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An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis.

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Title: An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis.
Authors: Vollheyde, Katharina1 (AUTHOR), Dudley, Quentin M.2 (AUTHOR), Yang, Ting1 (AUTHOR), Oz, Mehmet T.2 (AUTHOR), Mancinotti, Davide1 (AUTHOR), Fedi, Mariano Olivera2 (AUTHOR), Heavens, Darren2 (AUTHOR), Linsmith, Gareth2 (AUTHOR), Chhetry, Monika3 (AUTHOR), Smedley, Mark A.3 (AUTHOR), Harwood, Wendy A.3 (AUTHOR), Swarbreck, David2 (AUTHOR), Geu‐Flores, Fernando1 (AUTHOR) feg@plen.ku.dk, Patron, Nicola J.2 (AUTHOR) nicola.patron@earlham.ac.uk
Source: New Phytologist. Oct2023, Vol. 240 Issue 1, p302-317. 16p.
Subjects: Nicotiana benthamiana, Alkaloids, Enantiomeric purity, Biosynthesis, Nicotine, Berberine, CRISPRs, Pyridine
Abstract: Summary: The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high‐value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes.Here, we report a new assembly of the N. benthamiana genome as well as the generation of low‐nicotine lines by CRISPR/Cas9‐based inactivation of berberine bridge enzyme‐like proteins (BBLs). Triple as well as quintuple mutants accumulated three to four times less nicotine than the respective control lines.The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis.Our work delivers an improved N. benthamiana chassis for bioproduction and uncovers the crucial role of BBLs in the stereoselectivity of nicotine biosynthesis. [ABSTRACT FROM AUTHOR]
Copyright of New Phytologist is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
Database: Engineering Source
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PubType: Academic Journal
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  Data: An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis.
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  Data: <searchLink fieldCode="AR" term="%22Vollheyde%2C+Katharina%22">Vollheyde, Katharina</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Dudley%2C+Quentin+M%2E%22">Dudley, Quentin M.</searchLink><relatesTo>2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Yang%2C+Ting%22">Yang, Ting</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Oz%2C+Mehmet+T%2E%22">Oz, Mehmet T.</searchLink><relatesTo>2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Mancinotti%2C+Davide%22">Mancinotti, Davide</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Fedi%2C+Mariano+Olivera%22">Fedi, Mariano Olivera</searchLink><relatesTo>2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Heavens%2C+Darren%22">Heavens, Darren</searchLink><relatesTo>2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Linsmith%2C+Gareth%22">Linsmith, Gareth</searchLink><relatesTo>2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Chhetry%2C+Monika%22">Chhetry, Monika</searchLink><relatesTo>3</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Smedley%2C+Mark+A%2E%22">Smedley, Mark A.</searchLink><relatesTo>3</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Harwood%2C+Wendy+A%2E%22">Harwood, Wendy A.</searchLink><relatesTo>3</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Swarbreck%2C+David%22">Swarbreck, David</searchLink><relatesTo>2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Geu‐Flores%2C+Fernando%22">Geu‐Flores, Fernando</searchLink><relatesTo>1</relatesTo> (AUTHOR)<i> feg@plen.ku.dk</i><br /><searchLink fieldCode="AR" term="%22Patron%2C+Nicola+J%2E%22">Patron, Nicola J.</searchLink><relatesTo>2</relatesTo> (AUTHOR)<i> nicola.patron@earlham.ac.uk</i>
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  Data: <searchLink fieldCode="JN" term="%22New+Phytologist%22">New Phytologist</searchLink>. Oct2023, Vol. 240 Issue 1, p302-317. 16p.
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  Data: <searchLink fieldCode="DE" term="%22Nicotiana+benthamiana%22">Nicotiana benthamiana</searchLink><br /><searchLink fieldCode="DE" term="%22Alkaloids%22">Alkaloids</searchLink><br /><searchLink fieldCode="DE" term="%22Enantiomeric+purity%22">Enantiomeric purity</searchLink><br /><searchLink fieldCode="DE" term="%22Biosynthesis%22">Biosynthesis</searchLink><br /><searchLink fieldCode="DE" term="%22Nicotine%22">Nicotine</searchLink><br /><searchLink fieldCode="DE" term="%22Berberine%22">Berberine</searchLink><br /><searchLink fieldCode="DE" term="%22CRISPRs%22">CRISPRs</searchLink><br /><searchLink fieldCode="DE" term="%22Pyridine%22">Pyridine</searchLink>
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  Data: Summary: The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high‐value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes.Here, we report a new assembly of the N. benthamiana genome as well as the generation of low‐nicotine lines by CRISPR/Cas9‐based inactivation of berberine bridge enzyme‐like proteins (BBLs). Triple as well as quintuple mutants accumulated three to four times less nicotine than the respective control lines.The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis.Our work delivers an improved N. benthamiana chassis for bioproduction and uncovers the crucial role of BBLs in the stereoselectivity of nicotine biosynthesis. [ABSTRACT FROM AUTHOR]
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  Data: <i>Copyright of New Phytologist is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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      – Type: doi
        Value: 10.1111/nph.19141
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      – Code: eng
        Text: English
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        PageCount: 16
        StartPage: 302
    Subjects:
      – SubjectFull: Nicotiana benthamiana
        Type: general
      – SubjectFull: Alkaloids
        Type: general
      – SubjectFull: Enantiomeric purity
        Type: general
      – SubjectFull: Biosynthesis
        Type: general
      – SubjectFull: Nicotine
        Type: general
      – SubjectFull: Berberine
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      – SubjectFull: CRISPRs
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      – SubjectFull: Pyridine
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              Text: Oct2023
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