Astaxanthin Production Research Articles

Large-scale bioproduction of natural astaxanthin in Yarrowia lipolytica

Astaxanthin is a high-value chemical with strong antioxidant, anti-inflammatory and coloration properties. Here, a set of novel genetic engineering and culturing strategies for efficient astaxanthin bioproduction in Yarrowia lipolytica is reported. Modular pathway engineering and the fusion of two key enzymes (geranylgeranyl pyrophosphate synthase and bifunctional lycopene cyclase/phytoene synthase) yielded a recombinant strain with enhanced β-carotene and astaxanthin production, accumulating astaxanthin up to 131.9 mg/L in small-scale cultures with sucrose and 587.3 mg/L in a 3-L bioreactor with glucose as carbon sources, respectively. Notably, formation of intracellular carotenoid crystals was observed. Addition of oil overlayer at different regimes in 3-L bioreactors promoted secretion of astaxanthin and increased astaxanthin titer up to 973.4 g/L. Lastly, fed-batch fermentation in a 100-L bioreactor with glycerol as the carbon source resulted in a final astaxanthin titer of 812.3 mg/L after 114 h, representing the highest astaxanthin titer reported so far in large-scale cultures.

Biosynthesis of astaxanthin by using industrial yeast

AbstractAstaxanthin has been widely applied in feed additives and the medical industry due to its antioxidant, coloring, and health‐promoting properties. Traditional processes for astaxanthin production mainly include natural extraction and chemical synthesis; however, they cannot meet the growing market demand and consumers’ requirements for natural products. Microbial synthesis of astaxanthin offers a promising alternative. Many natural sources of astaxanthin, such as Xanthophyllomyces dendrorhous, and genetically modified yeasts, such as Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus have been isolated and contructed. Various strategies such as mutagenesis, genetic modification, and fermentation regulation have been developed to increase the astaxanthin production from industrial yeast. Due to the broad substrate spectrum of industrial yeast, many low‐cost resources have also been explored for astaxanthin production. Accordingly, this review will mainly summarize the progress of research into the production of astaxanthin with yeasts, analyze factors affecting astaxanthin synthesis in metabolic pathways, and propose methods and strategies to achieve high yields of astaxanthin from yeasts. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd.

Development of a 5-aminolevulinic acid feeding strategy capable of enhancing Haematococcus pluvialis biomass, astaxanthin, and fatty acid yields

Effective inducers play essential roles in the regulation of cell growth and astaxanthin production in Haematococcus pluvialis. Here, a novel 5-aminolevulinic acid (5-ALA) feeding strategy was developed and found to enhance H. pluvialis biomass, fatty acid, and astaxanthin yields. Specifically, 5-ALA feeding (4 μM) on day 1 caused respective 23.8 %, 24.8 %, and 20.3 % increases in biomass, fatty acid, and astaxanthin yields. The observed enhancement of biomass accumulation associated with the provision of 5-ALA during the early stages of growth was attributable to enhanced photosynthetic carbon assimilation. This increased biomass accumulation, in turn, contributed to the measured increases in both fatty acid and astaxanthin yields. Overall, these results provide new insight into the importance of photosynthetic carbon assimilation as a determinant of biomass accumulation and a regulator of both fatty acid and astaxanthin production, while offering an effective strategy capable of accelerating astaxanthin production by H. pluvialis in commercial agricultural contexts.

Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae

The accumulation of the intermediate zeaxanthin and canthaxanthin in the astaxanthin biosynthesis pathway catalyzed by β-carotene hydroxylase (crtZ) and β-carotene ketolase (crtW) decreases the content of the astaxanthin. Here, we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis in Saccharomyces cerevisiae. The results demonstrated that the fusion enzyme of crtZ-crtW with 2 X GGGGS peptides linker can effectively reduce the accumulation of intermediates and improves the content of astaxanthin. Compared with the control strain, the fusion enzyme of ketase and hydroxylase reduced zeaxanthin and canthaxanthin by 7 and 14 times and increased astaxanthin by 1.6 times, respectively. Moreover, 9 variant fusion mutants with improved astaxanthin production were generated through directed evolution. Combining these dominant mutants generated a variant, L95S + I206L, which increased the astaxanthin content of 3.8 times than the control strain. The AlphaFold2 assisted structural analysis indicated that these two mutations alter the interaction between the substrate and the enzymes pocket. Our research provided an efficient idea to reduce the accumulation of the intermediate products in complex biosynthesis pathway.

