Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli

Teegan A. Delli-Bovi, Maroya D. Spalding, Sean Taylor Prigge

Research output: Contribution to journalArticle

Abstract

Background: Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin.Results: In this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli.Conclusions: The strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.

Original languageEnglish (US)
Article number73
JournalBMC Biotechnology
Volume10
DOIs
StatePublished - Oct 11 2010

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Ligases
Biotin
Sulfur
Escherichia coli
Biotinylation
Affinity Chromatography
biotin synthetase
Acetyl-CoA Carboxylase
Decarboxylation
Coenzymes
Biosynthetic Pathways
Enzymes
Plasmodium falciparum
Hydrolysis
Genome

ASJC Scopus subject areas

  • Biotechnology

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Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli. / Delli-Bovi, Teegan A.; Spalding, Maroya D.; Prigge, Sean Taylor.

In: BMC Biotechnology, Vol. 10, 73, 11.10.2010.

Research output: Contribution to journalArticle

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