Search

[Display omitted] • ARTP-ethanol co-mutation for synchronous enhancement of lipid and astaxanthin. • Improved key enzyme activity, gene expression for lipid and astaxanthin biosynthesis. • Elucidated mechanisms of lipid and astaxanthin accumulation by ARTP and ethanol. • Upregulation of genes nubpl , Abcc 2, PAO related to lipid and astaxanthin accumulation. Haematococcus lacustris is a precious algal species renowned for its ability to simultaneous production of astaxanthin and lipid. However, its slow growth rate necessitates the development of appropriate mutagenesis methodologies to effectively enhance its synchronous production of both astaxanthin and lipid. This study introduced the co-mutation of Atmospheric and Room Temperature Plasma (ARTP) and ethanol. The performance and preliminary mechanisms underlying the combined accumulation of astaxanthin and lipid in H. lacustris under both mutations by ARTP and ethanol were comparatively analyzed. Combined astaxanthin and lipid contents relative to total cell mass in the 110–2 strain reached 54.4%, surpassing that of strain 0–3 and the control by 17.0% and 47.6% respectively. Transcriptome level analysis revealed how both ethanol and ARTP induction promote the expressions of carotenoid and lipid synthesis genes and related enzymatic activities. Upregulation of genes associated with cell activity contributed to lipid and astaxanthin metabolism in multi pathways. [ABSTRACT FROM AUTHOR]

[Display omitted] • 20 K-lux light/3x-nutrients exhibited max. astaxanthin 54.4 mg/L by C. zofingiensis. • 4–8 K-lux successive light increase and 5% CO 2 exhibited the best biomass yield. • The maximum lipid 37% accumulation was obtained at 5% CO 2 and 20 k-lux light. • 20 K-lux light/3x-nutrients exhibited max. astaxanthin content 16.7 mg/g DCW. • Two-stage designed bioprocess boosts astaxanthin yield of C. zofingiensis. Microalgal astaxanthin possesses numerous bioactivities and has several health applications. The current research focuses on designing and optimizing the two-stage mixotrophic bioprocess by Chlorella zofingiensis for astaxanthin production. Gradual increase in light intensity (4-8k-lux) and 3x micronutrient concentration were the key parameters for maximizing biomass yield of 2.5 g/L during 15 days of stage I. Furthermore, stress conditions (excessive CO 2, light, salinity, etc.) enhanced astaxanthin yield at stage II. 20k lux light, 3x nutrients, and 5% CO 2 were the best ranges for maximum astaxanthin production. Maximum biomass yield and astaxanthin content were 3.3 g/L and 16.7 mg/g, respectively, after 29 days of bioprocess. Astaxanthin biosynthesis was also affected by salinity, but less than other parameters. Astaxanthin bioprocess resulted in enhanced lipid yields of 35–37%, which could be used for biodiesel. This study shows promising scale-up potential with attractive sustainability features of Chlorella zofingiensis model for commercial astaxanthin-lipid biorefinery. [ABSTRACT FROM AUTHOR]

