Key Proteomics Tools for Fundamental and Applied Microalgal Research.

Microscopic, photosynthetic prokaryotes and eukaryotes, collectively referred to as microalgae, are widely studied to improve our understanding of key metabolic pathways (e.g., photosynthesis) and for the development of biotechnological applications. Omics technologies, which are now common tools in biological research, have been shown to be critical in microalgal research. In the past decade, significant technological advancements have allowed omics technologies to become more affordable and efficient, with huge datasets being generated. In particular, where studies focused on a single or few proteins decades ago, it is now possible to study the whole proteome of a microalgae. The development of mass spectrometry-based methods has provided this leap forward with the high-throughput identification and quantification of proteins. This review specifically provides an overview of the use of proteomics in fundamental (e.g., photosynthesis) and applied (e.g., lipid production for biofuel) microalgal research, and presents future research directions in this field.

Impact of nitrogen deficiency on succinic acid production by engineered strains of Yarrowia lipolytica.

Yarrowia lipolytica strains PGC01003 and PGC202 engineered for succinic acid production were studied and compared to the wild type strain W29. For the first time, these two strains were characterized in a chemically defined medium. Strain growth and organic acid production were investigated in fed-batch mode with glycerol as carbon and energy source. This study evaluated the impact of nitrogen deficiency strategy to redirect carbon flux toward succinic acid synthesis. Strain PGC01003 produced 19 g L-1 succinic acid with an overall yield of 0.23 g g-1 and an overall productivity of 0.23 g L-1 h-1, while strain PGC202 produced 33 g L-1 succinic acid with an overall yield of 0.12 g g-1 and a productivity of 0.57 g L-1 h-1. Nitrogen limitation effectively stopped biomass growth and increased succinic acid yield of PGC01003 and PGC202 by 18 % and 62 %, respectively. However, the specific succinic acid production rate was reduced by 77 % and 66 %, respectively.

A dansyl-derivatized phytic acid analogue as a fluorescent substrate for phytases: experimental and computational approach.

A new myo-inositol pentakisphosphate was synthesized, which featured a dansyl group at position C-5. The fluorescent tag was removed from the inositol by a 6-atom spacer to prevent detrimental steric interactions in the catalytic site of phytases. The PEG linker was used in order to enhance hydrophilicity and biocompatibility of the new artificial substrate. Computational studies showed a favorable positioning in the catalytic site of phytases. Enzymatic assays demonstrated that the tethered myo-inositol was processed by two recombinant phytases Phy-A and Phy-C, classified respectively as acid and alkaline phytases, with similar rates of phosphate release compared to their natural substrate.

Lipase-Catalyzed Ring-Opening Polymerization of Benzyl Malolactonate: An Unusual Mechanism?

The use of safe natural catalyst such as enzymes for ring opening polymerization (ROP) of ß-substituted ß-lactones such as benzyl malolactonate (MLABe) is an important objective considering the biomedical applications of the resulting (co)polymers. However, the preparation of well-defined polymeric materials using such systems requires an understanding of enzyme-substrate interactions. In this context, we investigated the mechanism of lipase-catalyzed ROP of MLABe, because it appears that it is probably not the same as the one widely described for other lactones such ε-caprolactone, propiolactone. and lactide. Enzymatic-catalyzed ROPs of MLABe in the presence of the lipase/acyltransferase CpLip2 and its serine knockout (serine KO) mutant (CpLip2_180A) have led to poly(benzyl malate) (PMLABe) terminated by a monobenzyl fumarate group with monomer conversion higher than 70% and weight-average molar mass of about 3600 g/mol (D = 1.42). On the other hand, only less than 7% of MLABe conversion and no polymer formation were observed when the polymerization reaction was conducted in the presence of inactivated CpLip2 (heated at 100 °C). Moreover, the ROP of MLABe in the presence of imidazole, a synthetic mimic of the catalytic histidine, led to a PMLABe terminated by a monobenzyl fumarate group. On the contrary, neither the enzymatic-catalyzed ROP of benzyl dimethylmalolactonate (diMeMLABe), a MLABe with two methyl groups instead of the two "acidic" protons on the lactone's ring, in the presence of CpLip2 and CpLip2_180A nor its chemical ROP in the presence of imidazole were successful. Together, all these results suggested that the lipase-catalyzed polymerization of malolactonates occurred through the abstraction of one of the two "acidic" protons of the lactone's ring by the histidine of the catalytic triad leading to the corresponding monobenzyl fumarate responsible for the polymerization of the remaining monomer. Finally, molecular modeling of the positioning of the monomer into the catalytic site of the CpLip2 and DFT quantum-chemical calculations highlighted an interaction of (R)- and (S)-MLABe with the catalytic histidine of the enzyme preferentially to serine, in the form of a strong hydrogen bond with one of the "acidic" protons of MLABe, thus, supporting the important role of the catalytic histidine in the polymerization of such cyclic lactones.

