Scale up production of astaxanthin by using recombinant yeast-Kluveromyces marxianus

doi:10.21203/rs.3.rs-7246484/v1

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Scale up production of astaxanthin by using recombinant yeast-Kluveromyces marxianus

https://doi.org/10.21203/rs.3.rs-7246484/v1

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Astaxanthin (AXT) is a dark-red ketocarotenoid from the xanthophyll group, renowned for its anti-inflammatory, antioxidant, and anti-proliferative properties, which is widely applied in animal feed, food, cosmeceuticals, and pharmaceuticals. Recently, the growing recognition of AXT’s numerous health benefits has significantly boosted both industrial and research interest in this remarkable compound. Synthetic biology represents a cost-effective strategy for the targeted production of specific optical isomers of natural compounds. This study explores the scale-up production of free-form 3S, 3'S astaxanthin (Asta-S) using recombinant probiotic yeast Kluyveromyces marxianus. Among various carbon sources tested, glucose was identified as the most practical substrate due to its cost-effectiveness, although galactose induced higher astaxanthin yields. Increasing glucose concentration from 20 to 80 g/L enhanced biomass but slightly reduced astaxanthin content in flask cultures. In 5 L fermenter trials, biomass growth increased with higher initial glucose levels, with the highest total astaxanthin yield of 41.06 mg/L achieved at 80 g/L glucose. To overcome carbon depletion and enhance productivity, a glucose fed-batch strategy was applied, resulting in the improved astaxanthin yield of 51.22 mg/L with a productivity of 0.711 mg/L/hr. The process was further scaled to a 30 L in-situ fermenter, where pressurized cultivation at 1 atm enhanced dissolved oxygen availability, resulting in higher astaxanthin content (4.38 mg/g) and improved biomass compared to atmospheric pressure fermentation. These results confirm the potential of recombinant K. marxianus 929 Asta strain D56 as a viable host for industrial astaxanthin production, particularly when leveraging fed-batch strategies and controlled pressure fermentation for scalability.

Astaxanthin

recombinant yeast

scale-up

Kluyveromyces marxianus

Astaxanthin (3,3'-dihydroxy-β-carotene-4,4'-dione) is a carotenoid pigment renowned for its superior antioxidant activity compared to other carotenoids and vitamin E. As a member of the xanthophyll family, astaxanthin is an oxygenated derivative of carotenoids, synthesized in plants from lycopene [1]. Numerous studies have demonstrated the health benefits of astaxanthin, particularly in the prevention and treatment of diseases such as cancer and cardiovascular conditions [2, 3]. Given its notable health benefits, astaxanthin holds substantial commercial potential across industries such as aquaculture, pharmaceuticals, cosmetics, and dietary supplements. To meet the rising global demand, microorganisms are increasingly considered ideal hosts for large-scale astaxanthin production, supported by advances in genetic engineering and the fast growth rates of microbial systems, which contribute to lower production costs [4].

Recently, there has been an enthusiastic push for the heterologous construction of a metabolic pathway for astaxanthin production through genetic engineering. By optimizing the activities of β-carotene ketolase and hydroxylase, an E. coli strain, ASTA-1, was engineered to produce astaxanthin as the predominant carotenoid (96.6%) with a specific content of 7.4±0.3 mg/g dry cell weight (DCW) without the need for any inducer [5]. The astaxanthin-producing fungal strain Xanthophyllomyces dendrorhous has also been studied, achieving astaxanthin content of 9.0 mg/g DCW in shake-flask cultures and 9.7 mg/g DCW in fermenter cultures [6]. In pilot-scale production using an 800 L fermenter, X. dendrorhous cultivated under continuous white light illumination reached a maximal astaxanthin content of 4.7 mg/g DCW (equivalent to the concentration of 420 mg/L) [7]. However, this fungus lacks efficient genetic tools for metabolic engineering, and the stereoisomerized form of astaxanthin (3R, 3'R) has lower antioxidant activity than the 3S, 3'S form derived from Haematococcus pluvialis [8].

