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Background The development of affordable strategy to concurrently enhance microalgal biomass and biocomponents is warranted for commercial applications. Here, we investigated the efficacy of a cheap and natural polyphenol, tannic acid, on regulating metabolic and physiological properties of oleaginous microalga Phaeodactylum tricornutum to overproduce biocompounds. Result Tannic acid provision regulated the key metabolic pathways to enhance algal biomass and lipids in a dose-dependent manner without direct impact on photosynthesis. It reduced oxidative stress and reallocated carbon precursors towards lipogenesis. Lipidomic analyses showed that tannic acid provision unprecedentedly regulated the key lipogenic pathways, enhanced glyco- and neutral-lipids by 1.29- and 1.54-fold, respectively, whereas phospholipids were significantly altered. Tannic acid facilitated polyunsaturated fatty acid overproduction with a specific increment of EPA and DHA by 1.18- and 2.25-fold, respectively. Transcriptomic analysis demonstrated that tannic acid upregulated the expression of multiple genes involved in lipogenesis. Conclusion Here, we dissected the potential of tannic acid, a natural and cheaper polyphenol, on concurrently enhancing lipids and PUFAs without impairing physiological properties in P. tricornutum. The findings provide novel insights into the mechanistic roles of polyphenol as a potential chemical modulator.

The established human health benefits of carotenoids along with the contemporary consumption of natural carotenoids bring the necessity to sustainable production of carotenoids. Among, marine diatoms have emerged as the potential biological resources for carotenoid production; however, their relatively lower yield in native strains provides the impetus to genetically improve the diatoms to cope with the burgeoning demand. In this study, we genetically improved the diatom Phaeodactylum tricornutum by overexpressing key carotenogenic genes involved in methylerythritol phosphate (MEP) pathway. The genes with lower relative transcript level under optimum conditions such as CMK and CMS were selected and overexpressed in P. tricornutum individually. Both CMK and CMS overexpressing lines exhibited elevated growth and photosynthesis. The expression of key carotenogenic genes such as PSY, PDS, ZDS, CRT, and LCYB was significantly upregulated. Furthermore, total carotenoid content was significantly increased; particularly, fucoxanthin content was increased by 1.83- and 1.82-fold in engineered lines CMK and CMS, respectively. Together, the results identify the potential metabolic targets and also uncover the crucial role of MEP pathway in redirecting metabolic precursors towards carotenogenesis. KEY POINTS: • Low abundant genes CMS and CMK of MEP pathway were overexpressed in the diatom • Total carotenoid content was increased, particularly fucoxanthin • Critical metabolic nodes were uncovered to accelerate fucoxanthin biosynthesis.

The unicellular freshwater green alga

Blooms of the dinoflagellate Prorocentrum donghaiense occur frequently along the East China coast and Yangtze River estuary and pose a potential threat to coastal ecosystems. However, information on the genetic diversity of P. donghaiense populations in the East China Sea remains limited despite its ecological significance. Here we developed nine microsatellite markers based on transcriptome sequencing and analyzed genetic diversity and structure of P. donghaiense population in the East China Sea. We found that the Nei’s genetic diversity index of P. donghaiense population varied from 0.019 to 0.551 with an average of 0.227, whereas Shannon’s index ranged from 0.055 to 0.968 with an average of 0.431. Moreover, four genetically distinct subpopulations were identified based on STRUCTURE analysis. These results clearly indicated that P. donghaiense population during the blooms had high genetic diversity in the East China Sea, which might form the basis for variability in phenotype of P. donghaiense population and empower its capability to adapt to changing environment. Collectively our findings uncovered the genetic distinctiveness in P. donghaiense populations between 2014 and 2016 and provide valuable insights into the genetic structure and diversity of P. donghaiense blooms.

The replacement of fossil fuels with clean and renewable biofuels is of both research and market interest for realising a circular economy. However, microalgae-based biofuels have shown promise as alternative low-carbon biofuels to other crop-based biofuels, some key obstacles in their production remain to be addressed, such as high costs and low lipid productivity. In this study, a Chlorella sp. CSH4 was cultivated using a combined light regime and carbon supply regulation strategy to enhance sugar industrial wastewater bioremediation, biomass accumulation and lipid production. Blue light irradiance of 200 μmol photons m -2 s-1 together with 10 g/L glucose and 9.2 g/L glycerol supply was found to effectively enhance the biomass accumulation and pollutant-removal capacity of Chlorella sp. during the growth phase and its lipid production during the stationary phase. Furthermore, the biodiesel properties of the lipid retrieved from Chlorella sp., as demonstrated by its fatty acid profile, were found to be suitable for commercial application. Possible mechanisms were explored to explain how this combined strategy caused this microalga to exhibit highly efficient biomass and lipid production together with efficient pollutant removal. Moreover, upscaled semi-continuous treatment using both sugar industry wastewater and negligible carbon sources (e.g., food waste hydrolysate and crude glycerol) with a mass balance analysis was conducted to initially validate the feasibility of applying our combined strategy for microalgae based wastewater treatment. In sum, this study demonstrated the feasibility of cultivating a microalga using a combined strategy comprising a light regime and carbon supply regulation to achieve both wastewater treatment and low-carbon biofuel production., peer-reviewed

