A Novel Bifunctional Wax Ester Synthase Involved in Early Triacylglycerol Accumulation in Unicellular Green Microalga Haematococcus pluvialis Under High Light Stress

doi:10.3389/fbioe.2021.794714

. 2022 Jan 17:9:794714.

doi: 10.3389/fbioe.2021.794714. eCollection 2021.

A Novel Bifunctional Wax Ester Synthase Involved in Early Triacylglycerol Accumulation in Unicellular Green Microalga Haematococcus pluvialis Under High Light Stress

Haiyan Ma ¹, Jie Zheng ^{1

2}, Yanhua Li ¹, Liang Zhao ¹, Song Zou ^{1

2}, Qiang Hu ^{1

3

4

5

6}, Danxiang Han ^{1

6}

Affiliations

¹ Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.
² University of Chinese Academy of Sciences, Beijing, China.
³ Institute for Advanced Study, Shenzhen University, Shenzhen, China.
⁴ Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
⁵ State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.
⁶ Key Laboratory for Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.

PMID: 35111735
PMCID: PMC8802113
DOI: 10.3389/fbioe.2021.794714

A Novel Bifunctional Wax Ester Synthase Involved in Early Triacylglycerol Accumulation in Unicellular Green Microalga Haematococcus pluvialis Under High Light Stress

Haiyan Ma et al. Front Bioeng Biotechnol. 2022.

. 2022 Jan 17:9:794714.

doi: 10.3389/fbioe.2021.794714. eCollection 2021.

Authors

Haiyan Ma ¹, Jie Zheng ^{1

2}, Yanhua Li ¹, Liang Zhao ¹, Song Zou ^{1

2}, Qiang Hu ^{1

3

4

5

6}, Danxiang Han ^{1

6}

Affiliations

¹ Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.
² University of Chinese Academy of Sciences, Beijing, China.
³ Institute for Advanced Study, Shenzhen University, Shenzhen, China.
⁴ Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
⁵ State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.
⁶ Key Laboratory for Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.

PMID: 35111735
PMCID: PMC8802113
DOI: 10.3389/fbioe.2021.794714

Abstract

The bulk of neutral lipids, including astaxanthin esters and triacylglycerols (TAGs), are accumulated in the green microalga Haematococcus pluvialis under high light (HL) stress. In this study, a novel bifunctional wax ester synthase (WS) gene was cloned from H. pluvialis upon HL stress. The overexpression of HpWS restored the biosynthesis of wax esters and TAGs in neutral lipid-deficient yeast mutant Saccharomyces cerevisiae H1246 fed with C18 alcohol and C18:1/C18:3 fatty acids, respectively. Under HL stress, HpWS was substantially upregulated at the transcript level, prior to that of the type I diacylglycerol:acyl-CoA acyltransferase encoding gene (HpDGAT1). HpDGAT1 is the major TAG synthase in H. pluvialis. In addition, the application of xanthohumol (a DGAT1/2 inhibitor) in the H. pluvialis cells did not completely eliminate the TAG biosynthesis under HL stress at 24 h. These results indicated that HpWS may contribute to the accumulation of TAGs in H. pluvialis at the early stage under HL stress. In addition, the overexpression of HpWS in Chlamydomonas reinhardtii bkt5, which is engineered to produce free astaxanthin, enhanced the production of TAGs and astaxanthin. Our findings broaden the understanding of TAG biosynthesis in microalgae and provide a new molecular target for genetic manipulation in biotechnological applications.

Keywords: Haematococcus pluvialis; bifunctional; biotechnological application; microalgae; triacylglycerol; wax synthase.

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Conflict of interest statement

LZ was employed by the company Demeter Bio-Tech Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1

FIGURE 1

Phylogenetic and alignment analysis of HpWS. (A) Phylogenetic tree of wax synthase (WS), diacylglycerol acyltransferase (DGAT), phytyl ester synthase (PES), and bifunctional WS/DGAT (WSD) from various organisms. (B) Conserved motifs in WS clade. Ab, Acinetobacter baylyi; Ac, Apis cerana; At, Arabidopsis thaliana; An, Aspergillus niger; Bn, Brassica napus; Car, Capsella rubella; Ce, Chlamydomonas eustigma; Cis, Citrus sinensis; Coe, Coffea eugenioides; Cr, Chlamydomonas reinhardtii; Cs, Chlorella sorokiniana; Cv, Chlorella vulgaris; Cz, Chromochloris zofingiensis; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster; Eg, Euglena gracilis; Es, Eutrema salsugineum; Hp, Haematococcus pluvialis; Lv, Leptolyngbya valderiana; Mm, Mus musculus; Mt, Mycobacterium tuberculosis; No, Nannochloropsis oceanica; Pt, Phaeodactylum tricornutum; Rs, Raphanus sativus; Sc, Saccharomyces cerevisiae; Ts, Tetrabaena socialis; Xt, Xenopus tropicalis; Zm, Zea mays. The accession numbers of these enzymes are listed in Supplementary Table S2.

