Photosynthetic microalgae are emerging as potential biomass feedstock for sustainable production of biofuels and value-added bioproducts. CO2 biomitigation through these organisms is considered as an eco-friendly and promising alternative to the existing carbon sequestration methods. Nonetheless, the inherent relatively low photosynthetic capacity of microalgae has hampered the practical use of this strategy for CO2 biomitigation applications.Here, we demonstrate the feasibility of improving photosynthetic capacity by the genetic manipulation of the Calvin cycle in the typical green microalga Chlorella vulgaris. Firstly, we fused a plastid transit peptide to upstream of the enhanced green fluorescent protein (EGFP) and confirmed its expression in the chloroplast of C. vulgaris. Then we introduced the cyanobacterial fructose 1,6-bisphosphate aldolase, guided by the plastid transit peptide, into C. vulgaris chloroplast, leading to enhanced photosynthetic capacity (~ 1.2-fold) and cell growth. Molecular and physiochemical analyses suggested a possible role for aldolase overexpression in promoting the regeneration of ribulose 1,5-bisphosphate in the Calvin cycle and energy transfer in photosystems.Our work represents a proof-of-concept effort to enhance photosynthetic capacity by the engineering of the Calvin cycle in green microalgae. Our work also provides insights into targeted genetic engineering toward algal trait improvement for CO2 biomitigation uses.

Photosynthetic microalgae are emerging as potential biomass feedstock for sustainable production of biofuels and value-added bioproducts. CO2 biomitigation through these organisms is considered as an eco-friendly and promising alternative to the existing carbon sequestration methods. Nonetheless, the inherent relatively low photosynthetic capacity of microalgae has hampered the practical use of this strategy for CO2 biomitigation applications.Here, we demonstrate the feasibility of improving photosynthetic capacity by the genetic manipulation of the Calvin cycle in the typical green microalga Chlorella vulgaris. Firstly, we fused a plastid transit peptide to upstream of the enhanced green fluorescent protein (EGFP) and confirmed its expression in the chloroplast of C. vulgaris. Then we introduced the cyanobacterial fructose 1,6-bisphosphate aldolase, guided by the plastid transit peptide, into C. vulgaris chloroplast, leading to enhanced photosynthetic capacity (~ 1.2-fold) and cell growth. Molecular and physiochemical analyses suggested a possible role for aldolase overexpression in promoting the regeneration of ribulose 1,5-bisphosphate in the Calvin cycle and energy transfer in photosystems.Our work represents a proof-of-concept effort to enhance photosynthetic capacity by the engineering of the Calvin cycle in green microalgae. Our work also provides insights into targeted genetic engineering toward algal trait improvement for CO2 biomitigation uses.

•Mixotrophy possessed much higher biomass productivity and biomass yield on glucose.•The reduced Rubisco activity indicated down-regulation of Calvin cycle in mixotrophy.•The reduced citrate synthase activity indicated down-regulation of TCA cycle in mixotrophy.•Mixotrophic cells accumulated more starch but less total fatty acid than heterotrophy.•A dedicated metabolic mechanism of mixotrophic cultivation was firstly proposedMicroalgal biofuels have attracted much attention in recent years. Nevertheless, the relatively low growth rate and low cell density limits its industrial application. Mixotrophic cultivation has been considered as a good strategy to increase the specific growth rate and biomass yield in some microalgal species. However, the metabolic mechanism has not been well elucidated. To address it, Chlorella zofingiensis was employed in the present study. The biomass productivity and maximum dry weight achieved under mixotrophic condition were greater than the sum of those under photoautotrophic and heterotrophic conditions. Interestingly, compared with photoautotrophic cultures, the RuBisCO activity was reduced for mixotrophic cultures, indicative of the down-regulation of the Calvin cycle. When compared with heterotrophic cultures, the citrate synthase activity was attenuated for mixotrophic cultures, indicative of the down-regulation of Tricarboxylic Acid cycle. Biochemical analysis showed that mixotrophic cells contained more starch but less total fatty acid content than heterotrophic cells. Besides, the biomass yield on glucose was 0.789 g g− 1 for mixotrophic cultures, 63.7% higher than that for heterotrophic cultures, and the enhancement effect of light under mixotrophic cultivation was 7.35-fold. Based on these results, a novel mechanism for the synergistic effects in mixotrophic cultivation was proposed, in which glucose was more efficiently utilized for biomass production by the possible coordination of energy and carbon metabolism between photosynthesis and aerobic respiration.