BACKGROUND: Microalgal lipid overproduction has been achieved greatly by stress treatments, which drastically impede cellular biomass and overall productivity, hence providing the impetus to develop a viable strategy to uncouple lipid enhancement from stress associated growth impairment. RESULTS: We designed a sequential strategy in Nannochloropsis oceanica, to enhance cellular biomass and carbon sink by providing optimized bicarbonate source during the growth phase, followed by low‐temperature treatment to harness the increased cellular biomass and carbon precursors for lipid hyperaccumulation. Provision of bicarbonate (1.0 g/L) remarkably promoted cellular physiological properties, which in turn enhanced cellular biomass and photosynthetic carbon sink. Subsequent low‐temperature treatment (16 °C) significantly redirected the carbon flux towards lipogenesis and significantly altered fatty acid composition. The designed strategy resulted in significant enhancement of lipid productivity by 2.9‐fold than that of control cells and resulted in lipid accumulation up to 57% of dry cell weight without impeding cellular biomass. Interestingly, the sequential treatment altered fatty acid profile, particularly a 8.8‐fold increase in the content of eicosapentaenoic acid with health significance. CONCLUSION: Our study exemplifies the feasible strategy to potentiate microalgal biomass, and also to circumvent stress associated growth impairment and consequent algal lipid productivity. © 2019 Society of Chemical Industry

Provision of chemical modulators has emerged as an effective strategy to govern cell growth and development. Here, the impact of flavonoid quercetin on algal growth, lipid accumulation and transcriptional patterns was investigated in the green microalga Chlorella vulgaris. These results demonstrated that quercetin (15 μg/l) significantly enhanced the cellular biomass and photosynthetic efficiency, with up to 2.5-fold in the biomass in the stationary phase. Lipidomic analyses revealed that lipid content was increased by 1.8-fold. Furthermore, the functional mechanism of quercetin on the molecular level was dissected by transcriptomic analysis. Results revealed that quercetin upregulated the expression pattern of key genes involved in cellular signaling mechanisms such as phosphatidylinositol 4-kinase α, thus consequently enhanced cell growth. Altogether, the data present in this study demonstrate the dramatic role of quercetin on enhancing microalgal biomass and lipid accumulation by unprecedented regulation, of key metabolic nodes, for the first time and provide a novel insight into microalgal metabolism and regulation.

Tobacco wastewater is too difficult to decontaminate which poses a significant environmental problem due to the harmful and toxic components. Chlorella pyrenoidosa is a typical microalgal species with potential in removal of organic/inorganic pollutants and proves to be an ideal algal-based system for wastewater treatment. However, the strategy of tobacco related wastewater treatment using microalgae is in urgent need of development. In this study, C. pyrenoidosa was used to evaluate the removal efficiency of artificial tobacco wastewater. Under various solid-to-liquid (g/L) ratios, 1:1 ratio and acidic pH 5.0 were optimal for C. pyrenoidosa to grow with high performance of removal capacity to toxic pollutants (such as COD, NH3-N, nicotine, nitrosamines and heavy metals) with the alleviation of oxidative damage. Algal biomass could reach up to 540.24 mg/L. Furthermore, carbon flux of C. pyrenoidosa was reallocated from carbohydrate and protein biosynthesis to lipogenesis with a high lipid content of 268.60 mg/L at pH 5.0. Overall, this study demonstrates an efficient and sustainable strategy for tobacco wastewater treatment at acidic pH with the production of valuable microalgal products, which provides a promising biorefinery strategy for microalgal-based wastewater bioremediation.

Molybdenum disulfide nanoparticles (MoS2 NPs) hold tremendous properties in wide domain of applications. In this study, the impact of MoS2 NPs was investigated on algal physiological and metabolic properties and a two-stage strategy was acquired to enhance the commercial potential of Dunaliella salina. With 50 µg/L of MoS2 NPs exposure, cellular growth and biomass production were promoted by 1.47- and 1.33-fold than that in control, respectively. MoS2 NPs treated cells were subject to high light intensity for 7 days after 30 days of normal light cultivation, which showed that high light intensity gradually increased β-carotene content by 1.48-fold. Furthermore, analyses of primary metabolites showed that combinatorial approach significantly altered the biochemical composition of D. salina. Together, these findings demonstrated that MoS2 NPs at an optimum concentration combined with high light intensity could be a promising approach to concurrently enhance biomass and β-carotene production in microalgae.

Waste streams have emerged as potential feedstocks for biofuel production via microbial bioconversion. Metabolic engineering of the microalga Phaeodactylum tricornutum in its lipid biosynthetic pathways has been conducted with an aim to improve lipid production. However, there has been only limited achievement in satisfying biofuel demands by utilising extracellular organic carbons from low-cost waste streams. Herein, we present a successive staged cultivation mode, based on a previously engineered strain that co-overexpresses two key triacylglycerol biosynthesis genes. We first optimised microalgal biomass and lipid production by using food waste hydrolysate and crude glycerol as the cultivation media. Food waste hydrolysate (5% v/v) is a low-cost organic carbon source for enhanced microalgal biomass production, and the resulting lipid concentration was 1.08-fold higher with food-waste hydrolysate than that of the defined medium. Additionally, the resultant lipid concentration after using crude glycerol (100 mM) was 1.24-fold higher than that using the defined medium. Two carbon feeding modes (hybrid and sequential) were also performed to investigate the potential of engineered P. tricornutum with preliminary mechanistic analyses. The biodiesel properties of lipids produced in the hybrid mode were evaluated for potential application prospects. Collectively, this study demonstrates a waste stream utilisation strategy for efficient and sustainable microalgal biofuel production.