In contrast, yeast cells have been widely used in industry for the production of heterologous proteins and natural products. Saccharomyces cerevisiae and Pichia pastoris have both been explored as potential yeast hosts for introducing the astaxanthin biosynthesis pathway. Supplementing S. cerevisiae cultures with 0.52 mM Fe²⁺ led to an astaxanthin content of 4.7 mg/g DCW, while the genetic overexpression of CrtE03M, CrtYB, CrtI, OCrtZ, and OBKTM in S. cerevisiae resulted in a high astaxanthin content of 8.10 mg/g DCW in shake-flask cultures [9, 10]. However, the production of hypermannosylated proteins by S. cerevisiae may pose a potential risk due to their antigenic properties. As a result, there is increasing interest in utilizing non-conventional yeasts, such as Kluyveromyces marxianus, for the biosynthesis of natural 3S, 3'S-astaxanthin (Asta-S). Additionally, the production of astaxanthin contributes to enhanced antioxidant capacity in the yeast host, promoting stress resistance and providing growth benefits [11]. K. marxianus offers several advantages over traditional yeast strains, including its Crabtree-negative nature, which allows for enhanced biomass production when supplemented with excess carbon sources. Additionally, some K. marxianus strains are thermotolerant, capable of fermenting at temperatures ranging from 25°C to 52°C. The first K. marxianus strain engineered for astaxanthin biosynthesis, Sm23, was constructed using a repeated-integration approach of bkt and the mutated Hpchyb genes. The resulting S3-2 strain produced 3S, 3'S-astaxanthin at a yield of 9.972 mg/g DCW in a 5 L fermenter [12].

This study aims to scale up the production of astaxanthin (Asta-S) using a recombinant yeast, K. marxianus, progressing from a 5 L bench-top bioreactor to a 30 L in-situ agitation reactor. Both batch and fed-batch fermentation processes were evaluated to optimize Asta-S production. The ultimate objective is to establish an efficient and scalable production process for astaxanthin using recombinant yeast, with the goal of enabling future commercialization.

Strain Development and Construction Details

The engineered strain K. marxianus 929 Asta strain D56 used in this study was kindly provided by Dr. Lance Chang. This strain was derived from the parental K. marxianus 4G5 Asta strain 6-13 a6, which had previously been genetically modified to introduce the astaxanthin biosynthesis pathway and to increase the copy numbers of the Hpchyb and CrBkt genes using the PGASO (Promoter-based Gene Assembly and Simultaneous Overexpression) technology [13].

Background of Parental Strain 6-13 a6

The K. marxianus 4G5 Asta strain 6-13 a6 underwent multiple rounds of genetic engineering to integrate astaxanthin biosynthesis gene cassettes, resulting in the accumulation of several antibiotic resistance markers, including G418, hygromycin, and zeocin. However, through continuous selection pressure using G418 alone, the strain’s hygromycin resistance was gradually attenuated. This outcome aligns with the design of the recombinant system, which utilizes galactose-inducible promoters (LAC4) to activate the astaxanthin biosynthetic pathway. This attenuation allowed hygromycin resistance to become a selectable marker again in subsequent engineering steps [14].

Genetic Construction of K. marxianus 929 Asta strain D56

The strain used in this study was engineered by sequentially and systematically introducing additional gene cassettes using the PGASO-based repeated integration method [13]. Each gene cassette consisted of a promoter-gene-terminator structure as follows: KmAdh1p-Hpchyb-ScAdh2t; KmAdh1p-XdCrtE-ScAdh2t, KmAdh1p-XdCrtyb-ScAdh2t, KmAdh1p-Hph-ScAdh2t (hygromycin resistance gene for selection); KmAdh1p-DrCrti-ScAdh2t; KmAdh1p-KmtHmg1-ScAdh2t; KmAdh1p-CrBkt-ScAdh2t. This outcome aligns with the design of the recombinant system, which utilizes galactose-inducible promoters (LAC4) and glucose-inducible promoters (Adh1) to activate the astaxanthin biosynthetic pathway.

Shake Flask Test Procedure

The shake flask test procedure involves cell activation and yeast cultivation using the engineered strain K. marxianus 929 Asta strain D56. An initial medium is prepared in a 50 mL Erlenmeyer flask, consisting of 1% yeast extract, 1% peptone, and 4% glucose. The strain K. marxianus is inoculated into the prepared initial medium and incubated in a shaking incubator at 30°C with continuous shaking for 24 hours.

