Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae
Abstract A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Δ5‐3β‐hydroxysteroid dehydrogenase gene from Digitalis...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2019-12-01
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| Series: | MicrobiologyOpen |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/mbo3.925 |
| _version_ | 1818361951597625344 |
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| author | Christoph Rieck Daniel Geiger Jennifer Munkert Katrin Messerschmidt Jan Petersen Juliane Strasser Nadine Meitinger Wolfgang Kreis |
| author_facet | Christoph Rieck Daniel Geiger Jennifer Munkert Katrin Messerschmidt Jan Petersen Juliane Strasser Nadine Meitinger Wolfgang Kreis |
| author_sort | Christoph Rieck |
| collection | DOAJ |
| description | Abstract A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Δ5‐3β‐hydroxysteroid dehydrogenase gene from Digitalis lanata, (b) a steroid Δ5‐isomerase gene from Comamonas testosteronii, (c) a mutated steroid‐5β‐reductase gene from Arabidopsis thaliana, and (d) a steroid 21‐hydroxylase gene from Mus musculus. A second plasmid bearing an ADR/ADX fusion gene from Bos taurus was also constructed. A Saccharomyces cerevisiae strain bearing these two plasmids was generated. This strain, termed “CARD II yeast”, was capable of producing 5β‐pregnane‐3β,21‐diol‐20‐one, a central intermediate in 5β‐cardenolide biosynthesis, starting from pregnenolone which was added to the culture medium. Using this approach, five consecutive steps in cardenolide biosynthesis were realized in baker's yeast. |
| first_indexed | 2024-12-13T21:24:51Z |
| format | Article |
| id | doaj.art-66f285ad80934f78860eb73c4d014a34 |
| institution | Directory Open Access Journal |
| issn | 2045-8827 |
| language | English |
| last_indexed | 2024-12-13T21:24:51Z |
| publishDate | 2019-12-01 |
| publisher | Wiley |
| record_format | Article |
| series | MicrobiologyOpen |
| spelling | doaj.art-66f285ad80934f78860eb73c4d014a342022-12-21T23:30:59ZengWileyMicrobiologyOpen2045-88272019-12-01812n/an/a10.1002/mbo3.925Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiaeChristoph Rieck0Daniel Geiger1Jennifer Munkert2Katrin Messerschmidt3Jan Petersen4Juliane Strasser5Nadine Meitinger6Wolfgang Kreis7Department Biology Friedrich‐Alexander‐University Erlangen‐Nuremberg Erlangen GermanyDepartment Biology Friedrich‐Alexander‐University Erlangen‐Nuremberg Erlangen GermanyDepartment Biology Friedrich‐Alexander‐University Erlangen‐Nuremberg Erlangen GermanyCell2Fab Research Unit University of Potsdam Potsdam GermanyDepartment Biology Friedrich‐Alexander‐University Erlangen‐Nuremberg Erlangen GermanyDepartment Biology Friedrich‐Alexander‐University Erlangen‐Nuremberg Erlangen GermanyDepartment Biology Friedrich‐Alexander‐University Erlangen‐Nuremberg Erlangen GermanyDepartment Biology Friedrich‐Alexander‐University Erlangen‐Nuremberg Erlangen GermanyAbstract A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Δ5‐3β‐hydroxysteroid dehydrogenase gene from Digitalis lanata, (b) a steroid Δ5‐isomerase gene from Comamonas testosteronii, (c) a mutated steroid‐5β‐reductase gene from Arabidopsis thaliana, and (d) a steroid 21‐hydroxylase gene from Mus musculus. A second plasmid bearing an ADR/ADX fusion gene from Bos taurus was also constructed. A Saccharomyces cerevisiae strain bearing these two plasmids was generated. This strain, termed “CARD II yeast”, was capable of producing 5β‐pregnane‐3β,21‐diol‐20‐one, a central intermediate in 5β‐cardenolide biosynthesis, starting from pregnenolone which was added to the culture medium. Using this approach, five consecutive steps in cardenolide biosynthesis were realized in baker's yeast.https://doi.org/10.1002/mbo3.925baker's yeastbiosynthesiscardenolidespathway engineeringsteroids |
| spellingShingle | Christoph Rieck Daniel Geiger Jennifer Munkert Katrin Messerschmidt Jan Petersen Juliane Strasser Nadine Meitinger Wolfgang Kreis Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae MicrobiologyOpen baker's yeast biosynthesis cardenolides pathway engineering steroids |
| title | Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae |
| title_full | Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae |
| title_fullStr | Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae |
| title_full_unstemmed | Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae |
| title_short | Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae |
| title_sort | biosynthetic approach to combine the first steps of cardenolide formation in saccharomyces cerevisiae |
| topic | baker's yeast biosynthesis cardenolides pathway engineering steroids |
| url | https://doi.org/10.1002/mbo3.925 |
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