FIGURE 2

FIGURE 2

Functional study of HpWS in the Saccharomyces cerevisiae H1246 system. (A) Expression of recombinant HpWS in yeast microsomes after induction for up to 24 h at 25°C. (B) Thin-layer chromatography (TLC) separation of wax esters produced in S. cerevisiae harboring empty vector (EV) (1) or HpWS (2) fed with 0.1% (w/v) C16:0 FA + C16 alcohol + C18 alcohol; S. cerevisiae harboring HpWS fed with 0.1% (w/v) C16:0 FA (3), C16:0 FA + C16 alcohol (4), or C16:0 FA + C18 alcohol (5). (C) TLC separation of TAGs produced in S. cerevisiae harboring HpWS fed with 0.1% (w/v) four species of fatty acid. Relative contents of WE and TAG were quantified by ImageJ. FA, fatty acid; WE, wax ester; TAG, triacylglycerol; DAG, diacylglycerol.

FIGURE 3

FIGURE 3

High-light-induced accumulation of neutral lipids in Haematococcus pluvialis NIES-144. (A) The relative gene expression of HpWS, HpDGAT1, and HpDGTT3. (B) Astaxanthin content. (C) TAG content. (D) Thin-layer chromatography (TLC) separation of sterol esters produced. Control, low light; HL, high light; TAG, Triacylglycerol; SE, sterol ester. Data are expressed as mean ± SD, n = 3. *p < 0.05 (Student’s t-test). The lipid residue was re-established in chloroform, and the volume was normalized to cell number. An equal volume of each sample was loaded on TLC for separation.

FIGURE 4

FIGURE 4

Effect of DGAT inhibitors on TAG biosynthesis in Haematococcus pluvialis under HL stress for 24 h. (A) TAG content in H. pluvialis fed with A922500 ranged from 7.5 to 30 μM. (B) TAG content in H. pluvialis fed with xanthohumol ranged from 5 to 40 μM. (C) Fatty acid content in H. pluvialis fed with A922500 ranged from 7.5 to 30 μM. (D) Fatty acid content in H. pluvialis fed with xanthohumol ranged from 5 to 40 μM. A922500, DGAT1-specific inhibitor; xanthohumol, DGAT1, and DGAT2 inhibitor. Data are expressed as mean ± SD, n = 3. *p < 0.05 (Student’s t-test).

FIGURE 5

FIGURE 5

Growth and lipid production in Chlamydomonas reinhardtii BKT5 transformants overexpressing HpWS under nitrogen-replete and nitrogen-depleted conditions. (A) Expression of HpWS in the C. reinhardtii transformants. (B) Dry weight. (C) Astaxanthin content. (D) TFA content. (E) Fatty acid composition in TFA after 3 days under nitrogen deprivation. (F) TAG content. (G) Fatty acid composition in TAG after 2 days under nitrogen deprivation. TFA, total fatty acid; FA, fatty acid. EV, C. reinhardtii BKT5 harboring pChamy_4 vector. HpWS1 and HpWS2 are two individual C. reinhardtii transformants overexpressing HpWS. D, culture under nitrogen repletion; N, culture under nitrogen deprivation. Data are expressed as mean ± SD, n = 4. *p < 0.05 (Student’s t-test).

