Elevated accumulation of lutein and zeaxanthin in a novel high-biomass strain Dunaliella sp. ZP-1 through EMS mutagenesis

doi:10.21203/rs.3.rs-3323661/v1

Download PDF

Research Article

Elevated accumulation of lutein and zeaxanthin in a novel high-biomass strain Dunaliella sp. ZP-1 through EMS mutagenesis

https://doi.org/10.21203/rs.3.rs-3323661/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

Dunaliella algae, such as Dunaliella salina rich in β-carotene and Dunaliella bardawil is rich in lutein and α-carotene, have been used in aquaculture, supplements, cosmetics and feed industries. The genus Dunaliella is diverse and secretive, so characterization of novel strains and breeding new varieties through mutagenesis technology will promote natural carotenoid bioproduction.

Results

Salt stress test demonstrated microalgae strain ZP-1 was a halotolerant strain. Morphology observation and phylogeny analysis indicated this unicellular green algae ZP-1 was a member of the genus Dunaliella. Biomass of ZP-1 in RAM medium is up to 2.45 g/L, showing an advantage over the common Dunaliella algae in terms of yield. Furthermore, a screen on ethyl methanesulfonate mutant library generated from this high-biomass strain was conducted to improve natural carotenoid productivity. A mutant strain selected through morphology observation combined with carotenoid quantification by HPLC was nominated as turn yellow dunaliella 4 (tyd4). The mutant tyd4 displayed an increased lutein productivity by 28.55% and an increased zeaxanthin productivity by 22.19%. Biomass of tyd4 was promoted by 17.40% through continuous culture under red light. Application of exogenous 1.0 µM melatonin on the mutant tyd4 lead to increased cell density and improved biomass.

Conclusions

The results together support that EMS mutagenesis was an effective breeding approach for improvement of Dunaliella sp. ZP-1, which was a high-biomass microalgae exhibiting potential to some extent overcome the bottleneck of low biomass of current commercial Dunaliella strains. The mutant tyd4 had higher contents of both lutein and zeaxanthin, whose yield could be further elevated by red light and melatonin at lab-scale culture. Our study provided new algae resources for scientific research and technical reference to bioproduction of natural carotenoids.

βcarotene

Dunaliella

ethyl methanesulfonate

lutein

microalgae

mutant

zeaxanthin

Photosynthetic pigments, which take part in absorption, transmission of light energy or cause primary photochemical reactions during photosynthesis[1, 2], mainly include chlorophylls, phycobilitins and carotenoids[3, 4]. Phycobilins and carotenoids could supplement chlorophylls to capture light energy[4]. Carotenoids consist of a kind of nitrogen-free tetraterpene polyene pigments, which exist almost universally in different kingdoms. Carotenes and xanthophylls are the two major types of carotenoids. Carotenes such as lycopene and β-carotene do not contain oxygen, while xanthophylls have oxygen-containing functional groups[5, 6]. Human body does not have de novo carotenoid synthesis pathway and rely on diet for carotenoids, which are antioxidants in lipophilic environment and precursors of retinol[7, 8]. Therefore, many nutritional health products related to carotenoids are also being developed and used.

Algae are photoautotrophic aquatic organisms with chloroplasts and photosynthetic pigments, so they can synthesize carbohydrates and lipids through photosynthesis. Microalgae have the advantages of short life cycle and strong reproductive ability, accomplish more than half of global CO₂ fixation, and are the most important primary producer on Earth[9, 10]. Diatom Phaeodactylum tricornutum is the best source of natural carotenoid fucoxanthin, and unicellular green microalgae Haematococcus pluvialis and Chromochloris zofingiensis are widely used in bioproduction of natural carotenoid astaxanthin[11–14].

The genus Dunaliella represented by Dunaliella salina, consist of a group of unicellular microalgae existed in salt lakes, salterns and sea all over the world[15]. The characteristics of cell-free wall make it a live feed for sustainable aquaculture of aquatic animals and a raw material for industrial extraction of natural carotenoids[16]. Large-scale aquaculture of Dunaliella algae in high salinity regions can greatly improve land use efficiency and increase economic benefits[17]. The industrialization of Dunaliella algae benefits from many research results, especially in the aspects of screening algae species, genetic manipulating carotenoid biosynthesis pathway, optimizing culture medium and improving production technology[18, 19]. It was found that increased biomass and lipid productivity of microalgae Dunaliella tertiolecta benefited from addition of high bicarbonate concentrations into hypersaline water-based medium[20]. A recent study reported red light increased biomass productivity and carotenoid content but decreased cell volume and cell weight of D. salina in raceway ponds[21]. Some research improved the yield and quality of β-carotene by optimizing extraction process[22, 23]. However, the yield of natural carotenoids cannot meet the growing market demand.