Next, 5 mL of the activated cell culture is transferred into 50 mL of new medium, which consists of 1% yeast extract, 1% peptone, and 4% carbon source (such as glucose, sucrose, lactose, galactose, and molasses). The inoculated medium is then incubated in a shaking incubator at 30°C with continuous shaking for 72 hours. During the experiment, the cell growth should be continuously monitored to ensure optimal growth conditions.

Fermentation Operation Procedure

The 5L and 30L fermentor test involves the large-scale cultivation of yeast using K. marxianus 929 Asta strain D56. First, prepare the medium in the fermentor with the following composition: 1% yeast extract, 1% peptone, and different glucose concentrations. Sterilize the medium at 121°C for 20 minutes. Next, add 5% of the pre-culture (obtained from the shake flask test) to 3 liters of the sterile medium in the fermentor.

Set the desk-top 5L and in-situ 30L stirred-tank stainless fermentation tank control parameters, including pH, temperature, agitation, and dissolved oxygen (DO) concentration, with the temperature maintained at 30°C. Adjust the agitation speed automatically to keep the DO concentration above 30% and ensure a fixed airflow rate to provide adequate oxygen supply. Maintain the pH at 6. Perform batch fermentation for 48 hours. Continuously monitor the parameters during fermentation, including pH, temperature, DO concentration, and cell growth, to ensure optimal cultivation conditions.

Astaxanthin Analysis Procedure

The astaxanthin analysis involves several steps to prepare and analyze the yeast samples. First, 10 mg of freeze-dried yeast is placed into a centrifuge tube, followed by the addition of 1 mL of acetone. The mixture is then processed using an ultrasonic processor for 5 minutes, with intervals of 30 seconds on and 20 seconds off. After sonication, the sample is left at room temperature for 1 hour. Next, the sample is centrifuged at 3500 rpm for 6 minutes. Following centrifugation, the sample is filtered to remove any impurities using a 0.22 µm filter.

The filtered sample is then analysed using high-performance liquid chromatography (HPLC) equipped with a C18 column. The mobile phase initially consists of 60% solvent A (acetonitrile/methanol, 20/80 v/v) and 40% solvent B (methanol/acetone, 80/20 v/v). Over a period of 15 minutes, the solvent composition is adjusted to 30% solvent A and 70% solvent B. The column is then returned to its original composition of 60% solvent A and 40% solvent B over the next 6 minutes prior to the injection of a new sample. The flow rate is set to 1.0 mL/min, and detection is performed using a UV detector (Hitachi) at 460 nm.

The screening of carbon sources and nitrogen sources on recombinant astaxanthin production

Several carbon sources were evaluated at a concentration of 40 g/L in flask cultures to support the growth of recombinant K. marxianus 929 Asta strain D56 and enhance astaxanthin accumulation. The tested carbon sources included glucose, sucrose, lactose, galactose, and molasses (Fig. 1). Among these, glucose supported the highest biomass growth, while galactose resulted in the greatest astaxanthin accumulation per unit of biomass. This outcome aligns with the design of the recombinant system, which utilizes galactose-inducible promoters (LAC4) and glucose-inducible promoters (Adh1) to activate the astaxanthin biosynthetic pathway. Even so, the enhancement of using galactose to be the carbon source didn’t create a significant difference on astaxanthin content as compared to using glucose.

The commercial application of galactose is severely limited due to its significantly higher cost—approximately twice that of glucose. From an industrial perspective, the trade-off between yield and production cost becomes a critical factor. Although galactose can trigger stronger pathway induction, the overall astaxanthin yield per cultivation volume may still be lower due to reduced biomass formation compared to glucose-based cultures. In contrast, glucose, despite its lower induction efficiency, supports much higher biomass production and is economically advantageous. This higher biomass compensates for the lower per-cell astaxanthin content, ultimately resulting in a competitive volumetric astaxanthin yield.

Taken together, considering the balance of pathway activation, biomass generation, and cost efficiency, glucose remains the most practical and commercially viable carbon source for large-scale astaxanthin production using recombinant K. marxianus. Further optimization of glucose-based fermentation, such as process control and feeding strategies, may help close the gap in induction efficiency while maximizing overall productivity.

