Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide

doi:10.1007/s10126-020-09978-z

. 2020 Aug;22(4):572-580.

doi: 10.1007/s10126-020-09978-z. Epub 2020 Jun 13.

Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide

Kang Wang¹, Yulin Cui^{2

3}, Yinchu Wang^{2

3}, Zhengquan Gao¹, Tianzhong Liu⁴, Chunxiao Meng⁵, Song Qin^{6

7}

Affiliations

¹ School of Life Sciences, Shandong University of Technology, Zibo, 255049, Shandong Province, China.
² Key Laboratory of Coastal Biology and Biological Resource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, 264003, China.
³ Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, 266071, China.
⁴ Microalgal Biotechnology Group, CAS key Laboratory of Biofuels and Shandong Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and bioprocess technology, Chinese Academy of Sciences, Qingdao, 266101, China.
⁵ School of Life Sciences, Shandong University of Technology, Zibo, 255049, Shandong Province, China. mengchunxiao@126.com.
⁶ Key Laboratory of Coastal Biology and Biological Resource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, 264003, China. sqin@yic.ac.cn.
⁷ Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, 266071, China. sqin@yic.ac.cn.

PMID: 32535692
DOI: 10.1007/s10126-020-09978-z

Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide

Kang Wang et al. Mar Biotechnol (NY). 2020 Aug.

. 2020 Aug;22(4):572-580.

doi: 10.1007/s10126-020-09978-z. Epub 2020 Jun 13.

Authors

Kang Wang¹, Yulin Cui^{2

3}, Yinchu Wang^{2

3}, Zhengquan Gao¹, Tianzhong Liu⁴, Chunxiao Meng⁵, Song Qin^{6

7}

Affiliations

¹ School of Life Sciences, Shandong University of Technology, Zibo, 255049, Shandong Province, China.
² Key Laboratory of Coastal Biology and Biological Resource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, 264003, China.
³ Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, 266071, China.
⁴ Microalgal Biotechnology Group, CAS key Laboratory of Biofuels and Shandong Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and bioprocess technology, Chinese Academy of Sciences, Qingdao, 266101, China.
⁵ School of Life Sciences, Shandong University of Technology, Zibo, 255049, Shandong Province, China. mengchunxiao@126.com.
⁶ Key Laboratory of Coastal Biology and Biological Resource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, 264003, China. sqin@yic.ac.cn.
⁷ Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, 266071, China. sqin@yic.ac.cn.

PMID: 32535692
DOI: 10.1007/s10126-020-09978-z

Abstract

The purpose of this study was to express an antimicrobial peptide in the chloroplast to further develop the plastid engineering of H. pluvialis. Homologous targeting of the 16S-trnI/trnA-23S region and four endogenous regulatory elements, including the psbA promoter, rbcL promoter, rbcL terminator, and psbA terminator in H. pluvialis, were performed to construct a chloroplast transformation vector for H. pluvialis. The expression of codon-optimized antimicrobial peptide piscidin-4 gene (ant1) and selection marker gene (bar, biolaphos resistance gene) in the chloroplast of H. pluvialis was controlled by the rbcL promoter and psbA promoter, respectively. Upon biolistic transformation and selection with phosphinothricin, integration and expression of ant1 in the chloroplast genome were detected using polymerase chain reaction (PCR), southern blotting, and western blotting. Using this method, we successfully expressed antimicrobial peptide piscidin-4 in H. pluvialis. Hence, our results showed H. pluvialis promises as a platform for expressing recombinant proteins for biotechnological applications, which will further contribute to promoting genetic engineering improvement of this strain.

Keywords: Antimicrobial peptide; Bar; Biolistic method; Chloroplast transformation; Haematococcus pluvialis.

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References

1. Bauer A, Minceva M (2019) Direct extraction of astaxanthin from the microalgae Haematococcus pluvialis using liquid–liquid chromatography. RSC Adv 9:22779–22789 - DOI
1. Bauman N, Akella S, Hann E, Morey R, Schwartz AS, Brown R, Richardson TH (2018) Next-generation sequencing of Haematococcus lacustris reveals an extremely large 1.35-megabase chloroplast genome. Genome Announc 6:e00181–e00118 - DOI - PubMed - PMC
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[1] Bauer A, Minceva M (2019) Direct extraction of astaxanthin from the microalgae Haematococcus pluvialis using liquid–liquid chromatography. RSC Adv 9:22779–22789 - DOI

[2] Bauer A, Minceva M (2019) Direct extraction of astaxanthin from the microalgae Haematococcus pluvialis using liquid–liquid chromatography. RSC Adv 9:22779–22789 - DOI

[3] Bauman N, Akella S, Hann E, Morey R, Schwartz AS, Brown R, Richardson TH (2018) Next-generation sequencing of Haematococcus lacustris reveals an extremely large 1.35-megabase chloroplast genome. Genome Announc 6:e00181–e00118 - DOI - PubMed - PMC

[4] Bauman N, Akella S, Hann E, Morey R, Schwartz AS, Brown R, Richardson TH (2018) Next-generation sequencing of Haematococcus lacustris reveals an extremely large 1.35-megabase chloroplast genome. Genome Announc 6:e00181–e00118 - DOI - PubMed - PMC

[5] Campos-Quevedo N, Rosales-Mendoza S, Paz-Maldonado LMT, Martínez-Salgado L, Guevara-Arauza JC, Soria-Guerra RE (2013) Production of milk-derived bioactive peptides as precursor chimeric proteins in chloroplasts of Chlamydomonas reinhardtii. Plant Cell Tissue Organ Cult 113:217–225 - DOI

[6] Campos-Quevedo N, Rosales-Mendoza S, Paz-Maldonado LMT, Martínez-Salgado L, Guevara-Arauza JC, Soria-Guerra RE (2013) Production of milk-derived bioactive peptides as precursor chimeric proteins in chloroplasts of Chlamydomonas reinhardtii. Plant Cell Tissue Organ Cult 113:217–225 - DOI

[7] Day A, Goldschmidt-Clermont M (2011) The chloroplast transformation toolbox: selectable markers and marker removal. Plant Biotechnol J 9(5):540–553 - DOI - PubMed

[8] Day A, Goldschmidt-Clermont M (2011) The chloroplast transformation toolbox: selectable markers and marker removal. Plant Biotechnol J 9(5):540–553 - DOI - PubMed

[9] Fan L, Vonshak A, Boussiba S (1994) Effect of temperature and irradiance on growth of Haematococcus pluvialis (chlorophyceae). J Phycol 30:829–833 - DOI

[10] Fan L, Vonshak A, Boussiba S (1994) Effect of temperature and irradiance on growth of Haematococcus pluvialis (chlorophyceae). J Phycol 30:829–833 - DOI

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Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide

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Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide

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