Recently, physical mutagenesis techniques such as heavy ion irradiation mutagenesis have been applied to D. salina[24]. However, most mutants carry multi-locus and large fragment mutations, which disturbs following identification of the causal mutation. Ethyl methanesulfonate (EMS) is an ideal chemical mutagen and usually only induces single nucleotide mutation, which is conducive to stable inheritance and genetic analysis[25]. Because EMS has the advantages of low-cost and easy-to-use, a number of genetic mutants have been generated through EMS mutagenesis and used[26–28]. Haematococcus cells exposed to EMS were further screened for mutants with altered total carotenoid and astaxanthin contents[29]. Eight Chlamydomonase reinhardtii mutants were selected from an EMS mutant library due to faster growth and higher lipid content[30]. Currently, high-throughput sequencing technologies are being applied to a wide variety of organisms to rapidly identify EMS mutations in organisms ranging from metazoans to microorganisms[31, 32]. A recent study on interaction between high light and green microalgae C. reinhardtii, whole-genome sequencing of the parental wildtype and two independent mutants successfully lead to identification of two single nucleotide polymorphisms within the same gene LRS1[33].

In response to adverse environments such as high salt concentration and high light intensity, the accumulation of photosynthetic pigment carotenoid in green microalgae is often promoted as a protective mechanism for algal cells[34, 35]. Some recent studies showed abiotic stresses allow D. salina to accumulate more β-carotene content[36–38]. It was reported that promoters of β-carotene biosynthesis key enzymes DbPSY, DbPDS, DbZDS and DbLCYB in D. bardawil contained hypoosmolarity-responsive element and/or salt-regulated element and were activiated by salt stress[39, 40]. A recent study revealed the positive correlation between β-carotene content and intracellular ROS level and the negative correlation between β-carotene content and maximum quantum efficiency of PSII under stress conditions[38]. Another interesting phenomenon was that D. salina cells moved to lower intensities or secrete pigments when exposed to high light intensities[41]. It was speculated that secreted photosynthetic pigment carotenoids could reduce the cellular damage caused by high light stress.

Genetic engineering is an effective and efficient approach to increase biomass productivity and carotenoid yield of industrial microalgae strains. The rapid development of Dunaliella genomics and genetic tool-box has laid a preliminary foundation for phenome-wide genetic-correlation analysis. Therefore, the purpose of this study is to create and screen high carotenoid mutants of high-biomass microalgae Dunaliella sp. ZP-1 through mutagenesis with ethyl methanesulfonate, and to provide new resources for breeding and functional genomics of the genus Dunaliella.

Strains and culture conditions

Dunaliella sp. ZP-1 was collected from Zhangpu saltern in Zhangzhou, China. Dunaliella salina CCAP 19/18 was gained from Culture Collection of Algae & Protozoa (CCAP) (http://www.ccap.ac.uk/). Dunaliella bardawil FACHB-847 were gained from Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences (http://algae.ihb.ac.cn/English/). All Dunaliella strains were cultured in RAM (RAMARAJ) medium. The growth chambers were maintained at 22 ± 1°C and 25 μmol photons/m²s under cool-white fluorescent lamps with a 16hrs-light/8hrs-dark cycle.

Salt stress test

Dunaliella algae cultured to exponential growth phase were inoculated into RAM medium containing 5 different concentrations of 0.5 M, 1.5 M, 2.5M, 3.5 M and 4.5M NaCl, respectively. Cell numbers was counted by a cell counting chamber every two days until the plateau growth phase.

Sequences of rbcL gene and 18S gene

Genomic DNA of Dunaliella. sp. ZP-1 was isolated with SteadyPure Plant Genomic DNA Extraction Kit (Accurate Biotechnology). The gene sequences of Dunaliella. sp. ZP-1 were gained by PCR with primers, which were designed according to conserved sequences of Dunaliella rbcL genes or 18S genes from National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/).

EMS mutagenesis and mutant screen

Algal cells of Dunaliella sp. ZP-1 were treated with 1% EMS for 20 min. The incubation was stopped by 5% sodium thiosulfate solution. Algal cells were washed with RAM medium (pH 7.5) for 3 times and then uniformly spread on solid RAM medium. To screen the mutant showing altered colony color, all treated cells were cultured under 300 μmol photons/m²s under cool-white light fluorescent lamps with a 16hrs-light/8hrs-dark cycle. Each candidate mutant was transferred to a new solid RAM medium for second round screen based on colony color. Mutant strains exhibiting constant colony color were subjected to the following carotenoid quantification by High Performance Liquid Chromatography (HPLC).

Growth measurement

Refer to methods of measurement of cell density, dry weight, protein content and chlorophyll content used by Liu, et al.[42].

Carotenoids extraction, identification and quantification

Harvested algal cells were dried in freeze-dryer (HNIA-10, Hunan Hengnuo Instrument Equipment Co., Ltd.) for 24 h. The carotenoids determination by HPLC analysis in algal cells was were processed, refering to Huang, et al.[43]. Commercial standards, such as lutein (HPLC≥90%), zeaxanthin (HPLC≥90%), α-carotene (HPLC≥90%) and β-carotene (HPLC≥90%) are from Shanghai Yuanye Bio-Technology Co., Ltd..

