Pharmaceutical removal from wastewater by introducing cytochrome P450s into microalgae.

Pharmaceuticals are of increasing environmental concern as they emerge and accumulate in surface- and groundwater systems around the world, endangering the overall health of aquatic ecosystems. Municipal wastewater discharge is a significant vector for pharmaceuticals and their metabolites to enter surface waters as humans incompletely absorb prescription drugs and excrete up to 50% into wastewater, which are subsequently incompletely removed during wastewater treatment. Microalgae present a promising target for improving wastewater treatment due to their ability to remove some pollutants efficiently. However, their inherent metabolic pathways limit their capacity to degrade more recalcitrant organic compounds such as pharmaceuticals. The human liver employs enzymes to break down and absorb drugs, and these enzymes are extensively researched during drug development, meaning the cytochrome P450 enzymes responsible for metabolizing each approved drug are well studied. Thus, unlocking or increasing cytochrome P450 expression in endogenous wastewater microalgae could be a cost-effective strategy to reduce pharmaceutical loads in effluents. Here, we discuss the challenges and opportunities associated with introducing cytochrome P450 enzymes into microalgae. We anticipate that cytochrome P450-engineered microalgae can serve as a new drug removal method and a sustainable solution that can upgrade wastewater treatment facilities to function as "mega livers".

Freshwater microalgae Nannochloropsis limnetica for the production of ß-galactosidase from whey powder.

This study investigated the first-ever reported use of freshwater Nannochloropsis for the bioremediation of dairy processing side streams and co-generation of valuable products, such as ß-galactosidase enzyme. In this study, N. limnetica was found to grow rapidly on both autoclaved and non-autoclaved whey-powder media (referred to dairy processing by-product or DPBP) without the need of salinity adjustment or nutrient additions, achieving a biomass concentration of 1.05-1.36 g L-1 after 8 days. The species secreted extracellular ß-galactosidase (up to 40.84 ± 0.23 U L-1) in order to hydrolyse lactose in DPBP media into monosaccharides prior to absorption into biomass, demonstrating a mixotrophic pathway for lactose assimilation. The species was highly effective as a bioremediation agent, being able to remove > 80% of total nitrogen and phosphate in the DPBP medium within two days across all cultures. Population analysis using flow cytometry and multi-channel/multi-staining methods revealed that the culture grown on non-autoclaved medium contained a high initial bacterial load, comprising both contaminating bacteria in the medium and phycosphere bacteria associated with the microalgae. In both autoclaved and non-autoclaved DPBP media, Nannochloropsis cells were able to establish a stable microalgae-bacteria interaction, suppressing bacterial takeover and emerging as dominant population (53-80% of total cells) in the cultures. The extent of microalgal dominance, however, was less prominent in the non-autoclaved media. High initial bacterial loads in these cultures had mixed effects on microalgal performance, promoting ß-galactosidase synthesis on the one hand while competing for nutrients and retarding microalgal growth on the other. These results alluded to the need of effective pre-treatment step to manage bacterial population in microalgal cultures on DPBP. Overall, N. limnetica cultures displayed competitive ß-galactosidase productivity and propensity for efficient nutrient removal on DPBP medium, demonstrating their promising nature for use in the valorisation of dairy side streams.

The enzyme encoded by Myrmecia incisa, a green microalga, phospholipase A2 gene preferentially hydrolyzes arachidonic acid at the sn-2 position of phosphatidylcholine.

