CLONING, SEQUENCING AND CHARACTERIZATION OF A FATTY-ACID SYNTHASE-ENCODING GENE FROM MYCOBACTERIUM-TUBERCULOSIS VAR BOVIS BCG

Mycobacterial cell walls contain unique lipids such as mycolic acids, very long chain fatty acids and multimethyl-branched fatty acids. A mu ltifunctional fatty acid synthase (Fas) with the unique capability of catalyzing both de novo synthesis and chain elongation of fatty acids has been purified and characterized from Mycobacterium tuberculosis va r, bovis BCG (Bacillus Calmette-Geurin) [Kikuchi et al., Arch. Biochem . Biophys. 295 (1992) 318-326]. To understand how the various domains that catalyze the reactions involved in both de novo synthesis and elo ngation are organized in the mycobacteria, a fas gene was cloned from a cosmid library of genomic DNA from M. bovis BCG. Sequencing of the c osmid clone revealed a contiguous sequence of 11577 bp of mycobacteria l genome containing a 8389-bp open reading frame that could code for a protein of 2797 amino acids (301 kDa). By comparing the Fas aa sequen ce with the sequences in the active site regions of known fas and poly ketide synthase-encoding genes, the functional catalytic domains in Fa s were identified. This analysis revealed that the domains are organiz ed in the following order: acyltransferase, enoyl reductase, dehydrata se, malonyl/palmitoyl transferase, acyl carrier protein, beta-keto red uctase, beta-ketoacyl synthase. This domain organization is like a hea d to tail fusion of the two yeast fas gene subunits. The results obtai ned constitute the first report of the cloning, sequencing and structu ral elucidation of a fas from the Mycobacteria.