As discussed earlier, glucose was identified as the most suitable carbon source for astaxanthin production in recombinant K. marxianus 929 Asta strain D56, primarily due to its cost-effectiveness. To further examine the impact of glucose concentration on astaxanthin biosynthesis, flask experiments were conducted using initial glucose concentrations of 20, 40, and 60 g/L. As illustrated in Fig. 2, biomass growth increased proportionally with higher glucose concentrations, indicating that glucose effectively supports cell proliferation. However, astaxanthin content (expressed as mg per g of biomass) exhibited an inverse relationship, decreasing as the glucose concentration increased. Conclusively, the results shown in Fig. 2 suggested 40 g/L of glucose can create the best astaxanthin production in the flask trials.

This phenomenon suggests that astaxanthin accumulation is influenced by factors beyond carbon availability, possibly linked to metabolic or environmental stresses. Specifically, a noticeable drop in final pH was observed with increasing initial glucose levels, with pH declining to around 6.0 at 60 g/L glucose. It is plausible that this acidification creates suboptimal conditions for the enzymes involved in the astaxanthin biosynthetic pathway, thereby suppressing its production despite robust biomass growth. These results highlight the delicate balance between substrate concentration, cellular growth, and metabolic output, emphasizing that optimizing pH and other fermentation conditions may be crucial to maximizing astaxanthin yield.

In addition to the carbon source, the choice of nitrogen source plays a crucial role in the cultivation of K. marxianus 929 Asta strain D56 for astaxanthin production. Peptone is commonly used as a nitrogen source in microbial cultivation due to its high nutritional value. However, its relatively high cost limits its industrial application. To explore more cost-effective alternatives, several organic and inorganic nitrogen sources—sodium nitrate (NaNO₃), ammonium sulfate ((NH₄)₂SO₄), urea, and soybean meal—were tested as partial substitutes. In these experiments, 50% of the peptone was replaced by each alternative nitrogen source, while glucose was maintained at 40 g/L.

The results of these flask trials are presented in Fig. 3. As illustrated, pure peptone remained the most effective nitrogen source for supporting the growth and astaxanthin production of engineered K. marxianus 929 Asta strain D56. Although some alternative nitrogen sources showed moderate performance, none matched the yield obtained with 100% peptone. This suggests that while partial replacement is feasible, peptone still provides the optimal nutritional balance for maximizing recombinant astaxanthin synthesis. Building on previous findings that identified glucose as the preferred carbon source, further experiments were conducted to optimize its concentration in a controlled fermentation setting. Initial flask trials indicated that while increasing glucose concentrations from 20 g/L to 60 g/L enhanced biomass production, high glucose levels led to significant pH drops, potentially inhibiting K. maximum growth.

Batch and Fed-batch operation for Astaxanthin production in a 5L-fermenter

To further evaluate the effects of initial glucose on astaxanthin production in recombinant K. marxianus 929 Asta strain D56, cultivation was carried out in a pH-controlled 5 L stirred-tank fermenter using initial glucose concentrations of 40, 60, and 80 g/L. As shown in Fig. 4, biomass increased with rising glucose levels, reaching a maximum of 16.1 g/L at 80 g/L glucose. The effect of initial glucose concentration on both biomass and astaxanthin content is summarized in Table 1. The astaxanthin content increased from 2.24 mg/g at 40 g/L glucose to 2.55 mg/g at 80 g/L glucose, demonstrating that higher glucose levels not only promote cell growth but also slightly enhance astaxanthin accumulation. A higher biomass obtained at high initial glucose resulted in greater total astaxanthin production, with the highest yield of 41.06 mg/L observed at 80 g/L glucose. Notably, glucose was completely consumed within 24 hours in all batches, indicating that carbon source depletion may limit biomass growth and, consequently, total astaxanthin output. To address this limitation, a glucose fed-batch strategy was implemented to enhance biomass accumulation and improve overall productivity.