Determination of catalase activity

Catalase (CAT) activity was measured by ammonium molybdate method using Catalase assay kit (Nanjing Jiancheng Bioengineering Institute).

Illuminant test

Dunaliella algae cultured to exponential growth phase were inoculated into RAM medium. The growth chamber for red light were maintained at 22 ± 1°C and 25 μmol photons/m²s under red light with a 16hrs-light/8hrs-dark cycle. The growth chamber for high light were maintained at 22 ± 1°C and 100 μmol photons/m²s under cool-white light fluorescent lamps with a 16hrs-light/8hrs-dark cycle.

Phytohormone treatment

The phytohormones abscisic acid (ABA), gibberellin 3 (GA) and melatonin (MT) were dissolved in ethanol and the diluted with double distilled H₂O. ABA, GA and MT were added into the RAM medium, respectively. Mock means the solvent without any phytohormone and serves as control group in each growth assay. ABA (A8060, Purity≥99.0%), GA3 (G8910, Purity≥90.0%) and MT (M8600, Purity≥99.0%) are gained from Beijing Solarbio Science & Technology Co.,Ltd..

Statistical analysis

Experiments were carried out with biological replicates included from three separate cultures independently, and data were presented as the mean with standard deviation (mean ± SD). For all of the data analysis, a p value < 0.01 was considered highly significantly different, a p value <0.05 represents statistically significant, while p > 0.05 means not significant.

Unicellular algae Dunaliella sp. ZP-1 is halotolerant.

Bioproduction of natural carotenoids by algal biotechnology has been established and continues to expand. The industrial-scale cultivation of green microalgae stores carbon dioxide and provides orange and red natural carotenoids and derivatives. At present, the identification of marine microalgae suitable for outdoor large-scale culture with high yield can promote the sustainable and stable development of algae industrialization. We isolated a green microalgae strain named as ZP-1 from Zhangpu saltern in Zhangzhou, China. Salt stress test showed that ZP-1 could live in RAM medium under a series of salinity (Fig. 1A and 1B). The biggest cell density (Fig. 1B) and highest biomass (Fig. 1C) was observed in regular RAM medium that contains 1.5 M NaCl. The cells of ZP-1 were oval or pear-shaped under 0.5 M, 1.5 M and 2.5 M NaCl (Fig. 1A), while the cells appeared circular under 3.5 M and 4.5 M NaCl. Besides, the biomass per cell decreased slightly with the increase of salt concentration (Fig. 1D). Together, these results reflected that 1.5 M NaCl was close to the optimal salt concentration for the growth of unicellular green algae ZP-1, which could adapt to different salt concentrations.

Dunaliella sp. ZP-1 is a high-biomass microalgae.

With genomic DNA of microalgae ZP-1 as template, the sequences of rbcL gene and 18S rRNA gene was gained by PCR with primer pair rbcL1-F/rbcL1-R and 18SrRNA-F/18SrRNA-R (Supplemental Table 1). BLAST results showed 18S rRNA sequence of ZP-1 was highly homologous to Dunaliella quartolecta CCAP 19/8 (KJ756824.1) and Dunaliella polymorpha CCAP 19/7A (KJ756821.1). Moreover, rbcL sequence of ZP-1 was homologous to Dunaliella granulata TAU-MAC 1120 (OQ446823.1). These results indicated ZP-1 was a strain of the genus Dunaliella. Dunaliella salina CCAP 19/18 and Dunaliella bardawil FACHB-847 are ideal aquatic food and the best industrial raw material for extraction of natural carotenoids. In lab-scale culture, cell density of ZP-1 was significantly higher than that of CCAP 19/18 or FACHB-84 (Fig. 2A). Consistently, ZP-1 exhibited higher biomass yield, more than double the biomass of commercial strains CCAP19/18 or FACHB-847 (Fig. 2B). Dunaliella sp. ZP-1 had the advantage of high biomass and was a promising bioengineering strain for natural carotenoid bioproduction.

The EMS mutant tyd4 has elevated lutein content and zeaxanthin content.

EMS mutagenesis is a chemical mutagenesis biotechnology widely used in crop breeding, to change metabolic pathway and improve germplasm resources. We established an EMS mutagenesis system for ZP-1 and screened the mutants under high light according to the colony color. Compared to the wild type Dunaliella sp. ZP-1, three algal colonies were always yellowish in two rounds of high light screen, so they were named as turn yellow dunaliella (tyd) mutants tyd4, tyd11 and tyd19. The EMS mutant tyd4 displayed similar cell density dynamics (Fig 3A), biomass (Fig 3B) and relative protein content (Fig 3C) as the wild type ZP-1. Interestingly, lutein content (Fig 4A) and zeaxanthin content (Fig 4B) in tyd4 increased without significant changes in cell number and biomass. At the same time, there was no decreases in β-carotene content and α-carotene content (Fig 4C and 4D). Notably, high light stress led to the increase of CAT activity of ZP-1 cells, which was less obvious in mutant tyd4. It suggested that higher lutein content and higher zeaxanthin content in tyd4 correlated with its adaption to high light stress.

