Mutational Analysis and Reconstituted Expression of the Biosynthetic Genes Involved in the Formation of 3-Amino-5-hydroxybenzoic Acid, the Starter Unit of Rifamycin Biosynthesis in Amycolatopsis mediterranei S699

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

Autoren

  • Tin Wein Yu
  • Rolf Müller
  • Michael Müller
  • Xiaohong Zhang
  • Gerald Draeger
  • Chun Gyu Kim
  • Eckhard Leistner
  • Heinz G. Floss

Organisationseinheiten

Externe Organisationen

  • University of Washington
  • Helmholtz-Zentrum für Infektionsforschung GmbH (HZI)
  • Forschungszentrum Jülich
  • Inje University
  • Rheinische Friedrich-Wilhelms-Universität Bonn
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Details

OriginalspracheEnglisch
Seiten (von - bis)12546-12555
Seitenumfang10
FachzeitschriftJournal of Biological Chemistry
Jahrgang276
Ausgabenummer16
PublikationsstatusVeröffentlicht - 20 Apr. 2001

Abstract

To investigate a novel branch of the shikimate biosynthesis pathway operating in the formation of 3-amino-5-hydroxybenzoic acid (AHBA), the unique biosynthetic precursor of rifamycin and related ansamycins, a series of target-directed mutations and heterologous gene expressions were investigated in Amycolatopsis mediterranei and Streptomyces coelicolor. The genes involved in AHBA formation were inactivated individually, and the resulting mutants were further examined by incubating the cell-free extracts with known intermediates of the pathway and analyzing for AHBA formation. The rifL, -M, and -N genes were shown to be involved in the step(s) from either phosphoenolpyruvate/D-erythrose 4-phosphate or other precursors to 3,4-dideoxy-4-amino-D-arabino-heptulosonate 7-phosphate. The gene products of the rifH, -G, and -J genes resemble enzymes involved in the shikimate biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998) Chem. Biol. 5, 69-79). Mutants of the rifH and -J genes produced rifamycin B at 1% and 10%, respectively, of the yields of the wild type; inactivation of the rifG gene did not affect rifamycin production significantly. Finally, coexpressing the rifG-N and -J genes in S. coelicolor YU105 under the control of the act promoter led to significant production of AHBA in the fermented cultures, confirming that seven of these genes are indeed necessary and sufficient for AHBA formation. The effects of deletion of individual genes from the heterologous expression cassette on AHBA formation duplicated the effects of the genomic rifG-N and -J mutations on rifamycin production, indicating that all these genes encode proteins with catalytic rather than regulatory functions in AHBA formation for rifamycin biosynthesis by A. mediterranei.

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Mutational Analysis and Reconstituted Expression of the Biosynthetic Genes Involved in the Formation of 3-Amino-5-hydroxybenzoic Acid, the Starter Unit of Rifamycin Biosynthesis in Amycolatopsis mediterranei S699. / Yu, Tin Wein; Müller, Rolf; Müller, Michael et al.
in: Journal of Biological Chemistry, Jahrgang 276, Nr. 16, 20.04.2001, S. 12546-12555.

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

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@article{b43326ea3eb241c1819c05e8e58d56d3,
title = "Mutational Analysis and Reconstituted Expression of the Biosynthetic Genes Involved in the Formation of 3-Amino-5-hydroxybenzoic Acid, the Starter Unit of Rifamycin Biosynthesis in Amycolatopsis mediterranei S699",
abstract = "To investigate a novel branch of the shikimate biosynthesis pathway operating in the formation of 3-amino-5-hydroxybenzoic acid (AHBA), the unique biosynthetic precursor of rifamycin and related ansamycins, a series of target-directed mutations and heterologous gene expressions were investigated in Amycolatopsis mediterranei and Streptomyces coelicolor. The genes involved in AHBA formation were inactivated individually, and the resulting mutants were further examined by incubating the cell-free extracts with known intermediates of the pathway and analyzing for AHBA formation. The rifL, -M, and -N genes were shown to be involved in the step(s) from either phosphoenolpyruvate/D-erythrose 4-phosphate or other precursors to 3,4-dideoxy-4-amino-D-arabino-heptulosonate 7-phosphate. The gene products of the rifH, -G, and -J genes resemble enzymes involved in the shikimate biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J., Ning, S., M{\"u}ller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998) Chem. Biol. 5, 69-79). Mutants of the rifH and -J genes produced rifamycin B at 1% and 10%, respectively, of the yields of the wild type; inactivation of the rifG gene did not affect rifamycin production significantly. Finally, coexpressing the rifG-N and -J genes in S. coelicolor YU105 under the control of the act promoter led to significant production of AHBA in the fermented cultures, confirming that seven of these genes are indeed necessary and sufficient for AHBA formation. The effects of deletion of individual genes from the heterologous expression cassette on AHBA formation duplicated the effects of the genomic rifG-N and -J mutations on rifamycin production, indicating that all these genes encode proteins with catalytic rather than regulatory functions in AHBA formation for rifamycin biosynthesis by A. mediterranei.",
author = "Yu, {Tin Wein} and Rolf M{\"u}ller and Michael M{\"u}ller and Xiaohong Zhang and Gerald Draeger and Kim, {Chun Gyu} and Eckhard Leistner and Floss, {Heinz G.}",
note = "Copyright: Copyright 2008 Elsevier B.V., All rights reserved.",
year = "2001",
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TY - JOUR

