Recovery of nutrients from fish sludge to enhance the growth of microalga Chlorella sorokiniana CMBB276.

Intensive aquaculture production generates large amounts of sludge. This waste could be considered as a potential source of nutrients that can be recovered and utilized. Little attention has been paid to nutrient recovery from fish sludge. In this study, bioconversion of sludge was evaluated in lab scale under anaerobic (AN), facultative anaerobic (FA) and aerobic (AE) conditions. After 40 days of fermentation, AN recovered the highest values of dissolved total nitrogen (82.7 mg L-1), while AE showed the highest dissolved total phosphorus (11.8 mg L-1) and the highest reduction of total suspended solids (36.0 %). Microbial analysis showed that AN exhibited a distinct bacterial community than that of FA and AE. Furthermore, C. sorokiniana grown in AN effluents collected after 12 days of fermentation achieved the highest biomass production (1.96 g L-1). These results suggest that AN has the best potential to recover nutrients from sludge for production of C. sorokiniana.

Advanced treatment and Resource recovery of brewery wastewater by Co-cultivation of filamentous microalga Tribonema aequale and autochthonous Bacteria.

To use unicellular microalgae to remove waste nutrients from brewery wastewater while converting them into algal biomass has been explored but high-cost treatment and low-value biomass associated with current technologies have prevented this concept from further attempts. In this study, a filamentous microalga Tribonema aequale was introduced and the alga can grow vigorously in brewery wastewater and algal biomass concentration could be as high as 6.45 g L-1 which can be harvested by a cost-effective filtration method. The alga together with autochthonous bacteria removed majority of waste nutrients from brewery wastewater. Specifically, 85.39% total organic carbon (TOC), 79.53% total dissolved nitrogen (TN), 93.38% ammonia nitrogen (NH3-N) and 71.33% total dissolved phosphorus (TP) in brewery wastewater were rapidly removed by co-cultivation of T. aequale and autochthonous bacteria. Treated wastewater met the national wastewater discharge quality, and resulting algal biomass contained large amounts of high-value products chrysolaminarin, palmitoleic acid (PLA) and eicosapentaenoic acid (EPA). It is anticipated that reduced cost of algal harvesting coupled with value-added biomass could make T. aequale as a promising candidate for brewery wastewater treatment and resource utilization.

Folate-mediated one-carbon metabolism as a potential antifungal target for the sustainable cultivation of microalga Haematococcus pluvialis.

BACKGROUND: Microalgae are widely considered as multifunctional cell factories that are able to transform the photo-synthetically fixed CO2 to numerous high-value compounds, including lipids, carbohydrates, proteins and pigments. However, contamination of the algal mass culture with fungal parasites continues to threaten the production of algal biomass, which dramatically highlights the importance of developing effective measures to control the fungal infection. One viable solution is to identify potential metabolic pathways that are essential for fungal pathogenicity but are not obligate for algal growth, and to use inhibitors targeting such pathways to restrain the infection. However, such targets remain largely unknown, making it challenging to develop effective measures to mitigate the infection in algal mass culture. RESULTS: In the present study, we conducted RNA-Seq analysis for the fungus Paraphysoderma sedebokerense, which can infect the astaxanthin-producing microalga Haematococcus pluvialis. It was found that many differentially expressed genes (DEGs) related to folate-mediated one-carbon metabolism (FOCM) were enriched in P. sedebokerense, which was assumed to produce metabolites required for the fungal parasitism. To verify this hypothesis, antifolate that hampered FOCM was applied to the culture systems. Results showed that when 20 ppm of the antifolate co-trimoxazole were added, the infection ratio decreased to ~ 10% after 9 days inoculation (for the control, the infection ratio was 100% after 5 days inoculation). Moreover, application of co-trimoxazole to H. pluvialis mono-culture showed no obvious differences in the biomass and pigment accumulation compared with the control, suggesting that this is a potentially algae-safe, fungi-targeted treatment. CONCLUSIONS: This study demonstrated that applying antifolate to H. pluvialis culturing systems can abolish the infection of the fungus P. sedebokerense and the treatment shows no obvious disturbance to the algal culture, suggesting FOCM is a potential target for antifungal drug design in the microalgal mass culture industry.