Sesamol is a strong antioxidant phenolic compound found in sesame seed. It possesses the ability to scavenge intracellular reactive oxygen species (ROS) and to inhibit malic enzyme activity and NADPH supply, resulting possibly in cell proliferation and alteration in the fatty acid composition. In the present study, the effect of sesamol on the growth and accumulation of docosahexaenoic acid (DHA) was investigated in the marine microalga Crypthecodinium cohnii, a prolific producer of DHA. C. cohnii showed a great decrease in the intracellular ROS level with the addition of sesamol. In contrast, the biomass concentration, DHA content (% of total fatty acids), and DHA productivity were significantly increased by 44.20, 11.25, and 20.00%, respectively (P < 0.01). Taken together, this work represents the first report of employing sesamol for enhanced production of DHA by C. cohnii, providing valuable insights into this alga for future biotechnological applications.

Microalgae are huge natural sources of high-value compounds with health-promoting properties. The carotenoids derived from microalgae have significant antioxidant and anti-inflammatory effects, which allow them to provide health benefits. In this article, the bioactivities of microalgal carotenoids are reviewed. Emphasis is placed on astaxanthin, a ketocarotenoid with extraordinary potential for protecting against a wide range of diseases.

Dissolved oxygen is a critical factor for heterotrophic cell growth and metabolite production. The aim of this study was to investigate the effects of an oxygen-involved protein on cell growth and fatty acid and astaxanthin production in the biologically important thraustochytrid Aurantiochytrium sp. The hemoglobin of the Vitreoscilla stercoraria (VHb) gene was fused upstream with a zeocin resistance gene (ble) and driven by the Aurantiochytrium tubulin promoter. The expression construct was introduced into two strains of Aurantiochytrium sp. by electroporation. Transgenic Aurantiochytrium sp. strains MP4 and SK4 expressing the heterologous VHb achieved significantly higher maximum biomass than their corresponding controls in microaerobic conditions. Furthermore, the transformants of Aurantiochytrium sp. SK4 produced 44% higher total fatty acid and 9-fold higher astaxanthin contents than the wild type control in aerobic conditions. The present study highlights the biotechnological application of VHb in high-cell density fermentation for enhanced biomass production as well as high-value metabolites.

Docosahexaenoic acid (DHA) plays important roles in the prevention of ageing and ageing-related diseases. In this work, the anti-ageing effect of Schizochytrium mangrovei (SM), a marine microalga with high contents of DHA was investigated for the first time. In vitro results showed that SM enhanced the vitality of healthy pheochromocytoma (PC12) cells, whereas it had no protective effect under H2O2-induced oxidative stress. Meanwhile, SM exerted neuroprotective actions in vivo: the supplementation of SM significantly extended the lifespan of both wild type and SODn108 mutant flies Drosophila melanogaster, and ameliorated the age-related decline of locomotor function. This lifespan-extending activity was associated with the mRNA up-regulation of endogenous stress-defense genes such as CuZn-superoxide dismutase (SOD1) and Mn-superoxide dismutase (SOD2), as well as the down-regulation of Methuselah (MTH) gene. Therefore, it was concluded that the anti-ageing capability of DHA-rich SM was mediated through the cooperation with endogenous stress-defense system rather than the direct interaction with oxidative free radicals.Highlights► The anti-ageing effect of DHA-rich microalga SM was investigated. ► SM significantly enhanced the viability of PC 12 cell. ► SM extended the lifespan of fruit flies. ► SM ameliorated the age-related decline of locomotor function. ► The observed neuroprotective activities were explained by mRNA regulations.