Red light increases cell density and biomass of tyd4.

EMS mutant tyd4 rich in lutein and zeaxanthin was obtained from the genetic background of high-biomass strain Dunaliella sp. ZP-1 and had potential to be used in the industrial bioproduction of natural carotenoids. High light had negative effects on cell density and biomass of tyd4 (Fig 5A). Moreover, long-term high light treatment will consume more electricity and increase costs. The appropriate light source can effectively improve the photosynthetic efficiency of microalgae, thereby achieving the increase of biomass and the accumulation of high value-added products. Red light of the same intensity significantly elevated the cell density of tyd4 (Fig 5A), which resulted in a significant increase in biomass (Fig 5B). It can be seen that high-density large-scale cultivation of tyd4 under red light has great potential for industrialization.

Melatonin positively regulates cell density and biomass of tyd4.

Phytohormone is a kind of important endogenous molecules that regulates the growth and metabolism of plant cells. Several studies demonstrated that abscisic acid, gibberellin and melatonin could affect cell division, photosynthesis and secondary metabolites accumulation of microalgae. Thus, in order to improve light energy utilization and increase yields, different concentrations of ABA (Fig 6C), GA (Fig 6D) and MT (Fig 6E) were added to RAM medium, respectively. The results of biomass determination showed that the effects of ABA and GA on mutant tyd6 were not obvious (Fig 6C and 6D). Whereas, 1.0 μM MT treatment resulted in a significant increase in biomass, compared to WT and 0.1 μM MT treatment (Fig 6E). It could not be ignored that 10.0 μM MT treatment decreased biomass (Fig 6E), suggesting excess MT instead inhibits growth and proliferation of the mutant tyd4. However, application of 1.0 μM MT positively regulated cell density (Fig 6A) and biomass (Fig 6B). This increase in productivity induced by melatonin treatment provides a promising strategy for optimization of industrial culture conditions for EMS mutant tyd4.

There are hundreds of uncharacterized Dunaliella species that exist worldwide, representing a rich phenotypic diversity. The β-carotene content in D. salina DF 15 (CCAP 19/41) is higher than that of D. salina CCAP 19/30[44]. Dunaliella sp. ABRIINW-I1 is rich in docosahexaenoic acid (DHA) and has potential to be a bioreactor to produce nutritional fatty acids[45]. Dunaliella sp. AL-1 has capacity of zinc biosorption and is an eco-friendly candidate for phycoremediation of zinc pollution of aquatic ecosystems[46]. Here, we isolated a novel strain ZP-1 from saltern in coastal areas and found it a halotolerant microalgae (Fig. 1A). Furthermore, Dunalliella sp. ZP-1 exhibited high biomass productivity beyond that of commercial strains D. salina CCAP 19/18 and D. bardawil FACHB-847 (Fig. 2B) at lab-scale culture. It appeared that ZP-1 is a high-biomass chassis strain suitable for integration with other ZP-1 is a high-biomass chassis strain suitable for integration with biosynthesis pathways of other Dunaliella microalgae. The focus of the next research is to combine the good traits of different Dunaliella species.

Genetic engineering technology has brought revolutionary development to the microalgae industry, and recombinant genes have greatly improved the accumulation of components of interest in transgenic microalgae. In a study revealing the underlying mechanism of lutein-rich D. bardawil FACHB-847, DbBCH and DbCYP97s were found involved in conversion from α-carotene into lutein[47]. Thus, with ZP-1 as the high-biomass chassis strain, heterologous expression of Dunalilla carotenoid biosynthesis genes such as DbBCH and/or DbCYP97s is one of the topics worth studying. Moreover, many promising candidate genes emerged in research with green microalgae model species C. reinhardtii are awaiting evaluation in genetic engineering of Dunaliella sp. ZP-1. Overexpression of endogenous 18S rRNA methyltransferase BUD23 significantly improved lutein accumulation and biomass productivity in C. reinhardtii[42]. Transgenic microalgae strains overexpressing a CrtY-type β-cyclase from Bacterium Pantoea agglomerans displayed enhanced β-carotene productivity[43]. It is interesting to integrate those candidate genes into genome of Dunaliella sp. ZP-1 and explore corresponding carotenoid productivity.