Fucoxanthin alleviated atherosclerosis by regulating PI3K/AKT and TLR4/NFκB mediated pyroptosis in endothelial cells.

Fucoxanthin, a type of natural xanthophyll carotenoid, is mainly present in seaweeds and various microalgae. This compound has been proved to possess multiple functions including antioxidation, anti-inflammation and anti-tumor. Atherosclerosis is widely deemed as a chronic inflammation disease, and as the basis of vascular obstructive disease. However, there is rare research about fucoxanthin's effects on atherosclerosis. In this study, we demonstrated that the plaque area of mice treated with fucoxanthin was significantly reduced compared to the group that did not receive fucoxanthin. In addition, Bioinformatics analysis showed that PI3K/AKT signaling might be involved in the protective effect of fucoxanthin, and this hypothesis was then verified in vitro endothelial cell experiments. Besides, our further results showed that endothelial cell mortality measured by TUNEL and flow cytometry was significantly increased in the oxidized low-density lipoprotein (ox-LDL) treatment group while significantly decreased in the fucoxanthin treatment group. In addition, the pyroptosis protein expression level in the fucoxanthin group was significantly lower than that in the ox-LDL group, which indicated that fucoxanthin improved the pyroptosis level of endothelial cells. Furthermore, it was revealed that TLR4/NFκB signaling were also participated in the protection of fucoxanthin on endothelial pyroptosis. Moreover, the protection of fucoxanthin on endothelial cell pyroptosis was abrogated when PI3K/AKT was inhibited or TLR4 was overexpressed, which further suggested the anti-pyroptosis effect of fucoxanthin was mediated through regulations of PI3K/AKT and TLR4/NFκB signaling.

Metabolomic analysis reveals astaxanthin biosynthesis in heterotrophic microalga Chromochloris zofingiensis.

The utilization of gibberellic acid-3, high carbon/nitrogen ratio and salinity concentration can effectively enhance astaxanthin biosynthesis in Chromochloris zofingiensis under the heterotrophic conditions, but the underlying mechanisms remained yet to be investigated. The metabolomics analysis revealed that enhancement of the glycolysis, pentose phosphate pathways (PPP), and tricarboxylic acid (TCA) cycle led to astaxanthin accumulation under the induction conditions. The increased fatty acids can significantly increase astaxanthin esterification. The addition of appropriate concentrations of glycine (Gly) and γ-aminobutyric acid (GABA) promoted astaxanthin biosynthesis in C. zofingiensis, as well as benefiting for biomass yield. With the addition of 0.5 mM GABA, the astaxanthin yield increased to 0.35 g·L-1, which was 1.97-fold higher than that of the control. This study advanced understanding about astaxanthin biosynthesis in heterotrophic microalga, and provided novel strategies for enhanced astaxanthin production in C. zofingiensis.

Microalgal glycerol-3-phosphate acyltransferase role in galactolipids and high-value storage lipid biosynthesis.

Glycerolipids are the most abundant lipids in microalgae, and glycerol-3-phosphate:acyl-CoA acyltransferase (GPAT) plays an important role in their biosynthesis. However, the biochemical and biological functions of algal GPAT remain poorly characterized. Here, we characterized the endoplasmic reticulum (ER)-associated GPAT of the model unicellular green alga Chlamydomonas reinhardtii (CrGPATer). Enzymatic assays indicated that CrGPATer is an sn-1 acyltransferase using a variety of acyl-CoAs as the acyl donor. Subcellular localization revealed that CrGPATer was associated with ER membranes and lipid droplets. We constructed overexpression (OE) and knockdown (KD) transgenic C. reinhardtii lines to investigate the in vivo function of CrGPATer. Lipidomic analysis indicated that CrGPATer OE enhanced the cellular content of galactolipids, especially monogalactosyldiacylglycerol, under nitrogen deficiency stress. Correspondingly, CrGPATer KD lines contained lower contents of galactolipids than the control. Feeding experiments with labeled phosphatidic acid revealed that the intermediate of the eukaryotic Kennedy pathway could be used for galactolipid biosynthesis in the chloroplasts. These results provided multiple lines of evidence that the eukaryotic Kennedy pathway mediated by CrGPATer may be involved in galactolipid biosynthesis in C. reinhardtii. OE of CrGPATer significantly increased the content of triacylglycerol and the yield of biomass. Moreover, the content and yield of 1, 3-olein-2-palmitin, a high-value lipid that can be used as an alternative for human milk fat in infant formula, were significantly enhanced in the OE transgenic lines. Taken together, this study provided insights into the biochemical and biological functions of CrGPATer and its potential as a genetic engineering target in functional lipid manufacturing.