Table 1
Effects of glucose concentrations of 40, 60 and 80 g/L in a 5L agitation fermenter
Glucose concentration (g/L)	Max. biomass (g/L)	Astaxanthin content (mg/g)	Astaxanthin Yield (mg/L)	Astaxanthin Productivity (mg/L hr)
40 g/L	13.51	2.24	30.26	0.42
60 g/L	14.77	2.49	36.78	0.51
80 g/L	16.10	2.55	41.06	0.56

The total glucose concentration was set up at 80 g/L for the batch and for the fed-batch operations of engineered K. marxianus 929 Asta strain D56 for astaxanthin production. Two different fed-batch strategies of glucose feeding were performed in this study for the comparison with batch operation. All the operations are based on the total glucose concentration of 80 g/L. The first fed-batch operation used the initial glucose of 60 g/L and fed another 20 g/L of glucose after 24 hours cultivation (marked as 60 + 20). The 2nd fed-batch operation used 40 g/L of initial glucose and fed 20 g/L and another 20 g/L at the 24th and 48th hours respectively (marked as 40 + 20 + 20). The results were shown in Fig. 5 and Table 2, which indicated the batch operation at 80 g/L can have the best performance of biomass of 16.1 g/L as compared to others, but the fed-batch operation of 40 + 20 + 20 has the higher astaxanthin content of 3.44 mg/g obtained. Overall, the fed-batch operation of 40 + 20 + 20 can lead to the highest total astaxanthin production of 51.22 mg/L.

Table 2
Comprehensive data of batch and fed-batch operation at final glucose concentration of 80 g/L in the 5L fermenter
Glucose concentration (g/L)	Max. biomass (g/L)	Astaxanthin content (mg/g)	Astaxanthin Yield (mg/L)	Astaxanthin Productivity (mg/L/hr)
80	16.10	2.55	41.06	0.570
60 + 20^*	14.01	2.89	40.49	0.562
40 + 20 + 20^**	14.89	3.44	51.22	0.711
*feeding at 24th hr
** feeding at 12th and 24th hr

It seems a low and stable glucose concentration will be beneficial to astaxanthin production, which is often observed in the recombinant microbial cell with rearranged metabolic network conferring improved biosynthetic capability of various bio-products. A key challenge on how to develop an efficient strategy to maximize target product synthesis without reducing cell growth is still a debate topic, since simple knockout or overexpression of essential genes can easily impair the intracellular metabolic network, resulting in metabolic disturbance, accumulation of intermediates and byproducts [11]. To address these problems, a so-called glucose repression strategy was adopted in the fed-batch operation to maximise the cell growth and the recombinant products production. Conclusively, the best way to perform the fed-batch operation for astaxanthin production is using the on-line glucose concentration monitoring system to maintain the designed glucose concentration by continuously feeding the glucose solution. Unfortunately, such a kind of glucose monitoring system is still not easy to obtain in the industry.

Comparison of Astaxanthin production in 5L and in 30L bioreactors

For the purpose of commercialized production of astaxanthin, the scale up fermentation of recombinant K. marxianus 929 Asta strain D56 was performed in a 30L in-situ fermenter. The major difference between in-situ 30L fermenter and 5L desk-top fermenter is the material of the reactor. It is well known that the high DO-level will lead to a high-cell density cultivation. The 5L fermenter is made of glass, which is not able to use tank pressure to increase dissolved oxygen. However, the in-situ 30L fermenter is a stainless tank that can tolerate the high tank pressure to reduce the agitation speed. To further confirm this recombinant K. marxianus can be a candidate for astaxanthin production in the industry. The batch fermentation of K. marxianus at 0 and 1 atm are examined in a 30L in-site fermenter as compared to that of bath operation in a 5L fermenter. The results of 30L operation were shown in Fig. 6, and the comparison data was shown in Table 3. Surprisingly, the biomass in the 5L and in the 30L fermenter didn’t yield a significant difference. However, the astaxanthin content in the 30L operation will be significantly higher than that of 5L operation. A high DO-level in the 30L fermenter is relatively easy to be maintained at the high level as compared to the 5L desk-top fermenter. It is suggested that a high level of DO might be beneficial to the formation of recombinant astaxanthin production. However, due to the limitation of glucose obtained in the batch operation, the biomass didn’t yield any differences among all batch operations in the 5L and in the 30L bioreactor. Conclusively, a high DO condition will be beneficial to the enhancement of astaxanthin content. However, if both the high biomass and the high astaxanthin content were being achieved, a fed-batch operation by maintaining glucose level during the fermentation is suggested.