Additional file 1: Fig. S1. In silico analyses of GPAT2 sequence. (A) Protein structure of GPAT2. (B) Phylogeny analysis of GPAT2 from the deduced amino acid sequence of GPAT2 and GPAT of various organisms. Phylogenetic tree was constructed by MEGA using Neighbor joining method. Percentage of the replicate trees where the taxa clustered together in the bootstrap value (500 replicates) are provided in the branches. Arrow mark denotes the GPAT2 of P. tricornutum. Fig. S2. Analyses of photosynthetic parameters in transgenics. (A) Maximum quantum yield of photosystem II as indicated by Fv/Fm. (B) Determination of chlorophyll α content (pg/cell). (C) Non-photo chemical quenching (NPQ). (D) Electron transport rate (ETR) (µmol e-m-2s-1). Significant difference between WT and transgenics is indicated at the p

Antifreeze proteins (AFP) are produced by certain plants, animals, fungi and bacteria that enable them to survive upon extremely low temperature. Perennial rye grass, Lolium perenne, was reported to possess AFP which protects them from cold environments. In the present investigation, we isolated AFP gene from L. perenne and expressed it in tomato plants to elucidate its role upon chilling stress. The T1 transgenic tomato lines were selected and subjected to molecular, biochemical and physiological analyses. Stable integration and transcription of LpAFP in transgenic tomato plants was confirmed by Southern blot hybridization and RT-PCR, respectively. Physiological analyses under chilling conditions showed that the chilling stress induced physiological damage in wild type (WT) plants, while the transgenic plants remained healthy. Total sugar content increased gradually in both WT and transgenic plants throughout the chilling treatment. Interestingly, transgenic plants exhibited remarkable alterations in terms of relative water content (RWC) and electrolyte leakage index (ELI) than those of WT. RWC increased significantly by 3-fold and the electrolyte leakage was reduced by 2.6-fold in transgenic plants comparing with WT. Overall, this report proved that LpAFP gene confers chilling tolerance in transgenic tomato plants and it could be a potential candidate to extrapolate the chilling tolerance on other chilling-sensitive food crops.

Microalgal metabolic engineering holds great promise for algal biofuels. However, identification of the key lipid metabolic target remains challenging due to its complex regulation. In this study, we advocated an alternative strategy that potentially rewired lipid metabolism by unprecedented mechanisms. PNPLA3, a human protein associated with non-alcoholic fatty liver disease (NAFLD), was firstly tested in microalgae for enhancing lipid accumulation. HsPNPLA3 was synthesized with a site mutation (I148M) and expressed in a model diatom Phaeodactylum tricornutum . Heterogeneous HsPNPLA3-I148M was successfully integrated, transcribed and expressed. Lipidomic analyses revealed that HsPNPLA3-I148M significantly elevated TAG content by 1.55-fold in algae, while algal growth and photosynthetic rate were not impaired. Fatty acid profile showed that content of C16:0, C18:1 and C20:4 was increased by 1.43-, 4.18- and 4.3-fold, respectively, which implied that HsPNPLA3-I148M might regulate the fatty acid substrate preference. Overall, the findings demonstrated that human PNPLA3 played a potential role in elevating TAG accumulation by regulating lipogenic enzymes and provide unprecedented insights into its functional significance.

Oleaginous microalgae have emerged as the promising feedstock for biofuels due to their various advantageous features over terrestrial plants. However, the microalgal fuel industry is still in its infancy due to the unavailability of robust algal strains with enhanced lipid content and biomass and economically feasible oil extraction strategies. Microalgal metabolic engineering shows the great potential to enhance lipid accumulation without impairing cell growth by targeted control of key metabolic nodes. Despite the recent advancements in synthetic biology, genetic improvement of microalgae without hindered cellular biomass remains an untapped tool for large-scale biofuel production. In this review, we therefore provide a comprehensive overview of various metabolic engineering strategies for lipid enhancement and the necessity of designing the appropriate strategy for scale-up processes. We have emphasized the need of streamlining the engineering strategies with the aid of recent advancements in DNA manipulation techniques to obviate the existing biological intricacies in lipogenesis. We outline the significance of recruiting potential engineering strategies such as transcription engineering, heterologous recombineering, and genome engineering to generate chassis algal strains successfully. Besides, this review highlights the designing of modular strategies by subjecting the engineered strains under sub-optimal conditions, mixotrophic cultivations and chemical treatment to overcome the existing bottlenecks in microalgal commercialization. This review also discusses the current large-scale algal cultivation units and the drawbacks. Overall, this review provides insights into employing the metabolic rewiring strategies to engineer the intricate lipogenic circuit in microalgae for enhancing biofuel potential.

Microalgae encounter a range of adverse conditions which restricts algal biomass, thereby the primary productivity is constrained. Death-specific proteins (DSP) play a crucial role in orchestrating cellular growth under adverse conditions. However, the functional role of DSPs in diatoms remains scant. Here, we identified a DSP (designated as PtDSP) in the model diatom Phaeodactylum tricornutum and elucidated its role in alleviating cellular growth and photosynthesis in a stress-dependent manner. Plastid-localized PtDSP upheld the cellular physiological properties under specific stress conditions. Overexpression of PtDSP potentiated the cells to withstand iron deprivation rather than nitrogen and phosphorus deprivation. Under iron deprivation, PtDSP orchestrated the expression of key genes involved in photosynthesis, thereby combated iron deprivation. This report epitomizes the role of DSP in governing diatom physiological properties in a stress-dependent manner and advances our understanding of the systematic response of diatoms to environmental changes.