Studies have shown that bulk sequencing approach combined with genetic analysis could facilitate identification of recessive mutations in animals and plants, such as Caenorhabditis. elegans and Arabidopsis thaliana. High quality genome is the prerequisite for genetic analysis of EMS mutants. Hundreds of microalgae genome have been sequenced since the genome of C. reinhardtii was released in 2007. Benefit from the development of third-generation sequencing technology and application of Hi-C assembly strategy, chromosome-level genome assembly of microalgae is providing powerful tools for synthetic biology of photosynthetic pigments. Based on chromosome-level genome of C. zofingiensis, mutations in β-ketolase (BKT) gene were identified as the causal mutations of astaxanthin biosynthesis deficient mutants[12]. A recent study published chromosome-level genome assembly of H. pluvialis and discussed duplications and clusters of astaxanthin biosynthesis key genes BKT and β-carotenoid hydroxylase (CHYB)[48]. Recently, the most exciting thing is the gap-free genome assembly of C. reinhardtii CC-5816 have been completed[49]. The draft genome of D. salina CCAP 19/18 was published in 2017[50], but incoherent assembly and inaccurate annotation caused a lot of difficulties in genetic analysis. On the other hand, it was proposed that Dunaliella. viridis SAG 44.89 could be used as the model species of halotolerant species of the genus Dunaliella, and Dunaliella. tertiolecta CCAP 19/6B (also known as UTEX 999 or SAG 13.86) as the model species of marine species of the genus Dunaliella[15]. With the efforts of scientific researchers, chromosome-level genome of marine strain D. tertiolecta and lutein-rich strain D. bardawil were de novo assembled and annotated[51]. Obviously, chromosome-level genome assembly and gene annotation of high-biomass strain Dunaliella sp. ZP-1 are also urgently needed.

The physiological state and growth rate of microalgae are affected by the amount of light received, so choosing the appropriate light source can increase yield. Red light enhanced algal growth and increased total carotenoid content of D. salina[52]. Also, red light promoted biomass productivity of D. salina in outdoor ponds[21]. In this study, we found that illumination with red light could boost biomass productivity of EMS mutant tyd4 (Fig. 5B). It appeared that red light signaling pathway in tyd4 was still similar to that of other Dunaliella microalgae, not disrupted by the EMS mutagenesis. Besides, red light positively regulated the β-carotene biosynthesis in a number of Dunaliella microalgae, such as D. salina[18] and Dunaliella sp. MACC/C43[53]. Interestingly, it was demonstrated that red light was involved in conversion between 9-cis β-carotene and 9-trans β-carotene in D. salina DF15[44]. Therefore, the effects of red light on carotenoid profiles of tyd4 should be investigated in the future.

In high plants, more and more results supported melatonin plays diverse roles in development and stress responses[54]. In microalgae Monoraphidium sp. QLY-1 cultured with normal BG-11 medium [55] and nitrogen-deficient medium[56], MT addition always resulted in higher lipid accumulation. Exogenous MT made C. reinhardtii accumulated more lipids[57]. Here, we found that red light increased biomass of tyd4 and that 1.0 µM MT positively affected biomass as well (Fig. 5B and 6B). In indicated that melatonin positively regulated growth of green microalgae. In addition to biomass and lipid accumulation, MT also regulates carotenoid metabolism. MT treatment stimulated astaxanthin biosynthesis of H. pluvialis[58, 59]. Therefore, whether exogenous melatonin will affect carotenoid content of tyd4 is another interesting question. Combined application of MT and 3-methyladenine activated carotenogenesis and lipogenesis at the transcriptional level, thereby promoting cell growth and astaxanthin biosynthesis in H. pluvialis[60]. Putrescine enhanced astaxanthin biosynthesis induced by MT in H. pluvialis under adverse conditions[61]. Simultaneous treatment of red light with MT significantly increased biomass productivity in Chlorella vulgaris [62] and carotenoid accumulation such as lutein, α-carotene and β-carotene in D. bardawil[63]. Thus, optimization of melatonin treatment is another valuable research direction of promoting carotenoid biosynthesis in microalgae. In the following study, the effects of combination of red light and melatonin should be further explored.

In this study, we collected a novel high-biomass Dunaliella algae designated as Dunaliella. sp. ZP-1, screened an EMS mutant library under high light, obtained an EMS mutant tyd4 containing more lutein and zeaxanthin without deduction in contents of both α-carotene and β-carotene, further characterized red light and melatonin positively regulated cell density and biomass of tyd4 at lab-scale culture. Our study proves the important value of microalgae germplasm resource mining, demonstrates the application potential of EMS mutagenesis technology in microalgae breeding, provides new genetic material for natural carotenoid synthetic biology, and opens the prelude to the in-depth study of the molecular mechanisms of red light and melatonin in Dunaliella algae.

ABA: abscisic acid

CAT: Catalase

CCAP: Culture Collection of Algae & Protozoa

EMS: Ethyl methanesulfonate

GA: gibberellin

GA₃: gibberellin 3

MT: melatonin

RAM: RAMARAJ

tyd: turn yellow dunaliella

Supplemental Information

The online version contains supplemental table 1.

Availability of data and materials

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Acknowledgements

The authors gratefully acknowledge the supports from Instrumental Analysis Center of Shenzhen University. The authors are grateful to Zeyu Hong and Qiaomian Zhou for technique supports on HPLC.

Funding

This research was supported by China National Key Research and Development Project (No. 2018YFA0902500), National Natural Science Foundation of China (No. 31970366), Guangdong Natural Science Foundation (No.2021A1515011590) and Guangdong Province Key Research and Development Project (No. 2022B1111070005).