The enzyme phospholipase A2 (PLA2) plays a crucial role in acyl remodeling of phospholipids via the Lands' cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In this study, a full-length cDNA sequence coding Myrmecia incisa phospholipase A2 (MiPLA2) was cloned using the technique of rapid amplification of cDNA ends. Comparison of the 1082-bp cDNA with its corresponding cloned DNA sequence revealed that MiPLA2 contained 3 introns. Mature MiPLA2 (mMiPLA2) had a conserved Ca2+-binding loop and a catalytic site motif that has been recognized in plant secretory PLA2 (sPLA2) proteins. Correspondingly, phylogenetic analysis illustrated that MiPLA2 was clustered within GroupXIA of plant sPLA2 proteins. To ascertain the function of MiPLA2, the cDNA coding for mMiPLA2 was subcloned into the vector pET-32a to facilitate the production of recombinant mMiPLA2 in Escherichia coli. Recombinant mMiPLA2 was purified and used for the in vitro enzyme reaction. Thin-layer chromatography profiles of the catalytic products generated by recombinant mMiPLA2 indicated a specificity for cleaving sn-2 acyl chains from phospholipids, thereby functionally characterizing MiPLA2. Although recombinant mMiPLA2 displayed a strong preference for phosphatidylethanolamine, it preferentially hydrolyzes arachidonic acid (ArA) at the sn-2 position of phosphatidylcholine. Results from the fused expression of p1300-sp-EGFP-mMiPLA2 illustrated that MiPLA2 was localized in the intercellular space of onion epidermis. Furthermore, the positive correlation between MiPLA2 transcription and free ArA levels were established. Consequently, the role of mMiPLA2 in the biosynthesis of ArA-rich TAG was elucidated. This study helps to understand how M. incisa preferentially uses ArA to synthesize TAG.

Metabolomics Reveals the Impact of Overexpression of Cytosolic Fructose-1,6-Bisphosphatase on Photosynthesis and Growth in Nannochloropsis gaditana.

Nannochloropsis gaditana, a microalga known for its photosynthetic efficiency, serves as a cell factory, producing valuable biomolecules such as proteins, lipids, and pigments. These components make it an ideal candidate for biofuel production and pharmaceutical applications. In this study, we genetically engineered N. gaditana to overexpress the enzyme fructose-1,6-bisphosphatase (cyFBPase) using the Hsp promoter, aiming to enhance sugar metabolism and biomass accumulation. The modified algal strain, termed NgFBP, exhibited a 1.34-fold increase in cyFBPase activity under photoautotrophic conditions. This modification led to a doubling of biomass production and an increase in eicosapentaenoic acid (EPA) content in fatty acids to 20.78-23.08%. Additionally, the genetic alteration activated the pathways related to glycine, protoporphyrin, thioglucosides, pantothenic acid, CoA, and glycerophospholipids. This shift in carbon allocation towards chloroplast development significantly enhanced photosynthesis and growth. The outcomes of this study not only improve our understanding of photosynthesis and carbon allocation in N. gaditana but also suggest new biotechnological methods to optimize biomass yield and compound production in microalgae.

Choline Dehydrogenase Contributes to Salt Tolerance in Dunaliella through Betaine Synthesis.

In Dunaliella tertiolecta, a microalga renowned for its extraordinary tolerance to high salinity levels up to 4.5 M NaCl, the mechanisms underlying its stress response have largely remained a mystery. In a groundbreaking discovery, this study identifies a choline dehydrogenase enzyme, termed DtCHDH, capable of converting choline to betaine aldehyde. Remarkably, this is the first identification of such an enzyme not just in D. tertiolecta but across the entire Chlorophyta. A 3D model of DtCHDH was constructed, and molecular docking with choline was performed, revealing a potential binding site for the substrate. The enzyme was heterologously expressed in E. coli Rosetta (DE3) and subsequently purified, achieving enzyme activity of 672.2 U/mg. To elucidate the role of DtCHDH in the salt tolerance of D. tertiolecta, RNAi was employed to knock down DtCHDH gene expression. The results indicated that the Ri-12 strain exhibited compromised growth under both high and low salt conditions, along with consistent levels of DtCHDH gene expression and betaine content. Additionally, fatty acid analysis indicated that DtCHDH might also be a FAPs enzyme, catalyzing reactions with decarboxylase activity. This study not only illuminates the role of choline metabolism in D. tertiolecta's adaptation to high salinity but also identifies a novel target for enhancing the NaCl tolerance of microalgae in biotechnological applications.

Editorial of Special Issue "Microalgal Molecules and Enzymes".

Microalgae are photosynthetic unicellular organisms that can be found in very different environments, both terrestrial and marine, including extreme environments such as cold, hot and high/low salinity [...].