Improvement of Astaxanthin Production in Aurantiochytrium limacinum by Overexpression of the Beta-Carotene Hydroxylase Gene.

Aurantiochytrium limacinum is a heterotrophic eukaryotic microorganism that can accumulate high levels of commercial products such as astaxanthin and docosahexaenoic acid. Due to its rapid growth and relatively simple extraction method, A. limacinum is considered a promising astaxanthin resource to replace the conventional microalgal production. However, the astaxanthin biosynthetic process in A. limacinum remains incompletely understood, especially in those catalysed by β-carotene hydroxylase (CrtZ) and ketolase. In this study, we overexpressed a crtZ candidate gene to increase astaxanthin production and expand our understanding of the conversion from beta-carotene to astaxanthin. The resultant transformant AlcrtZ#10 cultivated for 5days showed a significant increase in astaxanthin production per culture (2.8-fold) and per cell (4.5-fold) compared with that of the wild-type strain. Strikingly, longer light exposure increased astaxanthin production and decreased the beta-carotene content in the wild-type strain, suggesting that light exposure duration is important for astaxanthin production in A. limacinum. Among several predicted intermediates, furthermore, the cantaxanthin produced from β-carotene by ketolase activity were enhanced in the transformant AlcrtZ#10. Although the further investigation is needed, this result suggested that the main route of astaxanthin was via cantaxanthin. Thus, our findings will be valuable not only for its application, but also for understanding the astaxanthin biosynthetic process in A. limacinum.

Enhanced production of astaxanthin and co-bioproducts from microalga Haematococcus sp. integrated with valorization of industrial wastewater under two-stage LED light illumination strategy

Microalgal-based biochemicals and biofuels integrated with phycoremediation of industrial wastewater can potentially increase the profitability and sustainability of the food–water–energy–environment nexus. A key challenge in the high-density cultivation of microalgae is to provide light of efficient wavelengths in photobioreactors. In this study, specific narrow bands of light-emitting diodes (LEDs) and their intensities were manipulated to enhance astaxanthin and co-bioproduct production by the microalga Haematococcus sp. Among the strategies tested, the two-stage LED light illumination involving red LED for 5 days and then blue LED for the next 5 days, with a continuous light intensity of 40 μ mol m −2 s −1 , was shown to be the most efficient in improving the performance of Haematococcus sp. These conditions led to the highest astaxanthin production, of 2.55 mg/L, which was 1.22- to 2.07-fold higher than that by one-stage cultivation, with no significant effect on pigments (chlorophylls and carotenoids) and lipid production. Using a two-stage strategy integrated with the valorization of seafood processing effluent, Haematococcus sp. grew well in the secondary effluent from a seafood processing plant, giving 1.33 g/L of biomass, with 3.39 mg/L of astaxanthin, 14.3 mg/L of chlorophylls, 6.22 mg/L of carotenoids, and 0.41 g/L of lipids. The produced astaxanthin can be used as a sustainable source of bioactive ingredients with desirable antioxidative properties, giving ABTS activity of 5.24 mg of TE/g of astaxanthin, DPPH activity of 2.04 mg of GAE/g of astaxanthin, and PFRAP activity of 0.87 mg of GAE/g of astaxanthin. Meanwhile, lipids contain long-chain fatty acids (C16–C18> 96%) and their biodiesel characteristics were also satisfactory when compared to those required by international biodiesel standards, indicating the potential for use as biodiesel feedstock. Our results suggested that the integration process was cost effective and environmentally friendly for microalgal-based wastewater treatment and a microalgal-based multiproduct biorefinery. • Two-stage cultivation using red–blue LEDs was effective for astaxanthin production. • Industrial effluent was valorized into valuable products and effectively treated. • Microalgae also produce lipids with high potential as biodiesel feedstocks. • This integrated process would contribute to the sustainability of biofuel production.