Interactions between trans-resveratrol and CpLIP2 lipase/acyltransferase: Evidenced by fluorescence and in silico.

We have examined the trans-resveratrol/lipase interaction by quantitative and qualitative analyses of fluorescence spectra, molecular docking and quantum-chemical calculations at DFT level. Interactions of CpLIP2 from C. parapsilosis CBS 604 and trans-resveratrol were confirmed with a major contribution of tryptophan residues to fluorescence quenching. A thermodynamic study across a wide temperature range was consistent with the presence of a single binding site with a binding free energy of -24 kJ/mol. Nevertheless, trans-resveratrol competitively inhibited CpLIP2 activity. Molecular docking and quantum-chemical calculations were consistent with a strong binding of trans-resveratrol to the CpLIP2 catalytic site via electrostatic and hydrophobic forces. The structural analysis quantitatively revealed an energy transfer from W51 and W350 to trans-resveratrol with a distance of 32 Å. Precise understanding of trans-resveratrol/CpLIP2 interactions has important implications on lipases for screening of stilbenoid.

Effective Catalytic Delignification and Fractionation of Lignocellulosic Biomass in Water over Zn3V2O8 Mixed Oxide.

The conversion of poplar wood biomass to highly value-added chemicals and molecular building blocks was achieved by using the dispersed mixed oxide Zn3V2O8 (ZVO) in water under 100 kPa of 10% O2/N2 at 160, 180, and 200 °C for 4 h. This nanostructured mixed oxide was prepared via the precipitation process and then characterized by several techniques. The results showed that this mixed oxide has interesting catalytic properties and is a versatile catalyst for biomass delignification and lignin and hemicellulose depolymerization. ZVO exhibited high activity on poplar biomass delignification and fractionation (degree of delignification > 97%) and lignin and holocellulose conversion with high yield into aromatic and furan compounds (80 mg/g initial wood at 200 °C), with high selectivities for 5-hydroxymethylfurfural (HMF) (25 mg/g of initial wood), vanillin, and syringaldehyde.

Furanolysis with Menthofuran: A New Depolymerization Method for Analyzing Condensed Tannins.

An improved analytical depolymerization method for characterizing condensed tannins was developed with menthofuran (3,6-dimethyl-4,5,6,7-tetrahydro-1-benzofuran) as the nucleophilic trapping reagent. Herein, menthofuran was compared with routinely used nucleophiles, phloroglucinol and 2-mercaptoethanol. At 30 °C and in the presence of 0.1 M HCl, menthofuran displayed the outstanding ability to enable the fast and full depolymerization of procyanidin B2 using only a 1:1 molar ratio of both reactants. Under the same conditions, phloroglucinol and 2-mercaptoethanol led to a reaction equilibrium with significantly lower conversion yields. Application to commercial tannin extracts showed that a menthofuran-to-extract weight ratio of 1 gave the same yields of procyanidin constitutive units as 10-fold higher molecular equivalent phloroglucinol and 100-fold 2-mercaptoethanol. Finally, guidelines for implementing the menthofuran depolymerization method are proposed to assess the tannin content and composition of extracts as well as of plant materials without prior extraction.

Inhibition of CpLIP2 Lipase Hydrolytic Activity by Four Flavonols (Galangin, Kaempferol, Quercetin, Myricetin) Compared to Orlistat and Their Binding Mechanisms Studied by Quenching of Fluorescence.

