Astaxanthin Production Research Articles

Diversity, astaxanthin production, and genomic analysis of Rhodotorula paludigena SP9-15

Astaxanthin is a carotenoid known for its powerful antioxidant properties. This study focused on isolating yeast strains capable of producing astaxanthin from flower and fruit samples collected in Thailand. Out of 115 isolates, 11 strains were identified that produced astaxanthin. Molecular identification techniques revealed that these isolates belonged to two species: Rhodotorula paludigena (5 isolates) and Rhodosporidiobolus ruineniae (6 isolates). Whole-genome analysis of one representative strain, R. paludigena SP9-15, identified putative candidate astaxanthin synthesis-associated genes, such as CrtE, CrtYB, CrtI, CrtS, CrtR, CrtW, CrtO, and CrtZ. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) confirmed astaxanthin production. Further optimization of astaxanthin production was carried out by investigating the effects of various factors on the growth rate and astaxanthin production. The optimal conditions were 40 g/L glucose as a carbon source, pH 7.5, and cultivation at 25 °C with 200 rpm for 3 days. Under these conditions, R. paludigena SP9-15 synthesized biomass of 11.771 ± 0.003 g/L, resulting in astaxanthin with a content of 0.558 ± 0.018 mg/g DCW (dry cell weight), an astaxanthin yield of 6.565 ± 0.238 mg/L, and astaxanthin productivity of 2.188 ± 0.069 g/L/day. These findings provide insights into astaxanthin production using red yeast strains from Thailand and highlight the potential of R. paludigena SP9-15 for further application.

Astaxanthin production from the microalga Haematococcus lacustris with a dual substrate mixotrophy strategy.

This study investigates the development of dual-substrate mixotrophy strategy to cultivate the microalga Haematococcus lacustris for astaxanthin production. The influence of different concentrations of acetate and pyruvate on biomass productivity was first assessed individually, and then both substrates were used together to improve biomass growth in the green phase and astaxanthin accumulation in red the phase. The results showed that dual-substrates mixotrophy significantly increased the biomass productivity during green growth phase up to 2-fold compared to phototrophic controls. Furthermore, supplementation of dual-substrate to the red phase increased astaxanthin accumulation by 10% in the dual-substrate group compared to single-substrate acetate and no substrate. This dual-substrate mixotrophy approach shows promise for cultivating Haematococcus for commercial production of biological astaxanthin in indoor closed systems.

Screening of the Most Effective Media for Bioprospecting Three Indigenous Freshwater Microalgae Species

Microalgae are a natural source of lipids, carotenoids, and other value-added compounds. The combination of nutrients and their precise ratio have a significant impact on the productivity of microalgae-mediated compounds. The biomass, lipid, chlorophyll, and carotenoid production of three microalgae species, namely, Monoraphidium sp., Chlorella sorokiniana, and Scenedesmus obliquus, were investigated by employing standard photoautotrophic media, namely, BG-11, BBM, and HS CHU-10. BBM was found as the most effective medium since it obtained higher biomass, lipids, and carotenoids from microalgae. The lipids and carotenoids were identified using chromatographic and microscopy techniques. The findings showed that although the Monoraphidium sp. biomass productivity was the lowest, it emerged as a substantial producer of astaxanthin, whereas the Chlorella sorokiniana culture could grow in a variety of media and produced β-carotene as a major carotenoid. On the other hand, Scenedesmus obliquus was found to be a considerable source of lipids and β-carotene. This study provided a comprehensive understanding of the appropriate medium selection in order to extract an assortment of value-added compounds from freshwater microalgae species.

Enhancement of astaxanthin production from food waste by Phaffia rhodozyma screened by flow cytometry and feed application potential.