Metabolic engineering has been hailed as a promising tool to impart oil productivity in fast-growing microalgae, thereby generating industry-suitable strains. However, complex regulatory networks in lipogenesis hampered the success to unify the abundance of metabolic carbon precursors and reducing equivalents towards lipogenesis. To obviate the biological bottlenecks, we attempted to rewire the pyruvate pathway, which could enhance the supply of NADPH by heterologously overexpressing a malic enzyme from the oleaginous diatom Phaeodactylum tricornutum (PtME) in fast-growing microalga Tetradesmus obliquus (previously known as Scenedesmus obliquus). PtME overexpression did not impair physiological properties and upheld lipogenesis by facilitating the adequate provision of CO2 and NADPH, respectively, through malate decarboxylation. The malate-pyruvate pathway played a role in elevating lipid content by 2.4-fold via coupling the carbon precursors and reducing equivalents. The malic enzyme activity increased by 9.9-fold, thereby elevated lipogenic NADPH. Together, these data exemplify the crucial role of PtME in pyruvate cycle for amalgamating the provision of metabolic carbon precursors and NADPH towards lipid accumulation and lay a strong foundation for metabolic engineering in algal industrialization.

Lipid droplets (LDs) are lipid monolayer-enclosed organelles comprising a lipid core and surface associated-proteins. However, the protein components and their regulatory functions in LDs have remained largely unknown in oleaginous diatoms. In this study, we identified a gene encoding lipid droplet (LD)-associated protein ( PtLDP1 ) in Phaeodactylum tricornutum and examined its function. The PtLDP1 showed homology to the diatom-oleosome-associated protein 1 (DOAP1) from Fistulifera . Overexpression of the PtLDP1 gene elevated lipid content, enlarged LD size and increased relative expression levels of key genes involved in triacylglycerol (TAG) and fatty acid biosynthesis. In contrast, knockdown of PtLDP1 by RNAi decreased lipid and TAG content, and subsequently reduced LD size. In addition, LDs were isolated from P. tricornutum cells and the proteome of LDs was identified by mass spectrometry. We found that PtLDP1 was a significant protein in the LD proteome. Importantly, labeling of enhanced yellow fluorescent protein (EYFP) confirmed that the PtLDP1 was localized to the LDs. Altogether, our data suggest that the PtLDP1 could be an important LD-associated protein contributing to regulation of TAG synthesis and lipogenesis. The findings will provide new targets for genetic improvement of oleaginous microalgae.

Microalgal long-chain polyunsaturated fatty acids (LC-PUFAs) have emerged as promising alternatives to depleting fish oils. However, the overproduction of LC-PUFAs in microalgae has remained challenging. Here, we report a sequential metabolic engineering strategy that systematically overcomes the metabolic bottlenecks and overproduces LC-PUFAs. Malonyl CoA-acyl carrier protein transacylase, catalyzing the first committed step in type II fatty acid synthesis, and desaturase 5b, involved in fatty acid desaturation, were coordinately expressed in Phaeodactylum tricornutum. Engineered microalgae hyper-accumulated LC-PUFAs, with arachidonic acid (ARA) and docosahexaenoic acid (DHA) contents of up to 18.98 μg/mg and 9.15 μg/mg (dry weight), respectively. Importantly, eicosapentaenoic acid (EPA) was accumulated up to a highest record of 85.35 μg/mg by metabolic engineering. ARA and EPA were accumulated mainly in triacylglycerides, whereas DHA was found exclusively in phospholipids. Combinatorial expression of these critical enzymes led to the optimal increment of LC-PUFAs without unbalanced metabolic flux and demonstrated the practical feasibility of generating sustainable LC-PUFA production.

There is a huge interest in exploiting microalgae as an alternative bioenergy resource. Genetic improvement of microalgal strains with significant commercial potential is urgently demanded. Seipin has been shown to be associated with adipocyte differentiation and lipid droplet (LD) formation in mammals. However, very little is known about seipin in microalgae. Here, we identified a putative seipin in the oleaginous marine diatom Phaeodactylum tricornutum and developed a transgenic strain with overexpressed seipin. The transgenic strain possessed higher neutral lipid content and larger LDs than the wild type, and neutral lipid content was increased by 57%. Moreover, in this transgenic strain, the relative content of saturated fatty acids in total fatty acids was increased significantly, which improved lipid quality for the oil refinery. Our findings elucidated the significant potential role of seipin in regulating lipid accumulation and LD formation in microalgae for the first time.