The inhibition of recombinant CpLIP2 lipase/acyltransferase from Candida parapsiolosis was considered a key model for novel antifungal drug discovery and a potential therapeutic target for candidiasis. Lipases have identified recently as potent virulence factors in C. parapsilosis and some other yeasts. The inhibition effects of orlistat and four flavonols (galangin, kaempferol, quercetin and myricetin) characterized by an increasing degree of hydroxylation in B-ring, were investigated using ethyl oleate hydrolysis as the model reaction. Orlistat and kaempferol (14 µM) strongly inhibited CpLIP2 catalytic activity within 1 min of pre-incubation, by 90% and 80%, respectively. The relative potency of flavonols as inhibitors was: kaempferol > quercetin > myricetin > galangin. The results suggested that orlistat bound to the catalytic site while kaempferol interacted with W294 on the protein lid. A static mechanism of interactions between flavonols and CpLIP2 lipase was confirmed by fluorescence quenching analyses, indicating that the interactions were mainly driven by hydrophobic bonds and electrostatic forces. From the Lehrer equation, fractions of tryptophan accessibility to the quencher were evaluated, and a relationship with the calculated number of binding sites was suggested.

Characterization and Reshaping of a Large and Hydrophobic Nucleophile Pocket in Lipases/Acyltransferases.

Lipases/acyltransferases, such as CpLIP2 from Candida parapsilosis and CduLAc from Candida dubliniensis, catalyze acyl transfer preferentially over hydrolysis if a suitable nucleophile is present, even in a medium with a high thermodynamic activity of water (aW ). These enzymes are related to CAL-A from Moesziomyces antarcticus, which, in comparison, displays a lower acyl transfer ability. The 3D structures of wild types and mutants of CAL-A, CpLIP2, and CduLAc revealed differences in size and hydrophobicity of a large pocket located under the catalytic triad. The kinetic behavior of site-directed mutants confirmed the role of this pocket in competition between methanol and water as the nucleophile acceptor for the deacylation step. The mutations provided a better understanding of key structural determinants for variable levels of acyltransferase ability observed and supported the existence of a complex network of nucleophile interactions within the enzymes. The shape and size of the possible nucleophile pocket identified also suggested that multiple binding sites could exist, which supported the hypothesis of non-overlapping leaving and accepting nucleophile binding sites.

Fucoxanthin and Polyunsaturated Fatty Acids Co-Extraction by a Green Process.

By their autotrophic nature and their molecular richness, microalgae are serious assets in the context of current environmental and societal challenges. Some species produce both omega-3 long chain polyunsaturated fatty acids (PUFAs) and xanthophylls, two molecular families widely studied for their bioactivities in the fields of nutrition and cosmetics. Whereas most studies separately deal with the two families, synergies could be exploited with extracts containing both PUFAs and xanthophylls. The purpose of our work was to determine cost effective and eco-friendly parameters for their co-extraction. The effect of several parameters (solvent, solvent/biomass ratio, temperature, duration) were studied, using two microalgal species, the non-calcifying Haptophyta Tisochrysis lutea, and the diatom Phaeodactylum tricornutum, that presents a silicified frustule. Analyses of PUFAs and fucoxanthin (Fx), the main xanthophyll, allowed to compare kinetics and extraction yields between experimental protocols. Co-extraction yields achieved using 96% ethanol as solvent were 100% for Fx and docosahexaenoic acid (DHA) in one hour from T. lutea biomass, and respectively 95% and 89% for Fx and eicosapentaenoic acid (EPA) in eight hours from P. tricornutum. These conditions are compatible with industrial applications.

Mechano-Enzymatic Deconstruction with a New Enzymatic Cocktail to Enhance Enzymatic Hydrolysis and Bioethanol Fermentation of Two Macroalgae Species.

The aim of this study was to explore the efficiency of a mechano-enzymatic deconstruction of two macroalgae species for sugars and bioethanol production, by using a new enzymatic cocktail (Haliatase) and two types of milling modes (vibro-ball: VBM and centrifugal milling: CM). By increasing the enzymatic concentration from 3.4 to 30 g/L, the total sugars released after 72 h of hydrolysis increased (from 6.7 to 13.1 g/100 g TS and from 7.95 to 10.8 g/100 g TS for the green algae U. lactuca and the red algae G. sesquipedale, respectively). Conversely, total sugars released from G. sesquipedale increased (up to 126% and 129% after VBM and CM, respectively). The best bioethanol yield (6 geth/100 g TS) was reached after 72 h of fermentation of U. lactuca and no increase was obtained after centrifugal milling. The latter led to an enhancement of the ethanol yield of G. sesquipedale (from 2 to 4 g/100 g TS).