Myo-inositol facilitates astaxanthin and lipid coproduction in Haematococcus pluvialis by regulating oxidative stress and ethylene signalling

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.

Process model and techno-economic analysis of natural astaxanthin production from microalgae incorporating geospatial variabilities

Microalgae possess tremendous potential to meet the ever-increasing demand for food, feed, energy and fuels in a sustainable manner. Microalgae-based natural products are poised to compete with synthetic products in the market. A comprehensive theoretical approach was developed to determine economic feasibility of natural astaxanthin production from microalgae at various locations in the United States. A detailed economic analysis was conducted to determine natural astaxanthin production costs considering residual biomass utilization for various locations. Baseline production costs of astaxanthin were found to range from 1400 to 5000 $ kg−1 for the different locations. The results suggest that both microalgae productivity potential and astaxanthin production costs are heavily influenced by geographic location and type of bioreactor used. While production costs of natural astaxanthin are found to be higher than those of synthetic astaxanthin, economic analysis reveals significant opportunity for the economic viability of natural microalgae-based astaxanthin production in commercial scales.

Rutin Stimulates the Green Alga Chromochloris zofingiensis for Improved Biomass and Astaxanthin Production.

Chromochloris zofingiensis represents a potential algal producer of the value-added ketocarotenoid astaxanthin. Here, rutin, a low-cost flavonoid compound, was evaluated regarding its roles in C. zofingiensis production under astaxanthin-inducing conditions via physiological, biochemical, and transcriptomics analyses. The rutin treatment allowed C. zofingiensis to achieve 81.2% more biomass and 20.5% greater astaxanthin content under nitrogen deprivation, leading to more than doubled astaxanthin production. The rutin-treated C. zofingiensis had higher levels of chlorophylls, proteins, and lipids and lower carbohydrate level than the control. Rutin promoted the intracellular abscisic acid (ABA) level, which could be restored by the ABA biosynthesis inhibitor, accompanied by the restoration of biomass concentration and astaxanthin content. The application of exogenous ABA to C. zofingiensis also furthered biomass concentration and astaxanthin accumulation. Together with the comparative transcriptomics analysis, our study provides implications into the involvement of ABA in rutin-mediated stimulation of C. zofingiensis growth and astaxanthin accumulation and highlights a feasible strategy of combining stress and chemical induction for improved microalgal production.

High-density cultivation of Phaffia rhodozyma SFAS-TZ08 in sweet potato juice for astaxanthin production

BackgroundAstaxanthin is an important commercially valuable secondary metabolite produced during fermentation of the yeast Phaffia rhodozyma that can be used in aquatic feedstock. ResultsIn this study, we used sweet potato juice (SPJ), the by-product of sweet potato starch, to obtain a high-density culture of P. rhodozyma. We confirmed that hydrochloric acid deproteinized SPJ is suitable for the culture of P. rhodozyma, and using this as a substrate, supplemented with 0.05% yeast extract, we performed batch fermentation in a 5 L fermenter. Compared to shaking flask fermentation, we obtained an 18.86% increase in yeast biomass and a 32.5% increase in astaxanthin yield using the batch process. After culturing P. rhodozyma in a 5 L fermenter for 120 h, we achieved biomass and astaxanthin yield of 45.2 g/L and 19.465 mg/L, respectively. ConclusionsIn this study, we found that deproteinized SPJ was the most suitable for P. rhodozyma through experiments on different cultivars and different processing stages of SPJ. On the basis of deproteinized SPJ, supplemented with yeast extract, the biomass and astaxanthin yield reached a high level. The optimized system can substantially reduce the costs of raw material for astaxanthin yield by P. rhodozyma and enhance the comprehensive utilization of sweet potato resources.How to cite: Zhang C, Zhao X, Yao M, et al. High-density cultivation of Phaffia rhodozyma SFAS-TZ08 in sweet potato juice for astaxanthin production. Electron J Biotechnol 2023;61. https://doi.org/10.1016/j.ejbt.2022.09.007.