In vivo spatiotemporal dynamics of lipids and astaxanthin evolution in Haematococcus pluvialis mutant induced with 15% CO 2 and high light intensity were monitored with high spatial resolution in a non-destructive and label-free manner using single-cell Raman imaging. Astaxanthin intensity increased by 3.5 times within 12 h under 15% CO 2 , and the accumulation rate was 5.8 times higher than that under air. Lipids intensity under 15% CO 2 was 27% higher than that under air. The lipids initially concentrated in chloroplast under 15% CO 2 due to an increase of directly photosynthetic fatty acid, which was different from the whole-cell dispersed lipids under air. Astaxanthin produced in chloroplast first accumulated around nucleus and then spread in cytoplasmic lipids under both air and 15% CO 2 . The calculation results of kinetic models for lipids and astaxanthin evolutions showed that accumulation rate of lipids was much higher than that of astaxanthin in cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Myo -inositol (MI) application promotes astaxanthin and lipid coaccumulation. • MI upregulated carotenogenesis- and lipogenesis-associated genes and ethylene levels. • MI alleviated oxidative stress by increasing the levels of antioxidant enzymes. • Ethylene signalling had a positive effect on astaxanthin synthesis under MI induction. In the present study, exogenous myo -inositol (MI) was applied to induce natural astaxanthin and biolipid accumulation in Haematococcus pluvialis. Under 200 μM MI, algal cells exhibited 62.11 % and 34.67 % increases in astaxanthin and lipid content, respectively, compared to the control. The carotenogenesis and lipogenesis genes were upregulated by induction of MI. Interestingly, MI addition elevated the ethylene (ETH) content and activated antioxidant enzyme-associated gene levels, which could be involved in alleviating oxidative stress. Further data showed that the ETH signal played a positive function in stimulating astaxanthin biosynthesis under MI induction. Supplementation with ethephon plus MI boosted the astaxanthin content to 33.08 ± 0.03 mg g−1 by further upregulating astaxanthin biosynthesis genes and blocking reactive oxidative species (ROS) levels, and vice versa under ETH inhibition. This study provides a potential induction approach for natural astaxanthin production and explains the role of ethylene signalling in regulating astaxanthin synthesis by H. pluvialis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Phaffia rhodozyma was evaluated as a potential source of bioproducts. • Recovery and fractionation of astaxanthin, β-carotene, proteins and lipids. • Integration of a sequential operations in a biorefinery approach. • Carotenoids to produce sustainable bioactive plastics. Carotenoids over-producing yeast has become a focus of interest of the biorefineries, in which the integration of the bioproduction with the following downstream processing units for the recovery and purification of carotenoids and other value-added byproducts is crucial to improve the sustainability and profitability of the overall bioprocess. Aiming the future implementation of Phaffia rhodozyma -based biorefineries, in this work, an integrative process for fractionation of intracellular compounds from P. rhodozyma biomass using non-hazardous bio-based solvents was developed. After one-extraction step, the total amount of astaxanthin, β-carotene, lipids and proteins recovered was 63.11 µg/g DCW , 42.81 µg/g DCW , 53.75 mg/g DCW and 10.93 mg/g, respectively. The implementation of sequential back-extraction processes and integration with saponification and precipitation operations allowed the efficient fractionation and recovery (% w/w) of astaxanthin (∼72.5 %), β-carotene ∼90.17 %), proteins (21.04 %) and lipids (23.72 %). After fractionation, the manufacture of carotenoids-based products was demonstrated, through the mixture of carotenoids-rich extracts with bacterial cellulose to obtain biologically active bioplastics. [ABSTRACT FROM AUTHOR]

[Display omitted] • The combined application of SA and NaSH heightened astaxanthin and lipid yields. • SA and NaSH induction upregulated growth and carotenogenesis gene expression levels. • Cellular H 2 S mediated ROS signal transduction and enzymatic antioxidant levels. To obtain higher yield of natural astaxanthin, the present study aims to develop a viable and economic induction strategy for astaxanthin production comprising succinic acid (SA) combined with sodium hydrosulfide (NaHS). The biomass (1.33 g L-1), astaxanthin concentration (44.96 mg L-1), astaxanthin content (163.55 pg cell−1), and lipid content (55.34%) were achieved under 1.0 mM SA and 100 μM NaHS treatment. These results were concomitant with enhanced hydrogen sulfide (H 2 S) but diminished reactive oxide species (ROS). Further study discovered that endogenous H 2 S could improve astaxanthin and lipid coproduction under SA induction by mediating related gene transcript levels and ROS signalling. Additionally, the concentrations of biomass and astaxanthin increased to 2.14 g L-1 and 66.25 mg L-1, respectively, under the induction of SA and NaHS in a scaled-up bioreactor. Briefly, the work proposed a novel feasible strategy for high yields of biomass and astaxanthin by H. pluvialis. [ABSTRACT FROM AUTHOR]

