Astaxanthin Production Research Articles

Optimization of Physical Parameters of Astaxanthin Production from Haematococcus pluvialis

Haematococcus pluvialis, fresh water microalgae has been considered as a possible natural source for the production of astaxanthin and it has been widely studied. The general composition of Haematococcus algae consists of common carotenoids, fatty acids, proteins, carbohydrates, and minerals. Both of the main physical and chemical parameters, especially medium and light, directly control the growth rate of Haematococcus pluvialis Astaxanthin is a keto-carotenoid which is built from five carbon precursors isopentenyl diphosphate (IPP) and dimethyl allyldiphosphate. Astaxanthin has important metabolic functions in animals and humans ranging from protection against oxidation of essential polyunsaturated fatty acids, protection against UV light effects, pro vitamin A activity and vision, immune response, pigmentation and communication to reproductive behaviour and improved reproduction. The astaxanthin is the pigment that is considered as the most powerful antioxidants in nature. Due to the high production cost of synthetic astaxanthin and the market demand for natural astaxanthin, the biological sources of astaxanthin have long been widely exploited. To extract more astaxanthin from Haematococcus pluvialis, Growth optimization of the microalgae was done by using various medium such as BG-11, Basal, RM, BBM and TAP Medium. This comprehensive study on the determination of the culture medium and the light intensity was carried out to maximize the growth of Haematococcus pluvialis for batch cultivations. optimization was done by using different pH, temperature and light. the chlorophyll and protein content were analysed. And finally the astaxanthin was produced. The protein content of H. Pluvialis was found to be 338.27µg/ml.

A strategy for stimulating astaxanthin and lipid production in Haematococcus pluvialis by exogenous glycerol application under low light

In large-scale outdoor cultivation, Haematococcus pluvialis cultures are frequently exposed to low light caused by continuous rain conditions. Low light significantly restricts biomass and astaxanthin accumulation, thereby decreasing astaxanthin productivity. In this study, the effects of exogenous glycerol on the growth, photosynthesis, and astaxanthin and lipid accumulation in H. pluvialis under low light were investigated. A strategy was developed for promoting astaxanthin and lipid production in H. pluvialis under low light by the application of exogenous glycerol. Under low light, exogenous glycerol did not change the biomass and photosynthetic capacity of H. pluvialis. The addition of 1 mL L−1 glycerol increased total lipid content by 36.8% by increasing the formation of fatty acids during an 11-day growth period. Furthermore, exogenous glycerol could directly accelerate astaxanthin production under low light by increasing the formation of substrates (e.g. pyruvate). Meanwhile, the elevated fatty acid level due to the addition of glycerol facilitated the esterification of astaxanthin, thereby accelerating the biosynthesis of astaxanthin indirectly, which is indicated by the fact that exogenous glycerol enhanced astaxanthin esters content significantly under low light. All these results suggest that H. pluvialis has the ability to utilize glycerol for astaxanthin and lipid production under low light.

Primary metabolism is associated with the astaxanthin biosynthesis in the green algae Haematococcus pluvialis under light stress

Haematococcus pluvialis is a kind of native bioresource, which accumulate high abundance of astaxanthin, one of the most important bioactive compounds, under various biotic and abiotic stress conditions particularly under high light. However, the global metabolic responses to high light in general and the relationship between primary metabolism and astaxanthin accumulation in particular are still obscure. In this study, non-targeted metabolomics approach was first to be used to profile the dynamic metabolic changes of H. pluvialis along astaxanthin accumulation under different light intensities, in which >200 low molecular metabolites were identified. The integration of metabolomics data with previous transcriptomic data uncovered several metabolic pathways including raffinose family oligosaccharides biosynthesis pathway, GSH metabolism pathways, pyrimidine and purine metabolism pathways, TCA cycle, and phospholipids, that are important for astaxanthin biosynthesis under high light intensity, pinpointing a metabolic shift from primary metabolism to astaxanthin biosynthesis. Exogenous application of related metabolites such as nucleotides, carbohydrates and amino acids in algal medium confirmed the metabolic link between primary metabolism and astaxanthin biosynthesis. Our results provides with a solid metabolic base for the manipulation of astaxanthin production under high light stress, and reveals metabolic insights into the adaption strategy of H. pluvialis against light stress conditions.