Astaxanthin is widely used in food, aquaculture, cosmetics, and pharmaceuticals due to its strong antioxidant activity and coloring ability, but its production from Phaffia rhodozyma remains the main challenge due to the high fermentation cost and low content of carotenoid. In this study, the production of carotenoids from food waste (FW) by a P. rhodozyma mutant was investigated. P. rhodozyma mutant screened by UV mutagenesis and flow cytometry could stably produce high carotenoids at 25°C, with carotenoid production (32.9mg/L) and content (6.7mg/g), respectively, increasing by 31.6% and 32.3% compared with 25mg/L and 5.1mg/g of wild strain. Interestingly, the carotenoid production reached 192.6mg/L by feeding wet FW, which was 21% higher than batch culture. The 373g vacuum freeze-dried products were obtained from the fermentation of 1kg FW by P. rhodozyma, which contained 784mg carotenoids and 111mg astaxanthin. The protein, total amino acids, and essential amino acids content of the fermentation products were 36.6%, 40.5%, and 18.2% (w/w), respectively, and lysine-added fermentation products had the potential of high-quality protein feed source. This study provides insights for the high-throughput screening of mutants, astaxanthin production, and the development of the feed potential of FW.

Advancement of Carotenogenesis of Astaxanthin from Haematococcus pluvialis: Recent Insight and Way Forward.

The demand for astaxanthin has been increasing for many health applications ranging from pharmaceuticals, food, cosmetics, and aquaculture due to its bioactive properties. Haematococcus pluvialis is widely recognized as the microalgae species with the highest natural accumulation of astaxanthin, which has made it a valuable source for industrial production. Astaxanthin produced by other sources such as chemical synthesis or fermentation are often produced in the cis configuration, which has been shown to have lower bioactivity. Additionally, some sources of astaxanthin, such as shrimp, may denature or degrade when exposed to high temperatures, which can result in a loss of bioactivity. Producing natural astaxanthin through the cultivation of H. pluvialis is presently a demanding and time-consuming task, which incurs high expenses and restricts the cost-effective industrial production of this valuable substance. The production of astaxanthin occurs through two distinct pathways, namely the cytosolic mevalonate pathway and the chloroplast methylerythritol phosphate (MEP) pathway. The latest advancements in enhancing product quality and extracting techniques at a reasonable cost are emphasized in this review. The comparative of specific extraction processes of H. pluvialis biological astaxanthin production that may be applied to large-scale industries were assessed. The article covers a contemporary approach to optimizing microalgae culture for increased astaxanthin content, as well as obtaining preliminary data on the sustainability of astaxanthin production and astaxanthin marketing information.

Ultrasonic Treatment Enhanced Astaxanthin Production of Haematococcus pluvialis.

In this study, effects of ultrasonic treatment on Haematococcus pluvialis (H. pluvialis) were investigated. It has been confirmed that the ultrasonic stimulation acted as stress resources in the red cyst stage H. pluvialis cells containing astaxanthin, resulting in additional astaxanthin production. With the increase in production of astaxanthin, the average diameter of H. pluvialis cells increased accordingly. In addition, to determine how ultrasonic stimulation had an effect on the further biosynthesis of astaxanthin, genes related to astaxanthin synthesis and cellular ROS level were measured. As a result, it was confirmed that astaxanthin biosynthesis related genes and cellular ROS levels were increased, and thus ultrasonic stimulation acts as an oxidative stimulus. These results support the notion on the effect of the ultrasonic treatment, and we believe our novel approach based on the ultrasonic treatment would help to enhance the astaxanthin production from H. pluvialis.

Sustainable production of astaxanthin from Dilocarcinus pagei crab and optimisation of its extraction with edible oils

Dilocarcinus pagei is a South American crab commonly found in fishponds. As crabs are a source of astaxanthin (AST) and food inputs, this preliminary research aimed at studying the composition of females and males of this species to assess its potential for commercial applications, and at optimising the extraction of AST with edible oils to promote its use as an ingredient for the nutraceutical, pharmaceutical and feed industries. The chemical composition differed between males and females only in moisture, and the values obtained were: 65.4 ± 1.0% and 72.5 ± 3.1% moisture, 45.7–40.3% d.m. minerals, 22.0–24.1% d.m. fibres, 18.2–17.4% d.m. proteins, and 10.4–11.1% d.m. lipids. The Box-Behnken design was applied and validated for extraction with soya bean and sunflower oils, adjusting the oil:crab ratio, temperature, and extraction time. The optimal conditions found consisted of 140 mL/g, 90 °C and 170 min for soya bean oil, reaching an accumulation of AST of 50 ± 5 μg/g crab d.m. For sunflower oil, the conditions were 60 mL/g, 90 °C and 161 min, reaching 31 ± 3 μg/g crab d.m. Finally, the amounts of AST obtained using soya bean oil were higher than those obtained using sunflower oil; thus, soya bean oil would be recommended as a solvent to extract the pigment.