T1 - Mutational Analysis and Reconstituted Expression of the Biosynthetic Genes Involved in the Formation of 3-Amino-5-hydroxybenzoic Acid, the Starter Unit of Rifamycin Biosynthesis in Amycolatopsis mediterranei S699

AU - Yu, Tin Wein

AU - Müller, Rolf

AU - Müller, Michael

AU - Zhang, Xiaohong

AU - Draeger, Gerald

AU - Kim, Chun Gyu

AU - Leistner, Eckhard

AU - Floss, Heinz G.

N1 - Copyright: Copyright 2008 Elsevier B.V., All rights reserved.

PY - 2001/4/20

Y1 - 2001/4/20

N2 - To investigate a novel branch of the shikimate biosynthesis pathway operating in the formation of 3-amino-5-hydroxybenzoic acid (AHBA), the unique biosynthetic precursor of rifamycin and related ansamycins, a series of target-directed mutations and heterologous gene expressions were investigated in Amycolatopsis mediterranei and Streptomyces coelicolor. The genes involved in AHBA formation were inactivated individually, and the resulting mutants were further examined by incubating the cell-free extracts with known intermediates of the pathway and analyzing for AHBA formation. The rifL, -M, and -N genes were shown to be involved in the step(s) from either phosphoenolpyruvate/D-erythrose 4-phosphate or other precursors to 3,4-dideoxy-4-amino-D-arabino-heptulosonate 7-phosphate. The gene products of the rifH, -G, and -J genes resemble enzymes involved in the shikimate biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998) Chem. Biol. 5, 69-79). Mutants of the rifH and -J genes produced rifamycin B at 1% and 10%, respectively, of the yields of the wild type; inactivation of the rifG gene did not affect rifamycin production significantly. Finally, coexpressing the rifG-N and -J genes in S. coelicolor YU105 under the control of the act promoter led to significant production of AHBA in the fermented cultures, confirming that seven of these genes are indeed necessary and sufficient for AHBA formation. The effects of deletion of individual genes from the heterologous expression cassette on AHBA formation duplicated the effects of the genomic rifG-N and -J mutations on rifamycin production, indicating that all these genes encode proteins with catalytic rather than regulatory functions in AHBA formation for rifamycin biosynthesis by A. mediterranei.

AB - To investigate a novel branch of the shikimate biosynthesis pathway operating in the formation of 3-amino-5-hydroxybenzoic acid (AHBA), the unique biosynthetic precursor of rifamycin and related ansamycins, a series of target-directed mutations and heterologous gene expressions were investigated in Amycolatopsis mediterranei and Streptomyces coelicolor. The genes involved in AHBA formation were inactivated individually, and the resulting mutants were further examined by incubating the cell-free extracts with known intermediates of the pathway and analyzing for AHBA formation. The rifL, -M, and -N genes were shown to be involved in the step(s) from either phosphoenolpyruvate/D-erythrose 4-phosphate or other precursors to 3,4-dideoxy-4-amino-D-arabino-heptulosonate 7-phosphate. The gene products of the rifH, -G, and -J genes resemble enzymes involved in the shikimate biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998) Chem. Biol. 5, 69-79). Mutants of the rifH and -J genes produced rifamycin B at 1% and 10%, respectively, of the yields of the wild type; inactivation of the rifG gene did not affect rifamycin production significantly. Finally, coexpressing the rifG-N and -J genes in S. coelicolor YU105 under the control of the act promoter led to significant production of AHBA in the fermented cultures, confirming that seven of these genes are indeed necessary and sufficient for AHBA formation. The effects of deletion of individual genes from the heterologous expression cassette on AHBA formation duplicated the effects of the genomic rifG-N and -J mutations on rifamycin production, indicating that all these genes encode proteins with catalytic rather than regulatory functions in AHBA formation for rifamycin biosynthesis by A. mediterranei.

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U2 - 10.1074/jbc.M009667200

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