Cell Uptake and Biocompatibility of Nanoparticles Prepared from Poly(benzyl malate) (Co)polymers Obtained through Chemical and Enzymatic Polymerization in Human HepaRG Cells and Primary Macrophages.

The design of drug-loaded nanoparticles (NPs) appears to be a suitable strategy for the prolonged plasma concentration of therapeutic payloads, higher bioavailability, and the reduction of side effects compared with classical chemotherapies. In most cases, NPs are prepared from (co)polymers obtained through chemical polymerization. However, procedures have been developed to synthesize some polymers via enzymatic polymerization in the absence of chemical initiators. The aim of this work was to compare the acute in vitro cytotoxicities and cell uptake of NPs prepared from poly(benzyl malate) (PMLABe) synthesized by chemical and enzymatic polymerization. Herein, we report the synthesis and characterization of eight PMLABe-based polymers. Corresponding NPs were produced, their cytotoxicity was studied in hepatoma HepaRG cells, and their uptake by primary macrophages and HepaRG cells was measured. In vitro cell viability evidenced a mild toxicity of the NPs only at high concentrations/densities of NPs in culture media. These data did not evidence a higher biocompatibility of the NPs prepared from enzymatic polymerization, and further demonstrated that chemical polymerization and the nanoprecipitation procedure led to biocompatible PMLABe-based NPs. In contrast, NPs produced from enzymatically synthesized polymers were more efficiently internalized than NPs produced from chemically synthesized polymers. The efficient uptake, combined with low cytotoxicity, indicate that PMLABe-based NPs are suitable nanovectors for drug delivery, deserving further evaluation in vivo to target either hepatocytes or resident liver macrophages.

A glimpse into the specialization history of the lipases/acyltransferases family of CpLIP2.

Lipases/acyltransferases homologous to CpLIP2 from Candida parapsilosis belong to the α/ß hydrolase superfamily as lipase A from Moesziomyces antarcticus (Candida antarctica), and constitute a consistent phylogenetic subgroup with at least 56% identity. Lipases/acyltransferases share the phenotypic characteristic of a high acyltransfer activity even in aqueous media with very high water thermodynamic activity. Previous mutagenesis and evolution strategies have given insights into the role of key residues and protein subdomains in the reaction and substrate specificities of these enzymes. However, multiple mutations are often deleterious for the activity and the identification of all the residues that historically led to the function is complicated. A new complementary approach to elucidate structural determinant was conducted in this study, based on the resurrection of ancestral proteins to understand how the evolution led to the present properties of the biocatalysts. By doing so, the comparison with the extant proteins can lead to the identification of key residues involved in the enzymes' specialization. Using Ancestral Sequence Reconstruction, we have generated a putative ancestral lipases/acyltransferases, PaleoLAc. This enzyme shares a high level of identity with CpLIP2 but has a different catalytic behavior. PaleoLAc allowed the identification of putative key residues involved in acyltransfer ability and supports the hypothesis that this exceptional property within the lipases/acyltransferases family is linked to a cluster of residues in the vicinity of the active site. As a representative of the ancestral origin of the diversity of the catalytic behaviors observed in modern lipases/acyltransferases, PaleoLAc constitutes a powerful tool for further engineering toward targeted specialization.

Revealing the Roles of Subdomains in the Catalytic Behavior of Lipases/Acyltransferases Homologous to CpLIP2 through Rational Design of Chimeric Enzymes.

The lipases/acyltransferases homologous to CpLIP2 of Candida parapsilosis efficiently catalyze acyltransfer reactions in lipid/water media with high water activity (aW >0.9). Two new enzymes of this family, CduLAc from Candida dubliniensis and CalLAc8 from Candida albicans, were characterized. Despite 82 % sequence identity, the two enzymes have significant differences in their catalytic behaviors. In order to understand the roles played by the different subdomains of these proteins (main core, cap and C-terminal flap), chimeric enzymes were designed by rational exchange of cap and C-terminal flap, between CduLAc and CalLAc8. The results show that the cap region plays a significant role in substrate specificity; the main core was found to be the most important part of the protein for acyltransfer ability. Similar exchanges were made with CAL-A from Candida antarctica, but only the C-terminal exchange was successful. Yet, the role of this domain was not clearly elucidated, other than that it is essential for activity.