Table 3
Effects of tank pressure at 5L and 30L agitation fermenter on biomass and astaxanthin production
Fermentation type	Biomass (g/L)	Astaxanthin (mg/g)
5L fermenter at 0 atm	16.10 ± 0.75	2.55
30L fermenter at 0 atm	14.63 ± 0.88	4.03
30L fermenter at 1 atm	15.96 ± 0.36	4.38

Table 4
Comparison table of biomass, astaxanthin content and productivity in literature
Microorganism	Bioreactor type	Max biomass (g/L)	Astaxanthin content (mg/g)	Astaxanthin productivity (mg/L/d)	Reference
C. zofingiensis ATCC 30412	1 L agitation	12.9	1.1	1.7	[15]
C. zofingiensis ATCC 30412	3.7 L agitation	51.8	0.63	5.4	[16]
C. zofingiensis CCAP 211/14	Bubble column	7	6	1.3	[17]
C. glutamicum ASTA	2 L agitation	57	1.8	36	[18]
H. pluvialis NIES-144	1 L airlift	3.0	14	10.5	[19]
H. pluvialis M1	100 L flat	3.11	55.12	8.6	[20]
H. pluvialis SAG 34 − 7	1 L bubble-flat	1.59	32.1	9.7	[21]
K. marxianus Sm23	5 L agitation	11.42	9.97	22.77	[14]
X. dendrorhous ZJUT46	2000L agitation	17.42	2.27	7.2	[22]
K. marxianus 929 Asta D56	5 L agitation	16.10	2.55	13.68	this study
K. marxianus 929 Asta D56	30 L agitation	15.96	4.38	23.30	this study

Among the various carbon sources evaluated, glucose was identified as the most suitable carbon source for recombinant astaxanthin production using the engineered K. marxianus 929 Asta strain D56. This conclusion is based on both production efficiency and cost-effectiveness. Glucose not only supports robust cell growth but is also economically viable for large-scale applications. Experimental results indicate that a high initial glucose concentration is advantageous for promoting biomass accumulation. However, maintaining a lower glucose concentration during cultivation tends to enhance the intracellular astaxanthin content. This suggests that while high glucose levels stimulate cell proliferation, they may not necessarily be optimal for the synthesis of astaxanthin within the cells. To balance these two objectives—maximizing biomass and maximizing astaxanthin content—a fed-batch fermentation strategy is recommended. In fed-batch mode, glucose can be gradually supplied to the culture, which allows for sustained cell growth while preventing excessive glucose accumulation that could suppress astaxanthin biosynthesis. Additionally, when comparing fermentations conducted at different scales (5L and 30L), it was observed that higher dissolved oxygen (DO) levels positively influence astaxanthin production. Adequate DO supply appears to play a key role in enhancing the biosynthetic pathway of recombinant astaxanthin, likely by supporting active cell metabolism and reducing oxygen-limiting conditions that could hinder pigment synthesis.

Acknowledgements

The authors gratefully appreciate the financial support from Taiwan’s National Science and Technology Council under grant numbers NSTC 113-2321-B-029-002, 113-2221-E-029-032-MY3 and 114-2221E-029-012

Authors Contributions

HW Y., FS B. and JJ C. wrote the main manuscript. YC L., SS F. and HC W. jointly performed the experiments. YK L. and BY C. reviewed and edited the manuscript for the submission.

Competing interests

The authors declare that there are no competing interests.

Ethics approval

Not applicable.