Genomic Insight and Optimization of Astaxanthin Production from a New Rhodotorula sp. CP72-2

Astaxanthin is a carotenoid pigment extensively used in various industries. Rhodotorula sp. CP72-2, isolated from Calotropis gigantea, showed potential astaxanthin production. In this study, strain CP72-2 was identified as a putative new species in the genus Rhodotorula based on the 26S rRNA gene sequence (98% identity). It was first used as the microbial source for producing astaxanthin. Strain CP72-2 was screened for its astaxanthin production and was identified and quantified by High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and UV-Vis spectrophotometer. After a screening of astaxanthin production, various carbon sources, pH, temperature, and incubation period were evaluated for their effect on the astaxanthin production of strain CP72-2. Among the several experimental factors, the most efficient conditions for astaxanthin production were glucose (50 g/L), pH 4.5, 25 °C, and three days of cultivation. The assembly genome of strain CP72-2 has a total length of 21,358,924 bp and a GC content of 64.90%. The putative candidate astaxanthin biosynthesis-associated genes (i.e., CrtE, CrtYB, CrtI, CrtS, CrtR, CrtW, CrtO, and CrtZ) were found. This research presents the first report on the production and optimization of astaxanthin from strain CP72-2 and its genome analysis, focusing on the biotechnological potential of the astaxanthin producer.

Effect of Biosynthesized Silver Nanoparticles on the Growth of the Green Microalga Haematococcus pluvialis and Astaxanthin Synthesis.

Silver nanoparticles (AgNPs) are widely known for their antimicrobial activity in various systems from microorganisms to cell cultures. However, the data on their effects on microalgae are very limited. Unicellular green algae Haematococcus pluvialis is known for its ability to accumulate large amounts of astaxanthin under stress conditions. Therefore, it can be used as a suitable model system to test the influence of AgNPs on stress induction in unicellular algae, with visible phenotypic effects, such as astaxanthin synthesis and cell morphology. This study tested different AgNP concentrations (0-8 mg/L) effects on different growth stages (red and green) of H. pluvialis culture. Effects on cell morphology, culture productivity, and astaxanthin synthesis were evaluated. Data showed that the addition of high concentrations of AgNPs to the growing culture had a significant negative impact on culture productivity. Green-stage (HpG) cultures productivity was reduced by up to 85% by increasing AgNPs concentration to 8 mg/L while the impact on red-stage (HpR) culture was lower. Astaxanthin concentration measurements showed that AgNPs do not have any effect on astaxanthin concentration in HpG culture and caused decreased astaxanthin production rate in HpR culture. HpG culture astaxanthin concentration stayed constant at ~0.43% dry weight, while HpR culture astaxanthin concentration was significantly reduced from 1.89% to 0.60% dry weight by increasing AgNP concentration. AgNPs in the media lead to significant changes in cell morphology in both HpG and HpR cultures. Cell deformations and disrupted cytokinesis, as well as AgNPs and induced sexual reproduction, were observed.

Screening of Structurally Distinct Lycopene β-Cyclases for Production of the Cyclic C40 Carotenoids β-Carotene and Astaxanthin by Corynebacterium glutamicum

Lycopene β-cyclase (EC 5.5.1.19) is one of the key enzymes in the biosynthesis of β-carotene and derived carotenoids. It catalyzes isomerase reactions to form β-carotene from lycopene by β-cyclization of both of its ψ-ends. Lycopene β-cyclases are widespread in nature. We systematically analyzed the phylogeny of lycopene β-cyclases from all kingdoms of life and predicted their transmembrane structures. To this end, a collection of previously characterized lycopene β-cyclase polypeptide sequences served as bait sequences to identify their closest homologues in a range of bacteria, archaea, fungi, algae, and plant species. Furthermore, a DeepTMHMM scan was applied to search for the presence of transmembrane domains. A phylogenetic tree suggests at least five distinct clades, and the DeepTMHMM scan revealed that lycopene β-cyclases are a group of structurally different proteins: membrane-bound and cytosolic enzymes. Representative lycopene β-cyclases were screened in the lycopene-overproducing Corynebacterium glutamicum strain for β-carotene and astaxanthin production. This systematic screening facilitates the identification of new enzymes for carotenoid production. Higher astaxanthin production and less reduction of total carotenoids were achieved with the cytosolic lycopene β-cyclase CrtL from Synechococcus elongatus and the membrane-bound heterodimeric lycopene β-cyclase CrtYcd from Brevibacterium linens.

