The Spermidine Synthase Gene <i>SPD1</i>: A Novel Auxotrophic Marker for <i>Chlamydomonas reinhardtii</i> Designed by Enhanced CRISPR/Cas9 Gene Editing
Biotechnological application of the green microalga <i>Chlamydomonas reinhardtii</i> hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-02-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/11/5/837 |
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| author | Robert A. Freudenberg Luisa Wittemeier Alexander Einhaus Thomas Baier Olaf Kruse |
| author_facet | Robert A. Freudenberg Luisa Wittemeier Alexander Einhaus Thomas Baier Olaf Kruse |
| author_sort | Robert A. Freudenberg |
| collection | DOAJ |
| description | Biotechnological application of the green microalga <i>Chlamydomonas reinhardtii</i> hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only a few auxotrophic markers exist for <i>C. reinhardtii</i>. The recent improvements in gene editing via CRISPR/Cas allow directed exploration of new endogenous selectable markers. Since editing frequencies remain comparably low, a Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, which improved successful gene edits from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (<i>SPD1</i>) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic <i>SPD1</i> gene was achieved when the mature protein was expressed in the cytosol or targeted to the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in <i>C. reinhardtii</i>. |
| first_indexed | 2024-03-09T20:43:45Z |
| format | Article |
| id | doaj.art-7829a7904dd14130bd64361467bca99f |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-09T20:43:45Z |
| publishDate | 2022-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-7829a7904dd14130bd64361467bca99f2023-11-23T22:51:01ZengMDPI AGCells2073-44092022-02-0111583710.3390/cells11050837The Spermidine Synthase Gene <i>SPD1</i>: A Novel Auxotrophic Marker for <i>Chlamydomonas reinhardtii</i> Designed by Enhanced CRISPR/Cas9 Gene EditingRobert A. Freudenberg0Luisa Wittemeier1Alexander Einhaus2Thomas Baier3Olaf Kruse4Center for Biotechnology (CeBiTec), Faculty of Biology, Bielefeld University, Universitätsstrasse 27, 33615 Bielefeld, GermanyCenter for Biotechnology (CeBiTec), Faculty of Biology, Bielefeld University, Universitätsstrasse 27, 33615 Bielefeld, GermanyCenter for Biotechnology (CeBiTec), Faculty of Biology, Bielefeld University, Universitätsstrasse 27, 33615 Bielefeld, GermanyCenter for Biotechnology (CeBiTec), Faculty of Biology, Bielefeld University, Universitätsstrasse 27, 33615 Bielefeld, GermanyCenter for Biotechnology (CeBiTec), Faculty of Biology, Bielefeld University, Universitätsstrasse 27, 33615 Bielefeld, GermanyBiotechnological application of the green microalga <i>Chlamydomonas reinhardtii</i> hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only a few auxotrophic markers exist for <i>C. reinhardtii</i>. The recent improvements in gene editing via CRISPR/Cas allow directed exploration of new endogenous selectable markers. Since editing frequencies remain comparably low, a Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, which improved successful gene edits from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (<i>SPD1</i>) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic <i>SPD1</i> gene was achieved when the mature protein was expressed in the cytosol or targeted to the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in <i>C. reinhardtii</i>.https://www.mdpi.com/2073-4409/11/5/837microalgaeCRISPR/Cas9gene editingspermidine<i>SPD1</i>auxotrophy |
| spellingShingle | Robert A. Freudenberg Luisa Wittemeier Alexander Einhaus Thomas Baier Olaf Kruse The Spermidine Synthase Gene <i>SPD1</i>: A Novel Auxotrophic Marker for <i>Chlamydomonas reinhardtii</i> Designed by Enhanced CRISPR/Cas9 Gene Editing Cells microalgae CRISPR/Cas9 gene editing spermidine <i>SPD1</i> auxotrophy |
| title | The Spermidine Synthase Gene <i>SPD1</i>: A Novel Auxotrophic Marker for <i>Chlamydomonas reinhardtii</i> Designed by Enhanced CRISPR/Cas9 Gene Editing |
| title_full | The Spermidine Synthase Gene <i>SPD1</i>: A Novel Auxotrophic Marker for <i>Chlamydomonas reinhardtii</i> Designed by Enhanced CRISPR/Cas9 Gene Editing |
| title_fullStr | The Spermidine Synthase Gene <i>SPD1</i>: A Novel Auxotrophic Marker for <i>Chlamydomonas reinhardtii</i> Designed by Enhanced CRISPR/Cas9 Gene Editing |
| title_full_unstemmed | The Spermidine Synthase Gene <i>SPD1</i>: A Novel Auxotrophic Marker for <i>Chlamydomonas reinhardtii</i> Designed by Enhanced CRISPR/Cas9 Gene Editing |
| title_short | The Spermidine Synthase Gene <i>SPD1</i>: A Novel Auxotrophic Marker for <i>Chlamydomonas reinhardtii</i> Designed by Enhanced CRISPR/Cas9 Gene Editing |
| title_sort | spermidine synthase gene i spd1 i a novel auxotrophic marker for i chlamydomonas reinhardtii i designed by enhanced crispr cas9 gene editing |
| topic | microalgae CRISPR/Cas9 gene editing spermidine <i>SPD1</i> auxotrophy |
| url | https://www.mdpi.com/2073-4409/11/5/837 |
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