Disodium 2-oxoglutarate promotes carbon flux into astaxanthin and fatty acid biosynthesis pathways in Haematococcus

Improving carbon availability in astaxanthin production is pivotal in Haematococcus industry. In this study, disodium 2-oxoglutarate (2-OG-2Na) was observed to be a potential carbon regulator to increase the astaxanthin content. To illustrate its efficacy in astaxanthin production, key genes and enzyme were analyzed. Upon 2-OG-2Na treatment, genes ipi, bkt and crtR-b were up regulated, concomitantly, carotenoids and astaxanthin content increased by 15.4% and 14.0% at 120 h, respectively; additionally, Acetyl-CoA carboxylase was activated, consistent with 1.27-fold increase in fatty acids content. PUFAs increased earlier as fatty acids assembly gene fad was up-regulated to 20.56. It was also found that cell division was not compromised. Altogether, it was suggested that increased carbon skeletons were re-directed into the astaxanthin and fatty acids biosynthesis pathway. Furthermore, 2-OG-2Na was applied in ten Haematococcus strains. Of these strains, astaxanthin contents were accelerated with average net increase of 10.48%, exhibiting a scalable paradigm for commercial production.

Distinctive nutrient designs using statistical approach coupled with light feeding strategy to improve the Haematococcus pluvialis growth performance and astaxanthin accumulation

The optimization of the BG-11 culture medium nutrients using complex statistical design followed by incremental illumination was planned to stimulate the Haematococcus pluvialis growth and astaxanthin accumulation. Based on the Plackett-Burman design results, MgSO4·7H2O, H3BO3, and Na2CO3 were identified as critical components to improve the biomass and astaxanthin productivity. Using central composite design, their initial critical concentrations in the green stage were found as 57.5, 6.2 and 53.0 mg/L, while for the red stage, the 138.3, 8.5 and 41.0 mg/L, recorded as optimum respectively. Using the optimum media, growth and astaxanthin accumulation at the end of the phototrophic and photoinduction stages were boosted by 17 and 54% respectively. The results of scale-up coupled with incremental illumination in phototrophic stage revealed the biomass and astaxanthin concentration improved 50% and 60% over the BG-11 media under constant light intensity. Also, different optimum culture medium formula for green and red stages was proposed.

Linoleic-acid-enhanced astaxanthin content of Chlorella sorokiniana (Chlorophyta) under normal and light shock conditions

ABSTRACTResearch reports showed that linoleic acid is an allelochemical that can impact metabolic activity of microalgae. In this study, the effect of linoleic acid (LA) on astaxanthin accumulation in Chlorella sorokiniana was investigated under normal and light shock conditions. In normal autotrophic cultivation, maximum astaxanthin content was observed after 12 days in logarithmic growth with the addition of 32 µM LA; this was nearly twice as high as the control cultures without LA. After 15 days, chlorophyll a, astaxanthin to chlorophyll ratio, and biomass had changed by 0.6, 1.4 and 1.65 times relative to the control, respectively. When light stress was applied, addition of 32 µM LA enhanced astaxanthin concentration 1.3 times after 12 days, while chlorophyll a, astaxanthin to chlorophyll ratio and biomass had changed by 0.4, 1.8 and 2.6 times the control after 15 days, respectively. Accordingly, LA as a precursor to jasmonic and traumatic acids can have a role in the growth of C. sorokiniana, and production of astaxanthin and chlorophyll a in C. sorokiniana. This study demonstrates the potential of LA as an enhancer of algal astaxanthin production.

Enhancing the production of astaxanthin by mixotrophic cultivation of Haematococcus pluvialis in open raceway ponds

Mixotrophic cultivation of Haematococcus is efficient to increase astaxanthin productivity. However, application of this technique in open bioreactors is restricted because of bacterial contamination. In this study, mixotrophic cultivation of Haematococcus was carried out successfully in an outdoor raceway pond. The Haematococcus cells grew on light first, and then acetate/acetic acid was supplemented when nitrate was depleted from the culture media. Under such conditions, Haematococcus cells grew on intracellular nitrogen pool, while bacterial reproduction was limited. The average astaxanthin productivity reached 140 mg m−2day−1, which is about 1.2 times that of the simple phototrophic cultivation. This study explores a potential way to enhance astaxanthin productivity, and the results are helpful to the development of the Haematococcus industries.