Regulation of Light Spectra on Cell Division of the Unicellular Green Alga Haematococcus pluvialis: Insights from Physiological and Lipidomic Analysis.

Commercial scale production of natural astaxanthin is currently conducted through cultivation of the green alga Haematococcus pluvialis. This study comprehensively investigated the impact of seven different light spectra on the growth, morphology and photosynthesis of H. pluvialis vegetative cells. Further, the lipidomes of vegetative H. pluvialis grown under various light spectra were qualitatively and quantitatively analyzed using liquid chromatography/mass spectrometry (LC/MS). The results showed the existence of blue light-alone or with red light-promoted cell division, while pure red light or white light enabled increased cell sizes, cellular pigment, starch and lipid contents, and biomass production. Although the photosynthetic performance of H. pluvialis measured as chlorophyll a fluorescence was not significantly affected by light spectra, the lipid profiles, particularly chloroplast membrane lipids, showed remarkable changes with light spectra. The contents of most lipid species in the blue/red light 1/2 group, which showed the fastest cell division, remained at a moderate level compared with those under other light spectra, indicating the fastest dividing cells were featured by a fine-tuned lipid profile. From biotechnical perspective, this comprehensive study can provide insights into the development of appropriate light regimes to promote the cell density or biomass of H. pluvialis mass culture.

Assessment of Eicosapentaenoic Acid (EPA) Production from Filamentous Microalga Tribonema aequale: From Laboratory to Pilot-Scale Study.

It has long been explored to use EPA-rich unicellular microalgae as a fish oil alternative for production of the high-value omega-3 fatty acid eicosapentaenoic acid (EPA, 20:5, n-3). However, none of the efforts have ever reached commercial success. This study reported a filamentous yellow-green microalga Tribonema aequale that possesses the ability to grow rapidly and synthesize significant amounts of EPA. A series of studies were conducted in a glass column photobioreactor under laboratory culture conditions and in pilot-scale open raceway ponds outdoors. The emphasis was placed on the specific nutrient requirements and the key operational parameters in raceway ponds such as culture depth and mixing regimes. When optimized, T. aequale cells contained 2.9% of EPA (w/w) and reached a very high biomass concentration of 9.8 g L-1 in the glass column photobioreactor. The cellular EPA content was increased further to 3.5% and the areal biomass and EPA productivities of 16.2 g m-2 d-1 and 542.5 mg m-2 d-1, respectively, were obtained from the outdoor pilot-scale open raceway ponds, which were the record high figures reported thus far from microalgae-based EPA production. It was also observed that T. aequale was highly resistant to microbial contamination and easy for harvesting and dewatering, which provide two additional competitive advantages of this filamentous microalga over the unicellular counterparts for potential commercial production of EPA and other derived co-products.

Oxidative stress facilitates infection of the unicellular alga Haematococcus pluvialis by the fungus Paraphysoderma sedebokerense.