Strategies for the Microbial Carotenoids Production Competitiveness Improvement

The research and development of carotenoids production have a long history, and interest in this group of pigments has not decreased to this day. Among all existing carotenoids, six are considered industrially important: astaxanthin, β-carotene, lutein, zeaxanthin, canthaxanthin, and lycopene. These carotenoids have a wide range of application and are used as additives in food and beverage, feed, nutraceuticals, pharmaceuticals, and cosmetics due to their bioactive and colour properties. An undisputed leader in the global pigment market is chemically synthesized carotenoids. To a lesser extent, carotenoids derived from natural sources as plants and microorganisms. Currently, the market of natural carotenoids is mainly represented by microalgae Haematococcus pluvialis, Dunaliella salina, Botryococcus braunii, fungus Blakeslea trispora, yeast Phaffia rhodozyma and bacteria Paracoccus carotinifaciens. These microorganisms afford the production of astaxanthin, βcarotene, canthaxanthin, and lycopene. In turn, lutein is obtained by extracting marigold flowers Tagetes eracta L. and there is no other competitive source yet. Therefore, the potential of microorganisms to synthesize and accumulate lutein and other equally important carotenoids in their cells has been actively studied. Several yeast and bacteria species from Rhodosporidium, Rhodotorula, Sporobolomyces, Sphingomonas, Gordonia, and Sphingobacterium genus have a potential to replenish the diversity of sources of industrially important natural pigments, but available technologies still need improving. This paper reviews strategies for increasing of competitiveness of fungal and bacterial carotenoids production. Strategies such as selection of carotenogenic strain, use of low-cost substrates, simultaneous production of carotenoids and other value-added compounds, and optimization of fermentation medium and conditions are considered.

Expanded utilisation of microalgae in global aquafeeds

AbstractUtilising novel ingredients in aquafeeds is necessary for the continued expansion and intensification of aquaculture production. Microalgae are utilised widely in aquaculture for a variety of purposes; as live feed for fish and shellfish larvae and juveniles, for the production of algae‐based meals and oils to replace marine derived raw materials in aquafeeds, and for the production of astaxanthin, the carotenoid that gives salmon flesh its characteristic reddish colour. Notably, long‐chain polyunsaturated fatty acids, amino acids, proteins, fibres, antioxidant pigments, vitamins and minerals are among the compounds in microalgae with great biological value. A growing collection of experimental data has demonstrated that these substances are crucial for increasing larval survival and enhancing the growth and well‐being of fish and shellfish. In this review, the processing of microalgae utilised as aquafeed ingredient, the health promoting compounds in microalgae and their role in growth, immunity and disease resistance of fish and shellfish are described and discussed. Simultaneously, major hurdles in the commercial use of microalgae in aquafeed, and future research and development perspectives are all critically discussed.

Phenol phycoremediation by Haematococcus pluvialis coupled with enhanced astaxanthin and lipid production under rac-GR24 supplementation for enhanced biodiesel production

The present study evaluated the impact of rac-GR24 on biomass and astaxanthin production under phenol stress coupled with biodiesel recovery from Haematococcus pluvialis. Phenol supplementation showed negative impact on growth, where the lowest biomass productivity of 0.027 g L-1 day−1 was recorded at 10 µM phenol, while 0.4 µM rac-GR24 supplementation showed the highest recorded biomass productivity of 0.063 g L-1 day−1. Coupling 0.4 µM rac-GR24 at different phenol concentrations confirmed the potential of rac-GR24 to mitigate the toxic effect of phenol by enhancing yield of PSII yield, RuBISCo activity, and antioxidant efficiency, which resulted in improved phenol phycoremediation efficiency. In addition, results suggested a synergistic action by rac-GR24 supplementation under phenol treatment where rac-GR24 enhanced lipid accumulation, while phenol enhanced astaxanthin production. Dual supplementation of rac-GR24 and phenol showed the highest recorded FAMEs content, which was 32.6% higher than the control, with improved biodiesel quality. The suggested approach could enhance the economic feasibility of triple-purpose application of microalgae in wastewater treatment, astaxanthin recovery, and biodiesel production.