• Integration of MT and Put further promotes astaxanthin and lipids production. • MT and Put treatment upregulate carotenogenesis and lipogenesis. • MT and Put alleviate oxidative stress-caused by abiotic stresses in H. pluvialis. • MT and Put regulate GABA and ROS signallings. Coupling chemical induction and abiotic stresses is a beneficial strategy for astaxanthin (Asta) induction in Haematococcus pluvialis. The combined application of melatonin (MT) and putrescine (Put) induced Asta and lipid biosynthesis in H. pluvialis under adverse conditions. Under MT and Put inductions, the highest Asta and lipid contents were 3.64% and 55.84%, which were 1.71- and 1.17-times higher than the control group, respectively. The combination of MT and Put also enhanced the expression of carotenogenic, lipogenic and antioxidant enzyme genes. Additionally, this combined treatment increased the endogenous Put content while decreasing the reactive oxygen species (ROS) and γ-aminobutyric acid (GABA) levels. Further results proved that endogenous Put promoted Asta production and alleviated oxidative stress by regulating carotenogenesis and GABA and ROS signaling. This study describes a potential process for stimulating Asta and lipid coproduction and highlights the connections among MT, Put, signaling molecules, Asta and lipid synthesis in H. pluvialis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Haematococcus pluvialis is an amazing cell factory for astaxanthin. • Various stresses stimulate H. pluvialis to accumulate astaxanthin. • H. pluvialis has potential for co-production of multiple metabolites. • How H. pluvialis regulates astaxanthin biosynthesis remains largely unknown. • Novel technology is required for H. pluvialis to enhance astaxanthin production. Astaxanthin is one of the secondary carotenoids involved in mediating abiotic stress of microalgae. As an important antioxidant and nutraceutical compound, astaxanthin is widely applied in dietary supplements and cosmetic ingredients. However, most astaxanthin in the market is chemically synthesized, which are structurally heterogeneous and inefficient for biological uptake. Astaxanthin refinery from Haematococcus pluvialis is now a growing industrial sector. H. pluvialis can accumulate astaxanthin to ∼5% of dry weight. As productivity is a key metric to evaluate the production feasibility, understanding the biological mechanisms of astaxanthin accumulation is beneficial for further production optimization. In this review, the biosynthesis mechanism of astaxanthin and production strategies are summarized. The current research on enhancing astaxanthin accumulation and the potential joint-production of astaxanthin with lipids was also discussed. It is conceivable that with further improvement on the productivity of astaxanthin and by-products, the algal-derived astaxanthin would be more accessible to low-profit applications. [ABSTRACT FROM AUTHOR]

[Display omitted] • Succinic acid fed-batch strategy increases biomass, astaxanthin and lipid yields. • Succinic acid upregulates astaxanthin and lipids biosynthetic genes levels. • Succinic acid improves reactive oxygen species levels and antioxidase activities. Effects of succinic acid (SA) in fed-batch feeding mode on astaxanthin and lipids biopoduction of Haematococcus pluvialis against abiotic stresses were explored. By comparison with the control, the initial addition of SA on day 0 increased the production of astaxanthin by 71.61%. More importantly, the maximum values of astaxanthin (35.88 mg g−1) and lipid (54.79%) contents were obtained after supplementation of SA on day 7. Meanwhile, under SA treatment, the chlorophyll, carbohydrate, and protein levels were reduced, but the intracellular levels of SA and reactive oxygen species (ROS), the transcription levels of astaxanthin and fatty acids biosynthesis-, and antioxidant system-related genes were increased. Furthermore, scaling-up cultivation in bioreactor further enhanced the astaxanthin productivity from H. pluvialis. Generally, this study proved the intermittent SA feeding method in fed-batch culture as a potent strategy that facilitated massive astaxanthin and lipids production in algae. [ABSTRACT FROM AUTHOR]

• Cu induces astaxanthin and lipids accumulation in microalgae under adverse conditions. • Cu upregulates the transcriptional levels of carotenogenesis and lipogenesis genes. • Cu increases intracellular ROS and Ca2+ levels and activates the antioxidant system. • Cu activates CDPK and MAPK signaling. • Crosstalk among Ca2+, CDPK, MAPK and ROS signal transduction induced by Cu is speculated. This study investigated the effects of copper (Cu) on astaxanthin and lipid biological synthesis in unicellular alga Haematococcus pluvialis under high-light (HL) and nitrogen-deficiency (ND) conditions. During a 15-day cultivation period, the astaxanthin and lipid contents reached the peak values (3.32% and 47.72%) under 6 μM Cu treatment, which were increased by 66.87% and 34.99% compared to nontreated group, respectively. The application of Cu also increased the transcriptional expression of biosynthesis genes and antioxidant enzyme-related genes, as well as increased the intracellular calcium (Ca2+) level but led to a decrease in reactive oxygen species (ROS) levels. Additionally, Cu treatment induced the activation of calcium-dependent protein kinases (CDPKs) and mitogen-activated protein kinases (MAPKs). This approach simultaneously facilitated astaxanthin and lipid production, and the role of Cu were elucidated on the regulation of signal transduction (e.g., Ca2+, CDPK, MAPK and ROS) in the carotenogenesis and lipogenesis in H. pluvialis. [ABSTRACT FROM AUTHOR]