Producción de Astaxantina y expresión de genes en Haematococcus pluvialis (Chlorophyceae, Volvocales) bajo condiciones de estrés por deficiencia de nitrógeno y alta irradiancia: Producción de astaxantina y expresión de genes en H. pluvialis

Astaxanthin is an antioxidant carotenoid used in cosmetic, pharmaceutic and food industries that is produced by microalgae H. pluvialis in natural form; it is a secondary metabolite that increases production when microalgae grows under stress conditions, like food deficiency and high irradiation, among others. The goal of this research was to test microalgae growing under nitrogen deficiency conditions in a concentration of 4.0-5.0% and evaluate astaxanthin production and gene expression in dye biosynthesis. For this purpose, a culture in rm medium was carried out with 4.0 and 5.0% nitrogen, using the following conditions: pH 6.7; light/dark cycle 20h: 4h; temperature 25 oC; continuous agitation; 5% CO2; lighting with white fluorescent lamps (Tlt 20w/54rs Philips brand); and a high irradiance of 140 μE. The growth in the medium was used as a control with nitrogen in normal concentrations during 36 days (under the same conditions). Sampling was carried out every third day for quantification of astaxanthin and chlorophyll and the determination of gene expression. The anova performed established that there are no significant differences between treatments (P = 0.053). However, astaxanthin production with a nitrogen concentration of 4.0% and a high irradiance of 140 μE was 3.52x10-6 μg cell-1 x mL-1, increased by 40% when compared to that obtained with 5.0% nitrogen (2.08x10-6 ug cell-1 x mL-1). The psy, pds, lcy, bkt and chy genes were expressed with 4.0% nitrogen, while 5.0% nitrogen only allowed psy, pds and chy.

Time-resolved carotenoid profiling and transcriptomic analysis reveal mechanism of carotenogenesis for astaxanthin synthesis in the oleaginous green alga Chromochloris zofingiensis

BackgroundChromochloris zofingiensis is emerging as an industrially relevant alga given its robust growth for the production of lipids and astaxanthin, a value-added carotenoid with broad applications. Nevertheless, poor understanding of astaxanthin synthesis has limited engineering of this alga for rational improvements.ResultsTo reveal the molecular mechanism underlying astaxanthin accumulation in C. zofingiensis, here we conducted an integrated analysis by combining the time-resolved transcriptomes and carotenoid profiling in response to nitrogen deprivation (ND). A global response was triggered for C. zofingiensis to cope with the ND stress. Albeit the little variation in total carotenoid content, individual carotenoids responded differentially to ND: the primary carotenoids particularly lutein and β-carotene decreased, while the secondary carotenoids increased considerably, with astaxanthin and canthaxanthin being the most increased ones. The carotenogenesis pathways were reconstructed: ND had little effect on the carbon flux to carotenoid precursors, but stimulated astaxanthin biosynthesis while repressing lutein biosynthesis, thereby diverting the carotenoid flux from primary carotenoids to secondary carotenoids particularly astaxanthin. Comparison between C. zofingiensis and Haematococcus pluvialis revealed the distinctive mechanism of astaxanthin synthesis in C. zofingiensis. Furthermore, potential bottlenecks in astaxanthin synthesis were identified and possible engineering strategies were proposed for the alga.ConclusionsCollectively, these findings shed light on distinctive mechanism of carotenogenesis for astaxanthin biosynthesis in C. zofingiensis, identify key functional enzymes and regulators with engineering potential and will benefit rational manipulation of this alga for improving nutritional traits.

Transcriptomic and metabolic analysis of an astaxanthin-hyperproducing Haematococcus pluvialis mutant obtained by low-temperature plasma (LTP) mutagenesis under high light irradiation

Haematococcus pluvialis can accumulate sufficient levels of astaxanthin under various stress conditions. High light irradiation is essential for astaxanthin accumulation in Haematococcus pluvialis. However, a lack of genomic information limits the understanding of its physiological metabolism for high astaxanthin production. In this work, we investigated the wild-type (WT) strain and mutant strain (named as M3 which was obtained in our previous work) under high light irradiation and compared the difference in astaxanthin biosynthesis. We collected and analyzed the data of de novo transcriptome information of Haematococcus pluvialis at 24 h under high light stress. Our transcriptomic results indicated that M3 strain had higher utilization efficiency of CO2 to provide the precursors of carotenoid and fatty acid biosynthesis by increasing the expression levels of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), malate dehydrogenase (ME), ribulose bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) while decreasing the expression levels of fructose-1, 6-bisphosphatase (FBP). The analysis of pigments, chlorophyll fluorescence and the quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that M3 strain maintained higher photosynthetic activity by regulating chlororespiration pathway and elevating non-photosynthetic pigment (lutein, β-carotene, and astaxanthin) content to alleviate photooxidative damage. Moreover, the M3 strain showed higher fatty acid content than the WT strain. Overall, combinative analysis of de novo transcriptomic and physiological data provided information necessary for not only a better understanding of the difference in astaxanthin biosynthesis between WT and M3 strains but also the feasibility of genetic engineering of Haematococcus pluvialis in the future.