Higuera-Ciapara I, Felix-Valenzuela L, Goycoolea F (2006) Astaxanthin: a review of its chemistry and applications. Critical reviews in food science and nutrition 46, 185–196.
Mussagy CU, Dufossé L (2023) A review of natural astaxanthin production in a circular bioeconomy context using Paracoccus carotinifaciens. Bioresource technology 369, 128499.
Mussagy CU, Kot A, Dufossé L, Gonçalves CN, Pereira JF, Santos-Ebinuma VC, Raghavan V, Pessoa Jr A (2023) Microbial astaxanthin: from bioprocessing to the market recognition. Applied Microbiology and Biotechnology 107, 4199–4215.
Basiony M, Ouyang L, Wang D, Yu J, Zhou L, Zhu M, Wang X, Feng J, Dai J, Shen Y (2022) Optimization of microbial cell factories for astaxanthin production: Biosynthesis and regulations, engineering strategies and fermentation optimization strategies. Synthetic and Systems Biotechnology 7, 689–704.
Lu Q, Bu Y-F, Liu J-Z (2017) Metabolic engineering of Escherichia coli for producing astaxanthin as the predominant carotenoid. Marine Drugs 15, 296.
Ashokkumar V, Flora G, Sevanan M, Sripriya R, Chen W, Park J-H, Kumar G (2023) Technological advances in the production of carotenoids and their applications–A critical review. Bioresource technology 367, 128215.
Rodríguez-Sáiz M, de la Fuente JL, Barredo JL (2010) Xanthophyllomyces dendrorhous for the industrial production of astaxanthin. Applied microbiology and biotechnology 88, 645–658.
Li X, Wang X, Duan C, Yi S, Gao Z, Xiao C, Agathos SN, Wang G, Li J (2020) Biotechnological production of astaxanthin from the microalga Haematococcus pluvialis. Biotechnology Advances 43, 107602.
Zhou P, Ye L, Xie W, Lv X, Yu H (2015) Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt. Applied Microbiology and Biotechnology 99, 8419–8428.
Zhou P, Yue C, Zhang Y, Li Y, Da X, Zhou X, Ye L (2022) Alleviation of the byproducts formation enables highly efficient biosynthesis of rosmarinic acid in Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry 70, 5077–5087.
Zheng X, Shao C, Zhu J, Zhang L, Huang Q (2023) Study of the antioxidant capacity of astaxanthin in cells against radiation-induced strong oxidative stress. Aquaculture International 31, 2705–2725.
Zhang X, Cao Y, Liu Y, Lei Y, Zhai R, Chen W, Shi G, Jin J-M, Liang C, Tang S-Y (2023) Designing glucose utilization" highway" for recombinant biosynthesis. Metabolic Engineering 78, 235–247.
Chang J-J, Ho C-Y, Ho F-J, Tsai T-Y, Ke H-M, Wang CH, Chen H-L, Shih M-C, Huang C-C, Li W-H (2012) PGASO: A synthetic biology tool for engineering a cellulolytic yeast. Biotechnology for biofuels 5, 53.
Lin Y-J, Chang J-J, Lin H-Y, Thia C, Kao Y-Y, Huang C-C, Li W-H (2017) Metabolic engineering a yeast to produce astaxanthin. Bioresource Technology 245, 899–905.
Liu J, Huang J, Jiang Y, Chen F (2012) Molasses-based growth and production of oil and astaxanthin by Chlorella zofingiensis. Bioresource technology 107, 393–398.
Sun N, Wang Y, Li Y-T, Huang J-C, Chen F (2008) Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta). Process Biochemistry 43, 1288–1292.
Del Campo J, Rodriguez H, Moreno J, Vargas M, Rivas J, Guerrero M (2004) Accumulation of astaxanthin and lutein in Chlorella zofingiensis (Chlorophyta). Applied microbiology and biotechnology 64, 848–854.
Göttl VL, Meyer F, Schmitt I, Persicke M, Peters-Wendisch P, Wendisch VF, Henke NA (2024) Enhancing astaxanthin biosynthesis and pathway expansion towards glycosylated C40 carotenoids by Corynebacterium glutamicum. Scientific Reports 14, 8081.
Zhang Z, Wang B, Hu Q, Sommerfeld M, Li Y, Han D (2016) A new paradigm for producing astaxanthin from the unicellular green alga Haematococcus pluvialis. Biotechnology and bioengineering 113, 2088–2099.
Lee KH, Chun Y, Lee JH, Park C, Yoo HY, Kwak HS (2022) Improved productivity of astaxanthin from photosensitive Haematococcus pluvialis using phototaxis technology. Marine Drugs 20, 220.
Samhat K, Kazbar A, Takache H, Ismail A, Pruvost J (2023) Influence of light absorption rate on the astaxanthin production by the microalga Haematococcus pluvialis during nitrogen starvation. Bioresources and Bioprocessing 10, 78.
Hu Z-C, Zheng Y-G, Wang Z, Shen Y-C (2007) Production of astaxanthin by Xanthophyllomyces dendrorhous ZJUT46 with fed-batch fermentation in 2.0 m3 fermentor. Food Technology and Biotechnology 45, 209–212.

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Scale up production of astaxanthin by using recombinant yeast-Kluveromyces marxianus

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