Genome-based identification and comparative analysis of enzymes for carotenoid biosynthesis in microalgae.

Microalgae are potential feedstocks for the commercial production of carotenoids, however, the metabolic pathways for carotenoid biosynthesis across algal lineage are largely unexplored. This work is the first to provide a comprehensive survey of genes and enzymes associated with the less studied methylerythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway as well as the carotenoid biosynthetic pathway in microalgae through bioinformatics and comparative genomics approach. Candidate genes/enzymes were subsequently analyzed across 22 microalgae species of lineages Chlorophyta, Rhodophyta, Heterokonta, Haptophyta, Cryptophyta, and known Arabidopsis homologs in order to study the evolutional divergence in terms of sequence-structure properties. A total of 403 enzymes playing a vital role in carotene, lutein, zeaxanthin, violaxanthin, canthaxanthin, and astaxanthin were unraveled. Of these, 85 were hypothetical proteins whose biological roles are not yet experimentally characterized. Putative functions to these hypothetical proteins were successfully assigned through a comprehensive investigation of the protein family, motifs, intrinsic physicochemical features, subcellular localization, pathway analysis, etc. Furthermore, these enzymes were categorized into major classes as per the conserved domain and gene ontology. Functional signature sequences were also identified which were observed conserved across microalgal genomes. Additionally, the structural modeling and active site architecture of three vital enzymes, DXR, PSY, and ZDS catalyzing the vital rate-limiting steps in Dunaliella salina were achieved. The enzymes were confirmed to be stereochemically reliable and stable as revealed during molecular dynamics simulation of 100 ns. The detailed functional information about individual vital enzymes will certainly help to design genetically modified algal strains with enhanced carotenoid contents.

C4-monomethylsterol ß-glucoside and its synthase in Aurantiochytrium limacinum mh0186.

Thraustochytrids, unicellular marine protists, synthesize polyunsaturated fatty acids (PUFAs) and PUFA-containing phospholipids; however, little is known about their glycolipids and their associated metabolism. Here, we report two glycolipids (GL-A, B) and their synthases in Aurantiochytrium limacinum mh0186. Two glycolipids were purified from A. limacinum mh0186, and they were determined by gas chromatography, mass spectrometry and 2D nuclear magnetic resonance to be 3-O-ß-D-glucopyranosyl-stigmasta-5,7,22-triene (GL-A) and 3-O-ß-D-glucopyranosyl-4α-methyl-stigmasta-7,22-diene (GL-B), both of which are sterol ß-glucosides (ß-SGs); the structure of GL-B has not been reported thus far. Seven candidate genes responsible for the synthesis of these ß-SGs were extracted from the draft genome database of A. limacinum using the yeast sterol ß-glucosyltransferase (SGT; EC 2.4.1.173) sequence as a query. Expression analysis using Saccharomyces cerevisiae revealed that two gene products (AlSGT-1 and 2) catalyze the transfer of glucose from uridine diphosphate (UDP)-glucose to sterols, generating sterylglucosides (SGs). Compared to AlSGT-1, AlSGT-2 exhibited wide specificity for sterols and used C4-monomethylsterol to synthesize GL-B. The disruption of alsgt-2 but not alsgt-1 in strain mh0186 resulted in a decrease in the total SG and an almost complete loss of GL-B, indicating that AlSGT-2 is responsible for the synthesis of ß-SGs in A. limacinum mh0186, especially GL-B, which possesses a unique sterol structure.

Enhancing carbohydrate repartitioning into lipid and carotenoid by disruption of microalgae starch debranching enzyme.

Light/dark cycling is an inherent condition of outdoor microalgae cultivation, but is often unfavorable for lipid accumulation. This study aims to identify promising targets for metabolic engineering of improved lipid accumulation under outdoor conditions. Consequently, the lipid-rich mutant Chlamydomonas sp. KOR1 was developed through light/dark-conditioned screening. During dark periods with depressed CO2 fixation, KOR1 shows rapid carbohydrate degradation together with increased lipid and carotenoid contents. KOR1 was subsequently characterized with extensive mutation of the ISA1 gene encoding a starch debranching enzyme (DBE). Dynamic time-course profiling and metabolomics reveal dramatic changes in KOR1 metabolism throughout light/dark cycles. During light periods, increased flux from CO2 through glycolytic intermediates is directly observed to accompany enhanced formation of small starch-like particles, which are then efficiently repartitioned in the next dark cycle. This study demonstrates that disruption of DBE can improve biofuel production under light/dark conditions, through accelerated carbohydrate repartitioning into lipid and carotenoid.