Oleaginous microalgae hold great promises for biofuel production. However, commercialization of microalgal biofuels remains impracticable due to the lack of suitable industrial strains with high growth rate and lipid productivity. Engineering of metabolic pathways is a potential strategy for the improvement of microalgal strains for the production of lipids and also value-added products in microalgae. Malonyl CoA-acyl carrier protein transacylase (MCAT) has been reported to be involved in fatty acid biosynthesis. Here, we identified a putative MCAT in the oleaginous marine microalga Nannochloropsis oceanica. NoMCAT overexpressing N. oceanica showed a higher growth rate and photosynthetic efficiency. The neutral lipid content of engineered lines showed a significant increase by up to 31% compared to wild type. Gas chromatography-mass spectrometry analysis revealed that NoMCAT overexpression significantly altered the fatty acid composition. The composition of eicosapentaenoic acid (C20:5), which is a polyunsaturated fatty acid necessary for animal nutrition, increased by 8%. These results demonstrate the role of MCAT in enhancing fatty acid biosynthesis and growth in microalgae, and also provide an insight into metabolic engineering of microalgae with high industrial potential.

This study investigates the prospective of utilizing kitchen wastewater and food wastes, biofuels industry byproducts as alternative water and carbon sources. Kitchen wastewater did not impede cellular growth rate of the evolved Phaeodactylum strain E70, which indicates its potential as an alternative to freshwater resources. Among the organic wastes assessed, food waste hydrolysate significantly increased cell growth. Supplement of crude glycerol in cultivation medium enhances the total fatty acid content. Mixed food waste hydrolysate and crude glycerol remarkably increased both the cell density and total fatty acid content. Also, the supplement of butylated hydroxytoluene alleviated the oxidative stress induced by impurities in organic wastes and concomitantly increased microalgal total fatty acids and polyunsaturated fatty acids content. The experimental results reported in this study show that a waste-based biorefinery could lead to utilization of organic waste resources for the efficient production of value-added products.

Electroporation refers to the application of high strength electric pulse to create transient pores in the membrane, thereby enabling the passage of hydrophilic molecules into the cells. Based on the properties of cell and cell wall, the electroporation parameters vary among the algal species. Here, we demonstrated the optimized protocol for successful introduction of recombinant DNA (~5000 bp) into Nannochloropsis oceanica. The linearized recombinant plasmid that harbors eGFP and Bh-sle as the reporter and marker gene, respectively, was electroporated into the electrocompetent N. oceanica cells at voltage of 2200 V, 50 μF, resistance at 600 Ω using electroporator, and the transformed cells were then screened by molecular analysis. The report exemplifies a straightforward and reliable electroporation strategy for generating transgenic N. oceanica cells.

Electroporation refers to the application of high strength electric pulse to create transient pores in the membrane, thereby enabling the passage of hydrophilic molecules into the cells. Based on the properties of cell and cell wall, the electroporation parameters vary among the algal species. Here, we demonstrated the optimized protocol for successful introduction of recombinant DNA (~5000 bp) into Nannochloropsis oceanica. The linearized recombinant plasmid that harbors eGFP and Bh-sle as the reporter and marker gene, respectively, was electroporated into the electrocompetent N. oceanica cells at voltage of 2200 V, 50 μF, resistance at 600 Ω using electroporator, and the transformed cells were then screened by molecular analysis. The report exemplifies a straightforward and reliable electroporation strategy for generating transgenic N. oceanica cells.

Haematococcus pluvialis is a main biological resource for the antioxidant astaxanthin production, however, potential modulators and molecular mechanisms underpinning astaxanthin accumulation remain largely obscured. We discovered that provision of ethanol (0.4%) significantly triggered the cellular astaxanthin content up to 3.85% on the 4th day of treatment. Amongst, 95% of the accumulated astaxanthin was esterified, particularly enriched with monoesters. Ultrastructural analysis revealed that ethanol altered cell wall structure and physiological properties. Antioxidant analyses revealed that astaxanthin accumulation offset the ethanol induced oxidative stress. Ethanol treatment reduced carbohydrates while increased lipids and jasmonic acid production. Transcriptomic analysis uncovered that ethanol orchestrated the expression of crucial genes involved in carotenogenesis, e.g. PSY, BKT and CRTR-b were significantly upregulated. Moreover, methyl jasmonic acid synthesis was induced and played a major role in regulating the carotenogenic genes. The findings uncovered the novel viewpoint in the intricate transcriptional regulatory mechanisms of astaxanthin biosynthesis.

There has been growing interest in using microalgae as production hosts for a wide range of value-added compounds. However, microalgal genetic improvement is impeded by lack of genetic tools to concurrently control multiple genes. Here, we identified two novel strong promoters, designated Pt202 and Pt667, and delineated their potential role on simultaneously driving the expression of key lipogenic genes in Phaeodactylum tricornutum. In silico analyses of the identified promoter sequences predicted the presence of essential core cis elements such as TATA and CAAT boxes. Regulatory role of the promoters was preliminarily assessed by using GUS reporter which demonstrated strong GUS expression. Thereafter, two key lipogenic genes including malic enzyme (PtME) and 5-desaturase (PtD5b), were overexpressed by the two promoters Pt202 and Pt667, respectively, in P. tricornutum. Combinatorial gene overexpression did not impair general physiological performance, meanwhile neutral lipid content was remarkably increased by 2.4-fold. GC-MS analysis of fatty acid methyl esters revealed that eicosapentaenoic acid (EPA; C20:5) was increased significantly. The findings augment a crucial kit to microalgal genetic tools that could facilitate the multiple-gene expression driven by various promoters, and promote microalgae for industrial bioproduction.