• GABA treatment promoted astaxanthin and lipids production under abiotic stress. • GABA application upregulated carotenogenesis and lipogenesis. • Cellular GABA alleviated oxidative stress caused by abiotic stress in H. pluvialis. The effects of γ-aminobutyric acid (GABA) on the biomass and astaxanthin and lipids production in Haematococcus pluvialis under combined salinity stress and high-light stresses were investigated. The results showed that the highest biomass (1.65 g L−1), astaxanthin production (3.86 mg L−1 d−1) and lipids content (55.11%) in H. pluvialis LUGU were observed under the 0.25 mM GABA treatment. Moreover, compared with salinity and high-light stress, GABA treatment also increased the transcript levels of biosynthesis genes, the contents of endogenous GABA and carbohydrates but decreased reactive oxygen species (ROS) levels. Further evidence revealed that intracellular GABA could regulate cell growth, astaxanthin production and lipids synthesis by mediating carotenogenesis, lipogenesis and ROS signalling. Collectively, this study provides a combined strategy for promoting the coproduction of astaxanthin and lipids and sheds light on the regulatory mechanism through which GABA affects cell growth, astaxanthin production and lipids biosynthesis in H. pluvialis under unfavourable conditions. [ABSTRACT FROM AUTHOR]

• MT treatment promotes astaxanthin and lipids contents under adverse conditions. • MT treatment enhances H. pluvialis abiotic stress tolerance by regulating ROS. • Metabolomic mechanisms related to MT induction were elucidated in H. pluvialis. • The GABA shunt plays a key role in astaxanthin synthesis following treatment with MT. The effect of melatonin (MT) on the coproduction of astaxanthin and lipids was studied in Haematococcus pluvialis under inductive stress conditions. The contents of astaxanthin and lipids were enhanced by 1.78- and 1.3-fold, respectively. MT treatment upregulated the transcription levels of carotenogenic, lipogenic and antioxidant system-related genes and decreased the levels of abiotic stress-induced reactive oxidative species (ROS). Further metabolomic analysis suggested that the intermediates in glycolysis and TCA cycle facilitate the accumulation of astaxanthin and lipids in algae treated with MT. Meanwhile, MT treatment upregulated the metabolite levels of the γ-aminobutyric acid (GABA) shunt, which might regulate the carbon-nitrogen balance and the antioxidant system. After MT treatment, exogenous linoleic acid, succinate, and GABA further increased the astaxanthin content. This study may help to elucidate the specific responses to MT induction in H. pluvialis and to identify novel biomarkers that may be employed to further promote astaxanthin and lipids coproduction. [ABSTRACT FROM AUTHOR]

• Fed-batch strategy increased 41.5% lipid productivity without compromising biomass. • Carbon dependent kinetics under stress conferred cells with a higher biomass. • Central carbon metabolism offered energy and carbon availability for lipid synthesis. • Sufficient precursor and active synthetic pathway promoted lipid content. • Fed-batch strategy was attractive by understanding lipid metabolism in detail. To improve lipid and astaxanthin productivity without compromising biomass during the whole cultivation period, carbon-dependent kinetics involving nitrogen stress was applied under excess light to elevate intracellular carbon availability and metabolic activity of Chromochloris zofingiensis. Results suggested that fed-batch strategy proposed could increase lipid and astaxanthin productivity to 457.1 and 2.0 mg L−1 d−1, respectively. Biomass productivity at 1084.3 mg L−1 d−1 was comparable with that under suitable condition. Then 13C tracer-based metabolic flux analysis (13C-MFA) demonstrated that central carbon metabolism provided ATP, NADPH and carbon availability for lipid biosynthesis during the strategy. In combination with targeted metabolite analysis, 13C-MFA revealed that the strategy improved precursor content for lipid biosynthesis and elevated path rate to synthesize C16:0 and C18:0. The enhanced lipid content potentially accounted for the high biomass productivity. Therefore, comprehensively understanding relationships between carbon availability and carbon conversion could precisely design strategy for productivity improvements during cultivation. [ABSTRACT FROM AUTHOR]

• Exogenous MT improved astaxanthin and lipid synthesis in alga under abiotic stress. • Coupling of MT and CaCl 2 further enhanced the production of astaxanthin and lipids. • MT and CaCl 2 upregulated genes related to the astaxanthin and lipid synthesis. • MT and CaCl 2 alleviated oxidative stress by upregulating the level of GSH. • MT and CaCl 2 mediated GABA signalling in response to abiotic stress. This study focused on the influence of integrating melatonin (MT) and calcium (Ca2+) on the simultaneous accumulation of astaxanthin and lipids in Haematococcus pluvialis under abiotic stress conditions. Compared with the control condition, MT induction enhanced astaxanthin and lipid contents by 65.89% and 27.38%, respectively. The highest contents of astaxanthin and lipids under combined exposure to MT and Ca2+ were 3.8% and 49.53%, respectively, which were 1.13- and 1.21-fold higher than those of cells treated with MT alone. The application of MT and Ca2+ also promoted the expression of carotenogenic and lipogenic genes and increased the levels of Ca2+ and γ-aminobutyric acid (GABA) but decreased reactive oxygen species (ROS) levels. Further evidence indicated that the increased cellular Ca2+ could promote astaxanthin biosynthesis under MT induction by regulating carotenogenic gene levels and GABA and ROS signalling. The integrated strategy efficiently improved the coproduction of astaxanthin and lipids in H. pluvialis. [ABSTRACT FROM AUTHOR]