Author information

Authors and Affiliations

Guangdong Engineering Research Center for Marine Algal Biotechnology, Guangdong Provincial Key Laboratory for Plant Epigenetics, Shenzhen Engineering Laboratory for Marine Algal Biotechnology, Longhua Innovation Institute for Biotechnology, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China

Chenglong Liu, Danqiong Huang, Xinran Zhuo, Jinwei Feng, Xueer Wen, Zixin Liao, Runling Wu, Zhangli Hu, Sulin Lou, Hui Li

Institute of Biomedical Engineering, Shenzhen Bay Laboratory, Shenzhen, China

Jinwei Feng

School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, China

Runling Wu

Contributions

HL, SL and CL conceived the study, designed the experiments and revised the manuscript. CL, DH, XZ, JF, RW and ZL conducted the experiments. CL analyzed the data and wrote the draft. HL, ZH and SL participated in funding acquisition. HL and SL supervised the project.

Corresponding author

Correspondence to Sulin Lou and Hui Li.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Behrenfeld, M.J. and A.J. Milligan, Photophysiological expressions of iron stress in phytoplankton. Annual Review of Marine Science, 2013. p. 217-246.
Kana, T.M., R.J. Geider, and C. Critchley, Regulation of photosynthetic pigments in micro-algae by multiple environmental factors: a dynamic balance hypothesis. New Phytologist, 1997. 137(4): p. 629-638.
Koh, H.G., et al., Heterologous synthesis of chlorophyll b in Nannochloropsis salina enhances growth and lipid production by increasing photosynthetic efficiency. Biotechnology for Biofuels, 2019. 12.
Tomitani, A., et al., Chlorophyll b and phycobilins in the common ancestor of cyanobacteria and chloroplasts. Nature, 1999. 400(6740): p. 159-162.
Begum, H., et al., Availability and utilization of pigments from microalgae. Critical Reviews in Food Science and Nutrition, 2016. 56(13): p. 2209-2222.
Dall'Osto, L., et al., The Arabidopsis nox mutant lacking carotene hydroxylase activity reveals a critical role for xanthophylls in photosystem I biogenesis. Plant Cell, 2013. 25(2): p. 591-608.
BenAmotz, A. and Y. Levy, Bioavailability of a natural isomer mixture compared with synthetic all-trans beta-carotene in human serum. American Journal of Clinical Nutrition, 1996. 63(5): p. 729-734.
Liang, M., Zhu. J., and Jiang. J, Carotenoids biosynthesis and cleavage related genes from bacteria to plants. Critical Reviews in Food Science and Nutrition, 2018. 58(14): p. 2314-2333.
Yang, B., et al., Genetic engineering of the Calvin cycle toward enhanced photosynthetic CO₂ fixation in microalgae. Biotechnology for Biofuels, 2017. 10.
Kumar, A., et al., Enhanced CO₂ fixation and biofuel production via microalgae: recent developments and future directions. Trends in Biotechnology, 2010. 28(7): p. 371-380.
Butler, T.O., et al., Towards a Phaeodactylum tricornutum biorefinery in an outdoor UK environment. Bioresource Technology, 2022. 344.
Roth, M.S., et al., Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production. Proceedings of the National Academy of Sciences of the United States of America, 2017. 114(21): p. E4296-E4305.
Zhang, Y., et al., Astaxanthin is ketolated from zeaxanthin independent of fatty acid synthesis in Chromochloris zofingiensis. Plant Physiology, 2020. 183(3): p. 883-897.
Li, L., et al., Low-temperature plasma promotes growth of Haematococcus pluvialis and accumulation of astaxanthin by regulating histone H3 lysine 4 tri-methylation. Bioresource Technology, 2022. 343.
Polle, J.E.W., E. Jin, and A. Ben-Amotz, The alga Dunaliella revisited: Looking back and moving forward with model and production organisms. Algal Research-Biomass Biofuels and Bioproducts, 2020. 49.
Lamers, P.P., et al., Exploring and exploiting carotenoid accumulation in Dunaliella salina for cell-factory applications. Trends in Biotechnology, 2008. 26(11): p. 631-638.
Patel, A., et al., Biodiesel production from microalgae Dunaliella tertiolecta: a study on economic feasibility on large-scale cultivation systems. Biomass Conversion and Biorefinery, 2021.
Harvey, P.J. and A. Ben-Amotz, Towards a sustainable Dunaliella salina microalgal biorefinery for 9-cis beta-carotene production. Algal Research-Biomass Biofuels and Bioproducts, 2020. 50.
Monte, J., et al., Recycling of Dunaliella salina cultivation medium by integrated membrane filtration and advanced oxidation. Algal Research-Biomass Biofuels and Bioproducts, 2019. 39.
Hopkins, T.C., E.J.S. Graham, and A.J. Schuler, Biomass and lipid productivity of Dunaliella tertiolecta in a produced water-based medium over a range of salinities. Journal of Applied Phycology, 2019. 31(6): p. 3349-3358.
Nwoba, E.G., et al., Monochromatic light filters to enhance biomass and carotenoid productivities of Dunaliella salina in raceway ponds. Bioresource Technology, 2021. 340.
Ludwig, K., et al., Beta-Carotene extraction from Dunaliella salina by supercritical CO₂. Journal of Applied Phycology, 2021.
Marchal, L., et al., Centrifugal partition extraction of beta-carotene from Dunaliella salina for efficient and biocompatible recovery of metabolites. Bioresource Technology, 2013. 134: p. 396-400.
Xi, Y., et al., Photosynthetic profiling of a Dunaliella salina mutant DS240G-1 with improved beta-carotene productivity induced by heavy ions irradiation. International Journal of Agricultural and Biological Engineering, 2021. 14(2): p. 211-217.
Gady, A.L.F., et al., Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations. Plant Methods, 2009. 5.
Sattler, S.E., et al., Characterization of novel Sorghum brown midrib Mutants from an EMS-mutagenized population. G3-Genes Genomes Genetics, 2014. 4(11): p. 2115-2124.
Henry, I.M., et al., Efficient genome-wide detection and cataloging of EMS-induced mutations using exome capture and next-generation sequencing. Plant Cell, 2014. 26(4): p. 1382-1397.
Blumenstiel, J.P., et al., Identification of EMS-induced mutations in Drosophila melanogaster by whole-genome sequencing. Genetics, 2009. 182(1): p. 25-32.
Kamath, B.S., et al., Enhancement of carotenoids by mutation and stress induced carotenogenic genes in Haematococcus pluvialis mutants. Bioresource Technology, 2008. 99(18): p. 8667-8673.
Kim, H.S., et al., High-throughput droplet microfluidics screening platform for selecting fast-growing and high lipid-producing microalgae from a mutant library. Plant Direct, 2017. 1(3).