Increasing production and bio-accessibility of natural astaxanthin in Haematococcus pluvialis by screening and culturing red motile cells under high light condition

The thick cell wall and low astaxanthin productivity were two important bottlenecks limiting industrial production of astaxanthin via Haematococcus pluvialis. This study reports a strategy for increasing production and bio-accessibility of astaxanthin in H. pluvialis by screening and culturing red motile cells under high light condition. Compared with the original strain NBU489, the biomass of the novel isolated strain RMS10 increased by 31.9% under low light condition, and the astaxanthin content (44.6 mg/g) increased by 53.3% after 9-day high light induction, which were readily extracted and digested without cell disruption. Subsequent transcriptomic analysis confirmed the accumulation of astaxanthin and lipids in RMS10 cells as expression of genes associated with biosynthesis of fatty acid and astaxanthin were up-regulated, while those involved in thick cell wall biosynthesis and reactive oxygen species scavenging were down-regulated in RMS10. Collectively, this study provides a simple and effective method for economical production of natural astaxanthin.

Simple and rapid separation of Haematococcus pluvialis and ciliate based on the dean-coupled inertial microfluidics.

Astaxanthin with high antioxidant activity is of great practical value and Haematococcus pluvialis is recognized as the best natural astaxanthin producer. The yield of Haematococcus pluvialis was often affected by the ciliate during its production, however, the use of biochemical pesticides might have a great impact on Haematococcus pluvialis. Therefore, a simple microfluidic chip with the spiral microchannel was developed for continuous-flow physical separation of ∼10µm ciliate from ∼30µm Haematococcus pluvialis since their different sizes resulted in different equilibrium positions in the channel due to the Dean-coupled inertial migration. First, a spiral microchannel with a width of 700µm and a height of 130µm in the microfluidic chip was developed using three-dimensional printing and verified to completely separate polystyrene particles of 10µm from those of 30µm. Then, this microfluidic chip was used to separate the actual sample, and experimental results showed that ∼80% of ciliate was continuously separated from Haematococcus pluvialis at a flow rate of 2.8ml/min. More importantly, no additional biochemical reagents were used and the activity of Haematococcus pluvialis was not affected. This microfluidic chip featured with simple design, automatic operation, and small size is promising for purification and breeding of Haematococcus pluvialis.

Production of natural astaxanthin by Phaffia rhodozyma and its potential application in textile dyeing

Microbial astaxanthin are preferred over the synthetic equivalent, not only to impart color in a wide variety of products but also to provide bioactive antioxidants nutrients to human health. The yeast Phaffia rhodozyma is considered as one of the most important natural sources of astaxanthin. The present research work was designed to enhance the biosynthesis of astaxanthin by using synthetic substrates and moving from orbital shaker to 4 L stirred tank bioreactor (STB). Moreover, it was also evaluated the potential of astaxanthin-rich extracts in dyeing textile fabrics. Under optimal conditions in STB [carbon source (glucose/xylose) and aeration rate (1 vvm) with constant light irradiation], the process production achieved a 503.66 μg/gDCW (+ 48.99%) of astaxanthin compared to the orbital shakers (256.88 μg/gDCW). The in vitro antioxidant activity and potential cytotoxicity assessment of astaxanthin-rich extracts was evaluated, indicating the potential of astaxanthin to be used as a natural dyeing agent for textiles, envisioning the replacement of synthetic colorants by natural counterpart.