BACKGROUND: The green microalga Haematococcus pluvialis is used as a cell factory for producing astaxanthin, the high-value carotenoid with multiple biological functions. However, H. pluvialis is prone to the infection by a parasitic fungus Paraphysoderma sedebokerense, which is the most devastating threat to the mass culture of H. pluvialis all over the world. Through dissecting the mechanisms underlying the infection process, effective measures could be developed to mitigate the pathogen threatening for the natural astaxanthin industry. By far, understanding about the interaction between the algal host and fungal pathogen remains very limited. RESULTS: We observed that there were heat-stable substances with small molecular weight produced during the infection process and enhanced the susceptibility of H. pluvialis cells to the pathogen. The infection ratio increased from 10.2% (for the algal cells treated with the BG11 medium as the control) to 52.9% (for the algal cells treated with supernatant contained such substances) on the second day post-infection, indicating the yet unknown substances in the supernatant stimulated the parasitism process. Systematic approaches including multi-omics, biochemical and imaging analysis were deployed to uncover the identity of the metabolites and the underlying mechanisms. Two metabolites, 3-hydroxyanthranilic acid and hordenine were identified and proved to stimulate the infection via driving oxidative stress to the algal cells. These metabolites generated hydroxyl radicals to disrupt the subcellular components of the algal cells and to make the algal cells more susceptible to the infection. Based on these findings, a biosafe and environment-friendly antioxidant butylated hydroxyanisole (BHA) was selected to inhibit the fungal infection, which completely abolished the infection at 12 ppm. By applying 7 ppm BHA every 2 days to the algal cell culture infected with P. sedebokerense in the 100 L open raceway ponds, the biomass of H. pluvialis reached 0.448 g/L, which was comparable to that of the control (0.473 g/L). CONCLUSIONS: This study provides for the first time, a framework to dissect the functions of secondary metabolites in the interaction between the unicellular alga H. pluvialis and its fungal parasite, indicating that oxidative degradation is a strategy used for the fungal infest. Eliminating the oxidative burst through adding antioxidant BHA could be an effective measure to reduce parasitic infection in H. pluvialis mass culture.

Effective Two-Stage Heterotrophic Cultivation of the Unicellular Green Microalga Chromochloris zofingiensis Enabled Ultrahigh Biomass and Astaxanthin Production.

Chromochloris zofingiensis has obtained particular interest as a promising candidate for natural astaxanthin production. In this study, we established a two-stage heterotrophic cultivation process, by using which both the growth of C. zofingiensis and astaxanthin accumulation are substantially enhanced. Specifically, the ultrahigh biomass concentration of 221.3 g L-1 was achieved under the optimum culture conditions in 7.5 L fermenter during 12 days. When scaled-up in the 500 L fermentor, the biomass yield reached 182.3 g L-1 in 9 days, while the astaxanthin content was 0.068% of DW. To further promote astaxanthin accumulation, gibberellic Acid-3 (GA3) was screened from a variety of phytohormones and was combined with increased C/N ratio and NaCl concentration for induction. When C. zofingiensis was grown with the two-stage cultivation strategy, the astaxanthin yield reached 0.318 g L-1, of which the biomass yield was 235.4 g L-1 and astaxanthin content was 0.144% of DW. The content of the total fatty acids increased from 23 to 42% of DW simultaneously. Such an astaxanthin yield was 5.4-fold higher than the reported highest record and surpassed the level of Haematococcus pluvialis. This study demonstrated that heterotrophic cultivation of C. zofingiensis is competitive for industrial astaxanthin production.

Heterotrophically Ultrahigh-Cell-Density Cultivation of a High Protein-Yielding Unicellular Alga Chlorella With a Novel Nitrogen-Supply Strategy.

The unicellular green alga Chlorella is an ideal protein source. However, the high production cost and low production capability of the current main photoautotrophic culture mode limit its application especially as an alternative protein source for food and feed, which might be overcome through high-cell-density cultivation in fermenters. In this study, a Chlorella sorokiniana strain CMBB276 with high protein content was selected from five Chlorella strains by comprehensive evaluation of their growth rates, protein contents, and yields. The optimal cultural temperature, pH, and mole ratio of carbon and nitrogen (C/N) for C. sorokiniana CMBB276 growth were found to be 30°C, 6.5, and 18, respectively. Ammonium chloride was proved to be the best nitrogen (N) source for C. sorokiniana CMBB276 growth, whereas growth inhibition caused by the accumulation of salts was observed under fed-batch cultivation when maintaining a constant C/N ratio of 18 by controlling pH with sodium hydroxide solution. By simultaneously reducing the concentration of ammonium chloride in the feeding medium and controlling pH with ammonium hydroxide, we finally achieved the ultrahigh-cell-density cultivation of C. sorokiniana CMBB276. The highest biomass concentration and protein yield reached 232 and 86.55 g l-1, respectively, showing the great potential of culturing C. sorokiniana CMBB276 in fermenters for economic and large-scale protein source production.

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green alga Chlorella sorokiniana for biomass production.

