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 mediterraneiS699

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

doi:10.1074/jbc.m009667200

Tin-Wein Yu, Rolf Müller + Show 6 more

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https://doi.org/10.1074/jbc.m009667200

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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.

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF040570, AF040571, and AF335989
To investigate a novel branch of the shikimate biosynthesis pathway operating in the formation of 3-amino-5hydroxybenzoic 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
These three genes are located side by side and immediately upstream of rifK, the AHBA synthase gene. To relate their functions to AHBA biosynthesis, we constructed a mutant of A. mediterranei, HGF009, in which a 2496-bp NotI DNA fragment carrying the C terminus of rifG, TABLE III Characters of A. mediterranei S699-derived rif mutants and their effects on rifamycin productivity

Summary

Introduction

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF040570, AF040571, and AF335989. Based on the structure of the AHBAderived aromatic moiety, this family of compounds can be further subdivided into a benzenic and a naphthalenic subgroup The benzenic ansamycins, such as geldanamycin, ansatrienin A, and ansamitocin (Fig. 1), have been isolated from actinomycetes or higher plants and are mainly cytotoxic agents against eukaryotes (6 –13). Genetic investigations on aromatic amino acid-deficient mutants of Amycolatopsis mediterranei N813 further revealed that the mC7N unit of the rifamycin chromophore must be derived from early intermediates of the shikimate biosynthesis pathway (24 –27). Nitrogen is introduced at the earliest biosynthetic step to form an amino analog of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) Proposed intermediates such as 3,4-dideoxy-4amino-D-arabino-heptulosonate 7-phosphate (aDAHP), 5-deoxy-5-amino-3-dehydroquinate (aDHQ), and 5-deoxy-5-amino3-dehydroshikimate (aDHS) were synthesized and shown to be efficiently converted into AHBA in crude cell-free extracts of the rifamycin B producer, A. mediterranei S699, and the ansatrienin A producer, Streptomyces collinus Tu 1892. The normal shikimate biosynthesis pathway intermediate, DAHP, did not seem to give rise to AHBA under the same conditions, phosphoenolpyruvate plus erythrose 4-phosphate were converted into aDAHP and AHBA, albeit in very low yield

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