Microbial Astaxanthin Production from Agro-Industrial Wastes—Raw Materials, Processes, and Quality

The antioxidant and food pigment astaxanthin (AX) can be produced by several microorganisms, in auto- or heterotrophic conditions. Regardless of the organism, AX concentrations in culture media are low, typically about 10–40 mg/L. Therefore, large amounts of nutrients and water are necessary to prepare culture media. Using low-cost substrates such as agro-industrial solid and liquid wastes is desirable for cost reduction. This opens up the opportunity of coupling AX production to other existing processes, taking advantage of available residues or co-products in a biorefinery approach. Indeed, the scientific literature shows that many attempts are being made to produce AX from residues. However, this brings challenges regarding raw material variability, process conditions, product titers, and downstream processing. This text overviews nutritional requirements and suitable culture media for producing AX-rich biomass: production and productivity ranges, residue pretreatment, and how the selected microorganism and culture media combinations affect further biomass production and quality. State-of-the-art technology indicates that, while H. pluvialis will remain an important source of AX, X. dendrorhous may be used in novel processes using residues.

Metabolic pathway assembly using docking domains from type I cis-AT polyketide synthases

Engineered metabolic pathways in microbial cell factories often have no natural organization and have challenging flux imbalances, leading to low biocatalytic efficiency. Modular polyketide synthases (PKSs) are multienzyme complexes that synthesize polyketide products via an assembly line thiotemplate mechanism. Here, we develop a strategy named mimic PKS enzyme assembly line (mPKSeal) that assembles key cascade enzymes to enhance biocatalytic efficiency and increase target production by recruiting cascade enzymes tagged with docking domains from type I cis-AT PKS. We apply this strategy to the astaxanthin biosynthetic pathway in engineered Escherichia coli for multienzyme assembly to increase astaxanthin production by 2.4-fold. The docking pairs, from the same PKSs or those from different cis-AT PKSs evidently belonging to distinct classes, are effective enzyme assembly tools for increasing astaxanthin production. This study addresses the challenge of cascade catalytic efficiency and highlights the potential for engineering enzyme assembly.

Production of polyhydroxyalkanoates and astaxanthin from lignocellulosic biomass in high cell density membrane bioreactor

Microbial-derived bioplastics are receiving more attention and are a potential option for ecological sustainability. Lignocellulosic biomass is the ultimate resource for the production of second-generation biofuels and biochemicals. In this study, lignocellulosic biomass (corn cob) was first pretreated and hydrolyzed to produce fermentable sugars. Then, polyhydroxyalkanoates (PHA) and astaxanthin were produced through batch and cell retention cultures of two strains, Bacillus megaterium ALA2 and Paracoccus sp. LL1 using corn cob hydrolysate as a carbon source. Higher cell and PHA concentrations (101.7 g/L and 57.6 g/L, respectively) with a PHA productivity of 1.07 g/L/h were obtained by B. megaterium ALA2, but Paracoccus sp. LL1 could co-produce PHA and astaxanthin. PHA and astaxanthin concentrations were significantly increased by cell retention culture of Paracoccus sp. LL1 in high cell density membrane bioreactor compared to batch culture (9.04 and 13.1-folds, respectively). In addition, PHA and astaxanthin productivities by cell retention culture of Paracoccus sp. LL1 increased to 0.579 g/L/h and 0.385 mg/L/h, respectively, which were 4.02 and 10.1-folds higher than those of batch culture. Based on gas chromatography, 1H nuclear magnetic resonance, and Fourier transform infrared analysis, B. megaterium ALA2 produced a homopolymer of poly(3-hydroxybutyrate), whereas poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with 7.84 mol% of 3-hydroxyvalerate was accumulated by Paracoccus sp. LL1. This study demonstrates that bioplastic PHA and high value-added astaxanthin can be economically produced from lignocellulosic biomass as an inexpensive renewable resource. This is the first report on the enhanced co-production of PHA and astaxanthin from lignocellulosic biomass in a membrane bioreactor.