Heterotrophic cultivation of Chlorella has achieved commercial success, but the application of Chlorella biomass is still limited due to the high cost of biomass production. In this study, an effective and industrially scalable heterotrphic cultivation technology has been developed for a production strain Chlorella sorokiniana GT-1. Under the optimized culturing conditions, the ultrahigh biomass concentration of 271 and 247 g L-1 was achieved in 7.5 L bench-scale and 1000 L pilot-scale fermenters, respectively. Technoeconomic (TE) analysis indicated that the production cost of C. sorokiniana GT-1 could be reduced to $1601.27 per ton of biomass if the biomass concentration reached 200 g L-1 , which is 24.2% lower than that of the reported highest Chlorella biomass production through fermentation with the same TE model. Under the same growth conditions, the maximum biomass concentration of a low-starch mutant SLM2 was reduced to 93 g L-1 , which was 54% lower than that of the wild type, indicating the capabilities of C. sorokiniana GT-1 cells in accumulating large amounts of starch are essential for achieving the ultrahigh-cell-density under the heterotrophic conditions. In addition, the ultrahigh-cell-density growth potential of C. sorokiniana GT-1 cells was inferred to be related to the intrinsic biological characteristics including the tolerance to low dissolved oxygen and a moderate doubling time under the heterotrophic conditions as well. The breakthrough in cultivation technology is promising for Chlorella industry and would expand its applications in food and feed.

High-cell-density cultivation of the flagellate alga Poterioochromonas malhamensis for biomanufacturing the water-soluble ß-1,3-glucan with multiple biological activities.

The microalga Poterioochromonas malhamensis was found to be capable of accumulating the storage ß-1,3-glucan in soluble form under heterotrophic conditions. In this study, the highest biomass yield of 32.8 g L-1 was achieved by combining the utilization of ammonium chloride as the nitrogen source, simultaneous addition of vitamins B1 and B12 and maintenance of pH at 6.0. Sugar profiling and nuclear magnetic resonance analysis indicated that the P. malhamensis ß-1,3-glucan was composed of glucose with the ß-(1 â†’ 3) main chain and the ß-(1 â†’ 6) side chain. Under the optimal cultivation conditions, the cellular ß-1,3-glucan content was up to 55% of the cell dry weight. Moreover, the P. malhamensis ß-1,3-glucan could significantly promote the fin regeneration and improve the in vivo antioxidative activity of zebrafish. This study underpins the feasibility of culturing P. malhamensis under heterotrophic conditions for producing the highly water-soluble bioactive ß-1,3-glucans for food and pharmaceutical applications.

Trophic Transition Enhanced Biomass and Lipid Production of the Unicellular Green Alga Scenedesmus acuminatus.

Microalgal heterotrophic cultivation is an emerging technology that can enable producing high cell-density algal cell cultures, which can be coupled with photoautotrophic cultivation for valuable chemicals such as lipids manufacturing. However, how the heterotrophically grown algal cells respond to the lipid-inducing conditions has not been fully elucidated so far. In this study, when the heterotrophically grown Scenedesmus acuminatus cells were subjected to the high light (HL) and nitrogen-limited (NL) conditions, both the biomass and lipid productivity were enhanced as compared to that of the photoautotrophically grown counterparts. The chlorophyll a fluorometry analysis showed that the Fv/Fm and Y(II) of the heterotrophically grown cells subjected to the HL and NL conditions was recovered to the maximum value of 0.75 and 0.43, respectively, much higher than those of the photoautotrophically grown cells under the same stress conditions. Transcriptomic analysis revealed that heterotrophically grown cells fully expressed the genes coding for the photosystems proteins, including the key photoprotective proteins D1, PsbS, light-harvesting-complex (LHC) I and LHC II. Meanwhile, downregulation of the carotenoid biosynthesis and upregulation of the glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle and oxidative phosphorylation pathways were observed when the heterotrophically grown cells were subjected to the HL and N-limited conditions for lipid production. It was deduced that regulation of these pathways not only enhanced the light utilization but also provided the reducing power and ATP by which the biomass accumulation was significantly elevated. Besides, upregulation of the acetyl-CoA carboxylase/biotin carboxylase, digalactosyl diacylglycerol synthase and diacylglycerol acyltransferase 2 encoding genes may be attributable to the enhanced lipid production. Understanding the cellular responses during the trophic transition process could guide improvement of the strength of trophic transition enhancing microalgal biomass and lipid production.