Biodiesel production from crude jatropha oil catalyzed by immobilized lipase/acyltransferase from Candida parapsilosis in aqueous medium.

The lipase/acyltransferase from Candida parapsilosis (CpLIP2) immobilized on two synthetic resins (Accurel MP 1000 and Lewatit VP OC 1600) was used as catalyst for the production of biodiesel (fatty acid methyl esters, FAME) by transesterification of jatropha oil with methanol, in a lipid/aqueous system. The oil was dispersed in a buffer solution (pH 6.5) containing methanol in excess (2M in the biphasic system; molar ratio methanol/acyl chains 2:1). Transesterification was carried out at 30°C, under magnetic stirring, using 10% (w/w) of immobilized enzyme in relation to oil. The maximum FAME yields were attained after 8h reaction time: 80.5% and 93.8%, when CpLIP2 immobilized on Accurel MP 1000 or on Lewatit VP OC 1600 were used, respectively. CpLIP2 on both Accurel MP 1000 and Lewatit VP OC 1600 showed high operational stability along 5 consecutive 8h batches.

Elucidation of a key position for acyltransfer activity in Candida parapsilosis lipase/acyltransferase (CpLIP2) and in Pseudozyma antarctica lipase A (CAL-A) by rational design.

Performing transesterifications in aqueous media is becoming a priority challenge in lipid biotechnology in order to develop more eco-friendly and efficient biocatalytic processes in systems containing both polar and apolar substrates. In this context, our group has explored for several years the high potential of the lipase/acyltransferase CpLIP2 from Candida parapsilosis and of several of its homologs, that catalyze efficiently acyltransfer reactions in lipid/water media with high water activity (aw>0.9). The discovery of a new member of this group, CduLAc from Candida dubliniensis, with a higher acyltransferase activity than CpLIP2, has provided a new insight on structure-function relationships in this group. Indeed, the comparison of sequences and 3D models, especially of CpLIP2 and CduLAc, with those of the phylogenetically related lipase A from Pseudozyma antarctica (CAL-A), allowed elucidating a key structural determinant of the acyltransferase activity: serine S369 in CpLIP2 and its equivalents E370 in CAL-A and A366 in CduLAc. Mutants obtained by rational design at this key position showed significant changes in acyltransfer activity. Whereas mutation S369E resulted in an increase in the hydrolytic activity of CpLIP2, S369A increased alcoholysis. More strikingly, the single E370A mutation in CAL-A drastically increased the acyltransferase activity of this enzyme, giving it the character of a lipase/acyltransferase. Indeed, this single mutation lowered the methanol concentration for which the initial rates of alcoholysis and hydrolysis are equal from 2M in CAL-A down to 0.3M in its mutant, while the exceptional stability of the parental enzyme toward alcohol and temperature was conserved.

The 3D model of the lipase/acyltransferase from Candida parapsilosis, a tool for the elucidation of structural determinants in CAL-A lipase superfamily.

Because lipids are hydrophobic, the development of efficient bioconversions in aqueous media free of organic solvents is particularly challenging for green oleochemistry. Within this aim, enzymes exhibiting various abilities to catalyze acyltransfer reaction in water/lipid systems have been identified. Among these, CpLIP2 from Candida parapsilosis has been characterized as a lipase/acyltransferase, able to catalyze acyltransfer reactions preferentially to hydrolysis in the presence of particularly low acyl acceptor concentration and high thermodynamic activity of water (aw>0.9). Lipase/acyltransferases are thus of great interest, being able to produce new esters at concentrations above the thermodynamic equilibrium of hydrolysis/esterification with limited to no release of free fatty acids. Here, we present a 3D model of CpLIP2 based on homologies with crystallographic structures of Pseudozyma antarctica lipase A. Indeed, the two enzymes have 31% of identity in their primary sequence, yielding a same general structure, but different catalytic properties. The quality of the calculated CpLIP2 model was confirmed by several methods. Limited proteolysis confirmed the location of some loops at the surface of the protein 3D model. Directed mutagenesis also supported the structural model constructed on CAL-A template: the functional properties of various mutants were consistent with their structure-based putative involvement in the oxyanion hole, substrate specificity, acyltransfer or hydrolysis catalysis and structural stability. The CpLIP2 3D model, in comparison with CAL-A 3D structure, brings insights for the elucidation and improvement of the structural determinants involved in the exceptional acyltransferase properties of this promising biocatalyst and of homologous enzymes of the same family.