Phylogenetic and functional diversity of aldehyde-alcohol dehydrogenases in microalgae.

KEY MESSAGE: The study shows the biochemical and enzymatic divergence between the two aldehyde-alcohol dehydrogenases of the alga Polytomella sp., shedding light on novel aspects of the enzyme evolution amid unicellular eukaryotes. Aldehyde-alcohol dehydrogenases (ADHEs) are large metalloenzymes that typically perform the two-step reduction of acetyl-CoA into ethanol. These enzymes consist of an N-terminal acetylating aldehyde dehydrogenase domain (ALDH) and a C-terminal alcohol dehydrogenase (ADH) domain. ADHEs are present in various bacterial phyla as well as in some unicellular eukaryotes. Here we focus on ADHEs in microalgae, a diverse and polyphyletic group of plastid-bearing unicellular eukaryotes. Genome survey shows the uneven distribution of the ADHE gene among free-living algae, and the presence of two distinct genes in various species. We show that the non-photosynthetic Chlorophyte alga Polytomella sp. SAG 198.80 harbors two genes for ADHE-like enzymes with divergent C-terminal ADH domains. Immunoblots indicate that both ADHEs accumulate in Polytomella cells growing aerobically on acetate or ethanol. ADHE1 of ~ 105-kDa is found in particulate fractions, whereas ADHE2 of ~ 95-kDa is mostly soluble. The study of the recombinant enzymes revealed that ADHE1 has both the ALDH and ADH activities, while ADHE2 has only the ALDH activity. Phylogeny shows that the divergence occurred close to the root of the Polytomella genus within a clade formed by the majority of the Chlorophyte ADHE sequences, next to the cyanobacterial clade. The potential diversification of function in Polytomella spp. unveiled here likely took place after the loss of photosynthesis. Overall, our study provides a glimpse at the complex evolutionary history of the ADHE in microalgae which includes (i) acquisition via different gene donors, (ii) gene duplication and (iii) independent evolution of one of the two enzymatic domains.

Structural insight into a GH1 ß-glucosidase from the oleaginous microalga, Nannochloropsis oceanica.

Marine microalgae are promising sources of novel glycoside hydrolases (GHs), which have great value in biotechnical and industrial applications. Although many GH1 family ß-glucosidases have been extensively studied, studies on ß-glucosidases from microalgae are rare, and no structure of algal GH1 ß-glucosidase has been reported. Here, we report the biochemical and structural study of a GH1 ß-glucosidase BGLN1 from Nannochloropsis oceanica, an oleaginous microalga. Phylogenetic analysis of BGLN1, together with the known structures of GH1 ß-glucosidases, has indicated that BGLN1 is branched at the root of the eukaryotic part of the phylogenetic tree. BGLN1 showed higher activity against laminaribiose compared to cello-oligosaccharides. Unlike most of the other GH1 ß-glucosidases, BGLN1 is partially inhibited by metal ions. The crystal structure of BGLN1 revealed that BGLN1 adopts a typical (α/ß)8-barrel fold with variations in loops and N-terminal regions. BGLN1 contains extra residues at the N-terminus, which are essential for maintaining protein stability. BGLN1 has a more acidic substrate-binding pocket than other ß-glucosidases, and the variations beyond the conserved -1 site determine the substrate specificity. These results indicate that GH enzymes from microalgae may have unique structural and functional features, which will provide new insight into carbohydrate synthesis and metabolism in marine microalgae.

Heterologous Production of Polyunsaturated Fatty Acids in E. coli Using Δ5-Desaturase Gene from Microalga Isochrysis Sp.