Photosynthetic microalgae can accumulate a wide array of valuable components. However, higher titer of desired product usually occurs under sub-optimal conditions with the compromise of cellular biomass, which seriously hindered their commercial applications. Conventional metabolic engineering has been employed by targeted perturbation of selected genes in the metabolic pathway without compromising cellular growth. Nevertheless, previous studies have shown mixed and inconsistent success owing to the intricate nature of the target metabolic pathways. Transcriptional engineering represents a promising strategy to govern multiple metabolic pathways by regulation of critical transcription factors, thereby controlling the expression of target gene(s). It has exhibited potential significance and advancements in synthetic biology for microalgal strain improvement. In this chapter, we focus on the significance and status of transcriptional engineering strategies and also speculate on future development to enhance production of microalgal valuable components.

Molybdenum disulfide nanoparticles (MoS2 NPs) has emerged as the promising nanomaterial with a wide array of applications in the biomedical, industrial and environmental field. However, the potential effect of MoS2 NPs on marine organisms has yet to be reported. In this study, the effect of MoS2 NPs on the physiological index, subcellular morphology, transcriptomic profiles of the marine microalgae Dunaliella salina was investigated for the first time. exhibited “doping-like” effects on marine microalgae; Growth stimulation was 193.55%, and chlorophyll content increased 1.61-fold upon the addition of 50 μg/L MoS2 NPs. Additionally, exposure to MoS2 NPs significantly increased the protein and carbohydrate content by 2.03- and 1.56-fold, respectively. The antioxidant system was activated as well to eliminate the adverse influence of reactive oxygen species (ROS). Transcriptomic analysis revealed that genes involved in porphyrin synthesis, glycolysis/gluconeogenesis, tricarboxylic acid cycle and DNA replication were upregulated upon MoS2 NPs exposure, which supports the mechanistic role of MoS2 NPs in improving cellular growth and photosynthesis. The “doping-like” effects on marine algae suggest that the low concentration of MoS2 NPs might change the rudimentary ecological composition in the ocean.

Increasing population and urbanization bring along depletion of water resources with notable discharge of industrial and municipal wastewater which is rich in nutrients. Microalgae hold great promise for assimilating the nutrient compounds, particularly the intricate nitrogen (N) and phosphorus (P) compounds from wastewater, resulting in algal biomass enriched with various valuable biomolecules. However, development of strategies involving the cultivation of microalgae for wastewater treatment and production of commodity components is still in its infancy. In this review, the current status of microalgae-mediated wastewater treatment, the existing technological bottlenecks that hamper the effective use of photosynthetic microalgae, and innovative solutions to obviate the shortcomings are discussed. Also, this article highlights the potential use of genetic engineering technologies in overproducing algal biomolecules without impeding cellular biomass accumulation by regulating critical metabolic targets for the development of engineered algal strains. In this context, adaptive laboratory evolution (ALE) could be the promising tool to further empower the engineered strains to mitigate the adverse effect from the wastewater cultivation medium. In summary, this review provides novel insights into the application of genetic engineering strategies for overproducing algal biomass and valuable biomolecules such as lipids and polyunsaturated fatty acids using wastewater in a cost-effective manner.

"Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems.

Photosynthetic microalgae have garnered research attention due to their inherent advantageous characteristics, however, commercial exploitation of algae is still hampered by their relatively compromised overall productivity. Thus, it is of paramount significance to design a strategy to concurrently enhance the growth rate and compounds production. In this study, we interrogated the impact of fortifying bacterial growth medium components in the conventional f/2-medium for microalgae. Our results showed that supplement of yeast extract and tryptone significantly increased cell growth of the model diatom Phaeodactylum tricornutum. Particularly, cell biomass and triacylglyceride content increased up to 3.48-fold and 2.13-fold in the stationary phase, respectively. Besides, fucoxanthin content increased by 1.7-fold. Transcript abundance of key genes involved in carotenoid synthesis also increased significantly. Overall, the results exemplified a feasible and promising strategy to simultaneously enhance the biomass and accumulation of triacylglyceride and fucoxanthin for industrial applications of microalgae.

NobZIP1 governs the expression of various key genes, leading to lipid overproduction and secretion in Nannochloropsis oceanica., Commercialization of algal lipids and biofuels is still impractical owing to the unavailability of lipogenic strains and lack of economically viable oil extraction strategies. Because lipogenesis is governed by multiple factors, success in generating industrial-suitable algal strains using conventional strategies has been limited. We report the discovery of a novel bZIP1 transcription factor, NobZIP1, whose overexpression results in a remarkable elevation of lipid accumulation and lipid secretion in a model microalga Nannochloropsis oceanica, without impairing other physiological properties. Chromatin immunoprecipitation–quantitative PCR analysis revealed that the key genes up- and down-regulated by NobZIP1 are involved in lipogenesis and cell wall polymer synthesis, respectively, which, in turn, induce lipid overproduction and secretion. Among these regulated genes, UDP-glucose dehydrogenase was shown to alter cell wall composition, thus also boosting lipid secretion. In summary, these results offer a comprehensive strategy for concurrent lipid overproduction and secretion, strongly increasing the commercial potential of microalgae.