Biocompatible liquid-type carbon nanodots (C-paints) as light delivery materials for cell growth and astaxanthin induction of Haematococcus pluvialis.

In this study, we aimed to demonstrate the feasibility of the application of biocompatible liquid type fluorescent carbon nanodots (C-paints) to microalgae by improving microalgae productivity. C-paints were prepared by a simple process of ultrasound irradiation using polyethylene glycol (PEG) as a passivation agent. The resulting C-paints exhibited a carbonyl-rich surface with good uniformity of particle size, excellent water solubility, photo-stability, fluorescence efficiency, and good biocompatibility (<10.0mgmL-1 of C-paints concentration). In the practical application of C-paints to microalgae culture, the most effective and optimized condition leading to growth promoting effect was observed at a C-paints concentration of 1.0mgmL-1 (>20% higher than the control cell content). A C-paints concentration of 1-10.0mgmL-1 induced an approximately >1.8 times higher astaxanthin content than the control cells. The high light delivery effect of non-cytotoxic C-paints was applied as a stress condition for H. pluvialis growth and was found to play a major role in enhancing productivity. Notably, the results from this study are an essential approach to improve astaxanthin production, which can be used in various applications because of its therapeutic effects such as cancer prevention, anti-inflammation, immune stimulation, and treatment of muscle-soreness.

Construction of astaxanthin metabolic pathway in the green microalga Dunaliella viridis

Dunaliella viridis is a green microalga containing β-carotene, which is the precursor of astaxanthin, the most active antioxidant in Haematococcus pluvialis. Two key enzymes of H. pluvialis, β-carotene hydroxylase (CRTR-B) and β-carotenoid ketolase (BKT), are required for converting β-carotene to astaxanthin in D. viridis via the astaxanthin biosynthetic pathway. Considering the location of β-carotene in the chloroplast of D. viridis, the two modified genes encoding BKT and CRTR-B in H. pluvialis were integrated via homologous recombination into the chloroplast genome, in this study. In the chloroplast, the homologous recombination vector pMD-bkt-crtr (16S-TrnA-atpA-bkt-crtR-B-rbcL-psbA-bar-TrnI-23S), bkt and crtR-B were regulated by the atpA promoter in a polycistron. The presence of astaxanthin in the D. viridis mutant expressing BKT and CRTR-B was verified using high performance liquid chromatography (HPLC), and the maximum content of total astaxanthin and canthaxanthin after high light induction were 77.5±7.7 and 50.1±0.8μg g−1 in dry weight, respectively. Our results indicate that D. viridis can be used as a cell factory for astaxanthin production.

Metabolic engineering of Synechocystis sp. PCC6803 to produce astaxanthin

Astaxanthin is a carotenoid with wide applications because of its strong antioxidant properties. It is mainly produced from the green alga Haematococcus pluvialis, but large-scale production remains a challenge. To provide another source, here, Synechocystis sp. PCC6803 was adopted as the chassis cell, and its carotenoid biosynthesis pathway was genetically extended to produce astaxanthin. The β-carotene ketolase gene (bkt) and carotenoid hydroxylase gene (crtR-B) from H. pluvialis, which wild-type Synechocystis sp. PCC6803 lacks, were optimized based on the codon preference of Synechocystis sp. PCC6803 and heterologously expressed in that bacterium to create a strain capable of producing astaxanthin. The Synechocystis sp. PCC6803 mutant synthesized 4.81 ± 0.06 mg astaxanthin per gram of cells (dry weight) after nitrogen starvation for 14 days. More than 30% of the total astaxanthin was in the free form, and 82.61% was the strong antioxidant 3S, 3′S-astaxanthin isoform. These results show the industrial application potential of metabolically-engineered Synechocystis sp. PCC6803 for astaxanthin production.