The mitochondrial-targeted antioxidant, MitoQ, increases liver mitochondrial cardiolipin content in obesogenic diet-fed rats.

Cardiolipin (CL), a unique mitochondrial phospholipid, plays a key role in several processes of mitochondrial bioenergetics as well as in mitochondrial membrane stability and dynamics. The present study was designed to determine the effect of MitoQ, a mitochondrial-targeted antioxidant, on the content of liver mitochondrial membrane phospholipids, in particular CL, and its fatty acid composition in obesogenic diet-fed rats. To do this, twenty-four 6week old male Sprague Dawley rats were randomized into three groups of 8 animals and fed for 8weeks with either a control diet, a high fat diet (HF), or a HF diet with MitoQ (HF+MitoQ). Phospholipid classes and fatty acid composition were assayed by chromatographic methods in liver and liver mitochondria. Mitochondrial bioenergetic function was also evaluated. While MitoQ had no or slight effects on total liver fatty acid composition and phospholipid classes and their fatty acid composition, it had major effects on liver mitochondrial phospholipids and mitochondrial function. Indeed, MitoQ both increased CL synthase gene expression and CL content of liver mitochondria and increased 18:2n-6 (linoleic acid) content of mitochondrial phospholipids by comparison to the HF diet. Moreover, mitochondrial CL content was positively correlated to mitochondrial membrane fluidity, membrane potential and respiration, as well as to ATP synthase activity, while it was negatively correlated to mitochondrial ROS production. These findings suggest that MitoQ may decrease pathogenic alterations to CL content and profiles, thereby preserving mitochondrial function and attenuating the development of some of the features of metabolic syndrome in obesogenic diet-fed rats.

Grape seed and apple tannins: emulsifying and antioxidant properties.

Tannins are natural antioxidants found in plant-based foods and beverages, whose amphiphilic nature could be useful to both stabilize emulsions and protect unsaturated lipids from oxidation. In this paper, the use of tannins as antioxidant emulsifiers was studied. The main parameters influencing the stability of emulsions (i.e. tannins structure and concentration, aqueous phase pH, and ionic strength) were identified and optimized. Oil in water emulsions stabilized with tannins were compared with those stabilized with two commercial emulsifying agents, poly(vinyl alcohol) (PVA) and polyoxyethylene hydrogenated castor oil. In optimized conditions, the condensed tannins allowed to obtain a stability equivalent to that of PVA. Tannins presented good antioxidant activity in oil in water emulsion, as measured by the conjugated autoxidizable triene (CAT) assay.

Homologous yeast lipases/acyltransferases exhibit remarkable cold-active properties.

Lipases/acyltransferases catalyse acyltransfer to various nucleophiles preferentially to hydrolysis even in aqueous media with high thermodynamic activity of water (a w >0.9). Characterization of hydrolysis and acyltransfer activities in a large range of temperature (5 to 80 °C) of secreted recombinant homologous lipases of the Pseudozyma antarctica lipase A superfamily (CaLA) expressed in Pichia pastoris, enlighten the exceptional cold-activity of two remarkable lipases/acyltransferases: CpLIP2 from Candida parapsilosis and CtroL4 from Candida tropicalis. The activation energy of the reactions catalysed by CpLIP2 and CtroL4 was 18-23 kJ mol(-1) for hydrolysis and less than 15 kJ mol(-1) for transesterification between 5 and 35 °C, while it was respectively 43 and 47 kJ mol(-1) with the thermostable CaLA. A remarkable consequence is the high rate of the reactions catalysed by CpLIP2 and CtroL4 at very low temperatures, with CpLIP2 displaying at 5 °C 65 % of its alcoholysis activity and 45 % of its hydrolysis activity at 30 °C. These results suggest that, within the CaLA superfamily and its homologous subgroups, common structural determinants might allow both acyltransfer and cold-active properties. Such biocatalysts are of great interest for the efficient synthesis or functionalization of temperature-sensitive lipid derivatives, or more generally to lessen the environmental impact of biocatalytic processes.