Apoplastic expression of the xylanase and β(1–3, 1–4) glucanase domains of the xyn D gene from Ruminococcus flavefaciens leads to functional polypeptides in transgenic tobacco plants

Karin Herbers, Harry J Flint, Uwe Sonnewald

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

We are investigating the possibilities of transgenic plants as bioreactors for the production of industrial enzymes using cell wall-hydrolysing enzymes as first examples. Within the frame work of this work two distinct domains of the xynD gene from Ruminococcus flavefaciens encoding a xylanase (XYLD-A) and a beta(1-3,1-4)glucanase (XYLD-C) were separately cloned into a plant expression vector which would target the proteins into the apoplast. Transgenic tobacco plants were obtained expressing xylan-hydrolysing as well as lichenan-hydrolysing activities. Despite similar steady-state levels of the respective mRNAs xylan hydrolysis rates were between 40 and 170 mu mol min(-1) m(-2) leaf area depending on the transgenic plant while beta(1-3,1-4)glucan degradation was much more effective ranging between 200 and 2000 mu mol min(-1) m(-2). The high activity levels of the XYLD-C expressing plants were reflected on the protein level. XYLD-C accumulated in the intercellular space and was one of the most prominent bands in protein gels. Despite their apoplastic location as confirmed by activity measurements using intercellular fluids the transgenic plants had not undergone any phenotypic alteration.

Original languageEnglish
Pages (from-to)81-87
Number of pages7
JournalMolecular breeding
Volume2
Issue number1
DOIs
Publication statusPublished - 1 Mar 1996

Keywords

  • Ruminococcus flavefaciens
  • xynD gene
  • xylanase
  • beta(1-3,1-4)glucanase
  • lichenan
  • transgenic tobacco
  • primary-cell walls
  • proteins
  • growth

Cite this

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title = "Apoplastic expression of the xylanase and β(1–3, 1–4) glucanase domains of the xyn D gene from Ruminococcus flavefaciens leads to functional polypeptides in transgenic tobacco plants",
abstract = "We are investigating the possibilities of transgenic plants as bioreactors for the production of industrial enzymes using cell wall-hydrolysing enzymes as first examples. Within the frame work of this work two distinct domains of the xynD gene from Ruminococcus flavefaciens encoding a xylanase (XYLD-A) and a beta(1-3,1-4)glucanase (XYLD-C) were separately cloned into a plant expression vector which would target the proteins into the apoplast. Transgenic tobacco plants were obtained expressing xylan-hydrolysing as well as lichenan-hydrolysing activities. Despite similar steady-state levels of the respective mRNAs xylan hydrolysis rates were between 40 and 170 mu mol min(-1) m(-2) leaf area depending on the transgenic plant while beta(1-3,1-4)glucan degradation was much more effective ranging between 200 and 2000 mu mol min(-1) m(-2). The high activity levels of the XYLD-C expressing plants were reflected on the protein level. XYLD-C accumulated in the intercellular space and was one of the most prominent bands in protein gels. Despite their apoplastic location as confirmed by activity measurements using intercellular fluids the transgenic plants had not undergone any phenotypic alteration.",
keywords = "Ruminococcus flavefaciens, xynD gene, xylanase, beta(1-3,1-4)glucanase, lichenan, transgenic tobacco, primary-cell walls, proteins, growth",
author = "Karin Herbers and Flint, {Harry J} and Uwe Sonnewald",
year = "1996",
month = "3",
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doi = "10.1007/BF00171354",
language = "English",
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pages = "81--87",
journal = "Molecular breeding",
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TY - JOUR

T1 - Apoplastic expression of the xylanase and β(1–3, 1–4) glucanase domains of the xyn D gene from Ruminococcus flavefaciens leads to functional polypeptides in transgenic tobacco plants

AU - Herbers, Karin

AU - Flint, Harry J

AU - Sonnewald, Uwe

PY - 1996/3/1

Y1 - 1996/3/1

N2 - We are investigating the possibilities of transgenic plants as bioreactors for the production of industrial enzymes using cell wall-hydrolysing enzymes as first examples. Within the frame work of this work two distinct domains of the xynD gene from Ruminococcus flavefaciens encoding a xylanase (XYLD-A) and a beta(1-3,1-4)glucanase (XYLD-C) were separately cloned into a plant expression vector which would target the proteins into the apoplast. Transgenic tobacco plants were obtained expressing xylan-hydrolysing as well as lichenan-hydrolysing activities. Despite similar steady-state levels of the respective mRNAs xylan hydrolysis rates were between 40 and 170 mu mol min(-1) m(-2) leaf area depending on the transgenic plant while beta(1-3,1-4)glucan degradation was much more effective ranging between 200 and 2000 mu mol min(-1) m(-2). The high activity levels of the XYLD-C expressing plants were reflected on the protein level. XYLD-C accumulated in the intercellular space and was one of the most prominent bands in protein gels. Despite their apoplastic location as confirmed by activity measurements using intercellular fluids the transgenic plants had not undergone any phenotypic alteration.

AB - We are investigating the possibilities of transgenic plants as bioreactors for the production of industrial enzymes using cell wall-hydrolysing enzymes as first examples. Within the frame work of this work two distinct domains of the xynD gene from Ruminococcus flavefaciens encoding a xylanase (XYLD-A) and a beta(1-3,1-4)glucanase (XYLD-C) were separately cloned into a plant expression vector which would target the proteins into the apoplast. Transgenic tobacco plants were obtained expressing xylan-hydrolysing as well as lichenan-hydrolysing activities. Despite similar steady-state levels of the respective mRNAs xylan hydrolysis rates were between 40 and 170 mu mol min(-1) m(-2) leaf area depending on the transgenic plant while beta(1-3,1-4)glucan degradation was much more effective ranging between 200 and 2000 mu mol min(-1) m(-2). The high activity levels of the XYLD-C expressing plants were reflected on the protein level. XYLD-C accumulated in the intercellular space and was one of the most prominent bands in protein gels. Despite their apoplastic location as confirmed by activity measurements using intercellular fluids the transgenic plants had not undergone any phenotypic alteration.

KW - Ruminococcus flavefaciens

KW - xynD gene

KW - xylanase

KW - beta(1-3,1-4)glucanase

KW - lichenan

KW - transgenic tobacco

KW - primary-cell walls

KW - proteins

KW - growth

U2 - 10.1007/BF00171354

DO - 10.1007/BF00171354

M3 - Article

VL - 2

SP - 81

EP - 87

JO - Molecular breeding

JF - Molecular breeding

SN - 1380-3743

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ER -