A Lipid Bodies-Associated Galactosyl Hydrolase Is Involved in Triacylglycerol Biosynthesis and Galactolipid Turnover in the Unicellular Green Alga Chlamydomonas reinhardtii.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the main constituent lipids of thylakoid and chloroplast envelop membranes. Many microalgae can accumulate large amounts of triacylglycerols (TAGs) under adverse environmental conditions, which is accompanied by degradation of the photosynthetic membrane lipids. However, the process mediating the conversion from galactolipids to TAG remains largely unknown. In this study, we performed genetic and biochemical analyses of galactosyl hydrolases (CrGH) identified in the proteome of lipid bodies of the green microalga Chlamydomonas reinhardtii. The recombinant CrGH was confirmed to possess galactosyl hydrolase activity by using o-nitrophenyl-ß-D-galactoside as the substrate, and the Michaelis constant (Km) and Kcat of CrGH were 13.98 µM and 3.62 s-1, respectively. Comparative lipidomic analyses showed that the content of MGDG and DGDG increased by 14.42% and 24.88%, respectively, in the CrGH-deficient mutant as compared with that of the wild type cc4533 grown under high light stress conditions, and meanwhile, the TAG content decreased by 32.20%. Up-regulation of CrGH at both a gene expression and protein level was observed under high light stress (HL) conditions. In addition, CrGH was detected in multiple subcellular localizations, including the chloroplast envelope, mitochondria, and endoplasmic reticulum membranes. This study uncovered a new paradigm mediated by the multi-localized CrGH for the conversion of the photosynthetic membranes to TAGs.

Interaction between the cell walls of microalgal host and fungal carbohydrate-activate enzymes is essential for the pathogenic parasitism process.

Fungi can parasitize microalgae, exerting profound impacts on both the aquatic ecosystems and microalgal mass cultures. In this study, the unicellular green alga Haematococcus pluvialis and the blastocladialean fungus Paraphysoderma sedebokerense were used as a model system to address the mechanisms underlying the fungal parasitism on the algal host. High-throughput metabolic assay indicated that P. sedebokerense can utilize several carbon sources with a preference for mannose, glucose and their oligosaccharides, which was compatible with the profile of the host algal cell walls enriched with glucan and mannan. The results of dual transcriptomics analysis suggested that P. sedebokerense can upregulate a large number of putative carbohydrate-activate enzymes (CAZymes) encoding genes, including those coding for the endo-1,4-ß-glucanase and endo-1,4-ß-mannanase during the infection process. The cell walls of H. pluvialis can be decomposed by both P. sedebokerense and commercial CAZymes (e.g. cellulase and endo-1,4-ß-mannanase) to produce mannooligomers, while several putative parasitism-related genes of P. sedebokerense can be in turn upregulated by mannooligomers. In addition, the parasitism can be blocked by interfering the selected CAZymes including glucanase, mannanase and lysozyme with the specific inhibitors, which provided a framework for screening suitable compounds for pathogen mitigation in algal mass culture.

Functional divergence of diacylglycerol acyltransferases in the unicellular green alga Haematococcus pluvialis.

Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final committed step in triacylglycerol biosynthesis in eukaryotes. In microalgae, the copy number of DGAT genes is extraordinarily expanded, yet the functions of many DGATs remain largely unknown. This study revealed that microalgal DGAT can function as a lysophosphatidic acyltransferase (LPAAT) both in vitro and in vivo while losing its original function as DGAT. Among the five DGAT-encoding genes identified and cloned from the green microalga Haematococcus pluvialis, four encoded HpDGATs that showed triacylglycerol synthase activities in yeast functional complementation analyses; the exception was one of the type II DGAT encoding genes, HpDGTT2. The hydrophobic recombinant HpDGTT2 protein was purified in soluble form and was found to function as a LPAAT via enzymatic assay. Introducing this gene into the green microalga Chlamydomonas reinhardtii led to retarded cellular growth, enlarged cell size, and enhanced triacylglycerol accumulation, identical to the phenotypes of transgenic strains overexpressing CrLPAAT. This study provides a framework for dissecting uncharacterized DGATs, and could pave the way to decrypting the structure-function relationship of this large group of enzymes that are critical to lipid biosynthesis.

A Novel Bifunctional Wax Ester Synthase Involved in Early Triacylglycerol Accumulation in Unicellular Green Microalga Haematococcus pluvialis Under High Light Stress.

