. However, the low amount of produced protein will need further optimization of the growth conditions. Overall, this PhD thesis represents a contribution to the growing number of studies aimed to unravel the application of microalgae as an eco-friendly and eco-sustainable source of novel enzymes for human applications

Toward an effective use of microalgae: a study on Chlamydomonas reinhardtii to disentangle non photochemical quenching (NPQ) and to engineer ketocarotenoids biosynthesis

Photosynthetic organisms can use solar energy to produce organic biomass starting from simple elements as CO2 and water, releasing oxygen as side product. Algae are characterized by high growth rate, extremely rapid life cycle and intrinsic high photosynthetic efficiency. Moreover, microalgae can also be cultivated in a mixed autotrophic/heterotrophic condition, using reduced carbon sources. Several algal strains are characterized by high lipid accumulation or production of high value compounds. Thus, algae not only represent a valid alternative to plants, but they also play a central role considering the sustainability related to their cultivation. Wastewaters and flue gas can be used to ensure nutrients and CO2 for carbon fixation, and, after biomass harvesting, water can be reused leading to a far lower consumption with respect to plants (especially in closed photobioreactor in which the evaporation is low). Unfortunately, algae evolved in conditions extremely different compared to actual industrial ones which involves 24/24 hours of high irradiance, strong shaking as well as high CO2 concentration: all these elements ensure high photosynthetic rate and thus high biomass accumulation but make necessary a domestication of strains. Since this need became evident, engineers, biologists and biotechnologists had tried to overcome algae cultivation limitations in order to became it feasible and economically useful. From a biotechnological point of view several targets could be pointed. Optimization of absorption/dissipation of light energy is one of the most interesting and explored. This thesis reports the use of several approaches to investigate the heat dissipation mechanisms (NPQ) in green algae, mainly focusing on the model organism Chlamydomonas reinhardtii. The results obtained reveal the molecular mechanisms of energy conversion from excitation energy into heat by the activity of specific pigment binding proteins called LHCSR (Light Harvesting Stress Related), going deep into details of the protein domains and pigments involved in the quenching process and the protein interaction network necessary for NPQ. In particular, the regulation of the accumulation of LHCSR proteins in Chlamydomonas reinhardtii revealed to be a successful genetic engineering strategy to improve biomass productivity. Among the possible application of microalgae, one of the most promising one is their use as green factories to produce high value products: here, we report the metabolic engineering of Chlamydomonas reinhardtii as a bio-factory for ketocarotenoids production. The use of microalgae as host to produce high value metabolites, represents, indeed, an effective way to break down costs related to their cultivation with a potential high impact into the market. Astaxanthin is, currently, produce using Haematococcus. lacustris (recently renamed from Haematococcus pluvialis) in which, its accumulation causes a stop in growth. For that reason, in this thesis effects of astaxanthin accumulation of H. lacustris was investigated. This thesis presents, with different approaches, a leap forward in microalgae domestication both trough enrichment of knowledge about NPQ and trough application of metabolic engineering to develop green bio-factories