Metabolic mechanism of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous in response to sodium citrate treatment

Astaxanthin is an important ketocarotenoid widely used in industries. However, its application is limited because of its low yield. Sodium citrate (Na-citrate), one of the major carbon sources for microorganisms, can promote cell growth and product accumulation. The basidiomycetous red yeast Xanthophyllomyces dendrorhous was thus used to study the effect of Na-citrate on cell growth and astaxanthin synthesis. The highest biomass and astaxanthin yield (6.0 g/L and 22.5 mg/L) were obtained in shake-flask when 3 g/L Na-citrate was added at 24 h and were 1.8 and 2.0 times higher than those of the control group, respectively. Furthermore, metabolomics and real-time reverse transcription PCR (qRT-PCR) analysis were conducted to study the metabolic pathways of X. dendrorhous in response to Na-citrate. The qRT-PCR assay revealed that Na-citrate facilitated glucose consumption, promoted the metabolic flux from glycolysis, and regulated the tricarboxylic acid (TCA) cycle, providing more energy and substrates for the synthesis of astaxanthin. The gene analysis revealed that adding Na-citrate significantly upregulated the expression of six key genes (ICL, HMGS, crtE, crtYB, crtI, and crtS) involved in pathways related to astaxanthin biosynthesis. These results suggest that exogenous Na-citrate treatment is a potentially valuable strategy to stimulate astaxanthin production in X. dendrorhous.

Cell wall breaking of Haematococcus pluvialis biomass facilitated by Baijiu jiuqu fermentation with simultaneously production of beverages

The microalgae Haematococcus pluvialis is a commercial source of natural astaxanthin. However, mature cells develop rigid three-layer wall structures and a repulsive odor. This study applied a liquid static fermentation system to screen hydrolyzing microorganisms for cell wall hydrolysis. Baijiu jiuqu and Gutian hongqu were found to have promising potential for application. The fermentation using 2% baijiu jiuqu and 2% glucose for pre-activation achieved comparable recovery of carotenoids to homogenizer disruption methods and produced stable fragrance which may be attributed to ethyl octanoate, hexyl formate, and phenethyl butyrate, as revealed by gas chromatography-mass spectrometry analysis. The abundance of astaxanthin molecules was slightly affected by fermentation with fold change < 2, while molecules with higher fold change (>10) were mainly carbohydrates, lipids, and steroids proving the safety of the fermentation. This study provides a new scheme for the biorefining of Haematococcus. pluvialis, potentially contributing to the industrial production of natural astaxanthin.

Expression of fatty acid related gene promotes astaxanthin heterologous production in Chlamydomonas reinhardtii.

Natural astaxanthin is a high-value ketone carotenoid mainly derived from Haematococcus pluvialis, which is an excellent antioxidant for human consumption. To study the role of lipids in accumulation of astaxanthin, the H. pluvialis-derived astaxanthin synthesis pathway genes (β-carotene ketolase gene, BKT and β-carotene hydroxylase gene, BCH) and fatty acid elongation gene (mitochondrial trans-2-enoyl-coa reductase gene, MECR) were heterologously co-expressed in C. reinhardtii. Zeaxanthin, the precursor of astaxanthin synthesis, was significantly increased after BKT and BCH were expressed. In contrast, the α-carotene that competes with astaxanthin synthesis for lycopene decreased significantly. This redistribution of carbon flow was conducive to the synthesis of astaxanthin. In addition, the transformant only expressed astaxanthin metabolism related genes (BKT, BCH) would lead to an increase in total lipid, a decrease in monounsaturated fatty acids and an increase in polyunsaturated fatty acids. On this basis, the expression of MECR gene further increased the total lipid, and the relative content of different fatty acids also changed. The astaxanthin content of algal strains transformed with BKT and BCH genes was nearly 50% higher than that of the wild type. On this basis, the astaxanthin content of transformants expressing MECR gene related to long-chain fatty acid synthesis was increased by 227.5%. In this study, an astaxanthin production model similar to H. pluvialis by combining carotenoid metabolism and fatty acid metabolism was constructed in C. reinhardtii. The results suggest that the increase in astaxanthin is indeed linked to the regulation of fatty acid metabolism, and this link may involve the type of fatty acids and the dynamics of astaxanthin ester in cells. The strategy of promoting the synthesis of fatty acids has potential to achieve efficient production of astaxanthin in C. reinhardtii.