Tsuda, M., et al., Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing. Bmc Genomics, 2015. 16.
Zhu, Q., et al., High-throughput discovery of mutations in Tef semi-dwarfing genes by next-generation sequencing analysis. Genetics, 2012. 192(3): p. 819-+.
Schierenbeck, L., et al., Fast forward genetics to identify mutations causing a high light tolerant phenotype in Chlamydomonas reinhardtii by whole-genome-sequencing. Bmc Genomics, 2015. 16.
Wase, N., et al., Identification and metabolite profiling of chemical activators of lipid accumulation in green algae. Plant Physiology, 2017. 174(4): p. 2146-2165.
Sun, X.M., et al., Microalgae for the production of lipid and carotenoids: a review with focus on stress regulation and adaptation. Biotechnology for Biofuels, 2018. 11.
Liang, M., et al., Induction of carotenoid cleavage by salt stress and the effect of their products on cell growth and pigment accumulation in Dunaliella sp. FACHB-847. Algal Research-Biomass Biofuels and Bioproducts, 2020. 48.
Ramos, A.A., et al., The unicellular green alga Dunaliella salina Teod. as a model for abiotic stress tolerance: genetic advances and future perspectives. Algae, 2011. 26(1): p. 3-20.
Xi, Y., F. Kong, and Z. Chi, ROS Induce beta-carotene biosynthesis caused by changes of photosynthesis efficiency and energy metabolism in Dunaliella salina under stress conditions. Frontiers in Bioengineering and Biotechnology, 2021. 8.
Liang, M.-H., et al., The salt-regulated element in the promoter of lycopene -cyclase gene confers a salt regulatory pattern in carotenogenesis of Dunaliella bardawil. Environmental Microbiology, 2017. 19(3): p. 982-989.
Lao, Y.-M., et al., Hypoosmotic expression of Dunaliella bardawil zeta-carotene desaturase is attributed to a hypoosmolarity-responsive element different from other key carotenogenic genes. Plant Physiology, 2014. 165(1): p. 359-372.
Kleinegris, D.M.M., et al., The selectivity of milking of Dunaliella salina. Marine Biotechnology, 2010. 12(1): p. 14-23.
Liu, C.L., et al., Overexpression of 18S rRNA methyltransferase CrBUD23 enhances biomass and lutein content in Chlamydomonas reinhardtii. Frontiers in Bioengineering and Biotechnology, 2023. 11.
Huang, D.Q., et al., Enhancement of beta-carotene content in Chlamydomonas reinhardtii by expressing bacterium-driven lycopene beta-cyclase. Biotechnology for Biofuels and Bioproducts, 2023. 16(1).
Sui, Y., et al., A comparison of beta-carotene, phytoene and amino acids production in Dunaliella salina DF 15 (CCAP 19/41) and Dunaliella salina CCAP 19/30 using different light wavelengths. Foods, 2021. 10(11).
Gharajeh, N.H., et al., Dunaliella sp. ABRIINW-I1 as a cell factory of nutraceutical fatty acid pattern: An optimization approach to improved production of docosahexaenoic acid (DHA). Chemical Engineering and Processing-Process Intensification, 2020. 155.
Elleuch, J., et al., Zinc biosorption by Dunaliella sp. AL-1: Mechanism and effects on cell metabolism. Science of the Total Environment, 2021. 773.
Liang, M.-H., et al., Functional identification of two types of carotene hydroxylases from the green alga Dunaliella bardawil rich in lutein. Acs Synthetic Biology, 2020. 9(6): p. 1246-1253.
Bian, C., et al., A chromosome-level genome assembly for the astaxanthin-producing microalga Haematococcus pluvialis. Scientific Data, 2023. 10(1).
Payne, Z.L., et al., A gap-free genome assembly of Chlamydomonas reinhardtii and detection of translocations induced by CRISPR-mediated mutagenesis. Plant Communications, 2023. 4(2).
Polle, J.E.W., et al., Draft nuclear genome sequence of the halophilic and beta-carotene-accumulating green alga Dunaliella salina strain CCAP19/18. Microbiology Resource Announcements, 2017. 5(43).
Davidi, L., et al., Pumping iron: A multi-omics analysis of two extremophilic algae reveals iron economy management. Proceedings of the National Academy of Sciences of the United States of America, 2023. 120(30): p. e2305495120-e2305495120.
Li, Y., et al., Transcriptome analysis of carotenoid biosynthesis in Dunaliella salina under red and blue light. Journal of Oceanology and Limnology, 2020. 38(1): p. 177-185.
Li, Y., et al., Light absorption and growth response of Dunaliella under different light qualities. Journal of Applied Phycology, 2020. 32(2): p. 1041-1052.
Zhao, D., et al., Phytomelatonin: An emerging regulator of plant biotic stress resistance. Trends in Plant Science, 2021. 26(1): p. 70-82.
Li, D., et al., A strategy for promoting lipid production in green microalgae Monoraphidium sp QLY-1 by combined melatonin and photoinduction. Bioresource Technology, 2017. 235: p. 104-112.
Zhao, Y., et al., Melatonin enhances lipid production in Monoraphidium sp QLY-1 under nitrogen deficiency conditions via a multi-level mechanism. Bioresource Technology, 2018. 259: p. 46-53.
Meng, Y., et al., Melatonin facilitates the coordination of cell growth and lipid accumulation in nitrogen-stressed Chlamydomonas reinhardtii for biodiesel production. Algal Research-Biomass Biofuels and Bioproducts, 2020. 46.
Ding, W., et al., Melatonin enhances astaxanthin accumulation in the green microalga Haematococcus pluvialis by mechanisms possibly related to abiotic stress tolerance. Algal Research-Biomass Biofuels and Bioproducts, 2018. 33: p. 256-265.
Cui, J., et al., Melatonin and calcium act synergistically to enhance the coproduction of astaxanthin and lipids in Haematococcus pluvialis under nitrogen deficiency and high light conditions. Bioresource Technology, 2020. 305.
Zhao, Y.T., et al., A joint strategy comprising melatonin and 3-methyladenine to concurrently stimulate biomass and astaxanthin hyperaccumulation by Haematococcus pluvialis. Bioresource Technology, 2021. 341.
Xing, H., et al., Enhancing astaxanthin and lipid coproduction in Haematococcus pluvialis by the combined induction of plant growth regulators and multiple stresses. Bioresource Technology, 2022. 344.
Samani, M.S.M. and H. Mansouri, The novel strategy for enhancing growth and lipid accumulation in Chlorella vulgaris microalgae cultured in dairy wastewater by monochromatic LEDs and melatonin. Journal of Applied Phycology, 2022.
Xie, S.R., et al., A strategy to promote carotenoids production in Dunaliella bardawil by melatonin combined with photoinduction. Enzyme and Microbial Technology, 2022. 161.