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References

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1. Boyle N. R., Page M. D., Liu B., Blaby I. K., Casero D., Kropat J., et al. (2012). Three Acyltransferases and Nitrogen-Responsive Regulator Are Implicated in Nitrogen Starvation-Induced Triacylglycerol Accumulation in Chlamydomonas . J. Biol. Chem. 287 (19), 15811–15825. 10.1074/jbc.M111.334052 - DOI - PMC - PubMed
1. Chen G., Wang B., Han D., Sommerfeld M., Lu Y., Chen F., et al. (2015). Molecular Mechanisms of the Coordination between Astaxanthin and Fatty Acid Biosynthesis inHaematococcus pluvialis(Chlorophyceae). Plant J. 81 (1), 95–107. 10.1111/tpj.12713 - DOI - PubMed
1. Chi X., Hu R., Zhang X., Chen M., Chen N., Pan L., et al. (2014). Cloning and Functional Analysis of Three Diacylglycerol Acyltransferase Genes from Peanut (Arachis hypogaea L.). PloS one 9 (9), e105834. 10.1371/journal.pone.0105834 - DOI - PMC - PubMed
1. Chisti Y. (2007). Biodiesel from Microalgae. Biotechnol. Adv. 25 (3), 294–306. 10.1016/j.biotechadv.200702001 - DOI - PubMed

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[1] Boussiba S. (2000). Carotenogenesis in the green algaHaematococcus pluvialis: Cellular Physiology and Stress Response. Physiologia Plantarum 108 (2), 111–117. 10.1034/j.1399-3054.2000.108002111.x - DOI

[2] Boussiba S. (2000). Carotenogenesis in the green algaHaematococcus pluvialis: Cellular Physiology and Stress Response. Physiologia Plantarum 108 (2), 111–117. 10.1034/j.1399-3054.2000.108002111.x - DOI

[3] Boyle N. R., Page M. D., Liu B., Blaby I. K., Casero D., Kropat J., et al. (2012). Three Acyltransferases and Nitrogen-Responsive Regulator Are Implicated in Nitrogen Starvation-Induced Triacylglycerol Accumulation in Chlamydomonas . J. Biol. Chem. 287 (19), 15811–15825. 10.1074/jbc.M111.334052 - DOI - PMC - PubMed

[4] Boyle N. R., Page M. D., Liu B., Blaby I. K., Casero D., Kropat J., et al. (2012). Three Acyltransferases and Nitrogen-Responsive Regulator Are Implicated in Nitrogen Starvation-Induced Triacylglycerol Accumulation in Chlamydomonas . J. Biol. Chem. 287 (19), 15811–15825. 10.1074/jbc.M111.334052 - DOI - PMC - PubMed

[5] Chen G., Wang B., Han D., Sommerfeld M., Lu Y., Chen F., et al. (2015). Molecular Mechanisms of the Coordination between Astaxanthin and Fatty Acid Biosynthesis inHaematococcus pluvialis(Chlorophyceae). Plant J. 81 (1), 95–107. 10.1111/tpj.12713 - DOI - PubMed

[6] Chen G., Wang B., Han D., Sommerfeld M., Lu Y., Chen F., et al. (2015). Molecular Mechanisms of the Coordination between Astaxanthin and Fatty Acid Biosynthesis inHaematococcus pluvialis(Chlorophyceae). Plant J. 81 (1), 95–107. 10.1111/tpj.12713 - DOI - PubMed

[7] Chi X., Hu R., Zhang X., Chen M., Chen N., Pan L., et al. (2014). Cloning and Functional Analysis of Three Diacylglycerol Acyltransferase Genes from Peanut (Arachis hypogaea L.). PloS one 9 (9), e105834. 10.1371/journal.pone.0105834 - DOI - PMC - PubMed

[8] Chi X., Hu R., Zhang X., Chen M., Chen N., Pan L., et al. (2014). Cloning and Functional Analysis of Three Diacylglycerol Acyltransferase Genes from Peanut (Arachis hypogaea L.). PloS one 9 (9), e105834. 10.1371/journal.pone.0105834 - DOI - PMC - PubMed

[9] Chisti Y. (2007). Biodiesel from Microalgae. Biotechnol. Adv. 25 (3), 294–306. 10.1016/j.biotechadv.200702001 - DOI - PubMed

[10] Chisti Y. (2007). Biodiesel from Microalgae. Biotechnol. Adv. 25 (3), 294–306. 10.1016/j.biotechadv.200702001 - DOI - PubMed

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A Novel Bifunctional Wax Ester Synthase Involved in Early Triacylglycerol Accumulation in Unicellular Green Microalga Haematococcus pluvialis Under High Light Stress

Affiliations

A Novel Bifunctional Wax Ester Synthase Involved in Early Triacylglycerol Accumulation in Unicellular Green Microalga Haematococcus pluvialis Under High Light Stress

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

LinkOut - more resources

Full Text Sources