A strategy to maximize astaxanthin production from Haematococcus pluvialis in a cost-effective process by utilizing a PBR-LGP-PBR array (PLPA) hybrid system using light guide panel (LGP) and solar cells

This study evaluated economic feasibility through production efficiency, return on investment (ROI) and payout time of a hybrid system using a photobioreactor (PBR)-light guide panel (LGP)-PBR array (PLPA) and solar cells developed for astaxanthin and ω-3 FA simultaneous production of Haematococcus pluvialis. The economic feasibility of the PLPA hybrid system (8 PBRs) and the PBR-PBR-PBR array (PPPA) system (8 PBRs) was evaluated for producing high-value products while effectively reducing CO2. Introducing a PLPA hybrid system has increased the amount of culture per area by 1.6 times. Also, the shading effect was effectively suppressed with an LGP placed between each PBR, increasing biomass and astaxanthin productivity by 3.39-fold and 4.79-fold, respectively compared to the untreated H. pluvialis cultures. In addition, ROI increased by 6.55 and 4.71 times, and the payout time was reduced by 1.34 and 1.37 times, respectively in 10 and 100-ton scale processes.

Genetic Improvement to Obtain Specialized Haematococcus pluvialis Genotypes for the Production of Carotenoids, with Particular Reference to Astaxanthin

Nowadays, the search for natural substances with a high nutraceutical effect positively impact the world market. Among the most attractive macromolecules are antioxidants, capable of preventing the development of various pathologies. Astaxanthin (ASX) is antioxidant molecule produced by the microalga H. pluvialis as a response to different types of stress. Usually, astaxanthin production involves the first phase of accumulation of the biomass of H. pluvialis (green phase), which is then stressed to stimulate the biosynthesis and accumulation of ASX (red phase). In this study, the H. pluvialis wild-type strain was subjected to random mutagenesis by UV. Among the different mutant strains obtained, only two showed interesting bio-functional characteristics, such as a good growth rate. The results demonstrated that the HM1010 mutant not only has a higher growth trend than the WT mutant but accumulates and produces ASX even in the green phase. This innovative genotype would guarantee the continuous production of ASX, not linked to the two-step process and the uniqueness of the product obtained.

Fermentative production of astaxanthin from sorghum distillery residue by an indigenous Aurantiochytrium sp. CJ6 strain using a continuous-feeding fed-batch process

In this study, Aurantiochytrium sp. CJ6 was cultivated heterotrophically on a waste resource, sorghum distillery residue (SDR) hydrolysate without adding any nitrogen sources. Mild sulfuric acid treatment released sugars that supported the growth of CJ6. Optimal operating parameters (salinity, 2.5%; pH, 7.5; with light exposure) determined using batch cultivation attained biomass concentration and astaxanthin content of 3.72 g/L and 69.32 µg/g dry cell weight (DCW), respectively. Using continuous-feeding fed-batch (CF-FB) fermentation, the biomass concentration of CJ6 increased to 6.3 g/L with biomass productivity and sugar utilization rate of 0.286 mg/L/d and 1.26 g/L/d, respectively. Meanwhile, CJ6 obtained maximum astaxanthin content (93.9 µg/g DCW) and astaxanthin concentration (0.565 mg/L) after 20-day cultivation. Thus, the CF-FB fermentation strategy seems to have a high potential for the cultivation of thraustochytrids to produce the high-value product (astaxanthin) using SDR as the feedstock to achieve circular economy.

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.