Eicosapentaenoic acid (EPA) and arachidonic acid (ARA) are long-chain polyunsaturated fatty acids (PUFAs) that play a significant role in human growth and development, which deficiency can trigger several metabolic-related diseases. Since the availability of PUFA sources is limited, there arises a need to explore alternative sources. Therefore, the present study aimed to investigate whether an Escherichia coli which are engineered with Δ5Des-Iso gene isolated from Isochrysis sp. could be utilized to synthesize PUFAs. Full-length gene Δ5Des-Iso (1149 bp) was isolated from Isochrysis sp. that encodes 382 amino acids and identified as Δ5-desatruase gene using different bioinformatic analysis. Heterologous gene expression was carried out in E. coli having Δ5Des-Iso with precursor fatty acids. The Δ5Des-Iso produced novel fatty acids of EPA (ω-3) and ARA (ω-6) as respective products were identified by GC-MS. Gene expression and PUFA synthesis in E. coli were optimized by temperature, time, and concentrations of precursor fatty acid substrates. Δ5Des-Iso RNA transcript level was inversely proportional to the time and fatty acid synthesis. And, the significant production of EPA (4.1 mg/g) and ARA (8.3 mg/g) in total fatty acids was observed in E. coli grown at 37 °C for 24 h with 25 µM of external fatty acid substrate as an optimum growth conditions. E. coli could be used as alternative organism to synthesis PUFAs and widely applicable in many nutraceuticals and pharmaceuticals industry for human use.

Electrogenesis in Plasma Membrane Fraction of Halotolerant Microalga Dunaliella maritima and Effects of N,N'-Dicyclohexylcarbodiimide.

The effects of N,N'-dicyclohexylcarbodiimide (DCCD), non-specific inhibitor of various transport systems functioning in biological membranes, on Na+-transporting P-type ATPase of the green halotolerant microalga Dunaliella maritima were studied in the experiments with vesicular plasma membranes isolated from the alga cells. The effects of DCCD on electrogenic/ion transport function of the enzyme and its ATP hydrolase activity were investigated. Electrogenic/ion transport function of the enzyme was recorded as a Na+-dependent generation of electric potential on the vesicle membranes with the help of the potential-sensitive probe oxonol VI. It was found that unlike many other ion-transporting ATPases, the Na+-ATPase of D. maritima is insensitive to DCCD. This agent did not inhibit either ATP hydrolysis catalyzed by this enzyme or its transport activity. At the same time DCCD affected the ability of the vesicle membranes to maintain electric potential generated by the D. maritima Na+-ATPase. The observed effects can be explained based on the assumption that DCCD interacts with the Na+/H+ antiporter in the plasma membrane of D. maritima.

Toxicity of ten herbicides to the tropical marine microalgae Rhodomonas salina.

Herbicide contamination of nearshore tropical marine ecosystems is widespread and persistent; however, risks posed by most 'alternative' herbicides to tropical marine microalgae remain poorly understood. Experimental exposures of the important but understudied microalgae Rhodomonas salina to seven individual Photosystem II (PSII) inhibitor herbicides (diuron, metribuzin, hexazinone, tebuthiuron, bromacil, simazine, propazine) led to inhibition of effective quantum yield (ΔF/Fm') and subsequent reductions in specific growth rates (SGR). The concentrations which reduced ΔF/Fm' by 50% (EC50) ranged from 1.71-59.2 µg L-1, while the EC50s for SGR were 4-times higher, ranging from 6.27-188 µg L-1. Inhibition of ΔF/Fm' indicated reduced photosynthetic capacity, and this correlated linearly with reduced SGR (R2 = 0.89), supporting the application of ∆F/Fm' inhibition as a robust and sensitive indicator of sub-lethal toxicity of PSII inhibitors for this microalga. The three non-PSII inhibitor herbicides (imazapic, haloxyfop and 2,4-Dichlorophenoxyacetic acid (2,4-D)) caused low or no toxic responses to the function of the PSII or growth at the highest concentrations tested suggesting these herbicides pose little risk to R. salina. This study highlights the suitability of including R. salina in future species sensitivity distributions (SSDs) to support water quality guideline development for the management of herbicide contamination in tropical marine ecosystems.