Isolation of Dunaliella salina Microalgae from Pari Island, Jakarta, Indonesia

Dunaliella salina is a microalga from the Chlorophyta group which is reported to be found in mangrove forests. These microalgae are reported to have economic value as a producer of beta carotene, astaxanthin, and EPA fatty acids. The microalga was isolated from Pari Island, Jakarta. Sampling by streak plate method. This study aimed to obtain Dunaliella salina isolates for further exploration. The results obtained were, that three pure isolates MKA1, MKA2, and MKA3, were successfully purified. The three isolates showed oval-ovoid morphology and orange-pink pigmentation of the culture. Cultivation using seawater, glucose (10 g/L) and yeast extract (3 g/L) produced biomass of about 2.3 g/L and further optimization needs to be explored.

Static magnetic field improved growth and astaxanthin production in Haematococcus lacustris via the regulation of carbohydrate accumulation, H2O2 level, and antioxidant defense system

Static magnetic field improved growth and astaxanthin production in Haematococcus lacustris via the regulation of carbohydrate accumulation, H2O2 level, and antioxidant defense system

Insights into the extraction, characterization and antifungal activity of astaxanthin derived from yeast de-oiled biomass

The increasing popularity of bio-based oil in fuel, cosmetics and pharmaceutical industry has led the path of innovations where microbes are exploited as an alternative source. Thus, complete valorizationof left-over microbial biomass generated during these industrial fermentations will also address the environmental concern of its disposal. In line, current investigation utilizes residual de-oiled (lipid extracted) biomass of red oleaginous yeast, Rhodosporidium paludigenum for astaxanthin (AST) production and compares various cell disruption methods; conventional extraction, ultrasonic homogenization, cryogenic treatment for its efficient recovery. Among all, cryogenic treatment was found most promising and resulted in the highest AST yield ( ∼ 7.1 ± 0.72 mg/g wet cell weight) with improved extraction efficiency (47.94 %). The sub-zero temperature during this method facilitated complete homogenization of cells as well as helped to maintain the structural and functional integrity of recovered AST. Furthermore, the mechanistic insights in the antifungal action of extracted AST was also elucidated in C. albicans and C. glabrata . The data suggested that AST mediated reductive stress in biofilm cells interfered with intracellular signalling and resulted in membrane porosity, mitochondrial dysfunction leading eventual cell apoptosis. Conclusively, outcomes of this study propose a sustainable approach for AST extraction from de-oiled yeast biomass with detailed insight into its therapeutic activity. • Yeast de-oiled biomass used to produce astaxanthin (AST). • Cryogenic treatment and conventional methods are discussed for cell lysis. • Recovered AST showed comparable antioxidant activity with standard form. • Future directions on development of AST as antibiofilm agent are proposed.

Transcriptome Analysis of the Accumulation of Astaxanthin in Haematococcus pluvialis Treated with White and Blue Lights as well as Salicylic Acid

Haematococcus pluvialis is the most commercially valuable microalga for the production of natural astaxanthin, showing enhanced production of astaxanthin with the treatments of high-intensity light and hormones. The molecular mechanisms regulating the biosynthesis of astaxanthin in H. pluvialis treated with white light, blue light, and blue light with salicylic acid (SA) were investigated based on the transcriptome analysis. Results showed that the combined treatment with both blue light and SA generated the highest production of astaxanthin. A total of 109,443 unigenes were identified to show that the genes involved in the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway (PPP), and the astaxanthin biosynthesis were significantly upregulated to increase the production of the substrates for the synthesis of astaxanthin, i.e., pyruvate and glyceraldehyde-3-phosphate generated in the TCA cycle and PPP, respectively. Results of transcriptome analysis were further verified by the quantitative real-time PCR (qRT-PCR) analysis, showing that the highest content of astaxanthin was obtained with the expression of the bkt gene significantly increased. Our study provided the novel insights into the molecular mechanisms regulating the synthesis of astaxanthin and an innovative strategy combining the exogenous hormone and physical stress to increase the commercial production of astaxanthin by H. pluvialis.