The microalga Haematococcus pluvialis (HP) is the best natural producer of astaxanthin (AX), which is a potent antioxidant with broad health benefits. The present study investigated the antiaging potential of HP biomass using the fruit fly Drosophila melanogaster as the animal model. The results showed that in wild-type flies the treatment of HP induced the early mortality at a concentration of 20 mg/mL, which was associated with the decreased enzymatic activities of CuZn-superoxide dismutase (SOD1) and Mn-superoxide dismutase (SOD2) as well as the down-regulation of SOD1, SOD2, and catalase (CAT) at the transcriptional level. In SODn108 mutant flies, the supplementation of HP (10 or 20 mg/mL) significantly extended their lifespan and ameliorated the age-related decline in locomotor function. Further studies suggested that HP may play a role as a complement to the defective endogenous antioxidant system to exert such lifespan elongation effects. These results, taken together, strongly support the antiaging properties of HP and its therapeutic rather than preventive potential against aging-related diseases.

The present study examined the antiglycative and antioxidant properties of four edible sprouts popular in Chinese markets. In a protein-reducing sugar model, the sunflower sprout Helianthus annuus exhibited the strongest inhibitory effects against the formation of advanced glycation end products (AGEs). At a concentration of 1.0 mg/mL, its inhibitory rate achieved 83.29%, which is stronger than that of aminoguanidine (1 mM), a well-known synthetic antiglycative agent (with an inhibitory rate of 80.88%). The antioxidant capacity of H. annuus was also much stronger than other sprout samples in terms of free radical scavenging and reducing properties. An active ingredient contributing to the observed activities was identified as cynarin (1,5-dicaffeoylquinic acid). This is the first report of the novel function of cynarin to intervene against glycoxidation. Given the key roles of AGEs and oxidation in the pathogenesis of diabetes, the sunflower sprout H. annuus rich in cynarin may be regarded as a beneficial food choice for diabetic patients.

The green alga Chlorella zofingiensis can accumulate high level of oleic acid (OA, C18:1△9) rich oils in response to stress conditions. To understand the regulation of biosynthesis of fatty acid in particular OA at the molecular level, we cloned and characterized the stearoyl acyl carrier protein (ACP) desaturase (SAD) responsible for OA formation through desaturation of stearic acid (C18:0) from C. zofingiensis. Southern blot indicated that the C. zofingiensis genome contained a single copy of SAD, from which the deduced amino acid sequence shared high identity to the corresponding homologs from other microalgae and higher plants. The desaturation activity of SAD was demonstrated in vitro using C18:0-ACP as a substrate. Stress conditions such as high light (HL), nitrogen deficiency (N−), or combination of HL and N− (HL + N−) drastically up-regulated the transcripts of biotin carboxylase (BC, a subunit of ACCase) and SAD, and therefore induced considerably the cellular accumulation of total fatty acids including OA. Glucose (50 mM) gave rise to the similar up-regulation of the two genes and induction of fatty acid accumulation. The accumulation of intracellular reactive oxygen species was found to be associated with the up-regulation of genes. This is the first report of characterization of Chlorella-derived SAD and the results may contribute to understanding of the mechanisms involved in fatty acid/lipid biosynthesis in microalgae.

•A Crypthecodinium strain was isolated and named as Crypthecodinium sp. SUN.•Light was firstly employed on Crypthecodinium and increased its TFA and DHA contents.•The increase of TFA content was contributed by the decrease of starch.•Light increased the specific growth rate of Crypthecodinium sp. SUN.In the present study, light illumination was found to be efficient in elevating the total fatty acid content in a newly isolated heterotrophic microalga, Crypthecodinium sp. SUN. Under light illumination, the highest total fatty acid and DHA contents were achieved at 96 h as 24.9% of dry weight and 82.8 mg g−1 dry weight, respectively, which were equivalent to 1.46-fold and 1.68-fold of those under the dark conditions. The elevation of total fatty acid content was mainly contributed by an increase of neutral lipids at the expense of starches. Moreover, light was found to alter the cell metabolism and led to a higher specific growth rate, higher glucose consumption rate and lower non-motile cell percentage. This is the first report that light can promote the total fatty acids accumulation in Crypthecodinium without growth inhibition.