The bulk of neutral lipids, including astaxanthin esters and triacylglycerols (TAGs), are accumulated in the green microalga Haematococcus pluvialis under high light (HL) stress. In this study, a novel bifunctional wax ester synthase (WS) gene was cloned from H. pluvialis upon HL stress. The overexpression of HpWS restored the biosynthesis of wax esters and TAGs in neutral lipid-deficient yeast mutant Saccharomyces cerevisiae H1246 fed with C18 alcohol and C18:1/C18:3 fatty acids, respectively. Under HL stress, HpWS was substantially upregulated at the transcript level, prior to that of the type I diacylglycerol:acyl-CoA acyltransferase encoding gene (HpDGAT1). HpDGAT1 is the major TAG synthase in H. pluvialis. In addition, the application of xanthohumol (a DGAT1/2 inhibitor) in the H. pluvialis cells did not completely eliminate the TAG biosynthesis under HL stress at 24 h. These results indicated that HpWS may contribute to the accumulation of TAGs in H. pluvialis at the early stage under HL stress. In addition, the overexpression of HpWS in Chlamydomonas reinhardtii bkt5, which is engineered to produce free astaxanthin, enhanced the production of TAGs and astaxanthin. Our findings broaden the understanding of TAG biosynthesis in microalgae and provide a new molecular target for genetic manipulation in biotechnological applications.

Identification and Biotechnical Potential of a Gcn5-Related N-Acetyltransferase Gene in Enhancing Microalgal Biomass and Starch Production.

Microalgae are promising feedstocks for starch production, which are precursors for bioenergy and chemicals manufacturing. Though starch biosynthesis has been intensively studied in the green alga Chlamydomonas reinhardtii, regulatory mechanisms governing starch metabolism in this model species have remained largely unknown to date. We proposed that altering triacylglycerol (TAG) biosynthesis may trigger intrinsic regulatory pathways governing starch metabolism. In accordance with the hypothesis, it was observed in this study that overexpression of the plastidial lysophosphatidic acid acyltransferase gene (i.e. LPAAT1) in C. reinhardtii significantly enhanced TAG biosynthesis under nitrogen (N)-replete conditions, whereas the starch biosynthesis was enhanced in turn under N depletion. By the exploitation of transcriptomics analysis, a putative regulatory gene coding Gcn5-related N-acetyltransferase (GNAT19) was identified, which was up-regulated by 11-12 times in the CrLPAAT1 OE lines. Overexpression of the cloned full-length CrGNAT19 cDNA led to significant increase in the starch content of C. reinhardtii cells grown under both N-replete and N-depleted conditions, which was up to 4 times and 26.7% higher than that of the empty vector control, respectively. Moreover, the biomass yield of the CrGNAT19 OE lines reached 1.5 g L-1 after 2 days under N-depleted conditions, 72% higher than that of the empty vector control (0.87 g L-1). Overall, the yield of starch increased by 118.5% in CrGNAT19 OE lines compared to that of the control. This study revealed the great biotechnical potentials of an unprecedented GNAT19 gene in enhancing microalgal starch and biomass production.

PGRL1 overexpression in Phaeodactylum tricornutum inhibits growth and reduces apparent PSII activity.

Proton gradient regulation 5-like photosynthetic phenotype 1 (PGRL1)-dependent cyclic electron transport around photosystem I (PSI) plays important roles in the response to different stresses, including high light. Although the function of PGRL1 in higher plants and green algae has been thoroughly investigated, little information is available on the molecular mechanism of PGRL1 in diatoms. We created PGRL1 overexpression and knockdown transformants of Phaeodactylum tricornutum, the diatom model species, and investigated the impact on growth and photosynthesis under constant and fluctuating light conditions. PGRL1 over-accumulation resulted in significant decreases in growth rate and apparent photosystem II (PSII) activity and led to an opposing change of apparent PSII activity when turning to high light, demonstrating a similar influence on photosynthesis as a PSII inhibitor. Our results suggested that PGRL1 overexpression can reduce the apparent efficiency of PSII and inhibit growth in P. tricornutum. These findings provide physiological evidence that the accumulation of PGRL1 mainly functions around PSII instead of PSI.