• AGP locus in Tetraslemis sp. was mutated by CRISPR-Cas9 RNP. • AGP mutants showed enhanced lipid production and the absence of starch synthesis. • Fatty acids in AGP mutants increased by 274% and 314% compared to that in wild type. • AGP mutants showed 2.3- and 2.7-fold higher lipid productivity than the wild type. A marine green microalga, Tetraselmis sp., has been studied for the production of biomass and lipids in seawater culture. Since carbohydrate and lipid biosynthesis are competitive metabolic pathways, we attempted to increase lipid synthesis in Tetraselmis by inhibiting carbohydrate synthesis. The main regulatory enzyme in the starch synthesis pathway is ADP-glucose pyrophosphorylase (AGP). AGP loss-of-function mutants were developed using the CRISPR-Cas9 ribonucleoprotein (RNP) delivery system. AGP mutants showed a slight decrease in growth. However, the lipid content in two AGP mutants was significantly enhanced by 2.7 and 3.1 fold (21.1% and 24.1% of DCW), respectively, compared to that in the wild type (7.68% of DCW) under nitrogen starvation. This study is an example of metabolic engineering by genetic editing using the CRISPR-Cas9 RNP method in marine green microalgae. Consequently, starchless Tetraselmis mutants might be considered potential producers of lipids in seawater cultures. [ABSTRACT FROM AUTHOR]

• A two-stage screening strategy was established to identify positive phytohormones. • Auxins were the most efficient phytohormones for astaxanthin accumulation. • Maximum contents of astaxanthin (13.1 mg/g) and lipids (64.5% DW) were obtained. • Relations between phytohormones and astaxanthin and lipid biosynthesis were revealed. Phytohormones comprise a variety of trace bioactive compounds that can stimulate cell growth and promote metabolic shifts. In the present work, a two-stage screening strategy was innovatively established to identify positive phytohormones for enhancement of astaxanthin and lipid coproduction in microplate-based cultures of mixotrophic Chromochloris zofingiensis. The results showed that auxins were the most efficient stimulators for astaxanthin accumulation. The maximum content of 13.1 mg/g and yield of 89.9 mg/L were obtained using indole propionic acid (10 mg/L) and indoleacetic acid (7.8 mg/L), representing the highest levels of astaxanthin in this microalga reported to date. Total lipids with the highest content (64.5% DW) and productivity (445.7 mg/L/d) were coproduced with astaxanthin using indoleacetic acid. Statistical analysis revealed close relations between phytohormones and astaxanthin and lipid biosynthesis. This study provides a novel original strategy for improving astaxanthin and lipid coproduction in C. zofingiensis using the selected phytohormones as positive stimulators. [ABSTRACT FROM AUTHOR]

• BHA enhances astaxanthin and lipid levels under stress condition. • Physiological changes in the H. pluvialis were comparatively examined. • Metabolomic profiles about synthesis of astaxanthin and lipids were investigated. The major goal of this study was to explore the functions of butylated hydroxyanisole (BHA) combined with abiotic stress on the cultivation of the microalga Haematococcus pluvialis for astaxanthin and lipid production. Here, the effect of BHA on astaxanthin and lipid accumulation and physiological and metabolomic profiles was investigated. These results suggested that astaxanthin content was increased by 2.17-fold compared to the control. The lipid content was enhanced by 1.22-fold. BHA treatment simultaneously reduced carbohydrates and protein and delayed the decay of chlorophyll. Furthermore, metabolomic analysis demonstrated that BHA upregulated and activated the bioprocesses involved in cellular basal metabolism and signalling systems, such as glycolysis, the TCA cycle, amino acid metabolism and the phosphatidylinositol signalling system, thus enhancing astaxanthin and lipid accumulation. Altogether, this research shows the dramatic effects of BHA on algal metabolism in the regulation of key metabolic nodes and provides novel insights into microalgal regulation and metabolism. [ABSTRACT FROM AUTHOR]