Functional expression of polyethylene terephthalate-degrading enzyme (PETase) in green microalgae.

BACKGROUND: For decades, plastic has been a valuable global product due to its convenience and low price. For example, polyethylene terephthalate (PET) was one of the most popular materials for disposable bottles due to its beneficial properties, namely impact resistance, high clarity, and light weight. Increasing demand of plastic resulted in indiscriminate disposal by consumers, causing severe accumulation of plastic wastes. Because of this, scientists have made great efforts to find a way to biologically treat plastic wastes. As a result, a novel plastic degradation enzyme, PETase, which can hydrolyze PET, was discovered in Ideonella sakaiensis 201-F6 in 2016. RESULTS: A green algae, Chlamydomonas reinhardtii, which produces PETase, was developed for this study. Two representative strains (C. reinhardtii CC-124 and CC-503) were examined, and we found that CC-124 could express PETase well. To verify the catalytic activity of PETase produced by C. reinhardtii, cell lysate of the transformant and PET samples were co-incubated at 30 °C for up to 4 weeks. After incubation, terephthalic acid (TPA), i.e. the fully-degraded form of PET, was detected by high performance liquid chromatography analysis. Additionally, morphological changes, such as holes and dents on the surface of PET film, were observed using scanning electron microscopy. CONCLUSIONS: A PET hydrolyzing enzyme, PETase, was successfully expressed in C. reinhardtii, and its catalytic activity was demonstrated. To the best of our knowledge, this is the first case of PETase expression in green algae.

The combined toxicity influence of microplastics and nonylphenol on microalgae Chlorella pyrenoidosa.

Microplastics and nonylphenol (NP) are considered as emerging pollutant and have attracted wide attention, while their combined toxicity on aquatic organisms is barely researched. Therefore, the combined toxicity influence of NP with three types of microplastics containing polyethylene (PE1000, 13 µm and PE, 150 µm), polyamide (PA1000, 13 µm and PA, 150 µm) polystyrene (PS, 150 µm) on microalgae Chlorella pyrenoidosa was analyzed. Both growth inhibition, chlorophyll fluorescence, superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) were determined. We found that single microplastics and NP both inhibited algal growth, thereby causing oxidative stress. The order of inhibition effect in single microplastics experiment was PE1000 > PA1000 > PE ≈ PS > PA. The combined toxicity experiment results indicated that the presence of microplastics had positive effect in terms of alleviating NP toxicity to C. pyrenoidosa, and the microplastics adsorption capacity to NP was the dominant contributing factor for this effect. According to the independent action model, the combined toxicity was antagonistic. Because the negative effect of smaller size microplastics on algal growth was aggravated with prolonged exposure time, the optimum effect of microplastics alleviated NP toxicity was PA1000 at 48 h, while this effect was substituted by PA at 96 h during combined toxicity. Thus, the toxicity of smaller size microplastics has a nonnegligible influence on combined toxicity. This study confirms that microplastics significantly affected the toxicity of organic pollutants on microalgae. Further research on the combined toxicity of smaller size microplastics with pollutants in chronic toxicity is needed.

dEMBF v2.0: An Updated Database of Enzymes for Microalgal Biofuel Feedstock.

In light of increasing algal genomics data and knowledge of biosynthetic pathways responsible for biofuel production, an integrated resource for easy access to all information is essential to improve our understanding of algal lipid metabolism. Against this backdrop, dEMBF v2.0, a significantly updated and improved version of our database of microalgae lipid biosynthetic enzymes for biofuel production, has been developed. dEMBF v2.0 now contains a comprehensive annotation of 2018 sequences encoding 35 enzymes, an increase of over 7-fold as compared with the first version. Other improved features include an increase in species coverage to 32 algal genomes, analysis of additional metabolic pathways, expanded annotation thoroughly detailing sequence and structural features, including enzyme-ligand interactions, and integration of supporting experimental evidence to demonstrate the role of enzymes in increasing lipid content. Along with a complete redesign of the interface, the updated database provides several inbuilt tools and user-friendly functionalities for more interactive and dynamic visualization of data.

Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl-CoA binding protein.

Microalgal oils in the form of triacylglycerols (TAGs) are broadly used as nutritional supplements and biofuels. Diacylglycerol acyltransferase (DGAT) catalyzes the final step of acyl-CoA-dependent biosynthesis of TAG, and is considered a key target for manipulating oil production. Although a growing number of DGAT1s have been identified and over-expressed in some algal species, the detailed structure-function relationship, as well as the improvement of DGAT1 performance via protein engineering, remain largely untapped. Here, we explored the structure-function features of the hydrophilic N-terminal domain of DGAT1 from the green microalga Chromochloris zofingiensis (CzDGAT1). The results indicated that the N-terminal domain of CzDGAT1 was less disordered than those of the higher eukaryotic enzymes and its partial truncation or complete removal could substantially decrease enzyme activity, suggesting its possible role in maintaining enzyme performance. Although the N-terminal domains of animal and plant DGAT1s were previously found to bind acyl-CoAs, replacement of CzDGAT1 N-terminus by an acyl-CoA binding protein (ACBP) could not restore enzyme activity. Interestingly, the fusion of ACBP to the N-terminus of the full-length CzDGAT1 could enhance the enzyme affinity for acyl-CoAs and augment protein accumulation levels, which ultimately drove oil accumulation in yeast cells and tobacco leaves to higher levels than the full-length CzDGAT1. Overall, our findings unravel the distinct features of the N-terminus of algal DGAT1 and provide a strategy to engineer enhanced performance in DGAT1 via protein fusion, which may open a vista in generating improved membrane-bound acyl-CoA-dependent enzymes and boosting oil biosynthesis in plants and oleaginous microorganisms.

Identification and Functional Characterization of Two Novel Fatty Acid Genes from Marine Microalgae for Eicosapentaenoic Acid Production.

Marine microalgae such as Isochrysis sp. and Pavlova sp. are the predominant source of polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). EPA biosynthesis pathway is predominant in lower eukaryotes, and its biosynthetic gene expressions are not well established. Till date, the C18 elongation enzymes for EPA biosynthesis have not been identified from lower eukaryote. In the present study, we describe the identification of two microalgal genes Δ6-elongase and Δ5-desaturase involved for EPA biosynthesis. By PCR-based technique, a novel elongase gene (Δ6Elo-Iso) was isolated from Isochrysis sp., and 654 bp of full-length sequence was identified, which catalysed the conversion of SDA into ETr in E. coli. The identified gene displayed unique substrate specificity for both n-3 and n-6 C18-substrates for Δ6-elongation, with no activity towards Δ9-elongase. In addition, a novel Δ5-desaturase gene (Δ5Des-Pav) was isolated from Pavlova sp. and found an ORF of 1149 bp in length, which was capable of converting ETr into EPA in omega-3 pathway. For the first time, the heterologous expressions of two novel microalgal genes were successfully expressed in Escherichia coli. EPA production from E. coli is being considered as an alternative and economic source for industrial and pharmaceutical sectors.

Targeting TOR signaling for enhanced lipid productivity in algae.

Microalgae can produce large quantities of triacylglycerols (TAGs) and other neutral lipids that are suitable for making biofuels and as feedstocks for green chemistry. However, TAGs accumulate under stress conditions that also stop growth, leading to a trade-off between biomass production and TAG yield. Recently, in the model marine diatom Phaeodactylum tricornutum it was shown that inhibition of the target of rapamycin (TOR) kinase boosts lipid productivity by promoting TAG production without stopping growth. We believe that basic knowledge in this emerging field is required to develop innovative strategies to improve neutral lipid accumulation in oleaginous microalgae. In this minireview, we discuss current research on the TOR signaling pathway with a focus on its control on lipid homeostasis. We first provide an overview of the well characterized roles of TOR in mammalian lipogenesis, adipogenesis and lipolysis. We then present evidence of a role for TOR in controlling TAG accumulation in microalgae, and draw parallels between the situation in animals, plants and microalgae to propose a model of TOR signaling for TAG accumulation in microalgae.