Cooling coil led to the collapse of N-replete Haematococcus pluvialis cultures by releasing lethal levels of Cu2+

Photobioreactors for outdoor microalgal cultivation should be equipped with temperature control devices to avoid overheating. However, it is still not clear whether the cooling devices would release poisonous levels of Cu2+ that could affect the growth of microalga. In this study, the half maximal inhibitory concentration (IC50) of Cu2+ was determined by studying the effects of Cu2+ concentrations on the growth and photosynthesis of Haematococcus. Then, the Cu2+ release dynamics were studied by soaking copper cooling coils in water for different times. The results indicated that the IC50 of Cu2+ for Haematococcus was approximately 20 μM (16.9 μM) and, above this level, the growth and photosynthesis were obviously inhibited before astaxanthin production. Meanwhile, after being soaked in water for only 10 h, the copper cooling coil released 40 μM Cu2+, which was 100% higher than the IC50 and high enough to impose severe heavy metal stress on cells. These results indicated that Haematococcus was sensitive to Cu2+. Devices made with copper or copper-containing materials could release lethal levels of Cu2+ if exposed to a liquid environment and brought into direct contact with microalgal cells. This research sheds some light on the design and operation of cooling systems for outdoor large-scale microalgal cultivations.

Combinatorial expression of different β-carotene hydroxylases and ketolases in Escherichia coli for increased astaxanthin production.

In natural produced bacteria, β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) convert β-carotene into astaxanthin. To increase astaxanthin production in heterologous strain, simple and effective strategies based on the co-expression of CrtZ and CrtW were applied in E. coli. First, nine artificial operons containing crtZ and crtW genes from different sources were constructed and, respectively, introduced into E. coli ZF237T, a β-carotene producing host. Among the nine resulting strains, five accumulated detectable amounts of astaxanthin ranging from 0.49 to 8.07mg/L. Subsequently, the protein fusion CrtZ to CrtW using optimized peptide linkers further increased the astaxanthin production. Strains expressing fusion proteins with CrtZ rather than CrtW attached to the N-terminus accumulated much more astaxanthin. The astaxanthin production of the best strain ZF237T/CrtZAs-(GS)1-WBs was 127.6% and 40.2% higher than that of strains ZF237T/crtZAsWBs and ZF237T/crtZBsWPs, respectively. The strategies depicted here also will be useful for the heterologous production of other natural products.

Improved Astaxanthin Production with Corynebacterium glutamicum by Application of a Membrane Fusion Protein.

Astaxanthin is one of the strongest natural antioxidants and a red pigment occurring in nature. This C40 carotenoid is used in a broad range of applications such as a colorant in the feed industry, an antioxidant in cosmetics or as a supplement in human nutrition. Natural astaxanthin is on the rise and, hence, alternative production systems are needed. The natural carotenoid producer Corynebacterium glutamicum is a potent host for industrial fermentations, such as million-ton scale amino acid production. In C. glutamicum, astaxanthin production was established through heterologous overproduction of the cytosolic lycopene cyclase CrtY and the membrane-bound β-carotene hydroxylase and ketolase, CrtZ and CrtW, in previous studies. In this work, further metabolic engineering strategies revealed that the potential of this GRAS organism for astaxanthin production is not fully exploited yet. It was shown that the construction of a fusion protein comprising the membrane-bound β-carotene hydroxylase and ketolase (CrtZ~W) significantly increased astaxanthin production under high glucose concentration. An evaluation of used carbon sources indicated that a combination of glucose and acetate facilitated astaxanthin production. Moreover, additional overproduction of cytosolic carotenogenic enzymes increased the production of this high value compound. Taken together, a seven-fold improvement of astaxanthin production was achieved with 3.1 mg/g CDW of astaxanthin.

Single-Stage Astaxanthin Production Enhances the Nonmevalonate Pathway and Photosynthetic Central Metabolism in Synechococcus sp. PCC 7002.

The natural pigment astaxanthin is widely used in aquaculture, pharmaceutical, nutraceutical, and cosmetic industries due to superior antioxidant properties. The green alga Haematococcus pluvialis is currently used for commercial production of astaxanthin pigment. However, slow growing H.pluvialis requires a complex two-stage stress-induced process with high light intensity leading to increased contamination risks. In contrast, the fast-growing euryhaline cyanobacterium Synechococcus sp. PCC 7002 (Synechococcus 7002) is able to reach high density under stress-free phototrophic conditions, and is therefore a promising metabolic engineering platform for astaxanthin production. In the present study, genes encoding β-carotene hydroxylase and β-carotene ketolase, from the marine bacterium Brevundimonas sp. SD212, are integrated into the endogenous plasmid of Synechococcus 7002, and then expressed to biosynthesize astaxanthin. Although Synechococcus 7002 does not inherently produce astaxanthin, the recombinant ZW strain yields 3 mg/g dry cell weight astaxanthin from CO2 as the sole carbon source, with significantly higher astaxanthin content than previous cyanobacteria reports. Synechococcus 7002 astaxanthin productivity reached 3.35 mg/L/day after just 2 days in a continuous autotrophic process, which is comparable to the best H.pluvialis astaxanthin productivities when factoring in growth times. Metabolomics analysis reveals increases in fractions of hexose-, pentose-, and triose phosphates along with intermediates involved in the nonmevalonate pathway. Dynamic metabolomics analysis of 13C labeled metabolites clearly indicates flux enhancements in the Calvin cycle and glycolysis resulting from the overexpression of astaxanthin biosynthetic genes. This study suggests that cyanobacteria may enhance central metabolism as well as the nonmevalonate pathway in an attempt to replenish depleted pigments such as β-carotene and zeaxanthin.