Commercial production of biofuel from oleaginous microalgae is often impeded by their slow growth rate than other fast-growing algal species. A promising strategy is to genetically engineer the fast-growing algae to accumulate lipids by expressing key lipogenic genes from oleaginous microalgae. However, lacking of strong expression cassette to transform most of the algal species and potential metabolic target to engineer lipid metabolism has hindered its biotechnological applications. In this study, we engineered the oxidative pentose phosphate pathway (PPP) of green microalga Chlorella pyrenoidosa for lipid enhancement by expressing a glucose-6-phosphate dehydrogenase (G6PD) from oleaginous diatom Phaeodactylum tricornutum. Molecular characterization of transformed lines revealed that heterologous PtG6PD was transcribed and expressed successfully. Interestingly, subcellular localization analyses revealed that PtG6PD was targeted to chloroplasts of C. pyrenoidosa. PtG6PD expression remarkably elevated NADPH content and consequently enhanced the lipid content without affecting growth rate. Collectively, this report represents a promising candidate to engineer lipid biosynthesis in heterologous hosts with notable commercial significance, and it highlights the potential role of plastidial PPP in supplying lipogenic NADPH in microalgae.

Additional file 1: Figure S1. Protein structure of GPAT1 and LPAT1 obtained using SMART ( http://smart.embl-heidelberg.de ) and ChloroP ( http://www.cbs.dtu.dk/services/ChloroP/ ). TM, transmembrane; SP, signal peptide; LPLAT and PlsC represented the conserved domains in proteins. Figure S2. Schematic representation of the expression vectors employed in this study. GPAT1 and LPAT1 genes were cloned into the expression vectors pHY18 (A) and pHY21 (C), respectively under the control of promoter PfcpC. An omega leader sequence and “ACC” nucleotides were inserted between the promoter and the target gene for boosting protein translation. For subcellular localization, GPAT1-EGFP (B) and LPAT1-EGFP (D) were employed under the control of promoter ProPtAP. Figure S3. Subcellular localization of GPAT1 and LPAT1 in P. tricornutum cells. A, Microscopy images of a representative wild-type cell; B, Microscopy images of a representative transgenic line with co-overexpression of GPAT1 and EGFP; C, Microscopy images of a representative transgenic line with co-overexpression of LPAT1 and EGFP. From left to right, fluorescence of EGFP, autofluorescence of chloroplasts, differential interference contrast (DIC), fluorescence images overlaid on DIC image. Scale bars represent 5 μm. Figure S4. PCR validation by agarose gel electrophoresis to verify the antibiotic gene Shble (349 bp), CAT (500 bp) and endogenous gene 18s rDNA (498 bp) PCR product. V1, pHY 21 vector (containing Shble gene); V2, pHY 18 vector (containing CAT gene); OE1 and OE2, individually overexpressed transformants; WT, wild type; B, negative control; M, marker. Figure S5. Glycolipid content in overexpression lines (OE1 and OE2) and wild-type (WT) harvested at day 4 and 7. A significant difference between WT and OE lines is indicated at the p

Patatin-like phospholipase domain-containing protein 3 (PNPLA3) has been associated with nonalcoholic fatty liver disease which promoted hepatic lipid synthesis. With high lipid content, microalgae are found to be a potential source of biofuel. Phaeodactylum tricornutum , a fast-growing and oleaginous microalga, is a promising target for enhancing the biofuel by means of metabolic engineering. Here a putative PtPNPLA3 gene from P. tricornutum was cloned and characterized in transgenic P. tricornutum for the first time. Amino acid sequence analysis showed a high homology between PNPLA3 from P. tricornutum and other organisms. Overexpression of PtPNPLA3 increased the transcript level of PtPNPLA3 by 70% in transgenic microalgae compared to wild type. The neutral lipid content in transgenic microalgae was significantly increased by 70%, representing a notable enhancement of the lipid productivity in the transgenic microalgae. The fatty acid profile was also altered as determined by GC–MS analysis, with a significant increase of C20:4 in the transgenic microalgae compared to wild type. This work identified a microalgal PNPLA3 and proved it to be an important node in regulating lipid accumulation in microalgae, also demonstrating an efficient way to improve lipid productivity in microalgae by metabolic engineering.

Background Microalgae have emerged as a potential feedstock for biofuels and bioactive components. However, lack of microalgal strains with promising triacylglycerol (TAG) content and desirable fatty acid composition have hindered its commercial feasibility. Attempts on lipid overproduction by metabolic engineering remain largely challenging in microalgae. Results In this study, a microalgal 1-acyl-sn-glycerol-3-phosphate acyltransferase designated AGPAT1 was identified in the model diatom Phaeodactylum tricornutum. AGPAT1 contained four conserved acyltransferase motifs I–IV. Subcellular localization prediction and thereafter immuno-electron microscopy revealed the localization of AGPAT1 to plastid membranes. AGPAT1 overexpression significantly altered the primary metabolism, with increased total lipid content but decreased content of total carbohydrates and soluble proteins. Intriguingly, AGPAT1 overexpression coordinated the expression of other key genes such as DGAT2 and GPAT involved in TAG synthesis, and consequently increased TAG content by 1.81-fold with a significant increase in polyunsaturated fatty acids, particularly EPA and DHA. Moreover, besides increased lipid droplets in the cytosol, ultrastructural observation showed a number of TAG-rich plastoglobuli formed in plastids. Conclusion The results suggested that AGPAT1 overexpression could elevate TAG biosynthesis and, moreover, revealed the occurrence of plastidial TAG synthesis in the diatom. Overall, our data provide a new insight into microalgal lipid metabolism and candidate target for metabolic engineering. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0786-0) contains supplementary material, which is available to authorized users.