No competing interests reported.

SupplementalTable1.xls
Supplemental Table 1. Primers rbcL1-F/rbcL1-R and 18SrRNA-F/18SrRNA-R.

Download PDF

Editorial decision: Revision requested
13 Dec, 2023
Reviews received at journal
11 Oct, 2023
Reviewers agreed at journal
10 Oct, 2023
Reviewers agreed at journal
25 Sep, 2023
Reviewers invited by journal
25 Sep, 2023
Editor assigned by journal
05 Sep, 2023
Submission checks completed at journal
04 Sep, 2023
First submitted to journal
04 Sep, 2023

You are reading this latest preprint version

Elevated accumulation of lutein and zeaxanthin in a novel high-biomass strain Dunaliella sp. ZP-1 through EMS mutagenesis

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Materials and Methods

Strains and culture conditions

Salt stress test

Sequences of rbcL gene and 18S gene

EMS mutagenesis and mutant screen

Growth measurement

Carotenoids extraction, identification and quantification

Determination of catalase activity

Illuminant test

Phytohormone treatment

Statistical analysis

Results

Unicellular algae Dunaliella sp. ZP-1 is halotolerant.

Dunaliella sp. ZP-1 is a high-biomass microalgae.

The EMS mutant tyd4 has elevated lutein content and zeaxanthin content.

Red light increases cell density and biomass of tyd4.

Melatonin positively regulates cell density and biomass of tyd4.

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1