•SC by effective methods was used with a 1.16-fold increase of biomass.•Light, nutrient and metabolite were proved successively as limiting factors.•A mix of red and white light (2:1) was firstly proved to accelerate cell growth.•Replacement cultivation was proved to be more effective than fed-batch culture.An innovative staged cultivation (SC) method was proposed to overcome the limiting factors associated with the growth of Haematococcus pluvialis in the green growth phase. This strategy led to a 1.16-fold increase in biomass concentration. Light wavelength, nutrient concentration and extracellular metabolite were identified to be key limiting factors when cells of H. pluvialis were in the low, medium, and high cell density sub-phase, respectively. A mix of red and white light (2:1) was demonstrated for the first time to accelerate cell growth in the low cell density sub-phase. Shortage of nutrients during the medium density sub-phase was overcome with a fed-batch approach maintained at stable pH, while the inhibitory effect of extracellular metabolites during the high density sub-phase was overcome with replacement cultivation. The findings of the present study suggest SC in the green growth phase may be a promising approach to significantly enhance biomass accumulation in culturing microalgae.Staged cultivation (SC) in growth phase is effective to increase biomass by corresponding methods. A mix of red and white light (2:1) overcomes limitation associated with insufficient supply of light at low cell density. Shortage of nutrients during the medium density sub-phase is overcome with fed-batch culture or replacement cultivation; and excess accumulation of extracellular metabolites during the high density sub-phase is overcome with replacement cultivation.Download high-res image (65KB)Download full-size image

•Stresses attenuated pigments, proteins, and membrane polar lipids.•HL − N was more effective than HL and − N for TAG induction.•N. oculata involved prokaryotic pathway predominantly for TAG synthesis.•Key enzymes involved in lipid synthesis were up-regulated differentially by stresses.Abiotic stresses such as high light (HL) and/or nitrogen deficiency (− N) might induce triacylglycerol (TAG) biosynthesis in microalgae. However, little is known about the changes of lipid classes and the mechanism underlying the enhanced lipid production as imposed by irradiance and nitrogen starvation. In this work HL and − N were applied individually or in combination to Nannochloropsis oculata to investigate the underlying mechanism for stress-associated TAG synthesis. Stress conditions, HL − N in particular, inhibited cell growth and stimulated the accumulation of neutral lipids, predominantly in the form of TAG (402 mg/g DW). The membrane polar lipids, however, were attenuated to differential degrees depending on the stress conditions. Over 90% of TAG sn-2 position was occupied by C16 fatty acids, suggesting that N. oculata may involve the prokaryotic pathway mostly for TAG synthesis. Key enzymes of acetyl-CoA carboxylase, diacylglycerol acyltransferase and NADPH involved in lipid biosynthesis were up-regulated approximately 3–13 times by HL − N. Our results altogether demonstrated that the photosynthetically fixed carbon was partitioned into TAG as the major energy reservoir, and irradiance and nitrogen were the two important engineering factors that enhanced lipid production by pushing photosynthetic carbon flux to fatty acid biosynthesis and pulling fatty acids to TAG assembly.Download high-res image (118KB)Download full-size image

•HL + G − N was found efficient to induce astaxanthin accumulation in fermented cells.•A two-step cultivation strategy was proposed and conducted in C. zofingiensis.•Higher cell density and astaxanthin yield were achieved in the fermentation.•Astaxanthin content in fermented cells was increased during the outdoor induction.In the present study, high light and nitrogen starvation with glucose-fed to the culture was found efficient to induce astaxanthin accumulation in Chlorella zofingiensis. Therefore, a two-step cultivation strategy including high biomass yield fermentation and outdoor induction with an energy-free RFP was conducted. During the fermentation, the highest cell density of 98.4 g L−1 and astaxanthin yield of 73.3 mg L−1 were achieved, which were higher than those so far reported in C. zofingiensis. During the outdoor induction, astaxanthin content was further increased by 1.5-fold leading to the highest astaxanthin productivity of 5.26 mg L−1 day−1 under an optimal dilution of 5-fold. Our work thus provided an effective two-step cultivation strategy for production of astaxanthin by C. zofingiensis.