Additional file 2: Table S1. Acyltransferase motifs of AGPAT1/LPAT1 from various sources. Table S2. In silico prediction of four AGPAT1/LPAT1. Table S3. Proportion of fatty acids in total lipid extracts. Table S4. Proportion of fatty acids in TAGs isolated by TLC.

Additional file 1: Figure S1. Phylogenetic analysis and alignment of deduced amino-acid sequences of AGPAT/LPAT. a, Phylogenetic analysis of amino-acid sequences of the GPAT and AGPAT from several organisms. b, Sequence of AtLPAT1 (AT4G30580.1) was retrieved from TAIR database. RcLPAT1 (XP_002529386.1), CpuLPAT1 (ALM22868.1), and PtAGPAT1 (XP_002176893.1) were retrieved from NCBI database. Black boxes represent the acyltransferase motifs; arrowheads indicate the amino-acid residues that are not conserved. Figure S2. Prediction of transmembrane helix structure and topology of AGPAT1. a, Transmembrane helix predicted by SOSUI, TMHMM, HMMTOP, and their amino-acid sequences. b, Topology predicted by SOSUI. c, Topology predicted by TMHMM and HMMTOP. Figure S3. Subcellular localization of AGPAT1 in P. tricornutum. AGPAT1 was detected by immuno-gold labeling against c-Myc antibody. a & b, WT. c, AGPAT1-1. d, AGPAT1-2. Dense dots represent gold particles; Black arrows indicate the gold labeling of AGPAT1, the plastoglobulus (Pg) is indicated by red arrows. Ch: chloroplast; LD: lipid droplet. Bars: a, 2 μm; b, c & d, 1 μm; c1, 200 nm; d1 & d2, 100 nm.

Background Diatoms are able to acclimate to frequent and large light fluctuations in the surface ocean waters. However, the molecular mechanisms underlying these acclimation responses of diaotms remain elusive. Results In this study, we investigated the mechanism of high light protection in marine diatom Thalassiosira pseudonana using comparative proteomics in combination with biochemical analyses. Cells treated under high light (800 μmol photons m−2s−1) for 10 h were subjected to proteomic analysis. We observed that 143 proteins were differentially expressed under high light treatment. Light-harvesting complex proteins, ROS scavenging systems, photorespiration, lipid metabolism and some specific proteins might be involved in light protection and acclimation of diatoms. Non-photochemical quenching (NPQ) and relative electron transport rate could respond rapidly to varying light intensities. High-light treatment also resulted in increased diadinoxanthin + diatoxanthin content, decreased Fv/Fm, increased triacylglycerol and altered fatty acid composition. Under HL stress, levels of C14:0 and C16:0 increased while C20:5ω3 decreased. Conclusions We demonstrate that T. pseudonana has efficient photoprotective mechanisms to deal with HL stress. De novo synthesis of Ddx/Dtx and lipid accumulation contribute to utilization of the excess energy. Our data will provide new clues for in-depth study of photoprotective mechanisms in diatoms. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3335-5) contains supplementary material, which is available to authorized users.

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose-6-phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19-fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09-fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose-6-phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.

The expression of carrot antifreeze protein enhanced chilling tolerance in heterologous host system tomato and AFP can be a potential gene candidate for producing chilling tolerant crop plants. In an attempt to improve chilling tolerance, the carrot gene encoding the antifreeze protein (AFP) was cloned under the control of constitutive CaMV35S promoter and genetically transformed the tomato var. PKM1 using Agrobacterium-mediated genetic transformation. Putative transgenic plants were confirmed by PCR using AFP-specific primers and grown to maturity. The integration of AFP transgene in the tomato genome was confirmed by Southern blot analysis. The AFP gene expression in transgenic plants was determined using semi-quantitative reverse transcription PCR. Upon exposure to chilling stress (4 °C), a significant decrease in membrane injury index was observed in AFP transgenic tomato lines without any phenotypic aberrations when compared with WT plants. Hence, this study clearly proves that the development of chilling tolerant tomato plants will soon become a reality.

The aim of the present study was to compare the phytochemical and radical scavenging activities in the stem and leaf fractions of two species in Amaranthaceae family Amaranthus tristis and Alternanthera sessilis. Total flavonoids and phenolics were estimated using aluminium chloride and Folin-Ciocalteau methods, respectively; radical scavenging activities of the extracts were determined by 2, 2-diphenyl-1-picryl hydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assay. Our results showed that A. sessilis had higher levels of flavonoids (70.42 mg/100 g) and phenolics (103.75 mg/100 g), when compared with A. tristis that had relatively lower levels of flavonoids (62.87 mg/100 g) and phenolics (96.89 mg/100 g) in leaf fractions. The stem and leaf fractions of A. sessilis showed more DPPH and FRAP values indicating the higher radical scavenging activity of the extract, when compared to A. tristis. It was also found that the flavonoids and phenolics content are directly proportional to the radical scavenging activities of both vegetables. The results concluded that A. sessilis have relatively more phytochemicals and radical scavenging activities and it also reveals that the leaf fraction has more flavonoid and phenolic content than the stem fractions in both vegetables. It is clear that both plants have definitely more antioxidant properties making it an ideal dietary antioxidant supplement. Key words: Phytochemicals, antioxidants, total flavonoids, total phenolics, radical scavenging activity.