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

• Main Authors: Prigge Sean T, Spalding Maroya D, Delli-Bovi Teegan A
• Format: Article
• Language: English
• Published: BMC 2010-10-01
• Series: BMC Biotechnology
• Online Access: http://www.biomedcentral.com/1472-6750/10/73

<p>Abstract</p> <p>Background</p> <p>Biotin is an essential enzyme cofactor that acts as a CO₂carrier in carboxylation and decarboxylation reactions. The <it>E. coli </it>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.</p> <p>Results</p> <p>In this study, we produced [³⁵S]-biotin from Na³⁵SO₄and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (<it>Pf</it>BCCP-79) from the <it>Plasmodium falciparum </it>acetyl-CoA carboxylase (ACC) was expressed in <it>E. coli </it>as a biotinylation substrate. We found that overexpression of the <it>E. coli </it>biotin synthase, BioB, and biotin ligase, BirA, increased <it>Pf</it>BCCP-79 biotinylation 160-fold over basal levels. Biotinylated <it>Pf</it>BCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [<it>D</it>]-biotin that specifically labels biotinylated proteins through reuptake in <it>E. coli</it>.</p> <p>Conclusions</p> <p>The strategy described in our report provides a simple and effective method for the production of [³⁵S]-biotin in <it>E. coli </it>based on affinity chromatography.</p>