Physiological and Metabolomics Analyses Reveal the Roles of Fulvic Acid in Enhancing the Production of Astaxanthin and Lipids in Haematococcus pluvialis under Abiotic Stress Conditions.

In this study, it was found that fulvic acid (FA) enhanced the contents of astaxanthin and lipids in Haematococcus pluvialis under high light and nitrogen starvation conditions by 2- and 1.2-fold, respectively. Meanwhile, the carbohydrate and chlorophyll contents were decreased by FA induction, whereas the levels of reactive oxygen species (ROS) and glutathione (GSH) as well as the expression of astaxanthin and lipid biosynthetic genes were increased. To further explore the interrelation between FA and the biosynthesis of astaxanthin and lipids, a metabolomics analysis of H. pluvialis by combined FA and abiotic stress exposure was conducted by using LC-MS/MS. The contents of some cytoprotective metabolites and signal molecules, including d-maltose, succinate, malic acid, melatonin (MT), and some amino acids, were increased under FA induction and abiotic stress conditions. These metabolites are intermediates in the TCA cycle and Calvin cycle, providing more precursors for the synthesis of astaxanthin and lipids. Moreover, the signal molecules might contribute to enhancing the abiotic stress tolerance. This study provided new insights into the regulatory mechanism of FA on astaxanthin and lipid accumulation in H. pluvialis.

Enhancement of Astaxanthin Biosynthesis in Oleaginous Yeast Yarrowia lipolytica via Microalgal Pathway.

Astaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, and food industries. The astaxanthin produced chemically is costly and is not approved for human consumption due to the presence of by-products. The astaxanthin production by natural microalgae requires large open areas and specialized equipment, the process takes a long time, and results in low titers. Recombinant microbial cell factories can be engineered to produce astaxanthin by fermentation in standard equipment. In this work, an oleaginous yeast Yarrowia lipolytica was engineered to produce astaxanthin at high titers in submerged fermentation. First, a platform strain was created with an optimised pathway towards β-carotene. The platform strain produced 331 ± 66 mg/L of β-carotene in small-scale cultivation, with the cellular content of 2.25% of dry cell weight. Next, the genes encoding β-ketolase and β-hydroxylase of bacterial (Paracoccus sp. and Pantoea ananatis) and algal (Haematococcus pluvialis) origins were introduced into the platform strain in different copy numbers. The resulting strains were screened for astaxanthin production, and the best strain, containing algal β-ketolase and β-hydroxylase, resulted in astaxanthin titer of 44 ± 1 mg/L. The same strain was cultivated in controlled bioreactors, and a titer of 285 ± 19 mg/L of astaxanthin was obtained after seven days of fermentation on complex medium with glucose. Our study shows the potential of Y. lipolytica as the cell factory for astaxanthin production.

Enhanced Astaxanthin Production in Escherichia coli via Morphology and Oxidative Stress Engineering.

Astaxanthin is a carotenoid of high commercial value because of its excellent antioxidative, anti-inflammatory, and anticancer properties. Here, we developed a novel strategy for improving the production of astaxanthin via morphology and oxidative stress engineering. First, we identified the morphology-/membrane- and oxidative stress-related genes, which should be knocked down, using the CRISPRi system. Deleting the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) generated longer and larger cells with higher reactive oxygen species (ROS) levels, thus enhancing the production of astaxanthin and decreasing cell growth. To not only improve cell growth but also obtain longer and larger cells with higher ROS levels, a complementary expression system using a temperature-sensitive plasmid was established. Complementarily expressing the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) further improved the production of astaxanthin to 11.92 mg/g dry cell weight in shake flask cultures.