Removing a bottleneck in the Bacillus subtilis biotin pathway: BioA utilizes lysine rather than S-adenosylmethionine as the amino donor in the KAPA-to-DAPA reaction

• Published in: Biotechnology and bioengineering Vol. 91; no. 1; pp. 75 - 83
• Main Authors: Van Arsdell, Scott W., Perkins, John B., Yocum, R. Rogers, Luan, Linda, Howitt, C. Linda, Prasad Chatterjee, Nilu, Pero, Janice G.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 05-07-2005; Wiley; Wiley Subscription Services, Inc
• Subjects: Amino acids; Amino Acids - genetics; Amino Acids - metabolism; Amino Acids, Diamino - genetics; Amino Acids, Diamino - metabolism; Bacillus sphaericus; Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; bioA; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Biotin - biosynthesis; biotin biosynthetic pathway; DAPA aminotransferase; Enzymes; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial - physiology; Lysine - metabolism; metabolic engineering; Metabolism; Mission oriented research; Physiology and metabolism; Protein Engineering - methods; Recombinant Proteins - biosynthesis; S-Adenosylmethionine - metabolism; Serratia marcescens; Signal Transduction - physiology; Transaminases - genetics; Transaminases - metabolism; DAPA aminotransferase; Enzyme; Bacillus subtilis; Transferases; biotin biosynthetic pathway; Biosynthesis; Biotin; Metabolic pathway; Bacillaceae; Bacillales; Lysine; Transaminases; bioA; Bacteria; Metabolic engineering
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.20488

In biotin biosynthesis, DAPA aminotransferase encoded by the bioA gene catalyzes the formation of the intermediate 7,8‐diaminopelargonic acid (DAPA) from 7‐keto‐8‐aminopelargonic acid (KAPA). DAPA aminotransferases from Escherichia coli, Serratia marcescens, and Bacillus sphaericus use S‐adenosylmethionine (SAM) as the amino donor. Our observation that SAM is not an amino donor for B. subtilis DAPA aminotransferase led to a search for an alternative amino donor for this enzyme. Testing of 26 possible amino acids in a cell‐free extract assay revealed that only l‐lysine was able to dramatically stimulate the in vitro conversion of KAPA to DAPA by the B. subtilis DAPA aminotransferase. The Km for lysine and KAPA was estimated to be between 2 and 25 mM, which is significantly higher than the Km of purified E. coli BioA for SAM (0.15 mM). This higher requirement for lysine resulted in accumulation of KAPA during fermentation of B. subtilis biotin producing strains. However, this pathway bottleneck could be relieved by either addition of exogenous lysine to the medium or by introduction of lysine deregulated mutations into the production strains. © 2005 Wiley Periodicals, Inc.